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    Alcoholic liver disease. Lumeng L,Crabb D W Current opinion in gastroenterology Important mechanisms responsible for alcohol-induced liver injury include mitochondrial damage and loss of ATP, formation of acetaldehyde-and other aldehyde-protein adducts, release of reactive oxygen species (ROS) from mitochondrial electron transfer chain, CYP2E1 , and activated Kupffer cells (KCs); weakening of antioxidant defense systems; and increased intestinal permeability with endotoxemia. Endotoxin interacts with ethanol and/or acetaldehyde, and such interaction leads to a complex cascade of autocrine and paracrine pathways that involve the release of cytokines (proinflammatory, anti-inflammatory, and mutagenic), chemokines, and eicosanoids. These pathways are mediated by activation of KCs, induction of proliferation, and other phenotype changes in hepatic stellate cells (HSCs) leading to transformation to myofibroblasts (the latter is responsible for fibrogenesis, chemotaxis, and contractility, therefore contributing to portal hypertension, angiogenic response, and release of additional cytokines), and stimulation of sinusoidal cells (SECs) to release adhesive molecules and cytokines. Recent data implicate a likely role of apoptosis as a mechanism of hepatocyte cell death in alcoholic liver disease. 10.1097/00001574-200005000-00003
    Long-term expression of fibrogenic cytokines in radiation-induced damage to the internal anal sphincter. Gervaz P,Hennig R,Buechler M,Soravia C,Brigstock D R,Morel Ph,Egger J F,Friess H Swiss surgery = Schweizer Chirurgie = Chirurgie suisse = Chirurgia svizzera BACKGROUND:There is accumulating evidence, both quantitative and qualitative, that pelvic irradiation affects anorectal function. However, the molecular mechanisms responsible for radiation-induced damage to the anal sphincter remain unclear. AIM:To determine the expression of transforming growth factor-beta 1 (TGF-beta 1) and its downstream effector connective tissue growth factor (CTGF) in the anal sphincter of a patient irradiated for prostate cancer. PATIENT:A 82 year-old patient developed a rectal adenocarcinoma and underwent an abdomino-perineal resection (APR), four years after receiving pelvic irradiation for prostate carcinoma. METHODS:Tissue sections of the anal sphincter were processed for histology. Immunostaining for TGF-beta 1 and CTGF were performed. RESULTS:CTGF and TGF-beta 1 immunoreactivity was detected in the irradiated anal sphincter, and was absent in controls. Immunoreactivity for both cytokines predominated in the internal sphincter. CTGF and TGF-beta 1 were preferentially detected in endothelial cells, myofibroblasts and fibroblasts; in addition, there was strong immunoreactivity for TGF-beta 1, but not for CTGF in smooth muscle cells of the anal canal. CONCLUSION:Four years after pelvic irradiation, radiation-induced damage appeared to affect predominantly the smooth muscle layer of the anal canal. The molecular mechanisms responsible for radiation-induced fibrosis to these tissues involve prolonged activation of TGF-beta 1 and its downstream effector CTGF. 10.1024/1023-9332.9.4.193
    Hepatotoxicity of iron overload: mechanisms of iron-induced hepatic fibrogenesis. Ramm Grant A,Ruddell Richard G Seminars in liver disease While iron is a vital requirement for normal cellular physiology, excessive intestinal absorption of iron as seen in hemochromatosis leads to its deposition in parenchymal cells of various organs such as the liver, heart, and pancreas, resulting in cellular toxicity, tissue injury, and organ fibrosis. Cellular injury is induced by iron-generated oxyradicals and peroxidation of lipid membranes. In the liver, lipid peroxidation results in damage to hepatocellular organelles, such as mitochondria and lysosomes, which is thought to contribute to hepatocyte necrosis and apoptosis, and ultimately lead to the development of hepatic fibrogenesis. Hepatic stellate cells are central to the development of hepatic fibrosis, as they can be activated into collagen-producing myofibroblasts. Numerous potential stimuli associated with hepatic iron overload and iron-induced hepatocellular injury have been assessed in an attempt to explain stellate cell transformation in hemochromatosis. Stellate cell activation and fibrosis appear to be regulated by a series of events involving cellular interaction between resident and nonresident cells of the liver, the sequestration of free iron versus the transport and storage of mobilizable iron, and extracellular matrix remodeling as well as intracellular signaling events associated with inflammatory and fibrogenic cytokines. 10.1055/s-2005-923315
    Integrin α1 subunit is up-regulated in colorectal cancer. Boudjadi Salah,Carrier Julie C,Beaulieu Jean-François Biomarker research BACKGROUND:Colorectal cancer remains one of the leading causes of death from cancer in industrialized countries. Integrins are a family of heterodimeric glycoproteins involved in bidirectional cell signaling and participate in the regulation of cell shape, adhesion, migration, differentiation, gene transcription, survival and proliferation. The α1 subunit is known to be involved in RAS/ERK proliferative pathway activation and plays an important role in mammary carcinoma cell proliferation and migration. In the small intestine, α1 is present in the crypt proliferative compartment and absent in the villus, but nothing is known about its expression in the colon mucosa, or in colorectal cancer. RESULTS:In the present study, we demonstrated that in the colon mucosa, α1 is present in the basolateral domain of the proliferative cells of the crypt, and in the surrounding myofibroblasts. We found higher levels of α1 mRNA in 86% of tumours compared to their corresponding matched margin tissues. Immunohistochemical analysis showed that α1 staining was moderate to high in 65% of tumour cells and 97% of the reactive cells surrounding the tumour cells vs 23% of normal epithelial cells. CONCLUSION:Our findings suggest an active role for the α1β1 integrin in colorectal cancer progression. 10.1186/2050-7771-1-16
    Coordinate upregulation of interleukin-8 and growth-related gene product-alpha is present in the colonic mucosa of inflammatory bowel. Imada A,Ina K,Shimada M,Yokoyama T,Yokoyama Y,Nishio Y,Yamaguchi T,Ando T,Kusugami K Scandinavian journal of gastroenterology BACKGROUND:Although ãlpha-chemokines, such as interleukin (IL)-8 and epithelial neutrophil-activating peptide 78, are implicated in the pathogenesis of inflammatory bowel disease (IBD), little information is currently available on the expression and cellular source of growth-related gene product-alpha (GROalpha) and its functional relationship to other ãlpha-chemokines in the intestinal mucosa of patients with IBD. METHODS:The contents of IL-8 and GROalpha in organ cultures, the expression of IL-8 and GROalpha mRNA, and the modulatory effects of inflammatory mediators on IL-8 and GROalpha-producing cells were examined using colonic mucosal tissues. In vitro stimulatory effects of IL-8 and GROalpha on neutrophils were investigated in terms of chemotactic migration and superoxide anion generation. RESULTS:The contents of IL-8 and GROalpha in organ cultures were elevated in patients with IBD, especially in those with active ulcerative colitis (UC). Both IL-8 and GROalpha contents increased according to an increase in histological disease activity in patients with UC, but not in those with Crohn disease. In contrast, no significant correlation was found between the contents of these alpha-chemokines and clinical disease activity. In situ hybridization detected increased expression of IL-8 and GROalpha mRNA in macrophages, pericrypt myofibroblasts, and the epithelium of tissue specimens with active lesions of IBD. The secretion of IL-8 and GROalpha from macrophages and myofibroblasts obtained from control patients was upregulated by inflammatory cytokines and bacterial products. The concentrations of recombinant (r)-IL-8, which covered the levels of activity detected in individual organ cultures or cell cultures of fractionated mucosal cells, could induce chemotactic migration and superoxide anion generation in neutrophils in vitro, and r-GROalpha had synergistic effects on r-IL-8-induced neutrophil activation. CONCLUSIONS:A coordinate upregulation of IL-8 and GROalpha may be involved in the tissue injury in patients with IBD through their stimulatory effects on neutrophils.
    TGF-beta1 and radiation fibrosis: a master switch and a specific therapeutic target? Martin M,Lefaix J,Delanian S International journal of radiation oncology, biology, physics Radiation fibrosis is a frequent sequel of therapeutic or accidental radiation overexposure in normal human tissues. One of the main fundamental problems yet unsolved in fibrotic tissues is the origin of the chronic activation of myofibroblasts within these tissues. It has been postulated that this chronic activation results from a continuous production of activating factors. In this context, fibrosis could be defined as a wound where continuous signals for tissue repair are emitted. Cytokines and growth factors probably play a central role in this process. Among them, transforming growth factor-beta1 (TGF-beta1) is considered as a master switch for the fibrotic program. This review discusses recent evidence on the critical role played by TGF-beta in the initiation, development, and persistence of radiation fibrosis. It summarizes the results concerning this factor after irradiation of various tissues and cells, with an emphasis on superficial fibrosis, including skin and subcutaneous tissues. Finally, recent data concerning the treatment of established fibrotic disorders of various etiology are presented, as well as the possible mechanisms involved in fibrosis regression, which show that the TGF-beta pathway may constitute a specific target for antifibrotic agents. 10.1016/s0360-3016(00)00435-1
    Mesenchymal cells stimulate human intestinal intraepithelial lymphocytes. Roberts A I,Nadler S C,Ebert E C Gastroenterology BACKGROUND & AIMS:Intraepithelial lymphocytes (IELs) from human intestinal mucosa proliferate minimally to T-cell stimuli. Optimal growth may depend on factors that are missing in vitro, such as accessory cells. The aim of this study was to determine whether mesenchymal cells costimulate IELs. METHODS:IELs were isolated from human jejunum and cultured with fibroblasts or smooth muscle cells (mesenchymal cell models for mucosal myofibroblasts) and various T-cell stimuli. Proliferation was determined by [3H]thymidine incorporation, and interleukin 2 (IL-2) production was measured by enzyme-linked immunosorbent assay. Surface molecules were detected by immunofluorescence and flow cytometry. RESULTS:The proliferative responses of IELs to mitogen (phytohemagglutinin), superantigen (staphylococcal enterotoxin B), or anti-CD3 antibody were increased greatly by coculture with mesenchymal cells, while only slightly by peripheral-blood monocytes, the classical antigen-presenting cells. IL-2 production and receptor expression also increased. Mesenchymal cell costimulation of IEL growth required direct contact between the two cell types and was partly dependent on the integrin alpha4beta1 (very late activation 4[VLA-4]) and major histocompatibility complex (MHC) class I, as their respective antibodies blocked the effect. The surface molecules B7 (CD80), CD2, and MHC class II were not involved. CONCLUSIONS:Optimal IEL growth depends on their contact with mesenchymal cells, an interaction that is mediated by VLA-4 and MHC class I. In mucosal immunity, basement membrane myofibroblasts likely serve this role. 10.1016/s0016-5085(97)70089-1
    Human intestinal intraepithelial lymphocytes bind to mucosal mesenchymal cells through VLA4 and CD11A. Ebert E C,Roberts A I Cellular immunology Human intestinal intraepithelial lymphocytes (IEL), predominantly CD8+ T lymphocytes, are uniquely situated at the basolateral surfaces of epithelial cells in contact with the myofibroblasts that comprise the basement membrane. Since mesenchymal cells may anchor IEL in this location and may also serve as antigen-presenting cells, the mechanism of binding to IEL was investigated. Lymphocytes were radiolabeled with [51Cr] sodium chromate, cocultured with mesenchymal cell monolayers, and the nonadherent lymphocytes removed by washes. Those adherent to the monolayers were counted by measuring the amount of radiolabel retained in the well. A large fraction of IL-2-activated IEL bound to KD (lip fibroblast), HISM (jejunal smooth muscle), and JF (jejunal fibroblast) cell lines after a 2-hr incubation: 33 +/- 11, 37 +/-14, and 48 +/- 15%, respectively. When monoclonal antibodies directed at the alpha chains of the very late activation antigens (CD49) were added alone or combined with anti CD11a to assays measuring IEL binding to KD or JF monolayers, the greatest inhibition (33 to 38%) occurred with anti-alpha 4 combined with anti-CD11a. The majority of IEL expressed alpha 1 and alpha 4 before and after a 3-day culture with IL-2, with no change in surface density. VCAM-1, a binding partner to alpha 4, was not expressed on KD or JF cells, and anti-VCAM antibody had no effect on binding. In summary, alpha 4 and CD11a on IEL mediate binding to mesenchymal cells. 10.1006/cimm.1996.0013
    Matrix metalloproteinase pattern in celiac duodenal mucosa. Ciccocioppo Rachele,Di Sabatino Antonio,Bauer Michael,Della Riccia Daniela N,Bizzini Francesca,Biagi Federico,Cifone Maria G,Corazza Gino R,Schuppan Detlef Laboratory investigation; a journal of technical methods and pathology Matrix metalloproteinases (MMPs) are a family of endopeptidases playing a key role in tissue remodelling in both physiological and pathological conditions. Since little information is available about their role in celiac disease (CD), our aims were to quantify their expression/activity and to investigate their relation to proinflammatory cytokines in this condition. Duodenal biopsies from untreated, treated celiac patients and controls were used to quantify the expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-12, MMP-14, their inhibitor TIMP-1, IFN-gamma and TNF-alpha by using real-time reverse transcription-polymerase chain reaction and the gelatin/casein/elastin activities by gel zymography, and to isolate lamina propria mononuclear cells (LPMCs). These cells and myofibroblasts isolated from jejunal specimens were incubated in the absence or presence of IFN-gamma and TNF-alpha. MMP-1 and MMP-12 mRNA levels were significantly increased in active CD compared to treated (P<0.01 and P<0.0005, respectively) and normal mucosa (P<0.01 and P<0.0005, respectively), and this was paralleled by an upregulation of caseinolytic and elastolytic activities. Furthermore, MMP-12 levels significantly (P<0.05) correlated with those of IFN-gamma and the degree of villous flattening. MMP-2 turned out to be significantly (P<0.05) reduced in untreated and treated celiacs compared to controls. In active CD, transcripts of TIMP-1 were higher than in treated and controls (P<0.005 and P<0.05, respectively), such as those of IFN-gamma (P<0.05), whereas TNF-alpha levels were suppressed (P=0.0001). In physiological condition, myofibroblasts represent the main source of MMP-2, whereas LPMCs produce almost all MMPs only after cytokine stimulation. Conversely, cells isolated from active patients constitutively express MMPs without any increase after cytokine stimulation, while those from treated patients are in a resting condition. In conclusion, our results show the presence of a peculiar MMP pattern in active CD strongly dominated by MMP-12, correlating either with IFN-gamma or the degree of mucosal damage. 10.1038/labinvest.3700225
    Glycochenodeoxycholate Promotes Liver Fibrosis in Mice with Hepatocellular Cholestasis. Hohenester Simon,Kanitz Veronika,Kremer Andreas E,Paulusma Coen C,Wimmer Ralf,Kuehn Helen,Denk Gerald,Horst David,Elferink Ronald Oude,Beuers Ulrich Cells Hydrophobic bile salts are considered to promote liver fibrosis in cholestasis. However, evidence for this widely accepted hypothesis remains scarce. In established animal models of cholestasis, e.g., by knockout, cholestasis and fibrosis are both secondary to biliary damage. Therefore, to test the specific contribution of accumulating bile salts to liver fibrosis in cholestatic disease, we applied the unique model of inducible hepatocellular cholestasis in cholate-fed mice. Glycochenodeoxycholate (GCDCA) was supplemented to humanize the murine bile salt pool, as confirmed by HPLC. Biomarkers of cholestasis and liver fibrosis were quantified. Hepatic stellate cells (HSC) isolated from wild-type mice were stimulated with bile salts. Proliferation, cell accumulation, and collagen deposition of HSC were determined. In cholestatic mice, increased hepatic expression of αSMA and collagen1a mRNA and excess hepatic collagen deposition indicated development of liver fibrosis only upon GCDCA supplementation. In vitro, numbers of myofibroblasts and deposition of collagen were increased after incubation with hydrophobic but not hydrophilic bile salts, and associated with EGFR and MEK1/2 activation. We concluded that chronic hepatocellular cholestasis alone, independently of biliary damage, induces liver fibrosis in mice in presence of the human bile salt GCDCA. Bile salts may have direct pro-fibrotic effects on HSC, putatively involving EGFR and MEK1/2 signaling. 10.3390/cells9020281
    Urokinase plasminogen activator receptor on invasive cancer cells: a prognostic factor in distal gastric adenocarcinoma. Alpízar-Alpízar Warner,Christensen Ib Jarle,Santoni-Rugiu Eric,Skarstein Arne,Ovrebo Kjell,Illemann Martin,Laerum Ole Didrik International journal of cancer Gastric cancer is the second cancer causing death worldwide. The five-year survival for this malignancy is below 25% and few parameters have shown an impact on the prognosis of the disease. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micrometastasis and poor prognosis. Using immunohistochemistry, the prognostic significance of uPAR was evaluated in tissue samples from a retrospective series of 95 gastric cancer patients. uPAR was expressed by neoplastic cells, macrophages, myofibroblasts and neutrophils in both intestinal and diffuse subtypes. No association was demonstrated between the expression of uPAR on cancer cells and histological subtype (p = 0.64) or TNM stage (p = 0.75). Univariate analysis revealed a significant association between the expression of uPAR on tumor cells in the peripheral invasion zone and overall survival of gastric cancer patients (HR = 2.16; 95% CI: 1.13-4.14; p = 0.02). Multivariate analysis showed that uPAR immunoreactivity in cancer cells at the invasive front is an independent prognostic factor for overall survival in gastric cancer (HR = 2.39; 95% CI: 1.22-4.69; p = 0.011). In consequence, scoring of uPAR-positive cancer cells may be a direct measure for the invasive potential of gastric adenocarcinomas. 10.1002/ijc.26417
    Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1. Jaeger Emma,Leedham Simon,Lewis Annabelle,Segditsas Stefania,Becker Martin,Cuadrado Pedro Rodenas,Davis Hayley,Kaur Kulvinder,Heinimann Karl,Howarth Kimberley, ,East James,Taylor Jenny,Thomas Huw,Tomlinson Ian Nature genetics Hereditary mixed polyposis syndrome (HMPS) is characterized by apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma occurring in a high proportion of affected individuals. Here, we use genetic mapping, copy-number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a duplication spanning the 3' end of the SCG5 gene and a region upstream of the GREM1 locus. This unusual mutation is associated with increased allele-specific GREM1 expression. Whereas GREM1 is expressed in intestinal subepithelial myofibroblasts in controls, GREM1 is predominantly expressed in the epithelium of the large bowel in individuals with HMPS. The HMPS duplication contains predicted enhancer elements; some of these interact with the GREM1 promoter and can drive gene expression in vitro. Increased GREM1 expression is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel. 10.1038/ng.2263
    Alterations of mesenchymal and endothelial cells in inflammatory bowel diseases. Fiocchi Claudio,Ina Kenji,Danese Silvio,Leite André Z A,Vogel Jon D Advances in experimental medicine and biology The pathogenesis of complex chronic diseases like inflammatory bowel disease (IBD) can no longer be viewed as a one-way street in which classical immune cells have exclusive control over the initiation, duration and outcome of the disease. There is enough experimental evidence to demonstrate that nonimmune cells, among which are mucosal mesenchymal and endothelial cells, also play a decisive role by interacting with immune cells and establishing a two-way reciprocal exchange of signals and responses that dictate the ultimate outcome of inflammation. Smooth muscle cells and fibroblasts/myofibroblasts display a variety of immune functions and modulate the activity and survival of T-cells. Mucosal microvascular cells, through the expression of adhesion molecules and secretion of chemokines, regulate the quantity and quality of leukocytes transmigrating into the interstitial space. A number of receptor-ligand pairs are expressed by immune and nonimmune cells that control their functional interplay, but the CD40/CD40 ligand system may be the most effective because CD40 is expressed by activated muscle and endothelial cells, while the CD40 ligand is expressed by activated T-cells and platelets. The activation of this system in IBD can lead to the establishment of a continuous cycle of nonimmune cell-dependent, antigen-independent interactions that perpetuates gut inflammation. 10.1007/0-387-33778-4_11
    Dietary n-3 polyunsaturated fatty acids reduce disease and colonic proinflammatory cytokines in a mouse model of colitis. Whiting Christine V,Bland Paul W,Tarlton John F Inflammatory bowel diseases BACKGROUND:n-3 polyunsaturated fatty acids (PUFAs) reduce the severity of chronic inflammatory bowel disease, probably by means of reduction of immune cell activation or enhancement of the epithelial barrier. Using the severe combined immunodeficient (SCID) mouse model of colitis, this study examined the effect of dietary n-3 PUFAs on development of colitis and on immunologic, epithelial, and matrix parameters in the intestines of control and colitic animals. METHODS:SCID mice were fed n-3-enriched or control diet for 3 weeks before colitis induction by transplantation of CD45RB T cells and maintained on the same diet for 4 to 8 weeks. Phenotype of infiltrating cells, epithelial ZO-1 protein, and mucosal type I collagen were assessed by immunohistology and tissue cytokines by ELISA. RESULTS:Transplanted n-3-fed animals had significantly reduced pathology scores, colonic tumor necrosis factor-alpha, interleukin-12, and interleukin-1beta compared with animals fed standard diet. Proinflammatory cytokines were reduced despite a similar level of immune cell infiltration by T cells, CD11c cells, and CD11b cells. Neutrophil infiltration was significantly reduced in n-3-fed control and colitic mice, and other myeloid populations were reduced in mice on the n-3 diet. Epithelial ZO-1 expression was increased, and myofibroblast activation significantly decreased in transplanted n-3-fed animals compared with standard diet mice. Submucosal collagen synthesis was enhanced in n-3-fed mice. CONCLUSIONS:Dietary n-3 PUFAs reduced clinical colitis and colonic immunopathology in this model of colonic inflammation by decreasing proinflammatory cytokine synthesis, reducing myeloid cell recruitment and activation, and enhancing epithelial barrier function and mucosal wound healing mechanisms. 10.1097/01.mib.0000164016.98913.7c
    Insulin-like growth factor binding protein 5 enhances survival of LX2 human hepatic stellate cells. Sokolović Aleksandar,Sokolović Milka,Boers Willem,Elferink Ronald Pj Oude,Bosma Piter J Fibrogenesis & tissue repair BACKGROUND:Expression of insulin-like growth factor binding protein 5 (IGFBP5) is strongly induced upon activation of hepatic stellate cells and their transdifferentiation into myofibroblasts in vitro. This was confirmed in vivo in an animal model of liver fibrosis. Since IGFBP5 has been shown to promote fibrosis in other tissues, the aim of this study was to investigate its role in the progression of liver fibrosis. METHODS:The effect of IGFBP5 was studied in LX2 cells, a model for partially activated hepatic stellate cells, and in human primary liver myofibroblasts. IGFBP5 signalling was modulated by the addition of recombinant protein, by lentiviral overexpression, and by siRNA mediated silencing. Furthermore, the addition of IGF1 and silencing of the IGF1R was used to investigate the role of the IGF-axis in IGFBP5 mediated effects. RESULTS:IGFBP5 enhanced the survival of LX2 cells and myofibroblasts via a >50% suppression of apoptosis. This effect of IGFBP5 was not modulated by the addition of IGF1, nor by silencing of the IGF1R. Additionally, IGFBP5 was able to enhance the expression of established pro-fibrotic markers, such as collagen Ialpha1, TIMP1 and MMP1. CONCLUSION:IGFBP5 enhances the survival of (partially) activated hepatic stellate cells and myofibroblasts by lowering apoptosis via an IGF1-independent mechanism, and enhances the expression of profibrotic genes. Its lowered expression may, therefore, reduce the progression of liver fibrosis. 10.1186/1755-1536-3-3
    Neuroimmunophysiology of the gastrointestinal mucosa: implications for inflammatory diseases. Powell D W Transactions of the American Clinical and Climatological Association In conclusion, studies of the neuroimmunophysiology of the intestinal mucosa of the past 5-8 years have demonstrated an important role for the immune system in modulating water and electrolyte transport as well as intestinal motility in the gut. Activation of mast cells and phagocytes leads to heightened Cl- and water secretion, as well as changes in intestinal motility which leads to diarrheal states. These diarrheal responses are self-protective; they rid the intestine of offending microorganisms and antigens. Our investigation of this response has uncovered a new immune accessory cell Cz, the intestinal myofibroblast. This cell seems to play an important role in amplifying the immune signal. This cell is probably also important for the secretion of growth factors onto the epithelium and also the secretion of collagen which results in fibrosis under diseased states. These intestinal myofibroblasts are prolific prostaglandin producers, an important finding because prostaglandin synthesis inhibition has been shown to decrease the development of neoplasia in the gut. Thus, these intestinal myofibroblasts may have other important roles in addition to just modulating water and electrolyte secretion or gut motility. Our laboratory is now engaged in studying these intestinal myofibroblasts in some detail hoping to better understand the biology of these interesting cells.
    Transplantation of enteric neural stem/progenitor cells into the irradiated young mouse hippocampus. Osman Ahmed M,Zhou Kai,Zhu Changlian,Blomgren Klas Cell transplantation Radiotherapy is an effective treatment for brain tumors but often results in cognitive deficits in survivors. Transplantation of embryonic or brain-derived neural stem/progenitor cells (BNSPCs) ameliorated cognitive impairment after irradiation (IR) in animal models. However, such an approach in patients requires a clinically relevant source of cells. We show for the first time the utilization of enteric neural stem/progenitor cells (ENSPCs) from the postnatal intestinal wall as a source of autologous cells for brain repair after injury caused by IR. Cells were isolated from the intestinal wall and propagated in vitro for 1 week. Differentiation assays showed that ENSPCs are multipotent and generated neurons, astrocytes, and myofibroblasts. To investigate whether ENSPCs can be used in vivo, postnatal day 9 mice were subjected to a single moderate irradiation dose (6 or 8 Gy). Twelve days later, mice received an intrahippocampal injection of syngeneic ENSPCs. Four weeks after transplantation, 0.5% and 1% of grafted ENSPCs were detected in the dentate gyrus of sham and irradiated animals, respectively, and only 0.1% was detected after 16 weeks. Grafted ENSPCs remained undifferentiated but failed to restore IR-induced loss of BNSPCs and the subsequent impaired growth of the dentate gyrus. We observed microglia activation, astrogliosis, and loss of granule neurons associated with grafted ENSPC clusters. Transplantation of ENSPCs did not ameliorate IR-induced impaired learning and memory. In summary, while autologous ENSPC grafting to the brain worked technically, even in the absence of immunosuppression, the protocols need to be modified to improve survival and integration. 10.3727/096368913X674648
    Novel ent-Kaurane Diterpenoid from Rubus corchorifolius L. f. Inhibits Human Colon Cancer Cell Growth via Inducing Cell Cycle Arrest and Apoptosis. Chen Xuexiang,Wu Xian,Ouyang Wen,Gu Min,Gao Zili,Song Mingyue,Chen Yunjiao,Lin Yanyin,Cao Yong,Xiao Hang Journal of agricultural and food chemistry The tender leaves of Rubus corchorifolius L. f. have been consumed as tea for drinking in China since ancient times. In this study, a novel ent-kaurane diterpenoid was isolated and identified from R. corchorifolius L. f. leaves as ent-kaur-2-one-16β,17-dihydroxy-acetone-ketal (DEK). DEK suppressed the growth of HCT116 human colon cancer cells with an IC value of 40 ± 0.21 μM, while it did not cause significant growth inhibition on CCD-18Co human colonic myofibroblasts at up to100 μM. Moreover, DEK induced extensive apoptosis and S phase cell cycle arrest in the colon cancer cells. Accordingly, DEK caused profound effects on multiple signaling proteins associated with cell proliferation, cell death, and inflammation. DEK significantly upregulated the expression levels of pro-apoptotic proteins such as cleaved caspase-3, cleaved caspase-9, cleaved PARP, p53, Bax, and tumor suppressor p21, downregulated the levels of cell cycle regulating proteins such as cyclinD1, CDK2, and CDK4 and carcinogenic proteins such as EGFR and COX-2, and suppressed the activation of Akt. Overall, our results provide a basis for using DEK as a potential chemopreventive agent against colon carcinogenesis. 10.1021/acs.jafc.6b05376
    Keratinocyte growth factor-2 (FGF-10) promotes healing of experimental small intestinal ulceration in rats. Han D S,Li F,Holt L,Connolly K,Hubert M,Miceli R,Okoye Z,Santiago G,Windle K,Wong E,Sartor R B American journal of physiology. Gastrointestinal and liver physiology Keratinocyte growth factor-2 (KGF-2, repifermin) is a homolog of KGF-1 with epithelial mitogenic activities. We investigated the therapeutic role of KGF-2 in intestinal ulceration and its mechanisms of protection. KGF-2 (0.3-5 mg/kg) was administered before or after induction of small intestinal ulceration by indomethacin (Indo) in prevention and treatment protocols. In acute studies, KGF-2 was injected for up to 7 days before or daily for 5 days after Indo. In a 15-day chronic study, KGF-2 was injected intravenously daily beginning before or 7 days after Indo. Injury was evaluated by blinded macroscopic and microscopic inflammatory scores, epithelial BrdU staining, tissue IL-1beta, PGE(2), and hydroxyproline concentrations, and collagen type I RNA expression. In vitro effects of KGF-2 were evaluated by epithelial cellular proliferation, restitution of wounded monolayers, PGE(2) secretion, and expression of COX-2 and collagen mRNA. Intravenous KGF-2 significantly decreased acute intestinal injury by all parameters and significantly decreased chronic ulceration. Pretreatment, daily infusion, and delayed treatment were effective. KGF-2 promoted in vitro epithelial restitution with only modest effects on epithelial cell proliferation, stimulated COX-2 expression in cultured epithelial cells, and upregulated in vitro and in vivo PGE(2) production. KGF-2 did not affect in vivo fibrosis, although it induced collagen expression in cultured intestinal myofibroblasts. These results suggest that KGF-2 inhibits intestinal inflammation by stimulating epithelial restitution and protective PGs. 10.1152/ajpgi.2000.279.5.G1011
    Pathophysiology of Liver Fibrosis. Pinzani Massimo Digestive diseases (Basel, Switzerland) Progressive accumulation of fibrillar extracellular matrix (ECM) in the liver is the consequence of reiterated liver tissue damage due to infective (mostly hepatitis B and C viruses), toxic/drug-induced, metabolic and autoimmune causes, and the relative chronic activation of the wound-healing reaction. The process may result in clinically evident liver cirrhosis and hepatic failure. Although cirrhosis is the common result of progressive fibrogenesis, there are distinct patterns of fibrotic development related to the underlying disorders causing the fibrosis. These different patterns of fibrogenic evolution are related to different factors and particularly: (1) the topographic localization of tissue damage, (2) the relative concentration of profibrogenic factors and (3) the prevalent profibrogenic mechanism(s). The mechanisms responsible for the fibrogenic evolution of chronic liver diseases can be summarized in three main groups: chronic activation of the wound-healing reaction, oxidative stress-related molecular mechanisms, and the derangement of the so-called 'epithelial-mesenchymal' interaction leading to the generation of reactive cholangiocytes and peribiliary fibrosis. Most of the knowledge on the cell and molecular biology of hepatic fibrosis derives from in vitro studies employing culture of activated hepatic stellate cells isolated from rat, mouse or human liver. It is now evident that other ECM-producing cells, i.e. fibroblasts and myofibroblasts of the portal tract and circulating 'fibrocytes', are likely to contribute to liver fibrosis. More recently, the attention is progressively shifting to the profibrotic microenvironment of the liver with increasing interest for the role of immune cells and specific subsets of macrophages regulating the progression or the regression of fibrosis, the role of intestinal microbiota and the influence of tissue stiffness. Other major areas of development include the role of tissue hypoxia and the establishment of an anaerobic proinflammatory environment and the influence of epigenetic modification in conditioning the progression of fibrosis. 10.1159/000374096
    Ro60 Inhibits Colonic Inflammation and Fibrosis in a Mouse Model of Dextran Sulfate Sodium-Induced Colitis. Kim Se-Young,Park Min-Jung,Kwon Jeong-Eun,Choi Si-Young,Seo Hyeon-Beom,Jung Kyung Ah,Choi Jeong-Won,Baek Jin-Ah,Lee Han Hee,Lee Bo-In,Park Sung-Hwan,Cho Mi-La Immunology letters Inflammatory bowel disease (IBD) is caused by chronic inflammation of the gastrointestinal tract. The pathogenesis of IBD remains unclear. The inflammation is associated with activation of T helper (Th) lymphocytes and chronic production of inflammatory cytokines. Ro60 suppresses the expression of tumor necrosis factor α, interleukin (IL)-6, and interferon α by inhibiting Alu transcription; control of Ro60 mRNA expression may thus be therapeutically useful. However, few studies have evaluated the anti-inflammatory activity of Ro60. The Ro60 level is decreased in IBD patients; we thus hypothesized that Ro60 was involved in the development of this autoimmune disease. We subjected mice with dextran sodium sulfate (DSS)-induced colitis to gene therapy using a vector that overexpressed Ro60 threefold. We scored IBD progression by repeatedly weighing the mice. Ro60 ameliorated colitis severity and reduced the levels of tumor necrosis factor α, IL-6, IL-17, IL-8, and vascular endothelial growth factor. Ro60 overexpression decreased the levels of α-smooth muscle actin (a marker of activated myofibroblasts) and type I collagen. The anti-inflammatory and anti-fibrotic activities of Ro60 ameliorated the severity of DSS-induced colitis in mice by repressing inflammation, fibrosis, angiogenesis, and the production of reactive oxygen species. 10.1016/j.imlet.2018.11.001
    Expression of IL-24, an activator of the JAK1/STAT3/SOCS3 cascade, is enhanced in inflammatory bowel disease. Andoh Akira,Shioya Makoto,Nishida Atsushi,Bamba Shigeki,Tsujikawa Tomoyuki,Kim-Mitsuyama Shokei,Fujiyama Yoshihide Journal of immunology (Baltimore, Md. : 1950) IL-24 is a member of the IL-10 family of cytokines. In this study, we investigated IL-24 expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for IL-24 expression in human colonic subepithelial myofibroblasts (SEMFs). IL-24 expression in the IBD mucosa was evaluated by immunohistochemical methods. IL-24 mRNA and protein expression was determined by real-time PCR and ELISA, respectively. AP-1 and C/EBP DNA-binding activity and IL-24 promoter activity were assessed by EMSA analysis and a reporter gene assay, respectively. IL-24 mRNA expression was significantly elevated in active lesions from patients who have ulcerative colitis and Crohn's disease. Colonic SEMFs were identified as a major source of IL-24 in the mucosa. IL-1beta, but not IL-17A, TNF-alpha, or IFN-gamma, significantly enhanced IL-24 mRNA and protein expression in isolated colonic SEMFs. The IL-1beta-induced IL-24 mRNA expression was mediated by the activation of the transcription factors, AP-1 and C/EBP-beta. Induction of IL-24 mRNA stabilization was also involved in the effects of IL-1beta. IL-24 induced JAK1/STAT-3 phosphorylation and SOCS3 expression in HT-29 colonic epithelial cells. IL-24 did not modulate the proliferation of HT-29 cells, but significantly increased the mRNA expression of membrane-bound mucins (MUC1, MUC3, and MUC4). IL-24 derived from colonic SEMFs acts on colonic epithelial cells to elicit JAK1/STAT-3 activation and the expression of SOCS3 and mucins, supporting their suppressive effects on mucosal inflammation in IBD. 10.4049/jimmunol.0804169
    Mechanistic roles of epithelial and immune cell signaling during the development of colitis-associated cancer. Subramaniam Renuka,Mizoguchi Atsushi,Mizoguchi Emiko Cancer research frontiers To date, substantial evidence has shown a significant association between inflammatory bowel diseases (IBD) and development of colitis-associated cancer (CAC). The incidence/prevalence of IBD is higher in western countries including the US, Australia, and the UK. Although CAC development is generally characterized by stepwise accumulation of genetic as well as epigenetic changes, precise mechanisms of how chronic inflammation leads to the development of CAC are largely unknown. Preceding intestinal inflammation is one of the major influential factors for CAC tumorigenesis. Mucosal immune responses including activation of aberrant signaling pathways both in innate and adaptive immune cells play a pivotal role in CAC. Tumor progression and metastasis are shaped by a tightly controlled tumor microenvironment which is orchestrated by several immune cells and stromal cells including macrophages, neutrophils, dendritic cells, myeloid derived suppressor cells, T cells, and myofibroblasts. In this article, we will discuss the contributing factors of epithelial as well as immune cell signaling in initiation of CAC tumorigenesis and mucosal immune regulatory factors in the colonic tumor microenvironment. In depth understanding of these factors is necessary to develop novel anti-inflammatory and anti-cancer therapies for CAC in the near future. 10.17980/2016.1
    Collagen XVI in health and disease. Grässel Susanne,Bauer Richard J Matrix biology : journal of the International Society for Matrix Biology Collagen XVI, by structural analogy a member of the FACIT- (fibril-associated collagens with interrupted triple helices) family of collagens, is described as a minor collagen component of connective tissues. Collagen XVI is expressed in various cells and tissues without known occurrence of splice variants or isoforms. For skin and cartilage tissues its suprastructure is known. Presumably, there it acts as an adaptor protein connecting and organizing large fibrillar networks and thus modulates integrity and stability of the extracellular matrix (ECM). Collagen XVI is produced by myofibroblasts in the normal intestine and its synthesis is increased in the inflamed bowel wall where myofibroblasts develop increased numbers of focal adhesion contacts on collagen XVI. Consequently, recruitment of α1 integrin into the focal adhesions at the tip of the cells is induced followed by increased cell spreading on collagen XVI. This presumably adds to the maintenance of myofibroblasts in the inflamed intestinal regions and thus promotes fibrotic responses of the tissue. Notably, α1/α2 integrins interact with collagen XVI through an α1/α2β1 integrin binding site located in the COL 1-3 domains. Collagen XVI may act as a substrate for adhesion and invasion of connective tissue tumor cells. In glioblastoma it induces tumor invasiveness by modification of the β1-integrin activation pattern. Thus, altering the cell-matrix interaction through collagen XVI might be a molecular mechanism to further augment the invasive phenotype of glioma cells. In this line, in oral squamous cell carcinoma collagen XVI expression is induced which results in an upregulation of Kindlin-1 followed by an increased interaction with beta1-integrin. Consequently, collagen XVI induces a proliferative tumor phenotype by promoting an early S-phase entry. In summary, collagen XVI plays a decisive role in the interaction of connective tissue cells with their ECM, which is impaired in pathological situations. Alteration of tissue location and expression level of collagen XVI appears to promote tumorigenesis and to perpetuate inflammatory reactions. 10.1016/j.matbio.2012.11.001
    TLR4 enhances TGF-beta signaling and hepatic fibrosis. Seki Ekihiro,De Minicis Samuele,Osterreicher Christoph H,Kluwe Johannes,Osawa Yosuke,Brenner David A,Schwabe Robert F Nature medicine Hepatic injury is associated with a defective intestinal barrier and increased hepatic exposure to bacterial products. Here we report that the intestinal bacterial microflora and a functional Toll-like receptor 4 (TLR4), but not TLR2, are required for hepatic fibrogenesis. Using Tlr4-chimeric mice and in vivo lipopolysaccharide (LPS) challenge, we demonstrate that quiescent hepatic stellate cells (HSCs), the main precursors for myofibroblasts in the liver, are the predominant target through which TLR4 ligands promote fibrogenesis. In quiescent HSCs, TLR4 activation not only upregulates chemokine secretion and induces chemotaxis of Kupffer cells, but also downregulates the transforming growth factor (TGF)-beta pseudoreceptor Bambi to sensitize HSCs to TGF-beta-induced signals and allow for unrestricted activation by Kupffer cells. LPS-induced Bambi downregulation and sensitization to TGF-beta is mediated by a MyD88-NF-kappaB-dependent pathway. Accordingly, Myd88-deficient mice have decreased hepatic fibrosis. Thus, modulation of TGF-beta signaling by a TLR4-MyD88-NF-kappaB axis provides a novel link between proinflammatory and profibrogenic signals. 10.1038/nm1663
    Knockout of endothelin type B receptor signaling attenuates bleomycin-induced skin sclerosis in mice. Akashi Kengo,Saegusa Jun,Sendo Sho,Nishimura Keisuke,Okano Takuya,Yagi Keiko,Yanagisawa Masashi,Emoto Noriaki,Morinobu Akio Arthritis research & therapy BACKGROUND:Endothelin-1 (ET-1) is important in the pathogenesis of systemic sclerosis (SSc). ET-1 binds two receptors, endothelin type A (ETA) and endothelin type B (ETB). Dual ETA/ETB receptor antagonists and a selective ETA receptor antagonist are used clinically to treat SSc, and the effect of these antagonists on fibroblast activation has been described. However, the role of ETB receptor signaling in fibrogenesis is less clear. This study was conducted to evaluate the profibrotic function of ETB receptor signaling in a murine model of bleomycin (BLM)-induced scleroderma. METHODS:We used ETB receptor-knockout (ETBKO) mice, which are genetically rescued from lethal intestinal aganglionosis by an ETB receptor transgene driven by the human dopamine β-hydroxylase (DβH)-gene promoter, and wild-type mice with DβH-ETB (WT). BLM or phosphate-buffered saline (PBS) was administered subcutaneously by osmotic minipump, and skin fibrosis was assessed by dermal thickness, subcutaneous fat atrophy, and myofibroblast count in the dermis. Dermal fibroblasts isolated from ETBKO and WT mice were cultured in vitro, stimulated with BLM or ET-1, and the expression of profibrotic genes was compared by quantitative PCR. RESULTS:Dermal thickness, subcutaneous fat atrophy, and myofibroblast counts in the dermis were significantly reduced in ETBKO mice compared to WT mice, after BLM treatment. Compared with wild-type, dermal fibroblasts isolated from ETBKO mice showed lower gene expressions of α-smooth muscle actin and collagen 1α1 in response to BLM or ET-1 stimulation in vitro. CONCLUSIONS:ET-1-ETB receptor signaling is involved in skin sclerosis and in collagen synthesis by dermal fibroblasts. 10.1186/s13075-016-1011-4
    Fibrosis of liver, pancreas and intestine: common mechanisms and clear targets? Schuppan D,Koda M,Bauer M,Hahn E G Acta gastro-enterologica Belgica Chronic diseases of the liver, pancreas, intestine, kidneys, skin and lungs are usually accompanied by scarring. Loss of organ function is often progressive despite the use of immunosuppressive, antiviral or antiinflammatory agents. Therefore, well tolerated antifibrotic therapies are urgently needed. The targets for such therapies are activated mesenchymal cells that synthesize an excess of matrix proteins and resemble the myofibroblasts of healing wounds. These cells derive from normally quiescent fibroblasts or smooth muscle cells and from stellate cells of liver and pancreas. Their activation is triggered and maintained by mechanical stress and several fibrogenic modulators and cytokines. Some agents inhibit myofibroblast proliferation and collagen synthesis in vitro, but only few of them are effective in vivo. Potential antifibrotic drugs have been tested mainly in models of liver fibrosis. In the suitable rat model of biliary fibrosis, an antifibrotic effect was demonstrated for silymarin, a defined mixture of flavonoids, and to a lesser degree for pentoxifylline. A spin-off of the large multicenter trials for hepatitis C is the finding that interferon-alpha given for 6-12 months may halt or reverse fibrosis, even in virological non-responders. This has to be proven in prospective randomized trials. Specific inhibitors of the endothelin-A-receptor which are orally available can suppress liver collagen accumulation by 40-60%. Other strategies aim at inhibition of the profibrogenic cytokines TGF-beta or connective tissue growth factor. Effective drug targeting to the fibrogenic liver cells is now possible by use of cyclic peptides that bind to receptors which are specifically upregulated on activated stellate cells. Blockade of such activation receptors can induce stress-relaxation which reverts the fibrogenic cells to a fibrolytic, collagen degrading phenotype. Fibrosis has been discovered as a novel target for the pharmaceutical industry. This implies the use of combinatorial chemistry and an automatized screening machinery, greatly speeding up the design and selection of specific antifibrotic agents. Combined with the rapidly evolving validation of serological markers of fibrogenesis and fibrolysis unforeseen progress in the treatment of organ fibrosis can be expected.
    COMPARATIVE GUT PHYSIOLOGY SYMPOSIUM: Comparative physiology of glucagon-like peptide-2: Implications and applications for production and health of ruminants. Connor E E,Evock-Clover C M,Walker M P,Elsasser T H,Kahl S Journal of animal science Glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide derived from proteolytic cleavage of proglucagon by prohormone convertase 1/3 in enteroendocrine L cells. Studies conducted in humans, in rodent models, and in vitro indicate that GLP-2 is secreted in response to the presence of molecules in the intestinal lumen, including fatty acids, carbohydrates, amino acids, and bile acids, which are detected by luminal chemosensors. The physiological actions of GLP-2 are mediated by its G protein-coupled receptor expressed primarily in the intestinal tract on enteric neurons, enteroendocrine cells, and myofibroblasts. The biological activity of GLP-2 is further regulated by dipeptidyl peptidase IV, which rapidly cleaves the N-terminus of GLP-2 that is responsible for GLP-2 receptor activation. Within the gut, GLP-2 increases nutrient absorption, crypt cell proliferation, and mesenteric blood flow and decreases gut permeability and motility, epithelial cell apoptosis, and inflammation. Outside the gut, GLP-2 reduces bone resorption, can suppress appetite, and is cytoprotective in the lung. Thus, GLP-2 has been studied intensively as a therapeutic to improve intestinal function of humans during parenteral nutrition and following small bowel resection and, more recently, as a treatment for osteoporosis and obesity-related disorders and to reduce cellular damage associated with inflammation of the gut and lungs. Recent studies demonstrate that many biological actions and properties of GLP-2 in ruminants are similar to those in nonruminants, including the potential to reduce intestinal nitro-oxidative stress in calves caused by parasitic diseases such as coccidiosis. Because of its beneficial impacts on nutrient absorption, gut healing, and normal gut development, GLP-2 therapy offers significant opportunities to improve calf health and production efficiency. However, GLP-2 therapies require an extended time course to achieve desired physiological responses, as well as daily administration because of the hormone's short half-life. Thus, practical means of administration and alternative strategies to enhance basal GLP-2 secretion (e.g., through specific feed additives), which are more likely to achieve consumer acceptance, are needed. Opportunities to address these challenges are discussed. 10.2527/jas.2014-8577
    Inducible CD40 expression mediates NFkappaB activation and cytokine secretion in human colonic fibroblasts. Gelbmann C M,Leeb S N,Vogl D,Maendel M,Herfarth H,Schölmerich J,Falk W,Rogler G Gut BACKGROUND:CD40 has been shown to be a functional activation antigen on a variety of cell types involved in immune responses. As intestinal fibroblasts and myofibroblasts may play a role during mucosal inflammation, we investigated the functional consequences of CD40 induction in primary cultures of human colonic fibroblasts. METHODS:Primary colonic lamina propria fibroblasts (PCLF) were isolated from endoscopic biopsies and surgical specimens. Cultures were used between passages 3 and 9. CD40 surface display was determined by FACS analysis and mRNA expression by reverse transcription-polymerase chain reaction. Secretion of cytokines was determined by ELISA. Nuclear factor kappaB (NFkappaB) activation was shown by electrophoretic mobility shift assay (EMSA). RESULTS:After priming with interferon gamma (IFN-gamma) (200 U/ml) for 72 hours, five of eight tested PCLF cultures showed induction of CD40 surface display (up to 10-fold). Induction of CD40 mRNA expression was demonstrated by semiquantitative polymerase chain reaction. In the responder-PCLF cultures, IFN-gamma alone caused a 1.5-5-fold increase in interleukin (IL)-8 secretion. Addition of 1 ng/ml CD40L was sufficient to achieve a further increase in IL-8, IL-6, or monocyte chemotactic protein 1 (MCP-1) secretion (2.5-18-fold of controls). Incubation with CD40L alone without priming with IFN-gamma had no effect. The proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN 100 microM) reduced IFN-gamma/CD40L mediated cytokine induction, suggesting participation of NFkappaB, which was directly demonstrated by EMSA. CD4+ T cells induced MCP-1 secretion by PCLF, which was prevented by addition of an excess of CD40-IgG fusion protein. CD40 expression on PCLF could also be demonstrated in vivo by immunohistochemistry. CONCLUSION:The CD40-CD40L pathway augments mucosal inflammatory responses via mucosal PCLF. CD40-CD40L mediated T cell/PCLF interactions could play an important role during intestinal mucosal inflammation. 10.1136/gut.52.10.1448
    Recombinant Adenosine Deaminase Ameliorates Inflammation, Vascular Disease, and Fibrosis in Preclinical Models of Systemic Sclerosis. Zhang Yun,Zhu Honglin,Layritz Florian,Luo Hui,Wohlfahrt Thomas,Chen Chih-Wei,Soare Alina,Bergmann Christina,Ramming Andreas,Groeber Florian,Reuter Christian,Fornasini GianFranco,Soukhareva Nadejda,Schreiber Brian,Ramamurthy Santosh,Amann Kerstin,Schett Georg,Distler Jörg H W Arthritis & rheumatology (Hoboken, N.J.) OBJECTIVE:Systemic sclerosis (SSc) is characterized by fibrosis, vascular disease, and inflammation. Adenosine signaling plays a central role in fibroblast activation. We undertook this study to evaluate the therapeutic effects of adenosine depletion with PEGylated adenosine deaminase (PEG-ADA) in preclinical models of SSc. METHODS:The effects of PEG-ADA on inflammation, vascular remodeling, and tissue fibrosis were analyzed in Fra-2 mice and in a B10.D2→BALB/c (H-2 ) model of sclerodermatous chronic graft-versus-host disease (GVHD). The effects of PEG-ADA were confirmed in vitro in a human full-thickness skin model. RESULTS:PEG-ADA effectively inhibited myofibroblast differentiation and reduced pulmonary fibrosis by 34.3% (with decreased collagen expression) (P = 0.0079; n = 6), dermal fibrosis by 51.8% (P = 0.0006; n = 6), and intestinal fibrosis by 17.7% (P = 0.0228; n = 6) in Fra-2 mice. Antifibrotic effects of PEG-ADA were also demonstrated in sclerodermatous chronic GVHD (reduced by 38.4%) (P = 0.0063; n = 8), and in a human full-thickness skin model. PEG-ADA treatment decreased inflammation and corrected the M2/Th2/group 2 innate lymphoid cell 2 bias. Moreover, PEG-ADA inhibited proliferation of pulmonary vascular smooth muscle cells (reduced by 40.5%) (P < 0.0001; n = 6), and prevented thickening of the vessel walls (reduced by 39.6%) (P = 0.0028; n = 6) and occlusions of pulmonary arteries (reduced by 63.9%) (P = 0.0147; n = 6). Treatment with PEG-ADA inhibited apoptosis of microvascular endothelial cells (reduced by 65.4%) (P = 0.0001; n = 6) and blunted the capillary rarefication (reduced by 32.5%) (P = 0.0199; n = 6). RNA sequencing demonstrated that treatment with PEG-ADA normalized multiple pathways related to fibrosis, vasculopathy, and inflammation in Fra-2 mice. CONCLUSION:Treatment with PEG-ADA ameliorates the 3 cardinal features of SSc in pharmacologically relevant and well-tolerated doses. These findings may have direct translational implications, as PEG-ADA has already been approved by the Food and Drug Administration for the treatment of patients with ADA-deficient severe combined immunodeficiency disease. 10.1002/art.41259
    Stromal Lkb1 deficiency leads to gastrointestinal tumorigenesis involving the IL-11-JAK/STAT3 pathway. Ollila Saara,Domènech-Moreno Eva,Laajanen Kaisa,Wong Iris Pl,Tripathi Sushil,Pentinmikko Nalle,Gao Yajing,Yan Yan,Niemelä Elina H,Wang Timothy C,Viollet Benoit,Leone Gustavo,Katajisto Pekka,Vaahtomeri Kari,Mäkelä Tomi P The Journal of clinical investigation Germline mutations in the gene encoding tumor suppressor kinase LKB1 lead to gastrointestinal tumorigenesis in Peutz-Jeghers syndrome (PJS) patients and mouse models; however, the cell types and signaling pathways underlying tumor formation are unknown. Here, we demonstrated that mesenchymal progenitor- or stromal fibroblast-specific deletion of Lkb1 results in fully penetrant polyposis in mice. Lineage tracing and immunohistochemical analyses revealed clonal expansion of Lkb1-deficient myofibroblast-like cell foci in the tumor stroma. Loss of Lkb1 in stromal cells was associated with induction of an inflammatory program including IL-11 production and activation of the JAK/STAT3 pathway in tumor epithelia concomitant with proliferation. Importantly, treatment of LKB1-defcient mice with the JAK1/2 inhibitor ruxolitinib dramatically decreased polyposis. These data indicate that IL-11-mediated induction of JAK/STAT3 is critical in gastrointestinal tumorigenesis following Lkb1 mutations and suggest that targeting this pathway has therapeutic potential in Peutz-Jeghers syndrome. 10.1172/JCI93597
    Transgenic interleukin 11 expression causes cross-tissue fibro-inflammation and an inflammatory bowel phenotype in mice. Lim Wei-Wen,Ng Benjamin,Widjaja Anissa,Xie Chen,Su Liping,Ko Nicole,Lim Sze-Yun,Kwek Xiu-Yi,Lim Stella,Cook Stuart Alexander,Schafer Sebastian PloS one Interleukin 11 (IL11) is a profibrotic cytokine, secreted by myofibroblasts and damaged epithelial cells. Smooth muscle cells (SMCs) also secrete IL11 under pathological conditions and express the IL11 receptor. Here we examined the effects of SMC-specific, conditional expression of murine IL11 in a transgenic mouse (Il11SMC). Within days of transgene activation, Il11SMC mice developed loose stools and progressive bleeding and rectal prolapse, which was associated with a 65% mortality by two weeks. The bowel of Il11SMC mice was inflamed, fibrotic and had a thickened wall, which was accompanied by activation of ERK and STAT3. In other organs, including the heart, lung, liver, kidney and skin there was a phenotypic spectrum of fibro-inflammation, together with consistent ERK activation. To investigate further the importance of stromal-derived IL11 in the inflammatory bowel phenotype we used a second model with fibroblast-specific expression of IL11, the Il11Fib mouse. This additional model largely phenocopied the Il11SMC bowel phenotype. These data show that IL11 secretion from the stromal niche is sufficient to drive inflammatory bowel disease in mice. Given that IL11 expression in colonic stromal cells predicts anti-TNF therapy failure in patients with ulcerative colitis or Crohn's disease, we suggest IL11 as a therapeutic target for inflammatory bowel disease. 10.1371/journal.pone.0227505
    Frontiers in glucagon-like peptide-2: multiple actions, multiple mediators. Dubé Philip E,Brubaker Patricia L American journal of physiology. Endocrinology and metabolism Glucagon-like peptide-2 (GLP-2) is a pleiotropic hormone that affects multiple facets of intestinal physiology, including growth, barrier function, digestion, absorption, motility, and blood flow. The mechanisms through which GLP-2 produces these actions are complex, involving unique signaling mechanisms and multiple indirect mediators. As clinical trials have begun for the use of GLP-2 in a variety of intestinal disorders, the elucidation of such mechanisms is vital. The GLP-2 receptor (GLP-2R) is a G protein-coupled receptor, signaling through multiple G proteins to affect the cAMP and mitogen-activated protein kinase pathways, leading to both proliferative and antiapoptotic cellular responses. The GLP-2R also demonstrates unique mechanisms for receptor trafficking. Expression of the GLP-2R in discrete sets of intestinal cells, including endocrine cells, subepithelial myofibroblasts, and enteric neurons, has led to the hypothesis that GLP-2 acts indirectly through multiple mediators to produce its biological effects. Indeed, several studies have now provided important mechanistic data illustrating several of the indirect pathways of GLP-2 action. Thus, insulin-like growth factor I has been demonstrated to be required for GLP-2-induced crypt cell proliferation, likely involving activation of beta-catenin signaling. Furthermore, vasoactive intestinal polypeptide modulates the actions of GLP-2 in models of intestinal inflammation, while keratinocyte growth factor is required for GLP-2-induced colonic mucosal growth and mucin expression. Finally, enteric neural GLP-2R signaling affects intestinal blood flow through a nitric oxide-dependent mechanism. Determining how GLP-2 produces its full range of biological effects, which mediators are involved, and how these mediators interact is a continuing area of active research. 10.1152/ajpendo.00149.2007
    HGF and TSG-6 Released by Mesenchymal Stem Cells Attenuate Colon Radiation-Induced Fibrosis. Usunier Benoît,Brossard Clément,L'Homme Bruno,Linard Christine,Benderitter Marc,Milliat Fabien,Chapel Alain International journal of molecular sciences Fibrosis is a leading cause of death in occidental states. The increasing number of patients with fibrosis requires innovative approaches. Despite the proven beneficial effects of mesenchymal stem cell (MSC) therapy on fibrosis, there is little evidence of their anti-fibrotic effects in colorectal fibrosis. The ability of MSCs to reduce radiation-induced colorectal fibrosis has been studied in vivo in Sprague-Dawley rats. After local radiation exposure, rats were injected with MSCs before an initiation of fibrosis. MSCs mediated a downregulation of fibrogenesis by a control of extra cellular matrix (ECM) turnover. For a better understanding of the mechanisms, we used an in vitro model of irradiated cocultured colorectal fibrosis in the presence of human MSCs. Pro-fibrotic cells in the colon are mainly intestinal fibroblasts and smooth muscle cells. Intestinal fibroblasts and smooth muscle cells were irradiated and cocultured in the presence of unirradiated MSCs. MSCs mediated a decrease in profibrotic gene expression and proteins secretion. Silencing hepatocyte growth factor (HGF) and tumor necrosis factor-stimulated gene 6 (TSG-6) in MSCs confirmed the complementary effects of these two genes. HGF and TSG-6 limited the progression of fibrosis by reducing activation of the smooth muscle cells and myofibroblast. To settle in vivo the contribution of HGF and TSG-6 in MSC-antifibrotic effects, rats were treated with MSCs silenced for HGF or TSG-6. HGF and TSG-6 silencing in transplanted MSCs resulted in a significant increase in ECM deposition in colon. These results emphasize the potential of MSCs to influence the pathophysiology of fibrosis-related diseases, which represent a challenging area for innovative treatments. 10.3390/ijms22041790
    Up-regulation and activation of proteinase-activated receptor 2 in early and delayed radiation injury in the rat intestine: influence of biological activators of proteinase-activated receptor 2. Wang Junru,Zheng Huaien,Hollenberg Morley D,Wijesuriya Suranga J,Ou Xuemei,Hauer-Jensen Martin Radiation research Proteinase-activated receptor 2 (Par2, F2rl1, also designated PAR-2 or PAR2) is prominently expressed in the intestine and has been suggested as a mediator of inflammatory, mitogenic and fibrogenic responses to injury. Mast cell proteinases and pancreatic trypsin, both of which have been shown to affect the intestinal radiation response, are the major biological activators of Par2. Conventional Sprague-Dawley rats, mast cell-deficient rats, and rats in which pancreatic exocrine secretion was blocked pharmacologically by octreotide underwent localized irradiation of a 4-cm loop of small bowel. Radiation injury was assessed 2 weeks after irradiation (early, inflammatory phase) and 26 weeks after irradiation (chronic, fibrotic phase). Par2 expression and activation were assessed by in situ hybridization and immunohistochemistry, using antibodies that distinguished between total (preactivated and activated) Par2 and preactivated Par2. Compared to unirradiated intestine, irradiated intestine exhibited increased Par2 expression, particularly in areas of myofibroblast proliferation and collagen accumulation, after both single-dose and fractionated irradiation. The majority of Par2 expressed in fibrotic areas was activated. Postirradiation Par2 overexpression was greatly attenuated in both mast cell-deficient and octreotide-treated rats. The severity of acute mucosal injury did not affect postirradiation Par2 expression. Mast cells and pancreatic proteinases may exert their fibro-proliferative effects partly through activation of Par2. Par2 may be a potential target for modulating the intestinal radiation response, particularly delayed intestinal wall fibrosis.
    Development of ulcerative colitis and its associated colorectal neoplasia as a model of the organ-specific chronic inflammation-carcinoma sequence. Okayasu Isao Pathology international The organ-specific chronic inflammation-carcinoma sequence was summarized and proposed. As a typical model, the ulcerative colitis (UC)--UC-associated carcinoma sequence was selected, and the mechanism of development of UC and UC-associated carcinoma was reviewed, referring mainly to our data. Intestinal commensal bacteria, including Fusobacterium varium, obtained from the colonic mucosa of UC patients, can enter colonic epithelia and induce secretion of inflammatory cytokines, resulting in early inflammatory lesions consisting of cryptitis and crypt abscess. Inflammatory oxidative stress causes epithelial cell DNA-damage followed by p53 dependent G1 checkpoint activation and overloading, which causes p53 mutation. In long-standing UC, mucosal remodeling, including possible loss of crosstalk between epithelium and stroma may be critical for the development of UC-associated carcinoma, as well as accumulation of early p53 mutation at the crypt level and increase of other stem cell mutated crypts, telomere shortening, and genomic instability of epithelial and stromal cells, including subepithelial myofibroblasts (corresponding to colonic stellate cells or Ito cells) and interstitial cells. Thus, the stochastic (probabilistic) pathway to tumor development over time gains commonalty through chronic inflammation stimulation. For the prevention of cancer development, appropriate anti-inflammatory therapy is important with an accurate assessment of the inflammation status in the colorectum. 10.1111/j.1440-1827.2012.02807.x
    Expression and (Lacking) Internalization of the Cell Surface Receptors of Toxin B. Schöttelndreier Dennis,Seeger Katrin,Grassl Guntram A,Winny Markus R,Lindner Robert,Genth Harald Frontiers in microbiology Toxin-producing strains of and cause infections of the gastrointestinal tract in humans and ruminants, with the toxins being major virulence factors, essential for the infection, and responsible for the onset of severe symptoms. toxin A (TcdA) and toxin B (TcdB), and the large cytotoxin (TpeL) from are single chain bacterial protein toxins with an AB-like toxin structure. The C-terminal delivery domain mediates cell entry of the N-terminal glycosyltransferase domain by receptor-mediated endocytosis. Several cell surface proteins have been proposed to serve as toxin receptors, including chondroitin-sulfate proteoglycan 4 (CSPG4), poliovirus receptor-like 3 (PVRL3), and frizzled-1/2/7 (FZD1/2/7) for TcdB and LDL-receptor-related protein-1 (LRP1) for TpeL. The expression of the TcdB receptors was investigated in human intestinal organoids (HIOs) and in cultured cell lines. HIOs from four human donors exhibited a comparable profile of receptor expression, with PVRL3, LRP1, and FZD7 being expressed and CSPG4 and FZD2 not being expressed. In human epithelial Caco-2 cells and HT29 cells as well as in immortalized murine fibroblasts, either receptor FZD2/7, CSPG4, PVRL3, and LRP1 was expressed. The question whether the toxins take advantage of the normal turnover of their receptors (i.e., constitutive endocytosis and recycling) from the cell surface or whether the toxins activity induce the internalization of their receptors has not yet been addressed. For the analysis of receptor internalization, temperature-induced uptake of biotinylated toxin receptors into immortalized mouse embryonic fibroblasts (MEFs) and Caco-2 cells was exploited. Solely LRP1 exhibited constitutive endocytosis from the plasma membrane to the endosome, which might be abused by TpeL (and possibly TcdB as well) for cell entry. Furthermore, internalization of CSPG4, PVRL3, FZD2, and FZD7 was observed neither in MEFs nor in Caco-2 cells. FZD2/7, CSPG4, and PVRL3 did thus exhibit no constitutive recycling. The presence of TcdB and the p38 activation induced by anisomycin were not able to induce or enhance CSPG4 or PVRL3 uptake in MEFs. In conclusion, FZD2/7, CSPG4, and PVRL3 seem to serve as cell surface binding receptors rather than internalizing receptors of TcdB. 10.3389/fmicb.2018.01483
    Insulin-like growth factor binding protein-5 is a target of matrix metalloproteinase-7: implications for epithelial-mesenchymal signaling. Hemers Elaine,Duval Cedric,McCaig Catherine,Handley Mark,Dockray Graham J,Varro Andrea Cancer research Matrix metalloproteinase-7 (MMP-7) is localized to epithelial cells and is up-regulated in many cancers and in inflammation. We now report that MMP-7 targets a key mesenchymal cell type, the myofibroblast. Recombinant MMP-7 stimulated the proliferation and migration of human colonic myofibroblasts. These responses were partly attributable to activation of other MMPs, notably MMP-3 and MMP-8, and to stimulation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways. Using a proteomic approach, we identified insulin-like growth factor binding protein-5 (IGFBP-5) as a previously unsuspected target of MMP-7 produced by colonic myofibroblasts. We present evidence that the MMP-7 cleavage of IGFBP-5 liberates IGF-II that functions as an autocrine myofibroblast growth factor. Thus, MMP-7 may act as a signal from epithelial cells for local recruitment of myofibroblasts and stimulation of their proliferation. Similar effects of MMP-7 produced in epithelial tumors might account for the expansion of stroma through activation of myofibroblasts. 10.1158/0008-5472.CAN-05-0157
    IL-17 stimulates inflammatory responses via NF-kappaB and MAP kinase pathways in human colonic myofibroblasts. Hata Kazunori,Andoh Akira,Shimada Mitsue,Fujino Sanae,Bamba Shigeki,Araki Yoshio,Okuno Takafumi,Fujiyama Yoshihide,Bamba Tadao American journal of physiology. Gastrointestinal and liver physiology Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-kappaB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-kappaB activation within 45 min after stimulation. A blockade of NF-kappaB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1beta or IL-17 + tumor necrosis factor (TNF)-alpha enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-alpha on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation. 10.1152/ajpgi.00494.2001
    Novel agonist and antagonist radioligands for the GLP-2 receptor. Useful tools for studies of basic GLP-2 receptor pharmacology. British journal of pharmacology BACKGROUND:Glucagon-like peptide-2 (GLP-2) is a pro-glucagon-derived hormone secreted from intestinal enteroendocrine L cells with actions on gut and bones. GLP-2(1-33) is cleaved by DPP-4, forming GLP-2(3-33), having low intrinsic activity and competitive antagonism properties at GLP-2 receptors. We created radioligands based on these two molecules. EXPERIMENTAL APPROACH:The methionine in position 10 of GLP-2(1-33) and GLP-2(3-33) was substituted with tyrosine (M10Y) enabling oxidative iodination, creating [ I]-hGLP-2(1-33,M10Y) and [ I]-hGLP-2(3-33,M10Y). Both were characterized by competition binding, on-and-off-rate determination and receptor activation. Receptor expression was determined by target-tissue autoradiography and immunohistochemistry. KEY RESULTS:Both M10Y-substituted peptides induced cAMP production via the GLP-2 receptor comparable to the wildtype peptides. GLP-2(3-33,M10Y) maintained the antagonistic properties of GLP-2(3-33). However, hGLP-2(1-33,M10Y) had lower arrestin recruitment than hGLP-2(1-33). High affinities for the hGLP-2 receptor were observed using [ I]-hGLP-2(1-33,M10Y) and [ I]-hGLP-2(3-33,M10Y) with K values of 59.3 and 40.6 nM. The latter (with antagonistic properties) had higher B and faster on and off rates compared to the former (full agonist). Both bound the hGLP-1 receptor with low affinity (K of 130 and 330 nM, respectively). Autoradiography in wildtype mice revealed strong labelling of subepithelial myofibroblasts, confirmed by immunohistochemistry using a GLP-2 receptor specific antibody that in turn was confirmed in GLP-2 receptor knock-out mice. CONCLUSION AND IMPLICATIONS:Two new radioligands with different binding kinetics, one a full agonist and the other a weak partial agonist with antagonistic properties were developed and subepithelial myofibroblasts identified as a major site for GLP-2 receptor expression. 10.1111/bph.15766
    The Scientific Rationale for the Introduction of Renalase in the Concept of Cardiac Fibrosis. Frontiers in cardiovascular medicine Cardiac fibrosis represents a redundant accumulation of extracellular matrix proteins, resulting from a cascade of pathophysiological events involved in an ineffective healing response, that eventually leads to heart failure. The pathophysiology of cardiac fibrosis involves various cellular effectors (neutrophils, macrophages, cardiomyocytes, fibroblasts), up-regulation of profibrotic mediators (cytokines, chemokines, and growth factors), and processes where epithelial and endothelial cells undergo mesenchymal transition. Activated fibroblasts and myofibroblasts are the central cellular effectors in cardiac fibrosis, serving as the main source of matrix proteins. The most effective anti-fibrotic strategy will have to incorporate the specific targeting of the diverse cells, pathways, and their cross-talk in the pathogenesis of cardiac fibroproliferation. Additionally, renalase, a novel protein secreted by the kidneys, is identified. Evidence demonstrates its cytoprotective properties, establishing it as a survival element in various organ injuries (heart, kidney, liver, intestines), and as a significant anti-fibrotic factor, owing to its, and demonstrated pleiotropy to alleviate inflammation, oxidative stress, apoptosis, necrosis, and fibrotic responses. Effective anti-fibrotic therapy may seek to exploit renalase's compound effects such as: lessening of the inflammatory cell infiltrate (neutrophils and macrophages), and macrophage polarization (M1 to M2), a decrease in the proinflammatory cytokines/chemokines/reactive species/growth factor release (TNF-α, IL-6, MCP-1, MIP-2, ROS, TGF-β1), an increase in anti-apoptotic factors (Bcl2), and prevention of caspase activation, inflammasome silencing, sirtuins (1 and 3) activation, and mitochondrial protection, suppression of epithelial to mesenchymal transition, a decrease in the pro-fibrotic markers expression ('α-SMA, collagen I, and III, TIMP-1, and fibronectin), and interference with MAPKs signaling network, most likely as a coordinator of pro-fibrotic signals. This review provides the scientific rationale for renalase's scrutiny regarding cardiac fibrosis, and there is great anticipation that these newly identified pathways are set to progress one step further. Although substantial progress has been made, indicating renalase's therapeutic promise, more profound experimental work is required to resolve the accurate underlying mechanisms of renalase, concerning cardiac fibrosis, before any potential translation to clinical investigation. 10.3389/fcvm.2022.845878
    Coeliac Disease Pathogenesis: The Uncertainties of a Well-Known Immune Mediated Disorder. Dunne Margaret R,Byrne Greg,Chirdo Fernando G,Feighery Conleth Frontiers in immunology Coeliac disease is a common small bowel enteropathy arising in genetically predisposed individuals and caused by ingestion of gluten in the diet. Great advances have been made in understanding the role of the adaptive immune system in response to gluten peptides. Despite detailed knowledge of these adaptive immune mechanisms, the complete series of pathogenic events responsible for development of the tissue lesion remains less certain. This review contributes to the field by discussing additional mechanisms which may also contribute to pathogenesis. These include the production of cytokines such as interleukin-15 by intestinal epithelial cells and local antigen presenting cells as a pivotal event in the disease process. A subset of unconventional T cells called gamma/delta T cells are also persistently expanded in the coeliac disease (CD) small intestinal epithelium and recent analysis has shown that these cells contribute to pathogenic inflammation. Other unconventional T cell subsets may play a local immunoregulatory role and require further study. It has also been suggested that, in addition to activation of pathogenic T helper cells by gluten peptides, other peptides may directly interact with the intestinal mucosa, further contributing to the disease process. We also discuss how myofibroblasts, a major source of tissue transglutaminase and metalloproteases, may play a key role in intestinal tissue remodeling. Contribution of each of these factors to pathogenesis is discussed to enhance our view of this complex disorder and to contribute to a wider understanding of chronic immune-mediated disease. 10.3389/fimmu.2020.01374
    Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer. The Journal of pathology Loss-of-function mutations in RNF43 induce activation of Wnt ligand-dependent Wnt/β-catenin signaling through stabilization of the Frizzled receptor, which is often found in microsatellite instability (MSI)-type colorectal cancer (CRC) that develops from sessile serrated adenomas. However, the mechanism underlying how RNF43 mutations promote tumorigenesis remains poorly understood. In this study, we established nine human CRC-derived organoids and found that three organoid lines carried RNF43 frameshift mutations associated with MSI-high and BRAF mutations, suggesting that these CRCs developed through the serrated pathway. RNF43 frameshift mutant organoids required both Wnt ligands and R-spondin for proliferation, indicating that suppression of ZNRF3 and retained RNF43 function by R-spondin are required to achieve an indispensable level of Wnt activation for tumorigenesis. However, active β-catenin levels in RNF43-mutant organoids were lower than those in APC two-hit mutant CRC, suggesting a lower threshold for Wnt activation in CRC that developed through the serrated pathway. Interestingly, transplantation of RNF43-mutant organoids with intestinal myofibroblasts accelerated the β-catenin nuclear accumulation and proliferation of xenograft tumors, indicating a key role of stromal cells in the promotion of the malignant phenotype of RNF43-mutant CRC cells. Sequencing of subcloned organoid cell-expressed transcripts revealed that two organoid lines carried monoallelic RNF43 cis-mutations, with two RNF43 frameshift mutations introduced in the same allele and the wild-type RNF43 allele remaining, while the other organoid line carried two-hit biallelic RNF43 trans-mutations. These results suggest that heterozygous RNF43 frameshift mutations contribute to CRC development via the serrated pathway; however, a second-hit RNF43 mutation may be advantageous in tumorigenesis compared with a single-hit mutation through further activation of Wnt signaling. Finally, treatment with the PORCN inhibitor significantly suppressed RNF43-mutant cell-derived PDX tumor development. These results suggest a novel mechanism underlying RNF43 mutation-associated CRC development and the therapeutic potential of Wnt ligand inhibition against RNF43-mutant CRC. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. 10.1002/path.5868
    Pharmacological mechanisms of chitotriose as a redox regulator in the treatment of rat inflammatory bowel disease. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Although extensive development has been made in the treatment of inflammatory bowel disease (IBD), adverse effects and incomplete efficacy of currently used medications are continuous challenge. Accumulated reports on the benefits of chitosan oligosaccharides in intestinal disorders make chitotriose (COS) a breakthrough in the development of new IBD drugs. This study aimed to investigate the biosafety, efficacy and pharmacological mechanisms of COS in the treatment of experimental IBD in compare with the commercial 5-Aminosalicylic acid (5-ASA). In this study, COS effectively relieved active inflammation, restored epithelial function, and reduced intestinal fibrosis. Further investigation demonstrated that COS treatment regulated redox state of the colon tissue by stimulating the transcription factor nuclear factor E2-related factor 2 (Nrf2), increasing production of endogenous antioxidants, and alleviating oxidative stress. The offset of oxidative stress shut down the nuclear factor kappa-B (NF-ĸB) inflammatory pathway, mitophagy of epithelial cells, M2 macrophage polarization in pre-fibrotic inflammation, and myofibroblast activation in intestinal fibrogenesis. In conclusion, COS is a safe and effective therapeutic agent for experimental IBD as a redox regulator. Our results expand the current understanding of the pharmacology of chitosan oligosaccharides for IBD treatment and provides experimental basis for the medicinal development of small molecule carbohydrates. 10.1016/j.biopha.2022.112988
    Evidence for modulation of pericryptal sheath myofibroblasts in rat descending colon by transforming growth factor beta and angiotensin II. Thiagarajah Jay R,Griffiths Nina M,Pedley Kevin C,Naftalin Richard J BMC gastroenterology BACKGROUND:Absorption of water and Na+ in descending colonic crypts is dependent on the barrier function of the surrounding myofibroblastic pericryptal sheath. Here the effects of high and low Na+ diets and exposure to whole body ionising radiation on the growth and activation of the descending colonic pericryptal myofibroblasts are evaluated. In addition the effect of a post-irradiation treatment with the angiotensin converting enzyme inhibitor Captopril was investigated. METHODS:The levels of Angiotensin II type 1 receptor (AT1), ACE, collagen type IV, transforming growth factor-beta type 1 receptor (TGF-betaR1), OB cadherin and alpha-smooth muscle actin in both descending colon and caecum were evaluated, using immunocytochemistry and confocal microscopy, in rats fed on high and low Na+ diets (LS). These parameters were also determined during 3 months post-irradiation with 8Gy from a 60Co source in the presence and absence of the angiotensin converting enzyme inhibitor, Captopril. RESULTS:Increases in AT1 receptor (135.6% +/- 18.3, P < 0.001); ACE (70.1% +/- 13.1, P < 0.001); collagen type IV (49.6% +/- 15.3, P < 0.001); TGF-+/-beta1 receptors (291.0% +/- 26.5, P < 0.001); OB-cadherin (26.3% +/- 13.8, P < 0.05) and alpha-smooth muscle actin (82.5% +/- 12.4, P < 0.001) were observed in the pericryptal myofibroblasts of the descending colon after LS diet. There are also increases in AT1 receptor and TGF-beta1 receptor, smooth muscle actin and collagen type IV after irradiation. Captopril reduced all these effects of irradiation on the pericryptal sheath and also decreased the amount of collagen and smooth muscle actin in control rats (P < 0.001). CONCLUSIONS:These results demonstrate an activation of descending colonic myofibroblasts to trophic stimuli, or irradiation, which can be attenuated by Captopril, indicative of local trophic control by angiotensin II and TGF-beta release. 10.1186/1471-230x-2-4
    Cell-specific effects of insulin receptor substrate-1 deficiency on normal and IGF-I-mediated colon growth. Simmons J G,Ling Y,Wilkins H,Fuller C R,D'Ercole A J,Fagin James,Lund P K American journal of physiology. Gastrointestinal and liver physiology Insulin-like growth factor I (IGF-I) potently stimulates intestinal growth. Insulin receptor substrate-1 (IRS-1) mediates proliferative and antiapoptotic actions of IGF-I in cell lines, but its in vivo relevance in intestine is not defined. This study tested the hypothesis that there is cell type-specific dependence on IRS-1 as a mediator of IGF-I action. Length, mass, crypt cell proliferation, and apoptosis were measured in small intestine and colon of IRS-1-null mice and wild-type (WT) littermates and in colon of IRS-1-null or WT mice expressing IGF-I transgenes. Expression of IGF-I receptor and signaling intermediates was examined in intestine of WT and IRS-1-null mice, cultured intestinal epithelial cells, and myofibroblasts. Absolute IRS-1 deficiency reduced mucosal mass in jejunum and colon, but effects were more pronounced in colon. Muscularis mass was decreased in both segments. In IGF-I transgenics, IRS-1 deficiency significantly attenuated IGF-I-stimulated growth of colonic mucosa and abolished antiapoptotic but not mitogenic effects of IGF-I transgene on crypt cells. IGF-I-induced muscularis growth was unaffected by IRS-1 deficiency. In intestinal epithelial cells, IRS-1 was expressed at higher levels than IRS-2 and was preferentially activated by IGF-I. In contrast, IGF-I activated both IRS-1 and IRS-2 in intestinal myofibroblasts and IRS-2 activation was upregulated in IRS-1-null myofibroblasts. We conclude that the intestinal epithelium but not the muscularis requires IRS-1 for normal trophic actions of IGF-I and that IRS-1 is required for antiapoptotic but not mitogenic effects of IGF-I in the intestinal crypts in vivo. 10.1152/ajpgi.00537.2006
    IL-13 signaling via IL-13R alpha2 induces major downstream fibrogenic factors mediating fibrosis in chronic TNBS colitis. Fichtner-Feigl Stefan,Young Cheryl A,Kitani Atsushi,Geissler Edward K,Schlitt Hans-Jürgen,Strober Warren Gastroenterology BACKGROUND & AIMS:Previous studies have shown that fibrosis developing in chronic experimental colitis is driven by interleukin (IL)-13 signaling via IL-13R alpha(2) and the production of transforming growth factor (TGF)-beta1. In the present study, we sought to determine the fibrogenic downstream events set in motion by such signaling. METHODS:Experimental colitis with late-onset intestinal fibrosis was induced by weekly intrarectal administration of trinitrobenzene sulfonic acid (TNBS) to BALB/c mice. Blockade of IL-13 signaling via IL-13R alpha(2) and TGF-beta1 signaling was achieved by the administration of small interfering RNA or decoy oligonucleotides that target promoter sequences of signaling components of these receptors. Effects of blockade were determined by enzyme-linked immunosorbent assay or Western blotting detecting specific key fibrogenic factors and by measurement of collagen production. RESULTS:Initially, we showed that abrogation of IL-13 activity via blockade of IL-13R alpha(2) and TGF-beta1 signaling results in severe inhibition of expression of colonic insulin-like growth factor (IGF)-I and early growth response gene (Egr)-1, factors known to initiate and sustain fibrosis. We then showed that Egr-1 was necessary early in the fibrotic process for caspase-mediated apoptosis of myofibroblasts and the production of urokinase plasminogen activator, a protein that enhances TGF-beta1 activation. Finally, we showed that IGF-I (together with TGF-beta1) acts later in the process to stimulate myofibroblasts to deposit collagen in the colon. CONCLUSIONS:These studies establish that IL-13 signaling via the IL-13R alpha(2) is a key initiation point for a complex fibrotic program in the colon consisting of TGF-beta1 activation, IGF-I and Egr-1 expression, myofibroblast apoptosis, and myofibroblast production of collagen. 10.1053/j.gastro.2008.08.055
    Curcumin suppresses p38 mitogen-activated protein kinase activation, reduces IL-1beta and matrix metalloproteinase-3 and enhances IL-10 in the mucosa of children and adults with inflammatory bowel disease. Epstein Jenny,Docena Guillermo,MacDonald Thomas T,Sanderson Ian R The British journal of nutrition Inflammatory bowel disease (IBD) is a major source of morbidity in children and adults. Its incidence is rising, particularly in young people. IBD carries a lifelong risk of cancer, which is proportional to disease duration. Drug and surgical treatments rarely offer cure and often carry a high side effect burden. Dietary therapy is highly effective in Crohn's disease. For these reasons, there is much interest in developing novel dietary treatments in IBD. Curcumin, a component of the spice turmeric, and an anti-inflammatory and anti-cancer agent, shows preclinical and clinical potential in IBD. Its mechanisms of action are unknown. Our aim was to assess the effect of curcumin on key disease mediators p38 mitogen-activated protein kinase (MAPK), IL-1beta, IL-10 and matrix metalloproteinase-3 (MMP-3) in the gut of children and adults with IBD. Colonic mucosal biopsies and colonic myofibroblasts (CMF) from children and adults with active IBD were cultured ex vivo with curcumin. p38 MAPK, NF-kappaB and MMP-3 were measured by immunoblotting. IL-1beta and IL-10 were measured by ELISA. We show reduced p38 MAPK activation in curcumin-treated mucosal biopsies, enhanced IL-10 and reduced IL-1beta. We demonstrate dose-dependent suppression of MMP-3 in CMF with curcumin. We conclude that curcumin, a naturally occurring food substance with no known human toxicity, holds promise as a novel therapy in IBD. 10.1017/S0007114509992510
    IL-1 stimulates intestinal myofibroblast COX gene expression and augments activation of Cl- secretion in T84 cells. Hinterleitner T A,Saada J I,Berschneider H M,Powell D W,Valentich J D The American journal of physiology Because interleukin-1 (IL-1) is an important mediator in the inflamed intestine, its effects on enterocyte-subepithelial myofibroblast (SEMF) interaction were investigated in vitro. Acutely juxtaposing T84 cells with 18Co or P2JF SEMF preincubated with IL-1 alpha significantly enhanced T84 short-circuit current (Isc) responsiveness to secretagogues in comparison to SEMF not activated by IL-1 alpha. The sensitivity of T84 cell Isc to Ca(2+)-dependent, but not adenosine 3',5'-cyclic monophosphate-dependent, secretagogues was augmented by IL-1 alpha-treated SEMF. These effects of IL-1 alpha are directly correlated with SEMF prostaglandin E2 (PGE2) production. Both IL-1 alpha augmentation of Cl secretagogue responsiveness and PGE2 formation were inhibited by IL-1 receptor antagonist. Within 5 h, IL-1 alpha stimulated a 10-fold increase in cyclooxygenase (COX)-2 steady-state mRNA levels in 18Co cells. In contrast, COX-1 message levels increased more slowly to two- to threefold above control levels after 24 h incubation. These results demonstrate that the proinflammatory cytokine IL-1 alpha accentuates intestinal SEMF augmentation of enterocyte responsiveness to Ca(2+)-dependent CI-secretagogues. PGE2 is an important mediator of SEMF-enterocyte interaction. The effects of IL-1 alpha on SEMF PGE2 productions are, at least in part, due to stimulation of COX gene expression. 10.1152/ajpcell.1996.271.4.C1262
    Interleukin-1beta and tumor necrosis factor-alpha upregulate interleukin-23 subunit p19 gene expression in human colonic subepithelial myofibroblasts. Zhang Zhuobin,Andoh Akira,Yasui Hirofumi,Inatomi Osamu,Hata Kazunori,Tsujikawa Tomoyuki,Kitoh Katsuyuki,Takayanagi Atsushi,Shimizu Nobuyoshi,Fujiyama Yoshihide International journal of molecular medicine The recently identified cytokine interleukin-23 (IL-23) consists of p19 and p40 subunits. The major cellular source of IL-23 is dendritic cells and/or macrophages. We investigated the expression of IL-23 p19 mRNA in human colonic subepithelial myofibroblasts (SEMFs). p19 mRNA was not expressed in unstimulated SEMFs, but IL-1beta and TNF-alpha strongly induced p19 mRNA expression in these cells. The effects of IL-1beta were much stronger than those of TNF-alpha. These responses were observed in both a dose- and time-dependent manner. Furthermore, these cytokines acted synergistically when used in combination. A blockade of NF-kappaB activation by the overexpression of a stable form of IkappaBalpha completely blocked these responses, indicating that the induction of p19 mRNA expression by IL-1beta and TNF-alpha was mediated by the NF-kappaB activation pathway. In conclusion, this is the first report demonstrating that IL-23 p19 mRNA is inducible in colonic myofibroblasts by IL-1beta and TNF-alpha. The p19 expression in these cells might play a role in mucosal immune responses.
    Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells. Brun Paola,Pathak Surajit,Castagliuolo Ignazio,Palù Giorgio,Brun Paola,Zuin Matteo,Cavazzana Roberto,Martines Emilio PloS one Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a valid therapeutic approach for liver and gut lesions. 10.1371/journal.pone.0104397
    Extracellular Calcium-Sensing Receptor Inhibition of Intestinal EpithelialTNF Signaling Requires CaSR-Mediated Wnt5a/Ror2 Interaction. Kelly Jacqueline C,Lungchukiet P,Macleod R John Frontiers in physiology Tumor necrosis factor alpha (TNFα) and its receptor TNFR1 play a central role in the development of colitis-associated colon cancer. To understand a role for the extracellular calcium-sensing receptor (CaSR) and its non-canonical Wnt mediators, Wnt5a/Ror2, we used reductionistic systems. We added lipopolysaccharide (LPS) to mouse peritoneal macrophages, RAW264.7 cells, a murine macrophage cell line, and 18Co colonic myofibroblasts, to stimulate TNFα secretion and then activated endogenous CaSR. CaSR activation inhibited TNFα secretion, which in RAW264.7 cells knockdown of CaSR by short-interfering RNA (siRNA) duplex reversed. LPS-stimulated NFκB promoter activity in RAW264.7 cells was inhibited by CaSR activation with Ca(2+) or other polyvalent CaSR agonists. Reducing CaSR expression with siRNA duplex prevented this inhibition. Following LPS addition to CaSR-HEK cells or RAW264.7 macrophages, CaSR stimulation deneddylated Cullin1. Wnt5a added to HT-29 cells which overexpressed Ror2 or T84 monolayers treated with 3 mM Ca(2+) reduced TNFR1 protein expression ∼70%. TNFα/INFγ addition to high resistance T84 monolayers reduced transepithelial resistance 50% within 4 h. CaSR activation (3 mM Ca(2+)) together with rhWnt5a (200 ng/ml) prevented this reduction while Wnt3a addition had no effect. LPS-stimulated TNFα secretion from RAW264.7 cells was not effected by rhWnt5a but increased 10-fold by Wnt3a. Together our results suggest that following LPS challenge, CaSR activation will inhibit NFκB activity and reduce TNFα secretion from macrophages and stroma while Wnt5a/Ror2 engagement on intestinal epithelia reduces TNFR1 expression, allowing TNFα signaling to be titrated. Our results also suggest that canonical Wnt signaling may enhance TLR4 stimulation of TNFα secretion from murine macrophages. 10.3389/fphys.2011.00017
    Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases. Armaka Maria,Apostolaki Maria,Jacques Peggy,Kontoyiannis Dimitris L,Elewaut Dirk,Kollias George The Journal of experimental medicine Tumor necrosis factor (TNF) is key to the pathogenesis of various arthritic diseases and inflammatory bowel disease (IBD). Anti-TNF therapies have proved successful in the clinical treatment of these diseases, but a mechanistic understanding of TNF function is still lacking. We have investigated early cellular mechanisms of TNF function in these diseases using an established TNF transgenic model, which develops a spondyloarthritis-like disease characterized by peripheral joint arthritis, sacroiliitis, enthesitis, and Crohn's-like IBD. Bone marrow grafting experiments demonstrated that development of arthritis requires TNF receptor I (TNFRI) expression in the radiation-resistant compartment, which is also known to be a sufficient target of TNF in the development of Crohn's-like IBD in the same model. Early activation of synovial fibroblasts and intestinal myofibroblasts could also be demonstrated by perturbed expression of matrix metalloproteases and their inhibitors. Notably, selective Cre/loxP-mediated TNFRI expression in mesenchymal cells resulted in a fully arthritic-spondyloarthritic and intestinal phenotype, indicating that mesenchymal cells are primary and sufficient targets of TNF in these pathologies. Our results offer a novel mechanistic perspective for TNF function in gut and joint pathologies and indicate early common cellular pathways that may also explain the often observed synovial-gut axis in human disease. 10.1084/jem.20070906
    MicroRNA-489-3p Represses Hepatic Stellate Cells Activation by Negatively Regulating the JAG1/Notch3 Signaling Pathway. Li Juanjuan,Dong Shouquan,Ye Mingliang,Peng Ganjing,Luo Jie,Wang Chun,Wang Jing,Zhao Qiu,Chang Ying,Wang Hongling Digestive diseases and sciences BACKGROUND:The transformation of hepatic stellate cells (HSCs) into collagen-producing myofibroblasts is a key event in hepatic fibrogenesis. Recent studies have shown that microRNAs (miRNAs) play a critical role in the transformation of HSCs. However, the function of miR-489-3p in liver fibrosis remains unclear. METHODS:Here, we detected the levels of miR-489-3p and jagged canonical Notch ligand 1 (JAG1) in liver fibrosis by using CCl4-treated rats as an in vivo model and transforming growth factor-beta 1 (TGF-β1)-treated HSC cell lines LX-2 and HSC-T6 as in vitro models. The expression of profibrotic markers was affected by transfecting LX-2 cells with either miR-489-3p mimic or si-JAG1. A dual-luciferase reporter assay was carried out to study the interaction of JAG1 with miR-489-3p. RESULTS:We found that miR-489-3p was remarkably decreased while JAG1 was increased in liver fibrosis models both in vivo and in vitro. Overexpression of miR-489-3p reduced the expression of profibrotic markers and the activation of LX-2 cells induced by TGF-β1. Moreover, miR-489-3p decreased the expression of jagged canonical Notch ligand 1 (JAG1) in LX-2 cells by interacting with its 3'-UTR. As JAG1 is a Notch ligand, decreased JAG1 by miR-489-3p inhibited the Notch signaling pathway. Moreover, the downregulation of JAG1 inhibited the expression of fibrotic markers. CONCLUSION:Our results indicate that miR-489-3p can inhibit HSC activation by inhibiting the JAG1/Notch3 signaling pathway. 10.1007/s10620-020-06174-w
    Study of the colonic epithelial-mesenchymal dialogue through establishment of two activated or not mesenchymal cell lines: Activated and resting ones differentially modulate colonocytes in co-culture. PloS one Continuous and rapid renewal of the colonic epithelium is crucial to resist the plethora of luminal deleterious agents. Subepithelial fibroblasts contribute to this turnover by regulating epithelial proliferation and differentiation. However, when intestinal homeostasis is disturbed, fibroblasts can acquire an activated phenotype and play a major role in the progression of intestinal pathologies. To evaluate the involvement of fibroblasts in the regulation of colonocytes under homeostatic or pathological conditions, we established resting and activated conditionally immortalized fibroblast cell lines (nF and mF) from mouse colonic mucosa. We then studied the epithelial-mesenchymal interactions between activated or resting fibroblasts and the normal mouse colonocytes (Co) using a co-culture model. Both fibroblastic cell lines were characterized by RT-qPCR, western blot and immunofluorescence assay. Our results showed that nF and mF cells were positive for fibroblastic markers such as vimentin and collagen 1, and negative for cytokeratin 18 and E-cadherin, attesting to their fibroblastic type. They also expressed proteins characteristic of the epithelial stem cell niche such as Grem1, CD90 or Wnt5a. Only rare nF fibroblasts were positive for α-SMA, whereas all mF fibroblasts strongly expressed this marker, supporting that mF cells were activated fibroblasts/myofibroblasts. In coculture, nF fibroblasts and Co cells strongly interacted via paracrine exchanges resulting in BMP4 production in nF fibroblasts, activation of BMP signaling in Co colonocytes, and decreased growth of colonocytes. Activated-type mF fibroblasts did not exert the same effects on Co cells, allowing colonocytes free to proliferate. In conclusion, these two colonic fibroblast lines, associated with Co cells in coculture, should allow to better understand the role of mesenchymal cells in the preservation of homeostasis and the development of intestinal pathologies. 10.1371/journal.pone.0273858
    Interleukin-1beta and tumor necrosis factor-alpha induce chemokine and matrix metalloproteinase gene expression in human colonic subepithelial myofibroblasts. Okuno T,Andoh A,Bamba S,Araki Y,Fujiyama Y,Fujiyama M,Bamba T Scandinavian journal of gastroenterology BACKGROUND:Colonic subepithelial myofibroblasts may play a role in the inflammatory responses and in extracellular matrix (ECM) metabolism. In this study, we investigated the effects of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha on chemokine (IL-8 and monocyte chemoattractant protein (MCP)-1) and ECM turnover (proliferation of subepithelial myofibroblasts, and secretion of ECM and matrix metalloproteinases (MMPs)) in colonic subepithelial myofibroblasts. METHODS:Human colonic subepithelial myofibroblasts were isolated using the method described by Mahida et al. Chemokine and MMP expressions were determined by ELISA and Northern blotting. Nuclear factor (NF)-kappaB and NF-IL6 DNA binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA). RESULTS:IL-1beta and TNF-alpha did not affect the proliferation of subepithelial myofibroblasts, but stimulated the secretion of types I and IV collagens weakly. Unstimulated subepithelial myofibroblasts secreted a large amount of MMP-2, but a small amount of IL-8, MCP-1 and MMP-1. IL-1beta and TNF-alpha both induced a dose- and time-dependent increase in IL-8, MCP-1 and MMP-1 secretion, and weakly stimulated MMP-2 secretion. IL-1beta and TNF-alpha both rapidly evoked NF-kappaB DNA-binding activity. The inhibition of NF-kappaB activation markedly blocked both IL-1beta- and TNF-alpha-induced IL-8 and MCP-1 mRNA expression, but did not affect MMP-1 mRNA expression. CONCLUSIONS:These observations indicate that chemokine secretion and ECM metabolism are collectively regulated by the proinflammatory cytokines, IL-1beta and TNF-alpha, in colonic subepithelial myofibroblasts. Thus, colonic subepithelial myofibroblasts may play an important role in the pathophysiology of inflammation in the colon. 10.1080/003655202317284228
    Depletion of the colonic epithelial precursor cell compartment upon conditional activation of the hedgehog pathway. van Dop Willemijn A,Uhmann Anja,Wijgerde Mark,Sleddens-Linkels Esther,Heijmans Jarom,Offerhaus G Johan,van den Bergh Weerman Marius A,Boeckxstaens Guy E,Hommes Daan W,Hardwick James C,Hahn Heidi,van den Brink Gijs R Gastroenterology BACKGROUND & AIMS:The intestinal epithelium is a homeostatic system in which differentiated cells are in dynamic equilibrium with rapidly cycling precursor cells. Wnt signaling regulates intestinal epithelial precursor cell fate and proliferation. Homeostatic systems exist by virtue of negative feedback loops, and we have previously identified the Hedgehog (Hh) pathway as a potential negative feedback signal in the colonic epithelium. Indian hedgehog (Ihh) is produced by the differentiated enterocytes and negatively regulates Wnt signaling in intestinal precursor cells. We studied the role of members of the Hh signaling family in the intestine using a conditional genetic approach. METHODS:We inactivated the Hh receptor Patched1 (Ptch1) in adult mice, resulting in constitutive activation of the Hh signaling pathway. Effects on colonic mucosal homeostasis were examined. Colon tissues were examined by immunohistochemistry, in situ hybridization, transmission electron microscopy, and real-time polymerase chain reaction. RESULTS:Ihh but not Sonic hedgehog (Shh) was expressed in colonic epithelium. Expression of Ptch1 and Gli1 was restricted to the mesenchyme. Constitutive activation of Hh signaling resulted in accumulation of myofibroblasts and colonic crypt hypoplasia. A reduction in the number of epithelial precursor cells was observed with premature development into the enterocyte lineage and inhibition of Wnt signaling. Activation of Hh signaling resulted in induction of the expression of bone morphogenetic proteins (Bmp) and increased Bmp signaling in the epithelium. CONCLUSIONS:Hh signaling acts in a negative feedback loop from differentiated cells via the mesenchyme to the colonic epithelial precursor cell compartment in the adult mouse. 10.1053/j.gastro.2009.02.068
    Development, validation and implementation of an in vitro model for the study of metabolic and immune function in normal and inflamed human colonic epithelium. Pedersen Gitte Danish medical journal Ulcerative colitis (UC) and Crohn's disease (CD), collectively referred to as inflammatory bowel disease (IBD), are chronic immune disorders affecting the gastrointestinal tract. The aetiology of IBD remains an enigma, but increasing evidence suggests that the development of IBD may be triggered by a disturbance in the balance between gut commensal bacteria and host response in the intestinal mucosa. It is now known that epithelial cells have the capacity to secrete and respond to a range of immunological mediators and this suggests that these cells play a prominent role in the pathogenesis of IBD. Current knowledge about the intestinal epithelium has mainly been obtained using models based on animal cells, transformed human intestinal cell lines and isolated cells from resected colonic bowel segments. Species difference, malignant origin and confounders related to surgery, obviously make these cell models however less applicable for patophysiological studies. Consequently, there was a clear need for models of representative intestinal epithelial cells that would allow functional and dynamic studies of the differentiated human colonic epithelium in vitro. The primary purpose of this thesis was to explore and validate the optimal conditions for establishing a model based on short-term cultures of human colonic epithelial cells obtained from endoscopical biopsies. The cell cultures were accordingly used to describe the interplay between proinflammatory cytokines and colonic epithelium, with focus on alterations in viability, butyrate metabolism and secretion of a chemokine and metalloproteinases (MMP). Finally, the model was used to characterize expression and activation of receptors like toll like receptor (TLR)9 and peroxisome activated proliferators (PPAR)- known to be important players in regulation of innate and adaptive immune responses in human colonic epithelium. The results showed that it is possible to establish short-term cultures of representative, viable human colonic epithelial cells from endoscopic mucosal biopsies of patients with IBD. Short-time isolation by EGTA/EDTA from colonic biopsies allowed establishment of small scale cultures of epithelial cells which were viable and metabolic active for up to 48 hours in vitro. The cell model preserved important cellular metabolic and immunological functions of the human colonic epithelium, including the ability to oxidate butyrate, detoxificate phenolic compounds and secrete the chemokine interleukin (IL)-8 in vitro. Tumour necrosis factor (TNF)-α and interferon (IFN)-γ are pro-inflammatory cytokines, which are present in increased amounts in inflamed colonic mucosa. The precise mechanisms of cytokine-mediated mucosal injury are unknown, but one might be that TNF-α and IFN-γ directly impair epithelial cell function similar to effects seen on distinct target cells in other autoimmune diseases. Using the model, both cytokines were found directly to impair the viability of colonic epithelial cells and to induce secretion of IL-8 in vitro. Interestingly, the cells from inflamed IBD mucosa were less sensitive to cytokine-induced damage, which suggests that an intrinsic defense mechanism is triggered in these cells, perhaps as a result of exposure to toxic luminal factors or high local cytokine levels in vivo. TNF-α and IFN-γ may also be involved in regulation of intestinal inflammation through stimulation of MMP expression and proteolytic activity. We found that colonic epithelial cells express a range of MMPs and moreover that expression of distinct MMPs is increased in cells from inflamed IBD mucosa. Using a functional peptide cleavage assay it was shown that epithelial cells secreted proteolytic active enzymes and that the functional MMP activity was increased in inflamed IBD mucosa. This suggests that colonic epithelial cells, like myofibroblasts and immune cells, may contribute to local intestinal mucosal damage, through secretion of active MMPs. Disturbance of recognition and discrimination of potentially harmful pathogens from commensals in the intestinal mucosa have increasingly been implicated in the pathogenesis of IBD. Our results revealed that colonic epithelial cells express TLR9, a key pattern recognition receptor. Interestingly, the differentiated epithelial cells, which have been exposed to the luminal bacterial flora in vivo, were unresponsive to TLR9 ligand stimulation, contrasting findings in the epithelial cell line HT-29 that is cultured continuously in bacteria free environment. These findings suggest, theoretically, that colonic epithelium may regulate immune responses to microbial antigens including commensal bacterial DNA through modulation of the TLR9 pathway. Currently, the results are in line with the emerging view, that the epithelium represents an important frontline cellular component of the innate immune system in the gut. PPARγ is a nuclear receptor involved in the regulation of lipid and carbonhydrate metabolism. Recent studies in rodent colitis models suggest that PPARγ also is involved in modulation of inflammatory processes in the colon. Using the model, we characterise expression and activity of PPARs in human colonic epithelium and, additionally, evaluated the functional significance of a possible imbalanced PPARγ regulation in relation to inflammation. Our experiments showed that colonic epithelial cells express PPARγ and furthermore that PPARγ signalling was impaired in inflamed UC epithelium. It was possible to restore PPARγ signalling in the cell cultures by stimulation with rosiglitazone (a synthetic PPARγ ligand) in vitro. Hence, these experiments prompted us to design a small controlled, clinical study exploring the possible stimulatory effects of rosiglitazone (a PPAR ligand) in vivo. Interestingly, it was found that topical application of rosiglitazone in patients with active distal UC reduced clinical activity and mucosal inflammation similar to the effects measured in patients treated with mesalazine enemas. Moreover, rectal application of rosiglitazone induced PPARγ signalling in the epithelium in vivo, supporting the view that activation of PPARγ may be a new potential therapeutic target in the treatment of UC. Overall, the in vitro model of representative human colonic epithelial cells has shown to be a useful technique for detailed studies of metabolic and immunological functions that are important for homeostasis of the colonic epithelium. Currently, the findings support the view that intestinal epithelial cells actively participate in immunological processes in the colonic mucosa. Additionally, the model seems to be applicable for generating and evaluating new therapeutic approaches from laboratory bench to bed line as illustrated by the PPARγ study. It is therefore probable, that studies in models of representative colonic epithelial cells, as the one described here, could contribute with important knowledge about the pathogenesis of human inflammatory colonic diseases also in the future.
    Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development. Uchiyama Kazuhiko,Sakiyama Toshio,Hasebe Takumu,Musch Mark W,Miyoshi Hiroyuki,Nakagawa Yasushi,He Tong-Chuan,Lichtenstein Lev,Naito Yuji,Itoh Yoshito,Yoshikawa Toshikazu,Jabri Bana,Stappenbeck Thaddeus,Chang Eugene B Scientific reports Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. 10.1038/srep32094
    Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts. Andoh Akira,Zhang Zhuobin,Inatomi Osamu,Fujino Sanae,Deguchi Yasuyuki,Araki Yoshio,Tsujikawa Tomoyuki,Kitoh Katsuyuki,Kim-Mitsuyama Shokei,Takayanagi Atsushi,Shimizu Nobuyoshi,Fujiyama Yoshihide Gastroenterology BACKGROUND & AIMS:Interleukin (IL)-22, a member of the IL-10 subfamily, is a recently identified T-cell-derived cytokine. We investigated IL-22 expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD) and analyzed its biologic activities in human colonic subepithelial myofibroblasts (SEMFs). METHODS:Mucosal IL-22 expression was evaluated by immunohistochemical procedures. The effects of IL-22 on colonic SEMFs were investigated by cDNA microarrays, Northern blots, enzyme-linked immunosorbent assay, and electrophoretic gel mobility shift assays (EMSAs). RESULTS:IL-22 was not detectable in normal colonic mucosa. In IBD mucosa, IL-22 expression was detectable in CD4-positive T cells. IL-22-positive cells were increased in ulcerative colitis and even more so in Crohn's disease. IL-22 receptor expression colocalized with a marker of SEMFs. IL-22 did not modulate SEMF proliferation and collagen synthesis. cDNA microarray analyses demonstrated that, in colonic SEMFs, IL-22 increased the messenger RNA (mRNA) expression of inflammatory cytokines (IL-6, IL-8, IL-11, and leukemia inhibitory factor [LIF]), chemokines, and matrix metalloproteinases. IL-22 induced an activation of nuclear factor (NF)-kappaB and activating protein (AP)-1 within 1 hour, and a blockade of NF-kappaB and AP-1 activation markedly reduced IL-22 induction of IL-6, IL-8, IL-11, and LIF mRNA. MAP-kinase inhibitors (PD98059, U0216, and SB202190) significantly reduced IL-22 induction of cytokine secretion. The combination of either IL-17 plus IL-22 or IL-19 plus IL-22 additively up-regulated cytokine secretion. CONCLUSIONS:IL-22 derived from activated T cells acts on SEMFs to elicit expression of proinflammatory cytokines and matrix-degrading molecules indicating proinflammatory/remodeling roles in IBD. 10.1053/j.gastro.2005.06.071
    Inhibitory effects of short-chain fatty acids on matrix metalloproteinase secretion from human colonic subepithelial myofibroblasts. Kawamura Takato,Andoh Akira,Nishida Atsushi,Shioya Makoto,Yagi Yuhki,Nishimura Takashi,Hashimoto Takayoshi,Tsujikawa Tomoyuki,Yasui Hiroyuki,Fujiyama Yoshihide Digestive diseases and sciences BACKGROUND AND AIMS:Short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate, are the major by-product of bacterial fermentation of dietary fiber in the colon. In this report, we investigated how SCFAs modulate matrix metalloproteinase (MMP) secretion from human colonic subepithelial myofibroblasts (SEMFs). MATERIALS AND METHODS:SEMFs were identified by expression of alpha-smooth muscle actin and vimentin. Cytokine-induced MMP-1 and MMP-3 levels were determined by enzyme-linked immunosorbent assay. Cytokine-induced MMP mRNA expression was analyzed by RT-PCR and real-time PCR methods. RESULTS:Acetate had no effect on MMP secretion. Propionate and butyrate significantly attenuated IL-1 beta- and TNF-alpha-induced MMP-1 and MMP-3 secretion. Similar responses were also observed at the mRNA levels. Propionate and butyrate did not modulate IL-1 beta- and TNF-alpha-induced activation of mitogen-activated protein kinases (MAPKs), which play a crucial role in MMP induction. Trichostatin A, a histone-deacetylase inhibitor, reduced IL-1 beta-induced MMP-1 and MMP-3 mRNA expression, and suppressed TNF-alpha-induced MMP-3 mRNA expression. CONCLUSION:SCFAs play an anti-inflammatory role through suppression of MMP secretion in the colon. Inhibitory effects of SCFAs on MMP secretion might be associated with their action of histone hyperacetylation. 10.1007/s10620-008-0348-1
    Interleukin-33 expression is specifically enhanced in inflamed mucosa of ulcerative colitis. Kobori Ayako,Yagi Yuhki,Imaeda Hirotsugu,Ban Hiromitsu,Bamba Shigeki,Tsujikawa Tomoyuki,Saito Yasuharu,Fujiyama Yoshihide,Andoh Akira Journal of gastroenterology BACKGROUND:Interleukin (IL)-33 is a cytokine belonging to the IL-1 family. IL-33 has been shown to elicit a Th2-like cytokine response in immune cells. In this study, we investigated IL-33 expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for IL-33 expression in human colonic subepithelial myofibroblasts (SEMFs). METHODS:IL-33 mRNA expression was determined by real-time polymerase chain reaction (PCR). IL-33 expression in the IBD mucosa was evaluated by immunohistochemical methods. RESULTS:IL-33 mRNA expression was significantly elevated in active lesions from patients with ulcerative colitis (UC), but was not detected in inactive lesions from UC patients or in lesions from patients with either active or inactive Crohn's disease. Colonic SEMFs were identified as a major source of IL-33 in the mucosa. IL-1β and tumor necrosis factor-α (TNF-α) significantly enhanced IL-33 mRNA and protein expression in isolated colonic SEMFs. IL-1β and TNF-α did not affect IL-33 expression in intestinal epithelial cell lines (HT-29 and Caco-2 cells). This IL-1β- and TNF-α-induced IL-33 mRNA expression was mediated by p42/44 mitogen activated protein kinase (MAPK) pathway-dependent activation of nuclear factor (NF)-κB and activator protein (AP)-1. CONCLUSIONS:IL-33, derived from colonic SEMFs, may play an important role in the pathophysiology of UC. 10.1007/s00535-010-0245-1
    Endothelin-1 signaling promotes fibrosis in vitro in a bronchopulmonary dysplasia model by activating the extrinsic coagulation cascade. Kambas Konstantinos,Chrysanthopoulou Akrivi,Kourtzelis Ioannis,Skordala Marianna,Mitroulis Ioannis,Rafail Stavros,Vradelis Stergios,Sigalas Ioannis,Wu You-Qiang,Speletas Matthaios,Kolios George,Ritis Konstantinos Journal of immunology (Baltimore, Md. : 1950) Neonatal respiratory distress syndrome can progress to bronchopulmonary dysplasia (BPD), a serious pulmonary fibrotic disorder. Given the involvement of the extrinsic coagulation cascade in animal models of lung fibrosis, we examined its role in BPD. We observed a higher number of neutrophils expressing tissue factor (TF) in bronchoalveolar lavage fluid (BALF) from infants with BPD than from those with uncomplicated respiratory distress syndrome together with a parallel decrease in TF and connective tissue growth factor (CTGF) in BALF supernatants during the disease course. The involvement of coagulation in the fibrotic process associated with BPD was further evaluated by treating primary human colonic myofibroblasts with BALF supernatants from infants with BPD. These human colonic myofibroblasts demonstrated an enhanced C5a- and thrombin-dependent migration. Moreover, they expressed TF in an endothelin-1-dependent manner, with subsequent activation of the extrinsic coagulation cascade and CTGF production mediated by protease-activator receptor-1 signaling. These data provide a novel mechanism for the development of BPD and indicate that endothelin-1 signaling contributes to fibrosis by upregulating a TF/thrombin amplification loop responsible for CTGF production, and offer novel and specific therapeutic targets for pulmonary fibrotic disease. 10.4049/jimmunol.1003756
    Inflammatory responses induced by interleukin-17 family members in human colonic subepithelial myofibroblasts. Yagi Yuhki,Andoh Akira,Inatomi Osamu,Tsujikawa Tomoyuki,Fujiyama Yoshihide Journal of gastroenterology BACKGROUND:We investigated the potential role of interleukin (IL)-17 family members (IL-17A to IL-17F) in the induction of inflammatory responses in human colonic subepithelial myofibroblasts (SEMFs). METHODS:The expression of the inflammatory cytokines IL-6, IL-8, leukemia inhibitory factor (LIF), and matrix metalloproteinases (MMP)-1 and MMP-3 were evaluated by enzyme-linked immunosorbent assay and Northern blotting. Activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. RESULTS:IL-17A and IL-17F significantly enhanced IL-6, IL-8, LIF, MMP-1, and MMP-3 secretion. The effects of IL-17A were relatively stronger than those induced by IL-17F. The effects of IL-17B, IL-17C, IL-17D, and IL-17E were modest as compared with those induced by IL-17A and IL-17F. Both IL-17A and IL-17F augmented IL-1beta-induced secretion of IL-6, IL-8, LIF, MMP-1, and MMP-3. A similar augmentation was also observed in tumor necrosis factor (TNF)-alpha-induced cytokine and MMP secretion. IL-17A and IL-17F rapidly induced phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, p38 MAPKs, and c-Jun-NH(2)-terminal kinase (JNK) as early as 15 min after stimulation. Inhibitors for ERK (PD98059 and U0216) and p38 MAPK (SB203580) significantly reduced the IL-17F-induced IL-6, IL-8, LIF, MMP-1, and MMP-3 secretion. CONCLUSIONS:Among IL-17 family members, IL-17A and IL-17F strongly stimulate human colonic SEMFs, inducing inflammatory responses. 10.1007/s00535-007-2091-3
    Circulating Fibroblast Activation Protein as Potential Biomarker in Patients With Inflammatory Bowel Disease. Corsi Fabio,Sorrentino Luca,Albasini Sara,Colombo Francesco,Cigognini Maria,Massari Alessandro,Morasso Carlo,Mazzucchelli Serena,Piccotti Francesca,Ardizzone Sandro,Sampietro Gianluca M,Truffi Marta Frontiers in medicine A major concern in the management of Inflammatory Bowel Disease (IBD) is the absence of accurate and specific biomarkers to drive diagnosis and monitor disease status timely and non-invasively. Fibroblast activation protein (FAP) represents a hallmark of IBD bowel strictures, being overexpressed in stenotic intestinal myofibroblasts. The present study aimed at evaluating the potential of circulating FAP (cFAP) as an accessible blood biomarker of IBD. Quantitative determination of cFAP was performed by enzyme-linked immunosorbent assay on plasma samples prospectively collected from patients with IBD and control subjects. A discrimination model was established on a training set of 50% patients and validated on independent samples. Results showed that cFAP concentration was reduced in patients with IBD when compared to controls ( < 0.0001). Age, sex, smoking, disease location and behavior, disease duration and therapy were not associated with cFAP. The sensitivity and specificity of cFAP in discriminating IBD from controls were 70 and 84%, respectively, based on the optimal cutoff (57.6 ng mL, AUC = 0.78). Predictions on the test set had 57% sensitivity, 65% specificity, and 61% accuracy. There was no strong correlation between cFAP and routine inflammatory markers in the patients' population. A subgroup analysis was performed on patients with Crohn's disease undergoing surgery and revealed that cFAP correlates with endoscopic mucosal healing. In conclusion, cFAP deserves attention as a promising blood biomarker to triage patients with suspected IBD. Moreover, it might function as a biomarker of post-operative remission in patients with Crohn's disease. 10.3389/fmed.2021.725726
    Adherent invasive Escherichia coli (AIEC) strain LF82, but not Candida albicans, plays a profibrogenic role in the intestine. Chokr Dina,Cornu Marjorie,Neut Christel,Bortolus Clovis,Charlet Rogatien,Desreumaux Pierre,Speca Silvia,Sendid Boualem Gut pathogens BACKGROUND:Intestinal fibrosis is a frequent complication of Crohn's disease. However, the factors that cause chronicity and promote fibrogenesis are not yet understood. AIMS:In the present study, we evaluated the profibrotic effects of adherent-invasive Escherichia coli (AIEC) LF82 strain and Candida albicans in the gut. METHODS:Colonic fibrosis was induced in C57BL/6 mice by administration of three cycles of 2.5% (w/v) dextran sulfate sodium (DSS) for 5 weeks. LF82 and C. albicans were administered orally once at the start of each week or each cycle, respectively. Expression of markers of myofibroblast activation was determined in TGF-β1-stimulated human intestinal epithelial cells (IECs). RESULTS:LF82 administration exacerbated fibrosis in DSS-treated mice, revealed by increased colonic collagen deposition and expression of the profibrotic genes Col1a1, Col3a1, Fn1 and Vim. This was accompanied by enhanced gene expression of proinflammatory cytokines and chemokines, as well as more recruited inflammatory cells into the intestine. LF82 also potentiated TGF-β1-stimulated epithelial-mesenchymal transition and myofibroblast activation in IECs, by further inducing gene expression of the main mesenchymal cell markers FN1 and VIM and downregulating the IEC marker OCLN. Proinflammatory cytokines were overexpressed with LF82 in TGF-β1-stimulated IECs. Conversely, C. albicans did not affect intestinal fibrosis progression in DSS-treated mice or myofibroblast activation in TGF-β1-stimulated IECs. CONCLUSIONS:These results demonstrate that AIEC strain LF82, but not C. albicans, may play a major profibrogenic role in the gut. 10.1186/s13099-021-00401-z
    HETEs enhance IL-1-mediated COX-2 expression via augmentation of message stability in human colonic myofibroblasts. Di Mari J F,Saada J I,Mifflin R C,Valentich J D,Powell D W American journal of physiology. Gastrointestinal and liver physiology Proinflammatory cytokines and eicosanoids are central players in intestinal inflammation. IL-1, a key cytokine associated with intestinal mucosal inflammation, induces COX-2 expression in human colonic myofibroblasts (CMF) and increased prostaglandin E(2) secretion is associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC). We have previously demonstrated that IL-1alpha-induced cyclooxygenase-2 (COX-2) expression is the result of NF-kappaB- and ERK-mediated transcription, as well as COX-2 message stabilization, which depends on p38, MAPKAPK-2 (MK-2) and human antigen R (HuR) RNA binding protein activation. Lipoxygenase (LOX)-derived hydroxyeicosatetraenoic acids (HETEs) are elevated in IBD and colonic adenomas and "cross talk" has been observed between the COX and LOX pathways. Since COX-2 expression is primarily in CMFs in colonic adenomas, we examined the impact of LOX metabolites, particularly HETEs, on IL-1alpha-induced COX-2 expression in human CMFs. Although 5(S)-, 12(R)-, and 15(S)-HETEs alone had little to no effect on COX-2 expression, they enhanced IL-1-mediated COX-2 expression 3.6 +/- 0.5-fold. Studies utilizing heterogeneous nuclear RNA amplification and 5,6-dichloro-beta-d-ribofuranosylbenzimidazole treatment were undertaken to measure COX-2 transcription and message stabilization, respectively. We found that HETEs enhanced IL-1-induced COX-2 mRNA levels in CMF as the result of increased p38, MK-2, and HuR activity, increasing message stability greater than that observed with IL-1 alone. Thus HETEs can act synergistically with IL-1alpha to induce COX-2 expression in human CMFs. HETEs may play a role in both colonic inflammation and in increasing the risk of CRC in IBD independently and via induction of COX-2-mediated prostaglandin secretion. 10.1152/ajpgi.00117.2007
    EGF-R is expressed and AP-1 and NF-kappaB are activated in stromal myofibroblasts surrounding colon adenocarcinomas paralleling expression of COX-2 and VEGF. Konstantinopoulos Panagiotis A,Vandoros Gerasimos P,Karamouzis Michalis V,Gkermpesi Maria,Sotiropoulou-Bonikou Georgia,Papavassiliou Athanasios G Cellular oncology : the official journal of the International Society for Cellular Oncology BACKGROUND:COX-2 and VEGF are important triggers of colon cancer growth, metastasis and angiogenesis. Cox-2 promoter contains transcriptional regulatory elements for AP-1 and NF-kappaB transcription factors whilst vegf is a known AP-1 downstream target gene. We investigated whether stromal myofibroblasts surrounding colon adenocarcinomas express COX-2 and VEGF and whether activation of AP-1 and NF-kappaB, as well as expression of EGF-R parallel expression of COX-2 and VEGF in these cells. METHODS:Immunohistochemical methodology was performed on archival sections from 40 patients with colon adenocarcinomas. We evaluated c-FOS, p-c-JUN (phosphorylated c-JUN), p-IkappaB-alpha (phosphorylated IkappaB-alpha), EGF-R, COX-2, NF-kappaB and VEGF expression in stromal myofibroblasts surrounding colon adenocarcinomas. Double immunostaining with a-smooth muscle actin and each antibody was done to verify the expression of these molecules in stromal myofibroblasts. RESULTS:VEGF, p-IkappaB-alpha, NF-kappaB, c-FOS, p-c-JUN, EGF-R and COX-2 were expressed in stromal myofibroblasts surrounding colon adenocarcinomas in the majority of cases. EGF-R, p-IkappaB-alpha, NF-kappaB, c-FOS and p-c-JUN correlated positively with COX-2 and VEGF expression. CONCLUSION:Stromal myofibroblasts surrounding colon adenocarcinomas are an important source of VEGF and COX-2 production, while AP-1 and NF-kappaB transcription factors are activated and EGF-R is expressed in these cells and associated with COX-2 and VEGF production. 10.1155/2007/831416
    IL-1alpha-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCzeta-ROS pathway. Di Mari John F,Mifflin Randy C,Adegboyega Patrick A,Saada Jamal I,Powell Don W Gastroenterology BACKGROUND & AIMS:Intestinal myofibroblasts (IMFs) express cyclooxygenase 2 (COX-2) early on in polyp progression and respond to pro-inflammatory cytokines. Interleukin (IL)-1alpha induces COX-2 expression in IMF via mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and nuclear factor kappa B (NF-kappaB)-dependent pathways. Because NF-kappaB activity can be mediated by PKC activation and reactive oxygen species (ROS) generation, we examined the relationship of these pathways to IL-1alpha-induced COX-2 expression. METHODS:The effects of specific PKC inhibitors and antioxidants on PKC activation, ROS generation, and COX-2 expression were studied. RESULTS:Immunoprecipitation/kinase (IPK) analysis showed that IL-1alpha increased PKC alpha, delta, and zeta activity 4.5-, 3.1-, and 2.6-fold, respectively, within 5 minutes. Single-cell fluorescence microscopy of 2',7'-dichlorofluorescin diacetate (DCF)-loaded cells showed that IL-1alpha increased ROS levels 2-fold within 15 minutes and this increase was inhibited by 10 micromol/L bisindolylymaleimide I (BIS), a pan-specific PKC inhibitor that also inhibits COX-2 expression. Chelerythrine chloride (CC) (0.5 micromol/L) inhibited classic and novel PKC activity, but not PKCzeta, and enhanced IL-1alpha-mediated ROS generation 4.0-fold and COX-2 expression 1.8-fold. The use of a PKCzeta pseudosubstrate prevented IL-1 from increasing ROS greater than control levels and abolished IL-1alpha-induced COX-2 expression. Small inhibitory RNA (siRNA) for PKCzeta confirmed its role in COX-2 expression. Antioxidants inhibited ROS generation and diminished IL-1alpha-induced COX-2 expression by 80%, without affecting PKC activation. Neither the PKC inhibitors nor the antioxidants prevented NF-kappaB-mediated transcription as determined by reporter gene analysis. CONCLUSIONS:PKCzeta and threshold ROS generation are critical for IL-1alpha-induced COX-2 expression and act concomitantly with NF-kappaB translocation in IMF. 10.1016/s0016-5085(03)00399-8
    Molecular aspects of intestinal radiation-induced fibrosis. Gervaz Pascal,Morel Philippe,Vozenin-Brotons Marie-Catherine Current molecular medicine Radiation therapy is a key component of the management of various pelvic tumors, including prostate, gynecological, and anorectal carcinomas. Unfortunately, normal tissues located in the vicinity of target organs are radiosensitive, and long-term cancer survivors may develop late treatment-related injury, most notably radiation-induced fibrosis (RIF) of the small bowel. The cellular mediators of intestinal fibrosis are mesenchymal cells (i.e. myofibroblasts, fibroblasts and smooth muscle cells) which, when activated, serve as the primary collagen-producing cells, and are responsible for excess deposition of extracellular matrix components, eventually leading to intestinal loss of function. For decades, the underlying mechanisms involved in chronic activation of myofibroblasts within the normal tissues were unknown, and the fibrotic process, which ensued, was considered irreversible. Recent advances in the pathogenesis of RIF have demonstrated prolonged upregulation of fibrogenic cytokines, such as Transforming growth factor-beta1 (TGF-beta1) and its main downstream effector, Connective tissue growth factor (CTGF), in the myofibroblasts of irradiated small bowel. TGF-beta1-mediated activation of CTGF gene expression is controlled by Smads, but recently Rho/ROCK signaling has emerged as an alternative pathway involved in the control of CTGF expression in intestinal fibrosis. This article underlines the clinical relevance of RIF as it relates to damage to the small bowel, provides insight to its molecular biology, and finally unveils the potential role of Rho-ROCK inhibitors as emerging strategies to promote RIF reversal.
    Butyrate blocks interferon-gamma-inducible protein-10 release in human intestinal subepithelial myofibroblasts. Inatomi Osamu,Andoh Akira,Kitamura Ken-Ichi,Yasui Hirofumi,Zhang Zhuobin,Fujiyama Yoshihide Journal of gastroenterology BACKGROUND:Interferon (IFN)-gamma-inducible protein (IP)-10 is a chemoattractant for CXCR 3-expressing T lymphocytes and monocytes. IP-10 has been reported to mediate chronic inflammation such as that in inflammatory bowel disease (IBD). However, the local secretion of IP-10 in the intestine remains unclear. In this study, we investigated IP-10 secretion in human colonic subepithelial myofibroblasts (SEMFs). METHODS:IP-10 secretion was determined by enzyme-linked immunosorbent assay (ELISA), and IP-10 mRNA expression was evaluated by Northern blotting. RESULTS:Interleukin (IL)-10 mRNA was not detected in unstimulated SEMFs. Interferon (IFN)-gamma strongly induced IP-10 mRNA expression. Tumor necrosis factor (TNF)-alpha also stimulated IP-10 mRNA expression, but this was much weaker than that induced by IFN-gamma. The effects of IFN-gamma and TNF-alpha were detected in a dose- and time-dependent manner. These responses were also observed at the protein levels. The IFN-gamma-induced IP-10 secretion was not affected by acetate or propionate, but was significantly reduced by butyrate. Trichostatin A, a specific inhibitor of histone deacetylase, also blocked the IFN-gamma- and TNF-alpha-induced IP-10 mRNA expression, but the effects of trichostatin A were weaker than those of butyrate. The inhibitory effect of butyrate on IFN-gamma-induced IP-10 release was not associated with STAT (signaling transducer and activator of transcription)-1alpha activation. CONCLUSIONS:We demonstrated that human colonic SEMFs are the local site for the secretion IP-10. The regulation of IP-10 release by IFN-gamma and butyrate may play an important role in controlling chronic mucosal inflammation in pathological entities such as IBD. 10.1007/s00535-005-1573-4
    The Blockade of Tumoral IL1β-Mediated Signaling in Normal Colonic Fibroblasts Sensitizes Tumor Cells to Chemotherapy and Prevents Inflammatory CAF Activation. Díaz-Maroto Natalia Guillén,Garcia-Vicién Gemma,Polcaro Giovanna,Bañuls María,Albert Nerea,Villanueva Alberto,Molleví David G International journal of molecular sciences Heterotypic interactions between newly transformed cells and normal surrounding cells define tumor's fate in incipient carcinomas. Once homeostasis has been lost, normal resident fibroblasts become carcinoma-associated fibroblasts, conferring protumorogenic properties on these normal cells. Here we describe the IL1β-mediated interplay between cancer cells and normal colonic myofibroblasts (NCFs), which bestows differential sensitivity to cytotoxic drugs on tumor cells. We used NCFs, their conditioned media (CM), and cocultures with tumor cells to characterize the IL1β-mediated crosstalk between both cell types. We silenced IL1β in tumor cells to demonstrate that such cells do not exert an influence on NCFs inflammatory phenotype. Our results shows that IL1β is overexpressed in cocultured tumor cells. IL1β enables paracrine signaling in myofibroblasts, converting them into inflammatory-CAFs (iCAF). IL1β-stimulated-NCF-CM induces migration and differential sensitivity to oxaliplatin in colorectal tumor cells. Such chemoprotective effect has not been evidenced for TGFβ1-driven NCFs. IL1β induces the loss of a myofibroblastic phenotype in NCFs and acquisition of iCAF traits. In conclusion, IL1β-secreted by cancer cells modify surrounding normal fibroblasts to confer protumorogenic features on them, particularly tolerance to cytotoxic drugs. The use of IL1β-blocking agents might help to avoid the iCAF traits acquisition and consequently to counteract the protumorogenic actions these cells. 10.3390/ijms22094960
    Myofibroblasts. II. Intestinal subepithelial myofibroblasts. Powell D W,Mifflin R C,Valentich J D,Crowe S E,Saada J I,West A B The American journal of physiology Intestinal subepithelial myofibroblasts (ISEMF) and the interstitial cells of Cajal are the two types of myofibroblasts identified in the intestine. Intestinal myofibroblasts are activated and proliferate in response to various growth factors, particularly the platelet-derived growth factor (PDGF) family, which includes PDGF-BB and stem cell factor (SCF), through expression of PDGF receptors and the SCF receptor c-kit. ISEMF have been shown to play important roles in the organogenesis of the intestine, and growth factors and cytokines secreted by these cells promote epithelial restitution and proliferation, i.e., wound repair. Their role in the fibrosis of Crohn's disease and collagenous colitis is being investigated. Through cyclooxygenase (COX)-1 and COX-2 activation, ISEMF augment intestinal ion secretion in response to certain secretagogues. By forming a subepithelial barrier to Na(+) diffusion, they create a hypertonic compartment that may account for the ability of the gut to transport fluid against an adverse osmotic gradient. Through the paracrine secretion of prostaglandins and growth factors (e.g., transforming growth factor-beta), ISEMF may play a role in colonic tumorigenesis and metastasis. COX-2 in polyp ISEMF may be a target for nonsteroidal anti-inflammatory drugs (NSAIDs), which would account for the regression of the neoplasms in familial adenomatous polyposis and the preventive effect of NSAIDs in the development of sporadic colon neoplasms. More investigation is needed to clarify the functions of these pleiotropic cells. 10.1152/ajpcell.1999.277.2.C183
    Pathogenetic mechanisms in radiation fibrosis. Yarnold John,Brotons Marie-Catherine Vozenin Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology Deregulation of normal regenerative responses to physical, chemical and biological toxins in susceptible individuals leads to abnormal remodelling of extracellular matrix with pathological fibrosis. Processes deregulated after radiotherapy have much in common with processes associated with fibrotic diseases affecting the heart, skin, lungs, kidneys, gastro-intestinal tract and liver. Among the secreted factors driving fibrosis, transforming growth factor beta 1 (TGFβ1) produced by a wide range of inflammatory, mesenchymal and epithelial cells converts fibroblasts and other cell types into matrix-producing myofibroblasts. Even if required for the initiation of fibrosis, inflammation and the continued stimulus of TGFβ1 may not be needed to maintain it. After myofibroblast activation, collagen production can be perpetuated independently of TGFβ1 by autocrine induction of a cytokine called connective tissue growth factor. The role of inflammation, the origins and activation of myofibroblasts as biosynthetic cells and the downstream pathways of extracellular matrix synthesis in common fibrotic states are reviewed. Oxidative stress, hypoxia and microvascular damage are also considered, before examining the same processes in the context of radiotherapy. One of the main uncertainties is the relevance of very early events, including inflammatory responses in blood vessels, to fibrosis. Despite the power of animal models, including genetic systems, the potential contribution of research based on human tissue samples has never been greater. A closer interaction between scientists researching fibrosis and radiation oncologists holds enormous promise for therapeutic advances. 10.1016/j.radonc.2010.09.002
    Epimorphin(-/-) mice are protected, in part, from acute colitis via decreased interleukin 6 signaling. Shaker Anisa,Gargus Matthew,Fink Julie,Binkley Jana,Darwech Isra,Swietlicki Elzbieta,Levin Marc S,Rubin Deborah C Translational research : the journal of laboratory and clinical medicine Epimorphin (Epim), a member of the syntaxin family of membrane-bound, intracellular vesicle-docking proteins, is expressed in intestinal myofibroblasts and macrophages. We demonstrated previously that Epimorphin(-/-)(Epim(-/-)) mice are protected, in part, from dextran sodium sulfate (DSS)-induced colitis. Although interleukin (IL)-6/p-Stat3 signaling has been implicated in the pathogenesis of colitis, the myofibroblast contribution to IL-6 signaling in colitis remains unexplored. Our aim was to investigate the IL-6 pathway in Epim(-/-) mice in the DSS colitis model. Whole colonic tissue, epithelium, and stroma of WT and congenic Epim(-/-) mice treated with 5% DSS for 7 days were analyzed for IL-6 and a downstream effector, p-Stat3, by immunostaining and immunoblot. Colonic myofibroblast and peritoneal macrophage IL-6 secretion were evaluated by enzyme-linked immunosorbent assay. IL-6 and p-Stat3 expression were decreased in Epim(-/-) vs WT colon. A relative increase in stromal vs epithelial p-Stat3 expression was observed in WT mice but not in Epim(-/-) mice. Epim deletion abrogates IL-6 secretion from colonic myofibroblasts treated with IL-1β and decreases IL-6 secretion from peritoneal macrophages in a subset of DSS-treated mice. Epim deletion inhibits IL-6 secretion most profoundly from colonic myofibroblasts. Distribution of Stat3 activation is altered in DSS-treated Epim(-/-) mice. Our findings support the notion that myofibroblasts modulate IL-6/p-Stat3 signaling in DSS-treated Epim(-/-) mice. 10.1016/j.trsl.2014.03.007
    Multiple targets of carbon monoxide gas in the intestinal inflammation. Naito Yuji,Takagi Tomohisa,Uchiyama Kazuhiko,Katada Kazuhiro,Yoshikawa Toshikazu Archives of biochemistry and biophysics Inflammatory bowel diseases (IBDs) such as ulcerative colitis and Crohn's disease are chronic relapsing and remitting inflammatory disorders of the intestinal tract. It is important to investigate the precise pathogenesis of IBD, to evaluate new anti-inflammatory agents, and to develop novel drugs. Carbon monoxide (CO) has emerged as an important regulator of acute and chronic inflammation of the gastrointestinal tract. The mechanism underlying its anti-inflammatory effects is only partially understood. Recent reports have demonstrated that CO could play a role in the functional modulation of epithelial and immunological cells in the intestine. In this short review, we have highlighted the recent findings that CO stimulates the epithelial cell restitution and FGF production from myofibroblasts. CO was also shown to regulate T cell activation and differentiation, and to activate macrophages. Finally, we have discussed the direction of translational research with respect to launching a novel agent for releasing CO in the intestine. 10.1016/j.abb.2015.06.020
    Regulation of IL-11 expression in intestinal myofibroblasts: role of c-Jun AP-1- and MAPK-dependent pathways. Bamba Shigeki,Andoh Akira,Yasui Hirofumi,Makino Jin,Kim Shokei,Fujiyama Yoshihide American journal of physiology. Gastrointestinal and liver physiology IL-11 inhibits the activation of NF-kappaB and induces the Th2 polarization of CD4+ T cells. The clinical utility of IL-11 is being investigated in Crohn's disease. However, physiological secretion of IL-11 in the intestine remains unclear. In this study, we investigated IL-11 secretion in human intestinal subepithelial myofibroblasts (SEMFs). Intestinal SEMFs were isolated from the human colonic mucosa. IL-11 secretion and mRNA expression were determined by ELISA and Northern blot analysis. The activating protein (AP)-1-DNA binding activity was evaluated by EMSA. IL-11 secretion was induced by IL-1beta and transforming growth factor (TGF)-beta1. These were also observed at the mRNA level. The EMSAs demonstrated that both IL-1beta and TGF-beta1 induced AP-1 activation within 2 h after stimulation, and a blockade of AP-1 activation by the recombinant adenovirus containing a dominant negative c-Jun markedly reduced the IL-1beta- and TGF-beta1-induced IL-11 mRNA expression. IL-1beta and TGF-beta1 induced an activation of ERK p42/44 and p38 MAP kinases, and the MAP kinase inhibitors (SB-202190, PD-98059, and U-0216) significantly reduced the IL-1beta- and TGF-beta1-induced IL-11 secretion. The upregulation of IL-11 mRNA by IL-1beta- and TGF-beta1 was also mediated by a p38 MAP kinase-mediated mRNA stabilization. The combination of IL-1beta and TGF-beta1 additively enhanced IL-11 secretion. Intestinal SEMFs secreted IL-11 in response to IL-1beta- and TGF-beta1. Mucosal IL-11 secretion might be important as an anti-inflammatory response in the pathogenesis of intestinal inflammation. 10.1152/ajpgi.00050.2003
    Vitamin D prevents the intestinal fibrosis via induction of vitamin D receptor and inhibition of transforming growth factor-beta1/Smad3 pathway. Tao Qingsong,Wang Baochai,Zheng Yu,Jiang Xiaohua,Pan Zheng,Ren Jianan Digestive diseases and sciences BACKGROUND AND AIMS:Vitamin D deficiency in patients with inflammatory bowel disease (IBD) is associated with greater disease activity and lower quality of life. Intestinal fibrosis is a main complication of IBD. However, the effect of vitamin D on intestinal fibrosis remains unclear. We investigated the prophylactic effect and the underlying mechanism of vitamin D on the intestinal fibrosis in vitamin D-deficient mice with chronic colitis. METHODS:Vitamin D-deficient mice were randomized into two groups receiving the vitamin D-deficient or vitamin D-sufficient diet from weaning (week 4). Intestinal fibrosis was induced by six-weekly 2,4,6-trinitrobenzene sulfonic acid administrations from week 8. At week 14, the productions of extracellular matrix (ECM) and total collagen were measured in the colons, and TGF-β1/Smad3 signal transduction was examined in isolated colonic subepithelial myofibroblasts (SEMF). The expression of vitamin D receptor (VDR), α-SMA and Collagen I in normal SEMF and VDR-null SEMF exposed to TGF-β1 and/or 1,25(OH)2D3 was measured. RESULTS:Vitamin D significantly reduced the histological scoring, ECM and collagen productions in the colons and decreased the levels of TGF-β1, Smad-3, p-Smad3 and Collagen I in SEMF. 1,25(OH)2D3-induced VDR expression and decreased TGF-β1-stimulated α-SMA and Collagen I expressions in SEMF. Knocking down VDR expression in SEMF abolished the effect of 1,25(OH)2D3. CONCLUSIONS:Vitamin D has prophylactic effect on intestinal fibrosis in the vitamin D-deficient mice with chronic colitis, which may be associated with the inhibited activation of TGF-β1/Smad3 pathway in the SEMF via VDR induction. 10.1007/s10620-014-3398-6
    Mucosal healing and fibrosis after acute or chronic inflammation in wild type FVB-N mice and C57BL6 procollagen α1(I)-promoter-GFP reporter mice. Ding Shengli,Walton Kristen L W,Blue Randall Eric,McNaughton Kirk K,Macnaughton Kirk,Magness Scott T,Lund Pauline Kay PloS one BACKGROUND:Injury and intestinal inflammation trigger wound healing responses that can restore mucosal architecture but if chronic, can promote intestinal fibrosis. Intestinal fibrosis is a major complication of Crohn's disease. The cellular and molecular basis of mucosal healing and intestinal fibrosis are not well defined and better understanding requires well characterized mouse models. METHODS:FVB-N wild type mice and C57BL6 procollagen α1(I)-GFP reporter mice were given one (DSS1) or two (DSS2) cycles of 3% DSS (5 days/cycle) followed by 7 days recovery. Histological scoring of inflammation and fibrosis were performed at DSS1, DSS1+3, DSS1+7, DSS2, DSS2+3, and DSS2+7. Procollagen α1(I)-GFP activation was assessed in DSS and also TNBS models by whole colon GFP imaging and fluorescence microscopy. Colocalization of GFP with α-smooth muscle actin (α-SMA) or vimentin was examined. GFP mRNA levels were tested for correlation with endogenous collagen α1(I) mRNA. RESULTS:Males were more susceptible to DSS-induced disease and mortality than females. In FVB-N mice one DSS cycle induced transient mucosal inflammation and fibrosis that resolved by 7 days of recovery. Two DSS cycles induced transmural inflammation and fibrosis in a subset of FVB-N mice but overall, did not yield more consistent, severe or sustained fibrosis. In C57BL6 mice, procollagen α1(I)-GFP reporter was activated at the end of DSS1 and through DSS+7 with more dramatic and transmural activation at DSS2 through DSS2+7, and in TNBS treated mice. In DSS and TNBS models GFP reporter expression localized to vimentin(+) cells and much fewer α-SMA(+) cells. GFP mRNA strongly correlated with collagen α1(I) mRNA. CONCLUSIONS:One DSS cycle in FVB-N mice provides a model to study mucosal injury and subsequent mucosal healing. The procollagen α1(I)-GFP transgenic provides a useful model to study activation of a gene encoding a major extracellular matrix protein during acute or chronic experimental intestinal inflammation and fibrosis. 10.1371/journal.pone.0042568
    The extracellular calcium-sensing receptor reciprocally regulates the secretion of BMP-2 and the BMP antagonist Noggin in colonic myofibroblasts. Peiris Dinithi,Pacheco Ivan,Spencer Craig,MacLeod R John American journal of physiology. Gastrointestinal and liver physiology To understand whether postprandial extracellular Ca(2+) (Ca(o)(2+)) changes were related to intestinal epithelial homeostasis, we performed array analysis on extracellular calcium-sensing receptor (CaSR)-expressing colonic myofibroblasts (18Co cells) and observed increases in bone morphogenetic protein (BMP)-2 transcripts. The present experiments demonstrated that regulated secretion of BMP-2 occurs in response to CaSR activation of these cells and revealed a new property of BMP-2 on the intestinal barrier. Activation by Ca(o)(2+), spermine, GdCl(3), or neomycin sulfate of 18Co cells or primary isolates of myofibroblasts from the normal human colon stimulated both the synthesis (RT-PCR) and secretion (ELISA) of BMP-2. Transient transfection with short interfering RNA against CaSR completely inhibited BMP-2 secretion. Transient transfection with dominant negative CaSR (R185Q) increased the EC(50) of Ca(o)(2+) (5.7 vs. 2.3 mM). Upregulation of BMP-2 transcript and secretion occurring within 3 h of CaSR activation was prevented by actinomycin D. CaSR-mediated BMP-2 synthesis and secretion required phosphatidylinositol 3-kinase activation (as assessed by phospho-Akt generation). Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells accelerated restitution in wounded postconfluent Caco-2 cells. Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells increased the transepithelial resistance of low- and high-resistance T-84 epithelial monolayers. CaSR stimulation of T-84 epithelia and colonic myofibroblasts downregulated the BMP family antagonist Noggin, as assessed by RT-PCR and Western blot analysis. Together, our data suggest that the CaSR mediates the effective concentration of BMP-2 in the intestine, which leads to enhanced repair and barrier development. 10.1152/ajpgi.00225.2006
    Intestinal cancer progression by mutant p53 through the acquisition of invasiveness associated with complex glandular formation. Nakayama M,Sakai E,Echizen K,Yamada Y,Oshima H,Han T-S,Ohki R,Fujii S,Ochiai A,Robine S,Voon D C,Tanaka T,Taketo M M,Oshima M Oncogene Tumor suppressor TP53 is frequently mutated in colorectal cancer (CRC), and most mutations are missense type. Although gain-of-functions by mutant p53 have been demonstrated experimentally, the precise mechanism for malignant progression in in vivo tumors remains unsolved. We generated Apc Trp53 villin-CreER compound mice, in which mutant p53 was expressed in the intestinal epithelia upon tamoxifen treatment, and examined the intestinal tumor phenotypes and tumor-derived organoids. Mutant Trp53, but not Trp53-null mutation accelerated submucosal invasion with generation of desmoplastic microenvironment. The nuclear accumulation of p53 was evident in Apc Trp53 homozygous tumors like human CRC. Although p53 was distributed to the cytoplasm in Apc Trp53 heterozygous tumors, it accumulated in the nuclei at the invasion front, suggesting a regulation mechanism for p53 localization by the microenvironment. Importantly, mutant p53 induced drastic morphological changes in the tumor organoids to complex glandular structures, which was associated with the acquisition of invasiveness. Consistently, the branching scores of human CRC that carry TP53 mutations at codon 273 significantly increased in comparison with those of TP53 wild-type tumors. Moreover, allografted Apc Trp53 organoid tumors showed a malignant histology with an increased number of myofibroblasts in the stroma. These results indicate that nuclear-accumulated mutant p53 induces malignant progression of intestinal tumors through complex tumor gland formation and acquisition of invasiveness. Furthermore, RNA sequencing analyses revealed global gene upregulation by mutant p53, which was associated with the activation of inflammatory and innate immune pathways. Accordingly, it is possible that mutant p53 induces CRC progression, not only by a cell intrinsic mechanism, but also by the generation or activation of the microenvironment, which may synergistically contribute to the acceleration of submucosal invasion. Therefore, the present study indicates that nuclear-accumulated mutant p53 is a potential therapeutic target for the treatment of advanced CRCs. 10.1038/onc.2017.194
    Key role of aldosterone and pericryptal myofibroblast growth in colonic permeability. Cristià Esther,Moretó Miquel,Naftalin Richard J Journal of pediatric gastroenterology and nutrition When sodium intake diminishes, both the kidney and distal colon contribute directly to sodium homeostasis. In response to a diet with low amounts of sodium, the body hormonal profile changes to produce different effects on crypt-colon permeability and absorption and in the pericryptal sheath surrounding distal colonic crypts. This adaptation produces an increase in Na absorption, a decreased crypt-wall permeability, and an activation of the growth of pericryptal myofibroblasts. The separate roles of the 2 main hormones implicated in the process, aldosterone and angiotensin II, until now have been unclear. Experiments conducted on adrenalectomized rats on low- and high-sodium diets, implanted with osmotic pumps perfusing either aldosterone or angiotensin II, allow us to discriminate between the effects of these hormones. In the distal colon, aldosterone acts as a trophic agent on the myofibroblasts layer and is the key hormone controlling colonic permeability, but angiotensin II alone has no discernable direct role in the process. 10.1097/MPG.0b013e31812e680b
    Features of liver tissue remodeling in intestinal failure during and after weaning off parenteral nutrition. Mutanen Annika,Lohi Jouko,Sorsa Timo,Jalanko Hannu,Pakarinen Mikko P Surgery BACKGROUND:Intestinal failure is associated frequently with liver injury, which persists after weaning off parenteral nutrition. We compared features of liver remodeling in intestinal failure during and after weaning off parenteral nutrition. METHODS:Liver biopsies and serum samples were obtained from 25 intestinal failure patients at a median age of 9.7 years (interquartile range: 4.6-18) and from age-matched control patients. Seven patients had been receiving parenteral nutrition for 53 months (22-160), and 18 patients had been weaned off parenteral nutrition 6.3 years (2.4-17) earlier, after having received parenteral nutrition for 10 months (3.3-34). Expression of alpha-smooth muscle actin, collagen 1, proinflammatory cytokines, growth factors, and matrix metalloproteinases (MMPs) was measured. RESULTS:Significant increases in immunohistochemical expression of alpha-smooth muscle actin and collagen 1 were observed predominantly in portal areas and were similar to increases seen in patients currently receiving parenteral nutrition and in patients weaned off parenteral nutrition. Gene and protein expressions of alpha-smooth muscle actin and collagen were interrelated. Gene expression of ACTA2, encoding alpha-smooth muscle actin, was increased only in patients who were receiving parenteral nutrition currently. Comparable upregulation of interleukin-1 (α and ß), epidermal growth factor, integrin-ß6, and MMP9 gene expression was observed in both patient groups, irrespective of whether they were receiving parenteral nutrition currently. Liver expression and serum levels of TIMP1 and MMP7 were increased only in the patients on parenteral nutrition currently but were not increased after weaning off parenteral nutrition. CONCLUSION:Intestinal failure is characterized by abnormal activation of hepatic myofibroblast and accumulation of collagen both during and after weaning off parenteral nutrition. Persistent transcriptional upregulation of proinflammatory and fibrogenic cytokines after weaning off parenteral nutrition suggests that factors other than parenteral nutrition may contribute to intestinal failure-associated liver disease. 10.1016/j.surg.2016.04.025
    Differential activation of cytokine secretion in primary human colonic fibroblast/myofibroblast cultures. Rogler G,Gelbmann C M,Vogl D,Brunner M,Schölmerich J,Falk W,Andus T,Brand K Scandinavian journal of gastroenterology BACKGROUND:Fibroblasts and myofibroblasts are known to secrete a wide spectrum of cytokines, but the individual spectrum is tissue-specific. We investigated the effect of cell activation on cytokine secretion of isolated human colonic fibroblasts/myofibroblasts from control patients and patients with mucosal inflammation. METHODS:Primary cultures of human colonic submucosal fibroblasts/myofibroblasts were incubated with IL-1alpha (100 U/ml), IL-Ibeta (10 ng/ml), IL-10 (10 ng/ml), TNF (10 ng/ml), PMA (10 ng/ml), LPS (50 ng/ml), IL-4 (10 ng/ml), or a combination of IL-1 and TNF. Secreted cytokines were determined by ELISA. NF-kappaB activation was demonstrated by electrophoretic mobility-shift assays (EMSA). RESULTS:Incubation of colonic fibroblasts/myofibroblasts with IL-1, LPS, TNF and PMA induced secretion of IL-6, IL-8, M-CSF and GM-CSF. IL-8 and IL-6 secretion could be stimulated by IL-1alpha, IL-1beta, TNF, PMA and LPS within 6 h of incubation. IL-6 secretion was stimulated from 0.5 +/- 0.01 pg/h x microg fibroblast protein to 18.5 +/- 2.6 pg/h x microg fibroblast protein with IL-1beta (P < 0.01). IL-8 secretion was stimulated from 1.0 +/- 0.1 pg/h x microg fibroblast protein to 41.1 +/- 3.6 pg/h x microg (P < 0.005). IL-4 and IL-10 did not change cytokine secretion significantly. No significant differences between cultures from normal and inflamed mucosa were observed. TNF and IL-1 induced NF-kappaB activation. ALLN, a proteasome and NF-kappaB activation inhibitor, reduced TNF-mediated IL-8, GM-CSF and M-CSF induction significantly, whereas induction of IL-6 secretion remained unchanged. CONCLUSION:Human colonic myofibroblasts can secrete large amounts of IL-6, IL-8, M-CSF and GM-CSF upon stimulation. The induction of IL-8, M-CSF and GM-CSF, but not of IL-6 secretion, is mediated mainly by NF-kappaB activation. The cytokine profile and the total amounts of cytokines released suggest that colonic myofibroblasts can play a role in leukocyte recruitment and during mucosal inflammation. They therefore have to be regarded as an important part of the mucosal immune system.
    Pericryptal myofibroblast growth in rat descending colon induced by low-sodium diets is mediated by aldosterone and not by angiotensin II. Cristià E,Afzal-Ahmed I,Pérez-Bosque A,Amat C,Naftalin R J,Moretó M The Journal of membrane biology Pericryptal myofibroblast growth in descending colonic crypts correlates with the activation of the renin-angiotensin-aldosterone system. Earlier work showed that during the transition from a high-Na(+) (HS) to low-Na(+) (LS) diet there are changes in the colonic crypt wall and pericryptal sheath. As LS diet increases both aldosterone and angiotensin II, the aim here was to determine their individual contributions to the trophic changes in colonic crypts. Experiments were conducted on control and adrenalectomized Sprague-Dawley rats fed an HS diet and then switched to LS diet for 3 days and supplemented with aldosterone or angiotensin II. The actions of the angiotensin-converting enzyme inhibitor captopril, the angiotensin receptor antagonist losartan and the aldosterone antagonist spironolactone on extracellular matrix proteins, claudin 4 and E-cadherin myofibroblast proteins, alpha-smooth muscle actin (alpha-SMA) and OB-cadherin (cadherin 11), angiotensin type 1 and TGFbetar1 membrane receptors were determined by immunolocalization in fixed distal colonic mucosa. The LS diet or aldosterone supplementation following ADX in HS or LS increased extracellular matrix, membrane receptors and myofibroblast proteins, but angiotensin alone had no trophic effect on alpha-SMA. These results show that aldosterone stimulates myofibroblast growth in the distal colon independently of dietary Na(+) intake and of angiotensin levels. This stimulus could be a genomic response or secondary to stretch of the pericryptal sheath myofibroblasts accompanying enhanced rates of crypt fluid absorption. 10.1007/s00232-005-0773-4
    The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease. Di Sabatino A,Battista N,Biancheri P,Rapino C,Rovedatti L,Astarita G,Vanoli A,Dainese E,Guerci M,Piomelli D,Pender S L F,MacDonald T T,Maccarrone M,Corazza G R Mucosal immunology Activation of cannabinoid receptors (CBs) by endocannabinoids impacts on a number of gastrointestinal functions. Recent data indicate that CB1 agonists improve 2,4-dinitrobenzene sulfonic acid-induced colitis in mice, thus suggesting a role for the endocannabinoid agonist anandamide (AEA) in protecting the gut against inflammation. We here examined the gut endocannabinoid system in inflammatory bowel disease (IBD) patients, and investigated the ex vivo and in vitro effects of the non-hydrolysable AEA analog methanandamide (MAEA) on the mucosal proinflammatory response. The content of AEA, but not of 2-arachidonoyl-glycerol and N-palmitoylethanolamine, was significantly lower in inflamed than uninflamed IBD mucosa, and this was paralleled by lower activity of the AEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-specific phospholipase D and higher activity of the AEA-degrading enzyme fatty acid amide hydrolase. MAEA significantly downregulated interferon-γ and tumor necrosis factor-α secretion by both organ culture biopsies and lamina propria mononuclear cells. Although these results are promising, further studies are needed to determine the role of cannabinoid pathways in gut inflammation. 10.1038/mi.2011.18
    Protein kinase D mediates synergistic expression of COX-2 induced by TNF-{alpha} and bradykinin in human colonic myofibroblasts. Yoo James,Chung Christine,Slice Lee,Sinnett-Smith James,Rozengurt Enrique American journal of physiology. Cell physiology Myofibroblasts have recently been identified as major mediators of tumor necrosis factor-alpha (TNF-alpha)-associated colitis, but the precise mechanism(s) involved remains incompletely understood. In particular, the possibility that TNF-alpha signaling cross talks with other proinflammatory mediators, including bradykinin (BK), has not been examined in these cells. Here we show that treatment of 18Co cells, a model of human colonic myofibroblasts, with BK and TNF-alpha induced striking synergistic COX-2 protein expression that was paralleled by increases in the levels of transcripts encoding COX-2 and microsomal prostaglandin E synthase 1 (mPGES-1) and by the production of PGE(2). COX-2 expression in 18Co cells treated with BK and TNF-alpha was prevented by the B(2) BK receptor antagonist HOE-140, the preferential protein kinase C (PKC) inhibitors Ro31-8220 and GF-109203X, and Gö-6976, an inhibitor of conventional PKCs and protein kinase D (PKD). In a parallel fashion, TNF-alpha, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation induced by BK, as measured by PKD phosphorylation at its activation loop (Ser(744)) and autophosphorylation site (Ser(916)). BK-induced PKD activation was also inhibited by HOE-140, Ro31-8220, and Gö-6976. Transfection of 18Co cells with small interfering RNA targeting PKD completely inhibited the synergistic increase in COX-2 protein in response to BK and TNF-alpha, demonstrating, for the first time, a critical role of PKD in the pathways leading to synergistic expression of COX-2. Our results imply that cross talk between TNF-alpha and BK amplifies a PKD phosphorylation cascade that mediates synergistic COX-2 expression in colonic myofibroblasts. It is plausible that PKD increases COX-2 expression in colonic myofibroblasts to promote an inflammatory microenvironment that supports tumor growth. 10.1152/ajpcell.00184.2009
    Expression of proteinase-activated receptor 2 on human primary gastrointestinal myofibroblasts and stimulation of prostaglandin synthesis. Seymour Michelle L,Binion David G,Compton Steven J,Hollenberg Morley D,MacNaughton Wallace K Canadian journal of physiology and pharmacology It is known that subepithelial myofibroblast-derived prostaglandin (PG)E2 can regulate intestinal epithelial cell functions, and that proteinase-activated receptor-2 (PAR2) is abundantly expressed in the gastrointestinal tract. Since PAR2 activation has previously been associated with stimulation of PGE2 synthesis, we hypothesized that PAR2 expressed on primary human gastrointestinal myofibroblasts regulates PGE2 synthesis via cyclooxygenase (COX)-1 and (or) COX-2, and associated PGE synthases. Primary human myofibroblasts were isolated from the resection tissue of the esophagus, small intestine, and colon. Expression of functional PAR2 was determined by RT-PCR and by calcium mobilization in Fura-2/AM-loaded cells. Trypsin and the selective PAR2-activating peptide (PAR2-AP) SLIGRL-NH2 stimulated PGE2 synthesis in a concentration-dependent manner, as measured by enzyme immunoassay. Selective COX inhibition showed PAR2-induced PGE2 synthesis to be COX-1 dependent in esophageal myofibroblasts and both COX-1 and COX-2 dependent in colonic cells, consistent with the distribution of COX-1 and COX-2 expression. Although both cytosolic and microsomal PGE synthases were expressed in cells from all tissues, microsomal PGE synthases were expressed at highest levels in the colonic myofibroblasts. Activation of PAR2 on gastrointestinal myofibroblasts stimulates PGE2 synthesis via different pathways in the colon than in the esophagus and small intestine. 10.1139/y05-046
    Stromal Indian hedgehog signaling is required for intestinal adenoma formation in mice. Büller Nikè V J A,Rosekrans Sanne L,Metcalfe Ciara,Heijmans Jarom,van Dop Willemijn A,Fessler Evelyn,Jansen Marnix,Ahn Christina,Vermeulen Jacqueline L M,Westendorp B Florien,Robanus-Maandag Els C,Offerhaus G Johan,Medema Jan Paul,D'Haens Geert R A M,Wildenberg Manon E,de Sauvage Frederic J,Muncan Vanesa,van den Brink Gijs R Gastroenterology BACKGROUND & AIMS:Indian hedgehog (IHH) is an epithelial-derived signal in the intestinal stroma, inducing factors that restrict epithelial proliferation and suppress activation of the immune system. In addition to these rapid effects of IHH signaling, IHH is required to maintain a stromal phenotype in which myofibroblasts and smooth muscle cells predominate. We investigated the role of IHH signaling during development of intestinal neoplasia in mice. METHODS:Glioma-associated oncogene (Gli1)-CreERT2 and Patched (Ptch)-lacZ reporter mice were crossed with Apc(Min) mice to generate Gli1CreERT2-Rosa26-ZSGreen-Apc(Min) and Ptch-lacZ-Apc(Min) mice, which were used to identify hedgehog-responsive cells. Cyp1a1Cre-Apc (Apc(HET)) mice, which develop adenomas after administration of β-naphthoflavone, were crossed with mice with conditional disruption of Ihh in the small intestine epithelium. Apc(Min) mice were crossed with mice in which sonic hedgehog (SHH) was overexpressed specifically in the intestinal epithelium. Intestinal tissues were collected and analyzed histologically and by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction. We also analyzed levels of IHH messenger RNA and expression of IHH gene targets in intestinal tissues from patients with familial adenomatous polyposis (n = 18) or sessile serrated adenomas (n = 15) and normal colonic tissue from control patients (n = 12). RESULTS:Expression of IHH messenger RNA and its targets were increased in intestinal adenomas from patients and mice compared with control colon tissues. In mice, IHH signaling was exclusively paracrine, from the epithelium to the stroma. Loss of IHH from Apc(HET) mice almost completely blocked adenoma development, and overexpression of SHH increased the number and size of adenomas that developed. Loss of IHH from Apc(HET) mice changed the composition of the adenoma stroma; cells that expressed α-smooth muscle actin or desmin were lost, along with expression of cyclooxygenase-2, and the number of vimentin-positive cells increased. CONCLUSIONS:Apc mutant epithelial cells secrete IHH to maintain an intestinal stromal phenotype that is required for adenoma development in mice. 10.1053/j.gastro.2014.10.006
    Expression of Programmed Death-Ligand 1 by Human Colonic CD90 Stromal Cells Differs Between Ulcerative Colitis and Crohn's Disease and Determines Their Capacity to Suppress Th1 Cells. Beswick Ellen J,Grim Carl,Singh Abinav,Aguirre Jose E,Tafoya Marissa,Qiu Suimin,Rogler Gerhard,McKee Rohini,Samedi Von,Ma Thomas Y,Reyes Victor E,Powell Don W,Pinchuk Irina V Frontiers in immunology Background and Aims:The role of programmed cell death protein 1 (PD-1) and its ligands in the dysregulation of T helper immune responses observed in the inflammatory bowel disease (IBD) is unclear. Recently, a novel concept emerged that CD90 colonic (myo)fibroblasts (CMFs), also known as stromal cells, act as immunosuppressors, and are among the key regulators of acute and chronic inflammation. The objective of this study was to determine if the level of the PD-1 ligands is changed in the IBD inflamed colonic mucosa and to test the hypothesis that changes in IBD-CMF-mediated PD-1 ligand-linked immunosuppression is a mechanism promoting the dysregulation of Th1 cell responses. Methods:Tissues and cells derived from Crohn's disease (CD), ulcerative colitis (UC), and healthy individuals (N) were studied , and in culture. Results:A significant increase in programmed death-ligand 1 (PD-L1) was observed in the inflamed UC colonic mucosa when compared to the non-inflamed matched tissue samples, CD, and healthy controls. UC-CMFs were among the major populations in the colonic mucosa contributing to the enhanced PD-L1 expression. In contrast, PD-L1 expression was decreased in CD-CMFs. When compared to CD-CMFs and N-CMFs, UC-CMFs demonstrated stronger suppression of IL-2, Th1 transcriptional factor Tbet, and IFN-γ expression by CD3/CD28-activated CD4 T cells, and this process was PD-L1 dependent. Similar observations were made when differentiated Th1 cells were cocultured with UC-CMFs. In contrast, CD-CMFs showed reduced capacity to suppress Th1 cell activity and addition of recombinant PD-L1 Fc to CD-CMF:T cell cocultures partially restored the suppression of the Th1 type responses. Conclusion:We present evidence showing that increased PD-L1 expression suppresses Th1 cell activity in UC. In contrast, loss of PD-L1 expression observed in CD contributes to the persistence of the Th1 inflammatory milieu in CD. Our data suggest that dysregulation of the Th1 responses in the inflamed colonic mucosa of IBD patients is promoted by the alterations in PD-L1 expression in the mucosal mesenchymal stromal cell compartment. 10.3389/fimmu.2018.01125
    Decreased MAPK- and PGE2-dependent IL-11 production in Gialpha2-/- colonic myofibroblasts. Hoang Brian,Trinh Alice,Birnbaumer Lutz,Edwards Robert A American journal of physiology. Gastrointestinal and liver physiology Mice deficient in the G-protein alpha subunit G(i)alpha(2) spontaneously develop colitis and colon cancer. IL-11 is a pleiotropic cytokine known to protect the intestinal epithelium from injury in animal models of colitis and is produced by subepithelial myofibroblasts in response to inflammatory mediators including TGF-beta, IL-1beta, and PGE(2). Arachidonic acid release and subsequent PGE(2) production is significantly decreased in the colonic mucosa of G(i)alpha(2)-/- mice, and we hypothesized that this would affect mucosal IL-11 production. Mucosal levels of IL-11 were found to be significantly decreased in G(i)alpha(2)-/- mice despite the presence of mild colitis. Primary cultures of G(i)alpha(2)-/- intestinal and colonic myofibroblasts (IMF and CMF, respectively) produced less basal and TGF-beta or IL-1beta-stimulated IL-11 mRNA and protein than wild-type cells. Inhibitors of ERK or p38 MAPK activation dose dependently inhibited IMF and CMF IL-11 production in response to TGF-beta stimulation, whereas 16,16 dimethyl-PGE(2) and prostanoid receptor subtype-selective agonists induced IL-11 production. Treatment of animals with the EP4-specific agonist ONO-AE1-329 resulted in enhanced mucosal levels of IL-11, and increased IL-11 production by ex vivo cultured CMF. Modulation of cAMP levels produced diverging results, with enhancement of TGF-beta-induced IL-11 release in IMF pretreated with 8-Br-cAMP and inhibition in cells treated either with pertussis toxin or the PKA inhibitor H-89. These data suggest a physiological role for prostaglandins, MAPK signaling, and cAMP signaling for the production of myofibroblast-derived IL-11 in the mouse intestinal mucosa. 10.1152/ajpgi.00307.2006
    ABIN2 Function Is Required To Suppress DSS-Induced Colitis by a Tpl2-Independent Mechanism. Nanda Sambit K,Nagamori Tsunehisa,Windheim Mark,Amu Sylvia,Aviello Gabriella,Patterson-Kane Janet,Arthur J Simon C,Ley Steven C,Fallon Padraic,Cohen Philip Journal of immunology (Baltimore, Md. : 1950) The A20-binding inhibitor of NF-κB 2 (ABIN2) interacts with Met1-linked ubiquitin chains and is an integral component of the tumor progression locus 2 (Tpl2) kinase complex. We generated a knock-in mouse expressing the ubiquitin-binding-defective mutant ABIN2[D310N]. The expression of Tpl2 and its activation by TLR agonists in macrophages or by IL-1β in fibroblasts from these mice was unimpaired, indicating that the interaction of ABIN2 with ubiquitin oligomers is not required for the stability or activation of Tpl2. The ABIN2[D310N] mice displayed intestinal inflammation and hypersensitivity to dextran sodium sulfate-induced colitis, an effect that was mediated by radiation-resistant cells rather than by hematopioetic cells. The IL-1β-dependent induction of cyclooxygenase 2 (COX2) and the secretion of PGE was reduced in mouse embryonic fibroblasts and intestinal myofibroblasts (IMFs) from ABIN2[D310N] mice. These observations are similar to those reported for the Tpl2 knockout (KO) mice (Roulis et al. 2014. 111: E4658-E4667), but the IL-1β-dependent production of COX2 and PGE in mouse embryonic fibroblasts or IMFs was unaffected by pharmacological inhibition of Tpl2 in wild-type mice. The expression of ABIN2 is decreased drastically in Tpl2 KO mice. These and other lines of evidence suggest that the hypersensitivity of Tpl2 KO mice to dextran sodium sulfate-induced colitis is not caused by the loss of Tpl2 catalytic activity but by the loss of ABIN2, which impairs COX2 and PGE production in IMFs by a Tpl2 kinase-independent pathway. 10.4049/jimmunol.1700614
    Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF. Van Landeghem Laurianne,Chevalier Julien,Mahé Maxime M,Wedel Thilo,Urvil Petri,Derkinderen Pascal,Savidge Tor,Neunlist Michel American journal of physiology. Gastrointestinal and liver physiology Wound healing of the gastrointestinal mucosa is essential for the maintenance of gut homeostasis and integrity. Enteric glial cells play a major role in regulating intestinal barrier function, but their role in mucosal barrier repair remains unknown. The impact of conditional ablation of enteric glia on dextran sodium sulfate (DSS)-induced mucosal damage and on healing of diclofenac-induced mucosal ulcerations was evaluated in vivo in GFAP-HSVtk transgenic mice. A mechanically induced model of intestinal wound healing was developed to study glial-induced epithelial restitution. Glial-epithelial signaling mechanisms were analyzed by using pharmacological inhibitors, neutralizing antibodies, and genetically engineered intestinal epithelial cells. Enteric glial cells were shown to be abundant in the gut mucosa, where they associate closely with intestinal epithelial cells as a distinct cell population from myofibroblasts. Conditional ablation of enteric glia worsened mucosal damage after DSS treatment and significantly delayed mucosal wound healing following diclofenac-induced small intestinal enteropathy in transgenic mice. Enteric glial cells enhanced epithelial restitution and cell spreading in vitro. These enhanced repair processes were reproduced by use of glial-conditioned media, and soluble proEGF was identified as a secreted glial mediator leading to consecutive activation of epidermal growth factor receptor and focal adhesion kinase signaling pathways in intestinal epithelial cells. Our study shows that enteric glia represent a functionally important cellular component of the intestinal epithelial barrier microenvironment and that the disruption of this cellular network attenuates the mucosal healing process. 10.1152/ajpgi.00427.2010
    Intestinal myofibroblasts in innate immune responses of the intestine. Otte Jan-Michel,Rosenberg Ian M,Podolsky Daniel K Gastroenterology BACKGROUND & AIMS:Intestinal myofibroblasts are known to respond to inflammatory signals and may play a role in Crohn's disease-associated fibrosis. However, putative involvement by myofibroblasts in innate immune responses as part of intestinal host defense has not been characterized. We therefore analyzed expression and regulation of toll-like receptors (TLRs) in colonic human myofibroblasts (CCD-18) and primary human colonic myofibroblasts in comparison with human lung myofibroblasts (CCD-37). METHODS:Expression of TLRs (1-10) and NOD 1 and 2 was assessed before and after stimulation with either lipopolysaccharide (LPS) or lipoteichoic acid (LTA) by using a custom microarray, reverse-transcription polymerase chain reaction, Northern blot and Western blot analysis, and immunohistochemistry. Activation of signaling pathways, translocation of p65, and secretion of interleukin (IL)-8 were determined. RESULTS:Messenger RNAs encoding for TLR1-9, as well as NOD1 and NOD2, were amplified from cultured and primary human intestinal myofibroblasts. After stimulation with LPS or LTA, a 1.5-4.2-fold up-regulation of TLRs (2, 3, 4, 6, 7) and elements of the signaling cascade (MyD88, TIR domain-containing adapter protein [TIRAP]) was observed. CCD-18 and CCD-37 cells expressed TLR 2 and 4 protein, which were located primarily on the cell membrane. Stimulation with LTA or LPS resulted in activation of the mitogen-activated protein kinases pathway, nuclear translocation of p65, and significantly increased IL-8 secretion. CONCLUSIONS:Bacterial components directly activate intestinal myofibroblasts expressing TLRs. These cells may therefore participate in innate immune responses by sensing and responding to bacterial products that have penetrated into the subepithelial compartment. 10.1016/s0016-5085(03)00403-7
    Flavonol-rich fractions of yaupon holly leaves (Ilex vomitoria, Aquifoliaceae) induce microRNA-146a and have anti-inflammatory and chemopreventive effects in intestinal myofibroblast CCD-18Co cells. Noratto Giuliana D,Kim Youngmok,Talcott Stephen T,Mertens-Talcott Susanne U Fitoterapia Polyphenolics extracted from yaupon holly (Ilex vomitoria, Aquifoliaceae) (YH) leaves were investigated in human colon cells for their chemopreventive and anti-inflammatory activities. An activity-guided fractionation allowed the selection of YH flavonol-rich fraction due to its preferential inhibition of HT-29 colon cancer viability over the normal CCD-18Co colon cells. Quercetin and kaempferol 3-rutinosides, main components identified in this fraction, protected CCD-18Co cells against reactive oxidative species (ROS) in part due to increased activity of antioxidant enzymes. In addition, up-regulation of microRNA-146a (miR-146a) known as a negative regulator of pro-inflammatory NF-κB activation was the underlying molecular mechanism that protected CCD-18Co from inflammation. 10.1016/j.fitote.2011.01.013
    Intestinal epithelial cells as producers but not targets of chronic TNF suffice to cause murine Crohn-like pathology. Roulis Manolis,Armaka Maria,Manoloukos Menelaos,Apostolaki Maria,Kollias George Proceedings of the National Academy of Sciences of the United States of America TNF plays a crucial role in the pathogenesis of Crohn disease. Dysregulated TNF production in mice that bear the genetic deletion of the TNF AU-rich regulatory elements (ARE) (Tnf(ΔARE/+) mice) results in TNF receptor I (TNFRI)-dependent spontaneous Crohn-like pathology. Current concepts consider intestinal epithelial cell (IEC) responses to TNF to be critical for intestinal pathology, but the potential contribution of IEC-derived TNF in disease pathogenesis has not been addressed. In this study we examined whether IEC are sufficient as cellular targets or sources of TNF in the development of intestinal pathology. Using IEC-specific reactivation of a hypomorphic Tnf(ΔAREneo) allele in mice, we show that selective chronic overproduction of TNF by IEC suffices to cause full development of Crohn-like pathology. Epithelial TNF overexpression leads to early activation of the underlying intestinal myofibroblast, a cell type previously identified as a sufficient target of TNF for disease development in the Tnf(ΔARE) model. By contrast, restricted TNFRI expression on IEC although sufficient to confer IEC apoptosis after acute exogenous TNF administration, fails to induce pathology following chronic specific targeting of IEC by endogenous TNF in Tnf(ΔARE/+) mice. Our results argue against IEC being early and sufficient responders to chronic TNF-mediated pathogenic signals and suggest that proinflammatory aberrations leading to chronic TNF production by IEC may initiate pathology in Crohn disease. 10.1073/pnas.1007811108
    Epithelial expression of interleukin-37b in inflammatory bowel disease. Imaeda H,Takahashi K,Fujimoto T,Kasumi E,Ban H,Bamba S,Sonoda H,Shimizu T,Fujiyama Y,Andoh A Clinical and experimental immunology Interleukin (IL)-37 is a member of the IL-1 cytokine family. We investigated IL-37b expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, we analysed IL-37b expression in human colonic epithelial cells. The human colonic epithelial cell line T84 and human colonic subepithelial myofibroblasts (SEMFs) were used. IL-37b expression in the IBD mucosa was evaluated by immunohistochemistry. IL-37b mRNA and protein expression were determined by real time-polymerase chain reaction (PCR) and Western blotting, respectively. IL-37b was not detected in the normal colonic mucosa. In the inflamed mucosa of IBD patients, epithelial IL-37b expression was increased markedly. In ulcerative colitis (UC) and Crohn's disease (CD) patients, IL-37b expression was enhanced in the affected mucosa. In the intestinal epithelial cell line T84, the expression of IL-37b mRNA and protein was enhanced by tumour necrosis factor (TNF)-α. This IL-37b induction by TNF-α was mediated by nuclear factor (NF)-κB and activator protein (AP)-1 activation. Furthermore, IL-37b inhibited TNF-α-induced interferon-γ-inducible protein (IP)-10 expression significantly in human colonic SEMFs. Epithelial IL-37b expression was increased in IBD patients, especially UC patients. IL-37b may be involved in the pathophysiology of IBD as an anti-inflammatory cytokine and an inhibitor of both innate and acquired immune responses. 10.1111/cei.12061
    Interleukin-17 and lipopolysaccharides synergistically induce cyclooxygenase-2 expression in human intestinal myofibroblasts. Zhang Zhuobin,Andoh Akira,Inatomi Osamu,Bamba Shigeki,Takayanagi Atsushi,Shimizu Nobuyoshi,Fujiyama Yoshihide Journal of gastroenterology and hepatology BACKGROUND:Colonic subepithelial myofibroblasts (SEMF) play a role in the modulation of mucosal inflammatory responses via the secretion of various inflammatory mediators. In the present study the effects of interleukin (IL)-17 and lipopolysaccharides (LPS) on cyclooxygenase (COX) expression in colonic SEMF were investigated. METHODS:The expression of COX-1 and -2 proteins and mRNAs were determined by western and northern blotting, respectively. Nuclear factor (NF)-kappaB DNA binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA). RESULTS:The expression of COX-2 protein and mRNA was rapidly induced by the addition of IL-17 and LPS, whereas COX-1 expression was not affected by these factors. The effects of IL-17 and LPS were detected in a dose- and time-dependent manner. Furthermore, IL-17 and LPS synergistically induced COX-2 mRNA and protein expression. The EMSA demonstrated that the addition of IL-17 and LPS induced NF-kappaB activation within 1.5 h after stimulation, and a blockade of NF-kappaB activation by a recombinant adenovirus containing a stable form of IkappaBa markedly reduced the IL-17- and LPS-induced COX-2 mRNA expression. In these cells, the expression of Toll-like receptor (TLR)-4, which is a cellular receptor for LPS, was detected. CONCLUSION:Interleukin-17 and LPS play an important role in the induction of COX-2 in SEMF. These findings suggest that COX-2 expression and prostaglandin synthesis might be regulated by both T-cell-derived factor (IL-17) and bacterial products (LPS) in the inflamed mucosa. 10.1111/j.1440-1746.2004.03748.x
    Regulation of COX-2 expression in human intestinal myofibroblasts: mechanisms of IL-1-mediated induction. Mifflin Randy C,Saada Jamal I,Di Mari John F,Adegboyega Patrick A,Valentich John D,Powell Don W American journal of physiology. Cell physiology Elevated mucosal interleukin-1 (IL-1) levels are frequently seen during acute and chronic intestinal inflammation, and IL-1 neutralization lessens the severity of inflammation. One major effect of IL-1 is the increased release of eicosanoid mediators via induction of cyclooxygenase-2 (COX-2). One site of COX-2-derived prostaglandin synthesis during acute and chronic intestinal inflammation is the intestinal myofibroblast. COX-2 expression has also been documented in these cells in colonic neoplasms. Thus an understanding of the regulation of COX-2 expression in human intestinal myofibroblasts is important. As an initial step toward this goal we have characterized IL-1alpha signaling pathways that induce COX-2 expression in cultured human intestinal myofibroblasts. IL-1 treatment resulted in a dramatic transcriptional induction of COX-2 gene expression. Activation of nuclear factor-kappaB (NF-kappaB), extracellular signal-regulated protein kinase (ERK), p38, and protein kinase C (PKC) signaling pathways was each necessary for optimal COX-2 induction. In contrast to what occurs in other cell types, including other myofibroblasts such as renal mesangial cells, PKC inhibition did not prevent IL-1-induced NF-kappaB or mitogen activated protein kinase/ stress-activated protein kinase activation, suggesting a novel role for PKC isoforms during this process. The stimulatory effects of PKC, NF-kappaB, ERK-1/2, and presumably c-Jun NH(2)-terminal kinase activation were exerted at the transcriptional level, whereas p38 activation resulted in increased stability of the COX-2 message. We conclude that, in intestinal myofibroblasts, IL-1-mediated induction of COX-2 expression is a complex process that requires input from multiple signaling pathways. Each parallel pathway acts in relative autonomy, the sum of their actions culminating in a dramatic increase in COX-2 transcription and message stability. 10.1152/ajpcell.00388.2001
    CD147 Targeting by AC-73 Induces Autophagy and Reduces Intestinal Fibrosis Associated with TNBS Chronic Colitis. Journal of Crohn's & colitis BACKGROUND AND AIMS:Intestinal fibrosis is a common complication of inflammatory bowel diseases. Medical treatment of intestinal fibrosis is an unmet therapeutic need. CD147 overexpression can induce myofibroblast differentiation associated with extracellular matrix deposition, favouring the development of fibrosis. To understand whether CD147 may promote intestinal fibrosis, we analysed its expression and blocked its function by using its specific inhibitor AC-73 [3-{2-[([1,1'-biphenyl]-4-ylmethyl) amino]-1-hydroxyethyl} phenol] in the murine TNBS [trinitrobenzenesulfonic acid]-chronic colitis model associated with intestinal fibrosis. METHODS:TNBS chronic colitis was induced by weekly intrarectal administration of escalating doses of TNBS. Ethanol-treated and untreated mice were used as controls. Separated groups of TNBS, ethanol-treated or untreated mice received AC-73 or vehicle administered intraperitoneally from day 21 to day 49. At day 49, mice were killed, and colons collected for histological analysis, protein and RNA extraction. CD147, α-SMA and activated TGF-β1 protein levels, CD147/ERK/STAT3 signalling pathway and autophagy were assessed by Western blot, collagen and inflammatory/fibrogenic cytokines mRNA tissue content by quantitative PCR. RESULTS:In mice with chronic TNBS colitis, CD147 protein level increased during fibrosis development in colonic tissue, as compared to control mice. CD147 inhibition by AC-73 treatment reduced intestinal fibrosis, collagen and cytokine mRNA tissue content, without significant modulation of activated TGF-β1 protein tissue content. AC-73 inhibited CD147/ERK1/2 and STAT3 signalling pathway activation and induced autophagy. CONCLUSIONS:CD147 is a potential new target for controlling intestinal fibrosis and its inhibitor, AC-73, might represent a potential new anti-fibrotic therapeutic option in IBD. 10.1093/ecco-jcc/jjac084
    TLR4 activation enhances the PD-L1-mediated tolerogenic capacity of colonic CD90+ stromal cells. Beswick Ellen J,Johnson Jameel R,Saada Jamal I,Humen Martin,House Jenifer,Dann Sara,Qiu Suimin,Brasier Allan R,Powell Don W,Reyes Victor E,Pinchuk Irina V Journal of immunology (Baltimore, Md. : 1950) Signaling via programmed death ligand-1 (PD-L1) and PD-L2 is crucial for maintaining peripheral tolerance. CD90(+) myofibroblasts/fibroblasts (CMFs) are major programmed cell death-1 (PD-1) ligand-expressing cells in normal human colonic mucosa. CMFs suppress activated CD4(+) T cell proliferation via PD-1 ligands. It is not known whether signaling through TLRs contribute to the regulation PD-1 ligands on CMFs upon colonic mucosal tolerance. In this study, we demonstrated that stimulation of TLR4 on human CMFs upregulates PD-L1, but not PD-L2, and reinforces CMF-mediated suppression of CD4(+) T cell proliferation and IFN-γ production. TLR4-mediated upregulation of PD-L1 on CMFs involved NF-κB pathways and was JAK2 and MyD88 dependent. MyD88-dependent stimulation of TLR1/2 and TLR5 also upregulated PD-L1 expression on CMFs in culture. PD-L1 expression was drastically decreased in vivo in the colonic mucosa of mice devoid of MyD88. Induction of MyD88 deficiency in CMFs in fibroblast-specific MyD88 conditional knockout mice resulted in a strong increase in a mucosal IFN-γ expression concomitantly with the abrogation of PD-L1 expression in CMFs under homeostasis and epithelial injury induced by dextran sodium sulfate. Together, these data suggest that MyD88-dependent TLR stimulation of CMFs in the normal colonic mucosa may reinforce these cells' anti-inflammatory capacity and thus contribute to the maintenance of mucosal tolerance. 10.4049/jimmunol.1203441
    Constitutive IKK2 activation in intestinal epithelial cells induces intestinal tumors in mice. Vlantis Katerina,Wullaert Andy,Sasaki Yoshiteru,Schmidt-Supprian Marc,Rajewsky Klaus,Roskams Tania,Pasparakis Manolis The Journal of clinical investigation Many cancers display increased NF-κB activity, and NF-κB inhibition is known to diminish tumor development in multiple mouse models, supporting an important role of NF-κB in carcinogenesis. NF-κB activation in premalignant or cancer cells is believed to promote tumor development mainly by protecting these cells from apoptosis. However, it remains unclear to what extent NF-κB activation exhibits additional protumorigenic functions in premalignant cells that could be sufficient to induce spontaneous tumor development. Here we show that expression of constitutively active IκB kinase 2 (IKK2ca) in mouse intestinal epithelial cells (IECs) induced spontaneous tumors in aged mice and also strongly enhanced chemical- and Apc mutation-mediated carcinogenesis. IECs expressing IKK2ca displayed altered Wnt signaling and increased proliferation and elevated expression of genes encoding intestinal stem cell-associated factors including Ascl2, Olfm4, DLK1, and Bmi-1, indicating that increased IKK2/NF-κB activation synergized with Wnt signaling to drive intestinal tumorigenesis. Moreover, IECs expressing IKK2ca produced cytokines and chemokines that induced the recruitment of myeloid cells and activated stromal fibroblasts to become myofibroblasts, thus creating a tumor-promoting microenvironment. Taken together, our results show that constitutively increased activation of IKK2/NF-κB signaling in the intestinal epithelium is sufficient to induce the full spectrum of cell-intrinsic and stromal alterations required for intestinal tumorigenesis. 10.1172/JCI45349
    Sphingosine-1-phosphate enhances IL-1{beta}-induced COX-2 expression in mouse intestinal subepithelial myofibroblasts. Ohama Takashi,Okada Muneyoshi,Murata Takahisa,Brautigan David L,Hori Masatoshi,Ozaki Hiroshi American journal of physiology. Gastrointestinal and liver physiology Intestinal subepithelial myofibroblasts (SEMFs) is a specific population of cells involved in intestinal inflammation and carcinogenesis via an elaborate network of cytokines, chemokines and other inflammatory factors, including PGE(2). Sphingosine-1-phosphate (S1P) has been implicated as an important mediator of inflammation and cancer and in certain cell types increases cyclooxygenase-2 (COX-2) expression. In the present study, we aimed to assess involvement of S1P in COX-2 expression by SEMFs. Primary SEMFs were obtained from C57BL/6J mouse and their identity was verified by fluorescent staining of specific marker proteins. Expression of S1P receptors 1, 2, 3 and sphingosine kinases 1 and 2 in SEMFs were determined by RT-PCR analysis. COX-2 expression and PGE(2) production were assayed by Western blotting and ELISA, respectively. COX-2 mRNA stability was assayed by Northern blotting. S1P produced dose-dependent increase in COX-2 expression, resulting in increased PGE(2) release from SEMFs. Using specific inhibitors, we show that actions of p38, ERK, IKK, and PKC were involved in S1P-induced COX-2 expression. On the other hand, p38 and PKC had lesser roles in IL-1beta-induced COX-2 expression. Inhibition of sphingosine kinase to block S1P production did not affect IL-1beta-induced COX-2 expression, but S1P amplified IL-1beta-induced p38 activation and COX-2 expression. PKC inhibition blocked S1P amplified COX-2 expression. S1P addition increased COX-2 mRNA stability. In SEMFs, S1P amplifies IL-1beta-induced COX-2 expression through increased mRNA stability. These observations point to involvement of S1P in activation of SEMFs that may contribute to intestinal inflammation and carcinogenesis. 10.1152/ajpgi.90423.2008
    Tumor necrosis factor (TNF) alpha increases collagen accumulation and proliferation in intestinal myofibroblasts via TNF receptor 2. Theiss Arianne L,Simmons James G,Jobin Christian,Lund P Kay The Journal of biological chemistry Intestinal fibrosis is an incurable complication of Crohn's disease involving increased numbers of collagen-producing myofibroblasts. Tumor necrosis factor (TNF) alpha has defined proinflammatory roles in Crohn's disease but its role in fibrosis is unclear. We tested the hypothesis that TNFalpha increases collagen accumulation and proliferation in intestinal myofibroblasts and has additive effects in combination with insulin-like growth factor (IGF) I. The mechanisms, TNF receptor isoform, and downstream signaling pathways were examined. Intestinal myofibroblasts from wild-type (WT) mice or mice homozygous for disruption of genes encoding TNFR1 (TNFR1-/-), TNFR2 (TNFR2-/-), or both (TNFR1/2-/-), were treated with TNFalpha, IGF-I, or both. In WT cells, TNFalpha and IGF-I stimulated type I collagen accumulation and DNA synthesis in an additive manner. IGF-I, but not TNFalpha, stimulated type I collagen gene activation. TNFalpha, but not IGF-I, induced tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and reduced matrix metalloproteinases-2 activity and collagen degradation. TNFalpha also activated ERK1/2. These responses to TNFalpha were absent in TNFR2-/- and TNFR1/2-/- myofibroblasts, whereas TNFR1-/- cells showed similar responses to WT. Inhibition of ERK1/2 diminished TNFalpha induced DNA synthesis in WT and TNFR1-/- cells. Differences in TNFalpha-induced STAT3/DNA binding activity and not NFkappaB and AP-1 transcriptional activation correlated with impaired collagen accumulation/TIMP-1 induction in TNFR2(-/-) cells. Constitutively active STAT3 rescued TIMP-1 expression in TNFR2-/- cells. We conclude that TNFalpha and IGF-I may additively contribute to fibrosis during intestinal inflammation. TNFR2 is a primary mediator of fibrogenic actions of TNFalpha acting through ERK1/2 to stimulate proliferation and through STAT3 to stimulate TIMP-1 and inhibit collagen degradation. 10.1074/jbc.M505291200
    Functional modulation of Crohn's disease myofibroblasts by anti-tumor necrosis factor antibodies. Di Sabatino Antonio,Pender Sylvia L F,Jackson Claire L,Prothero Joanna D,Gordon John N,Picariello Lucia,Rovedatti Laura,Docena Guillermo,Monteleone Giovanni,Rampton David S,Tonelli Francesco,Corazza Gino R,MacDonald Thomas T Gastroenterology BACKGROUND & AIMS:Infliximab induces immune cell apoptosis by outside-to-inside signaling through transmembrane tumor necrosis factor-alpha (mTNF). However, in inflamed gut, myofibroblasts also produce TNF-alpha, and the affects of anti-TNF antibodies on these structural cells are unknown. We investigated the action of infliximab on apoptosis, the production of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP)-1, and migration of Crohn's disease (CD) myofibroblasts. METHODS:Colonic myofibroblasts were isolated from patients with active CD and controls. mTNF was evaluated by Western blotting and flow cytometry. Infliximab-treated myofibroblasts were analyzed for apoptosis by Annexin V staining and caspase-3. TIMP-1 and MMPs were measured by Western blotting, and fibroblast migration was assessed by using an in vitro wound-healing scratch assay. RESULTS:CD myofibroblasts showed higher mTNF expression than control myofibroblasts. Infliximab had no effect on CD myofibroblast apoptosis, caspase-3 activation, and production of MMP-3 and MMP-12. However, infliximab induced a significant dose-dependent increase in TIMP-1 production, which was inhibited by the p38 mitogen-activated protein kinase inhibitor SB 203580. The anti-TNF agents adalimumab, etanercept, and p55 TNF-receptor-human IgG fusion protein also increased TIMP-1 production. The migration of CD myofibroblasts was enhanced significantly by infliximab and recombinant human TIMP-1, and infliximab-induced migration was inhibited by anti-TIMP-1 neutralizing antibody. Infliximab also decreased CD myofibroblast collagen production. CONCLUSIONS:Our findings show a novel therapeutic pathway for anti-TNF therapies in enhancing TIMP-1 production and myofibroblast migration, which may reduce MMP activity and facilitate the wound healing. 10.1053/j.gastro.2007.04.069
    Myofibroblast matrix metalloproteinases activate the neutrophil chemoattractant CXCL7 from intestinal epithelial cells. Kruidenier Laurens,MacDonald Thomas T,Collins Jane E,Pender Sylvia L F,Sanderson Ian R Gastroenterology BACKGROUND & AIMS:The up-regulation of matrix metalloproteinases (MMPs) in the inflamed gut has mainly been associated with mucosal degradation and ulceration. However, their in vitro capacity to specifically cleave inflammatory mediators indicates that MMPs may have a profound immunoregulatory impact. We hypothesized that MMPs proteolytically modify intestinal epithelial chemokine signaling. METHODS:Interleukin-1beta-stimulated Caco-2 cells were exposed basolaterally to nanomolar concentrations of activated MMP-3 or cocultured with interleukin-1beta-stimulated, MMP-producing, colonic myofibroblasts (CCD-18co). The conditioned media were subjected to chemotaxis assays. In addition, epithelial cells from patients with colitis were examined by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULTS:MMP-3 dose-dependently induced the neutrophil (up to 5-fold) but not monocyte chemoattractant capacity of Caco-2 cells. A similar Caco-2 chemotactic response was obtained in the Caco-2/CCD-18co cocultures. The principal mediator of these protease-related effects was identified as the potent neutrophil chemokine CXCL7 (neutrophil activating peptide 2), a proteolytic cleavage product of chemotactically inert platelet basic protein (PBP), not previously identified in the intestine. Antibodies against CXCL7 inhibited the MMP-induced chemotactic response by 84%, and PBP mRNA and protein were detected in stimulated Caco-2 but not in CCD-18co cells. Furthermore, PBP transcript and protein levels were low in the mucosa and in isolated epithelial cells from patients with Crohn's disease and from normal intestine but increased up to 13-fold in patients with ulcerative colitis. CONCLUSIONS:These findings identify a novel proinflammatory action of MMPs in inflammation and suggest that lamina propria myofibroblasts are required to achieve maximal intestinal epithelial immune activation. 10.1053/j.gastro.2005.09.032
    The chemokines CXCL9, CXCL10, and CXCL11 differentially stimulate G alpha i-independent signaling and actin responses in human intestinal myofibroblasts. Kouroumalis Andreas,Nibbs Robert J,Aptel Herve,Wright Karen L,Kolios George,Ward Stephen G Journal of immunology (Baltimore, Md. : 1950) Intestinal myofibroblasts have been implicated in the pathogenesis of chronic inflammatory conditions such as Crohn's disease via interactions with an elaborate network of cytokines, growth factors, and other inflammatory mediators. CXCR3 is a Galpha(i) protein-coupled receptor that binds the proinflammatory chemokines CXCL9, CXCL10, and CXCL11, which are released from the intestinal epithelium. The three CXCR3 ligands shared the ability to activate biochemical (e.g., PI3K and MAPK activation) and functional events (actin reorganization) in intestinal myofibroblasts. However, CXCL11 is unique in its ability to elevate intracellular calcium. Surprisingly, although CXCR3 mRNA is detectable in these myofibroblasts, there is no detectable surface expression of CXCR3. Furthermore, the biochemical responses and actin reorganization stimulated by the CXCR3 ligands in intestinal myofibroblasts are insensitive to the Galpha(i) inhibitor, pertussis toxin. This suggests either the existence of differential receptor coupling mechanisms in myofibroblasts for CXCR3 that are distinct from those observed in PBLs and/or that these cells express a modified or variant CXCR3 compared with the CXCR3 expressed on PBLs. 10.4049/jimmunol.175.8.5403
    Reelin expression is up-regulated in mice colon in response to acute colitis and provides resistance against colitis. Carvajal Ana E,Vázquez-Carretero María D,García-Miranda Pablo,Peral María J,Calonge María L,Ilundain Anunciación A Biochimica et biophysica acta. Molecular basis of disease Reelin is an extracellular matrix protein first known for its key role in neuronal migration. Studies in rodent small intestine suggested that reelin protects the organism from intestinal pathology. Here we determined in mice colon, by real time-PCR and immunological assays, the expression of the reelin signalling system; its response to dextran sulphate sodium (DSS) and the response of wild-type and reeler mice to DSS-treatment. DNA methylation was determined by bisulfite modification and sequencing of genomic DNA. In the colon mucosa reelin expression is restricted to the myofibroblasts, whereas both epithelial cells and myofibroblasts express reelin receptors (ApoER2 and VLDLR) and its effector protein Dab1. The muscle layer also expresses reelin. DSS-treatment reduces reelin expression in the muscle but it is activated in the mucosa. Activation of mucosal reelin is greater in magnitude and is delayed until after the activation of the myofibroblasts marker, α-SMA. This indicates that the DSS-induced reelin up-regulation results from changes in the reelin gene expression rather than from myofibroblasts proliferation. DSS-treatment does not modify Sp1 or Tbr1 mRNA abundance, but increases that of TGF-β1 and ApoER2, decreases that of CASK and DNMT1 and it also decreases the reelin promoter methylation. Finally, the reeler mice exhibit higher inflammatory scores than wild-type mice, indicating that the mutation increases the susceptibility to DSS-colitis. In summary, this data are the first to demonstrate that mouse distal colon increases reelin production in response to DSS-colitis via a DNMT1-dependent hypo-methylation of the gene promoter region and that reelin provides protection against colitis. 10.1016/j.bbadis.2016.11.028
    Lipopolysaccharide activates innate immune responses in murine intestinal myofibroblasts through multiple signaling pathways. Walton Kristen L W,Holt Lisa,Sartor R Balfour American journal of physiology. Gastrointestinal and liver physiology Myofibroblasts (MF) play an important role in intestinal wound healing. A compromised epithelial barrier exposes intestinal subepithelial MF to luminal bacterial products. However, responses of murine intestinal MF to bacterial adjuvants and potential roles of intestinal MF in innate immune responses are not well defined. Our aims in this study were to determine innate immune responses and intracellular signaling pathways of intestinal MF exposed to LPS, a prototypic Toll-like receptor (TLR) ligand. Expression of TLR4 in primary murine intestinal MF cultures was confirmed by RT-PCR and Western blotting. LPS-induced secretion of prostaglandin E2 (PGE2), interleukin (IL)-6, and keratinocyte-derived chemokines (KC) was measured by ELISA. Intracellular responses to LPS were assessed by Western blotting for NF-kappaB p65, Ikappa-Balpha, Akt, p38 MAP kinase, and cyclooxygenase-2 (COX-2). LPS induced rapid phosphorylation of NF-kappaB p65, Akt, and p38 MAPK and degradation of Ikappa-Balpha. LPS induced expression of COX-2 and secretion of PGE2 (2.0+/-0.8-fold induction vs. unstimulated cells), IL-6 (6.6+/-0.4-fold induction), and KC (12.5+/-0.4-fold induction). Inhibition of phosphoinositide-3 (PI3)-kinase, p38 MAPK, or NF-kappaB pathways reduced LPS-induced PGE2, IL-6, and KC secretion. These studies show that primary murine intestinal MF respond to LPS, evidenced by activation of NF-kappaB, PI3-kinase, and MAPK signaling pathways and secretion of proinflammatory molecules. Inhibition of these pathways attenuated LPS-dependent PGE2, IL-6, and KC production, indicating that LPS activates MF by multiple signaling pathways. These data support the hypothesis that MF are a component of the innate immune system and may exert paracrine effects on adjacent epithelial and immune cells by responding to luminal bacterial adjuvants. 10.1152/ajpgi.00022.2008
    Cellular and molecular mechanisms of intestinal fibrosis. Speca Silvia,Giusti Ilaria,Rieder Florian,Latella Giovanni World journal of gastroenterology Fibrosis is a chronic and progressive process characterized by an excessive accumulation of extracellular matrix (ECM) leading to stiffening and/or scarring of the involved tissue. Intestinal fibrosis may develop in several different enteropathies, including inflammatory bowel disease. It develops through complex cell, extracellular matrix, cytokine and growth factor interactions. Distinct cell types are involved in intestinal fibrosis, such as resident mesenchymal cells (fibroblasts, myofibroblasts and smooth muscle cells) but also ECM-producing cells derived from epithelial and endothelial cells (through a process termed epithelial- and endothelial-mesenchymal transition), stellate cells, pericytes, local or bone marrow-derived stem cells. The most important soluble factors that regulate the activation of these cells include cytokines, chemokines, growth factors, components of the renin-angiotensin system, angiogenic factors, peroxisome proliferator-activated receptors, mammalian target of rapamycin, and products of oxidative stress. It soon becomes clear that although inflammation is responsible for triggering the onset of the fibrotic process, it only plays a minor role in the progression of this condition, as fibrosis may advance in a self-perpetuating fashion. Definition of the cellular and molecular mechanisms involved in intestinal fibrosis may provide the key to developing new therapeutic approaches. 10.3748/wjg.v18.i28.3635
    Aspirin-mediated COX-2 transcript stabilization via sustained p38 activation in human intestinal myofibroblasts. Mifflin Randy C,Saada Jamal I,Di Mari John F,Valentich John D,Adegboyega Patrick A,Powell Don W Molecular pharmacology Acetylsalicylic acid (aspirin) is a cyclooxygenase (COX) inhibitor, yet some of its therapeutic effects are thought to derive from mechanisms unrelated to prostaglandin synthesis inhibition. In human intestinal myofibroblasts, aspirin, at therapeutic doses, had the unexpected effect of inducing prolonged COX-2 expression. This induction was especially pronounced when cells were treated with interleukin-1alpha (IL-1) plus aspirin for 24 h. Sodium salicylate, a poor COX inhibitor, likewise enhanced IL-1-mediated COX-2 gene expression whereas 5-aminosalicylic acid (5-ASA) or indomethacin had no effect. The COX-2 transcriptional rate, measured by nuclear runoff analysis and heterogeneous nuclear RNA reverse transcription-polymerase chain reaction, was only modestly elevated by aspirin treatment. In contrast, aspirin treatment dramatically stabilized the COX-2 message. The COX-2 mRNA half-life in IL-1 treated cells was 1 h and was increased in excess of 5 h in IL-1 + aspirin-treated cells. Phosphorylation of p38 MAPK was enhanced in aspirin-treated cells (but not in cells treated with 5-ASA or indomethacin) for up to 24 h after treatment. Inhibition of p38 activity negated aspirin-mediated COX-2 mRNA stabilization and the resultant increase in COX-2 mRNA and protein levels. The modest transcriptional response seen in aspirin treated cells was also abolished by p38 inhibition. We conclude that aspirin enhances COX-2 expression via sustained activation of p38, which results in prolonged stabilization of the COX-2 message and a slightly elevated transcription rate. Aspirin also enhanced steady-state mRNA levels of other IL-1 modulated genes (IL-1beta, IL-6, groalpha, and TNFalpha) that are likewise regulated at the level of message stability via p38 activation. 10.1124/mol.65.2.470
    Human colonic myofibroblasts promote expansion of CD4+ CD25high Foxp3+ regulatory T cells. Pinchuk Irina V,Beswick Ellen J,Saada Jamal I,Boya Gushyalatha,Schmitt David,Raju Gottumukkala S,Brenmoehl Julia,Rogler Gerhard,Reyes Victor E,Powell Don W Gastroenterology BACKGROUND & AIMS:Regulatory T (Treg) cells (CD4+ CD25high FoxP3+) regulate mucosal tolerance; their adoptive transfer prevents or reduces symptoms of colitis in mouse models of inflammatory bowel disease. Colonic CD90+ mesenchymal myofibroblasts and fibroblasts (CMFs) are abundant, nonprofessional antigen-presenting cells in the normal human colonic mucosa that suppress proliferation of activated CD4+ effector T cells. We studied CMF suppressive capacity and evaluated the ability of CMF to induce Treg cells. METHODS:Allogeneic cocultures of CD4+ T cells and CMFs, derived from normal mucosa of patients undergoing colectomy for colon cancer or inflamed colonic tissues from patients with ulcerative colitis or Crohn's disease, were used to assess activation of the Treg cells. RESULTS:Coculture of normal CMF with resting or naïve CD4+ T cells led to development of cells with a Treg phenotype; it also induced proliferation of a CD25+ CD127- FoxP3+ T cells, which expressed CTLA-4, interleukin-10, and transforming growth factor-β and had suppressive activities. In contrast to dendritic cells, normal CMFs required exogenous interleukin-2 to induce proliferation of naturally occurring Treg cells. Induction of Treg cells by normal CMFs required major histocompatibility complex class II and prostaglandin E2. CMFs from patients with inflammatory bowel diseases had reduced capacity to induce active Treg cells and increased capacity to transiently generate CD4+CD25+/- CD127+ T cells that express low levels of FoxP3. CONCLUSIONS:CMFs suppress the immune response in normal colon tissue and might therefore help maintain colonic mucosal tolerance. Alterations in CMF-mediated induction of Treg cells might promote pathogenesis of inflammatory bowel diseases. 10.1053/j.gastro.2011.02.059
    Integrin alpha1beta1 is involved in the differentiation into myofibroblasts in adult reactive tissues in vivo. Rodriguez Alejandro,Karen Jakob,Gardner Humphrey,Gerdin Bengt,Rubin Kristofer,Sundberg Christian Journal of cellular and molecular medicine Connective tissue cell activation is of importance during reactive conditions such as solid tumour growth, wound healing and pannus formation in rheumatoid arthritis. Here, we have compared connective tissue cells of mesenchymal origin in human tissues from these conditions and their normal counterparts using a panel of cell-type-specific markers. In particular, we investigated variations of integrin expression among connective tissue cell phenotypes. Connective tissue cell populations were defined based on their association with the microvasculature and their expression of activation markers. The phenotype of these cells varied according to the type of pathological connective tissue examined. Our morphological data from human tissues suggested that the alpha(1)beta(1) integrin, a collagen/laminin receptor, is involved in the differentiation of precursor cells into myofibroblasts. To mechanistically investigate this hypothesis, we employed experimental models for carcinoma growth and wound healing utilizing alpha(1) integrin-deficient mice. The data confirmed that the alpha(1)beta(1) integrin is of importance not only for the differentiation of mesenchymal cells into myofibroblasts but also for the neovascularization and connective tissue organization and emphasize the importance of myofibroblasts in the pathophysiology of tissue repair, inflammation and tumour growth. 10.1111/j.1582-4934.2008.00638.x
    Mesenchymal Cells in Colon Cancer. Koliaraki Vasiliki,Pallangyo Charles K,Greten Florian R,Kollias George Gastroenterology Mesenchymal cells in the intestine comprise a variety of cell types of diverse origins, functions, and molecular markers. They provide mechanical and structural support and have important functions during intestinal organogenesis, morphogenesis, and homeostasis. Recent studies of the human transcriptome have revealed their importance in the development of colorectal cancer, and studies from animal models have provided evidence for their roles in the pathogenesis of colitis-associated cancer and sporadic colorectal cancer. Mesenchymal cells in tumors, called cancer-associated fibroblasts, arise via activation of resident mesenchymal cell populations and the recruitment of bone marrow-derived mesenchymal stem cells and fibrocytes. Cancer-associated fibroblasts have a variety of activities that promote colon tumor development and progression; these include regulation of intestinal inflammation, epithelial proliferation, stem cell maintenance, angiogenesis, extracellular matrix remodeling, and metastasis. We review the intestinal mesenchymal cell-specific pathways that regulate these processes, with a focus on their roles in mediating interactions between inflammation and carcinogenesis. We also discuss how increasing our understanding of intestinal mesenchymal cell biology and function could lead to new strategies to identify and treat colitis-associated cancers. 10.1053/j.gastro.2016.11.049
    Platelet-Specific Deletion of Cyclooxygenase-1 Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice. Sacco Angela,Bruno Annalisa,Contursi Annalisa,Dovizio Melania,Tacconelli Stefania,Ricciotti Emanuela,Guillem-Llobat Paloma,Salvatore Tania,Di Francesco Luigia,Fullone Rosa,Ballerini Patrizia,Arena Vincenzo,Alberti Sara,Liu Guizhu,Gong Yanjun,Sgambato Alessandro,Patrono Carlo,FitzGerald Garret A,Yu Ying,Patrignani Paola The Journal of pharmacology and experimental therapeutics Inflammatory bowel disease (IBD) is associated with an increased risk for thromboembolism, platelet activation, and abnormalities in platelet number and size. In colitis, platelets can extravasate into the colonic interstitium. We generated a mouse with a specific deletion of cyclooxygenase (COX)-1 in megakaryocytes/platelets [(COX-1 conditional knockout (cKO)] to clarify the role of platelet activation in the development of inflammation and fibrosis in dextran sodium sulfate (DSS)-induced colitis. The disease activity index was assessed, and colonic specimens were evaluated for histologic features of epithelial barrier damage, inflammation, and fibrosis. Cocultures of platelets and myofibroblasts were performed. We found that the specific deletion of COX-1 in platelets, which recapitulated the human pharmacodynamics of low-dose aspirin, that is, suppression of platelet thromboxane (TX)A production associated with substantial sparing of the systemic production of prostacyclin, resulted in milder symptoms of colitis, in the acute phase, and almost complete recovery from the disease after DSS withdrawal. Reduced colonic accumulation of macrophages and myofibroblasts and collagen deposition was found. Platelet-derived TXA enhanced the ability of myofibroblasts to proliferate and migrate in vitro, and these effects were prevented by platelet COX-1 inhibition or antagonism of the TXA receptor. Our findings allow a significant advance in the knowledge of the role of platelet-derived TXA in the development of colitis and fibrosis in response to intestinal damage and provide the rationale to investigate the potential efficacy of the antiplatelet agent low-dose aspirin in limiting the inflammatory response and fibrosis associated with IBD. SIGNIFICANCE STATEMENT: Inflammatory bowel disease (IBD) is characterized by the development of a chronic inflammatory response, which can lead to intestinal fibrosis for which currently there is no medical treatment. Through the generation of a mouse with specific deletion of cyclooxygenase-1 in megakaryocytes/platelets, which recapitulates the human pharmacodynamics of low-dose aspirin, we demonstrate the important role of platelet-derived thromboxane A in the development of experimental colitis and fibrosis, thus providing the rationale to investigate the potential efficacy of low-dose aspirin in limiting the inflammation and tissue damage associated with IBD. 10.1124/jpet.119.259382
    IL-33 activates tumor stroma to promote intestinal polyposis. Maywald Rebecca L,Doerner Stephanie K,Pastorelli Luca,De Salvo Carlo,Benton Susan M,Dawson Emily P,Lanza Denise G,Berger Nathan A,Markowitz Sanford D,Lenz Heinz-Josef,Nadeau Joseph H,Pizarro Theresa T,Heaney Jason D Proceedings of the National Academy of Sciences of the United States of America Tumor epithelial cells develop within a microenvironment consisting of extracellular matrix, growth factors, and cytokines produced by nonepithelial stromal cells. In response to paracrine signals from tumor epithelia, stromal cells modify the microenvironment to promote tumor growth and metastasis. Here, we identify interleukin 33 (IL-33) as a regulator of tumor stromal cell activation and mediator of intestinal polyposis. In human colorectal cancer, IL-33 expression was induced in the tumor epithelium of adenomas and carcinomas, and expression of the IL-33 receptor, IL1RL1 (also referred to as IL1-R4 or ST2), localized predominantly to the stroma of adenoma and both the stroma and epithelium of carcinoma. Genetic and antibody abrogation of responsiveness to IL-33 in the Apc(Min/+) mouse model of intestinal tumorigenesis inhibited proliferation, induced apoptosis, and suppressed angiogenesis in adenomatous polyps, which reduced both tumor number and size. Similar to human adenomas, IL-33 expression localized to tumor epithelial cells and expression of IL1RL1 associated with two stromal cell types, subepithelial myofibroblasts and mast cells, in Apc(Min/+) polyps. In vitro, IL-33 stimulation of human subepithelial myofibroblasts induced the expression of extracellular matrix components and growth factors associated with intestinal tumor progression. IL-33 deficiency reduced mast cell accumulation in Apc(Min/+) polyps and suppressed the expression of mast cell-derived proteases and cytokines known to promote polyposis. Based on these findings, we propose that IL-33 derived from the tumor epithelium promotes polyposis through the coordinated activation of stromal cells and the formation of a protumorigenic microenvironment. 10.1073/pnas.1422445112
    Preventive effect of mesenchymal stem cell culture supernatant on luminal stricture after endoscopic submucosal dissection in the rectum of pigs. Tsuda Momoko,Ohnishi Shunsuke,Mizushima Takeshi,Hosono Hidetaka,Yamahara Kenichi,Ishikawa Marin,Abiko Satoshi,Katsurada Takehiko,Shimizu Yuichi,Sakamoto Naoya Endoscopy BACKGROUND:Mesenchymal stem cells (MSCs) are valuable in regenerative medicine, and MSC culture supernatant (MSC-CS) reportedly inhibits inflammation and fibrosis. We investigated whether colorectal luminal stricture develops after circumferential endoscopic submucosal dissection (ESD) in the colorectum, and whether the development of luminal stricture could be prevented by using MSC-CS enema. METHODS:In the first experiment, we performed circumferential ESD in the rectums or distal colons of pigs (n = 4 in each group). We sacrificed the pigs on Day 22 and measured the degree of luminal stricture. In the second experiment, we performed circumferential ESD in the rectums of pigs and administered an MSC-CS gel or a control gel enema after ESD for 4 days. We sacrificed the pigs on Day 8 (n = 3 in each group) or 22 (n = 3 in each group) to measure the degree of luminal stricture, and performed histological analysis. RESULTS:Severe luminal stricture was observed in the rectum but not in the distal colon. Moreover, fiber accumulation in the submucosa and hypertrophy of the muscularis propria were observed in the rectum but not in the distal colon. The degree of luminal stricture in the rectum was significantly lower in the MSC-CS group than in the control group. Furthermore, MSC-CS attenuated myofibroblast activation and hypertrophy of the muscularis propria on Day 22, and reduced inflammatory cell infiltration on Day 8. CONCLUSIONS:Luminal stricture after ESD developed only in the rectum because of the difference in myofibroblast activation and fiber accumulation. In addition, MSC-CS enema prevented luminal stricture after ESD, possibly by inhibiting the inflammatory reaction and fibrosis. 10.1055/a-0584-7262
    Role of intestinal subepithelial myofibroblasts in inflammation and regenerative response in the gut. Andoh Akira,Bamba Shigeki,Brittan Mairi,Fujiyama Yoshihide,Wright Nicholas A Pharmacology & therapeutics Inflammatory bowel disease (IBD) is characterized by an ongoing mucosal inflammation caused by a dysfunctional host immune response to commensal microbiota and dietary factors. In the pathophysiology of IBD, mesenchymal cells such as intestinal subepithelial myofibroblasts (ISEMF) affect the recruitment, retention and activation of immune cells. Mesenchymal cells also promote resolution of inflammatory activity accompanied with balanced repair processes. The transient appearance of mesenchymal cells is a feature of normal wound healing, but the persistence of these cells is associated with tissue fibrosis. Recent studies suggest that mesenchymal cells derived from bone marrow (BM) stem cells play a crucial role in intestinal repair and fibrosis. This article focuses on recent knowledge about ISEMF in the field of immune response inflammation and repair. Two major topics were documented: interaction between interleukin (IL)-17-secreting CD4+ cells (Th-17 cells) and about role of BM-derived stem cells in mucosal regenerative response via differentiation to ISEMF. Recent therapeutic strategies targeting BM stem cells for IBD patients were also documented. 10.1016/j.pharmthera.2006.12.004
    Interleukin-32α expression in human colonic subepithelial myofibroblasts. Yagi Yuhki,Andoh Akira,Imaeda Hirotsugu,Aomatsu Tomoki,Ohsaki Rie,Inatomi Osamu,Bamba Shigeki,Tsujikawa Tomoyuki,Shimizu Tomoharu,Fujiyama Yoshihide International journal of molecular medicine Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by induction of nuclear factor (NF)-κB activation. We studied IL-32α expression in human colonic subepithelial myofibroblasts (SEMFs). Colonic SEMFs were isolated from normal human colon tissue. IL-32α protein expression was evaluated by Western blot analyses, and IL-32α mRNA expression was analyzed by real-time PCR. IL-32α mRNA was weakly expressed without a stimulus, and its expression was markedly enhanced by IL-1ß and TNF-α. IL-1ß and TNF-α enhanced intracellular accumulation of IL-32α protein, but IL-32α was not detected in supernatants. Each cytokine dose- and time-dependently induced IL-32α mRNA expression. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed IL-1ß- and TNF-α-induced IL-32α mRNA expression, although MAPK inhibitors had no effect. Akt activation in response to these cytokines was confirmed by Western blotting. Blockade of NF-κB activation by an adenovirus expressing a stable mutant form of IκBα markedly suppressed IL-1ß- and TNF-α-induced IL-32α mRNA expression. Human colonic SEMFs expressed IL-32α in response to IL-1ß and TNF-α. IL-32α mRNA expression depends on the phosphatidylinositol 3-kinase and the NF-κB system. 10.3892/ijmm.2010.575
    CaSR stimulates secretion of Wnt5a from colonic myofibroblasts to stimulate CDX2 and sucrase-isomaltase using Ror2 on intestinal epithelia. Pacheco Ivan I,Macleod R John American journal of physiology. Gastrointestinal and liver physiology To understand whether extracellular calcium-sensing receptor (CaSR) expression on colonic myofibroblast cells (18Co) contributed to epithelial homeostasis, we activated the CaSR with 5 mM Ca(2+), screened by RT-PCR Wnt family members, and measured their secretion. Transcripts for Wnt 1, 2, 2b, 3a, 4, and 7a were either absent or unchanged whereas Wnt3 decreased and Wnt5a increased. We assessed Wnt5a secretion by Western blot. High Ca(2+) (5 mM) substantially increased Wnt5a secretion; small interfering RNA (siRNA) against the CaSR reduced this to constitutive amounts. Expression of Wnt5a plasmid but not Wnt1 or Wnt3a increased caudal homeodomain factor CDX2 transcripts and protein in HT-29 adenocarcinoma cells. Wnt5a increased activity of a sucrase-isomaltase (SI) promoter in Caco-2BBE cells. Wnt5a protein stimulation of CDX2 transcripts and protein and SI reporter were increased by overexpression of wild-type Ror2, a Wnt5a receptor, and reduced with siRNA against Ror2. CaSR activation of HT-29 cells increased Ror2 protein expression. Ror2 protein was expressed in mouse jejunum from crypt base to villus tip and in the colon on surface epithelia. Our results show that activation of a G protein-coupled receptor, the CaSR, stimulates secretion of Wnt5a from myofibroblasts. Stimulation of epithelia by the CaSR increased the expression of a receptor for Wnt5a, the tyrosine kinase Ror2, suggesting existence of a unique paracrine relationship for CDX2 homoeostasis in the intestine and revealing new contributions of CaSR-activated myofibroblasts to intestinal stem cell niche microenvironments. 10.1152/ajpgi.00560.2007
    Tissue factor-thrombin signaling enhances the fibrotic activity of myofibroblasts in systemic sclerosis through up-regulation of endothelin receptor A. Chrysanthopoulou Akrivi,Mitroulis Ioannis,Kambas Konstantinos,Skendros Panagiotis,Kourtzelis Ioannis,Vradelis Stergios,Kolios George,Aslanidis Spyros,Doumas Michael,Ritis Konstantinos Arthritis and rheumatism OBJECTIVE:The extrinsic coagulation cascade is involved in the fibrotic process, via thrombin-dependent induction of CCN2 (connective tissue growth factor) expression. Given the previously reported activation of the coagulation system in systemic sclerosis (SSc), we undertook the present study to investigate the involvement of cross-talk between the tissue factor (TF)-thrombin axis and endothelin 1 (ET-1) signaling in the fibrotic activity of SSc. METHODS:Human colonic myofibroblasts (HCMFs) from 6 patients with SSc and gastrointestinal symptoms and from 6 control subjects were isolated and cultured under various conditions. Messenger RNA and protein levels of TF, CCN2, and endothelin receptor A (ET(A) ) were investigated. Collagen production and migratory activity of HCMFs were further assessed. RESULTS:HCMFs from SSc patients demonstrated increased basal CCN2 production, collagen deposition, and migration rate, in a thrombin-dependent manner. Increased TF expression was also observed in SSc HCMFs. Subsequent activation of the extrinsic coagulation system resulted in thrombin-dependent enhancement of ET(A) expression. ET(A) overexpression led to further increases in both TF expression and fibrotic activity in HCMFs. Moreover, inhibition of ET-1 signaling by bosentan abolished the TF-mediated fibrotic capacity of HCMFs. CONCLUSION:Tissue factor-thrombin signaling is involved in the increased fibrotic activity of HCMFs from patients with SSc. Moreover, the up-regulation of ET(A) expression by thrombin and the effect of ET-1 in the induction of TF expression indicate an amplification loop for enhanced collagen deposition. Therapeutic interventions targeting the extrinsic coagulation system or ET-1 signaling may provide clinical benefit by breaking this vicious circle. 10.1002/art.30586
    Vitamin D and Wnt3A have additive and partially overlapping modulatory effects on gene expression and phenotype in human colon fibroblasts. Ferrer-Mayorga Gemma,Niell Núria,Cantero Ramón,González-Sancho José Manuel,Del Peso Luis,Muñoz Alberto,Larriba María Jesús Scientific reports The Wnt/β-catenin signalling pathway is essential for intestinal epithelium homeostasis, but its aberrant activation is a hallmark of colorectal cancer (CRC). Several studies indicate that the bioactive vitamin D metabolite 1α,25-dihydroxyvitamin D (1,25(OH)D) inhibits proliferation and promotes epithelial differentiation of colon carcinoma cells in part through antagonism of the Wnt/β-catenin pathway. It is now accepted that stromal fibroblasts are crucial in healthy and pathologic intestine: pericryptal myofibroblasts are constituents of the stem cell niche and cancer-associated fibroblasts (CAFs) contribute to CRC progression. However, studies on the combined action of 1,25(OH)D and Wnt factors in colon fibroblasts are lacking. Here we show by global transcriptomic studies that 1,25(OH)D and Wnt3A have profound, additive, partially overlapping effects on the gene expression profile of CCD-18Co human colon myofibroblasts. Moreover, 1,25(OH)D and Wnt3A inhibit CCD-18Co cell proliferation and migration, while 1,25(OH)D reduces, but Wnt3A increases, their capacity to contract collagen gels (a marker of fibroblast activation). These data were largely confirmed in patient-derived primary colon normal fibroblasts and CAFs, and in fibroblasts from other origins. Our results indicate that 1,25(OH)D and Wnt3A are strong regulators of colon fibroblast biology and contribute to a better knowledge of intestinal homeostasis and stromal fibroblast action in CRC. 10.1038/s41598-019-44574-9
    Stable and scalable SERS tags conjugated with neutravidin for the detection of fibroblast activation protein (FAP) in primary fibroblasts. Talamona Federica,Truffi Marta,Caldarone Alessandro Aldo,Ricciardi Alessandra,Corsi Fabio,Pellegrini Giovanni,Morasso Carlo,Taglietti Angelo Nanotechnology SERS tags are a class of nanoparticles with great potential in advanced imaging experiments. The preparation of SERS tags however is complex, as they suffer from the high variability of the SERS signals observed even at the slightest sign of aggregation. Here, we developed a method for the preparation of SERS tags based on the use of gold nanostars conjugated with neutravidin. The SERS tags here obtained are extremely stable in all biological buffers commonly employed and can be prepared at a relatively large scale in very mild conditions. The obtained SERS tags have been used to monitor the expression of fibroblast activation protein alpha (FAP) on the membrane of primary fibroblasts obtained from patients affected by Crohn's disease. The SERS tags allowed the unambiguous identification of FAP on the surface of cells thus suggesting the feasibility of semi-quantitative analysis of the target protein. Moreover, the use of the neutravidin-biotin system allows to apply the SERS tags for any other marker detection, for example, different cancer cell types, simply by changing the biotinylated antibody chosen in the analysis. 10.1088/1361-6528/abf5fd
    Periostin Promotes Colorectal Tumorigenesis through Integrin-FAK-Src Pathway-Mediated YAP/TAZ Activation. Ma Handong,Wang Jing,Zhao Xueli,Wu Tiantian,Huang Zhengjie,Chen Dafan,Liu Yingfu,Ouyang Gaoliang Cell reports Periostin is a multifunctional extracellular matrix protein involved in various inflammatory diseases and tumor metastasis; however, evidence regarding whether and how periostin actively contributes to inflammation-associated tumorigenesis remains elusive. Here, we demonstrate that periostin deficiency significantly inhibits the occurrence of colorectal cancer in azoxymethane/dextran sulfate sodium-treated mice and in Apc mice. Moreover, periostin deficiency attenuates the severity of colitis and reduces the proliferation of tumor cells. Mechanistically, stromal fibroblast-derived periostin activates FAK-Src kinases through integrin-mediated outside-in signaling, which results in the activation of YAP/TAZ and, subsequently, IL-6 expression in tumor cells. Conversely, IL-6 induces periostin expression in fibroblasts by activating STAT3, which ultimately facilitates colorectal tumor development. These findings provide the evidence that periostin promotes colorectal tumorigenesis, and identify periostin- and IL-6-mediated tumor-stroma interaction as a promising target for treating colitis-associated colorectal cancer. 10.1016/j.celrep.2019.12.075
    Novel PPARγ Modulator GED-0507-34 Levo Ameliorates Inflammation-driven Intestinal Fibrosis. Speca Silvia,Rousseaux Christel,Dubuquoy Caroline,Rieder Florian,Vetuschi Antonella,Sferra Roberta,Giusti Ilaria,Bertin Benjamin,Dubuquoy Laurent,Gaudio Eugenio,Desreumaux Pierre,Latella Giovanni Inflammatory bowel diseases BACKGROUND:Intestinal fibrosis is mainly associated with Crohn's disease and is defined as a progressive and excessive deposition of extracellular matrix components. No specific antifibrotic therapies are available. In this study, we evaluate the antifibrotic effect of a novel 5-ASA analog able to activate the peroxisome proliferator-activated receptor γ, named GED-0507-34 Levo. METHODS:Colonic fibrosis was induced in 110 C57BL/6 mice by 3 cycles of 2.5% (wt/vol) dextran sulfate sodium administration for 6 weeks. The preventive effects of oral daily GED (30 mg · kg(-1) · d(-1)) administration were evaluated using a macroscopic and histological score and also through biological endpoints. Expression of main markers of myofibroblasts activation was determined in transforming growth factor (TGF-β)-stimulated intestinal fibroblasts and epithelial cells. RESULTS:GED improved macroscopic and microscopic intestinal lesions in dextran sulfate sodium-treated animals and reduced the profibrotic gene expression of Acta2, COL1a1, and Fn1 by 1.48-folds (P < 0.05), 1.93-folds (P < 0.005), and 1.03-fold (P < 0.05), respectively. It reduced protein levels of main markers of fibrosis (α-SMA and Collagen I-II) and the main TGF-β/Smad pathway components. GED also decreased the interleukin-13 and connective tissue growth factor expression by 1.89-folds (P < 0.05) and 2.2-folds (P < 0.005), respectively. GED inhibited TGF-β-induced activation of both fibroblast and intestinal epithelial cell lines, by regulating mRNA expression of α-SMA and fibronectin, and restoring the TGF-β-induced loss of intestinal epithelial cell markers. GED treatment also reduced the TGF-β and ACTA1 expression in primary human intestinal fibroblasts from ulcerative colitis patients. CONCLUSIONS:GED ameliorates intestinal fibrosis in dextran sulfate sodium-induced chronic colitis in mice and regulates major profibrotic cellular and molecular mechanisms. 10.1097/MIB.0000000000000618
    Hedgehog signaling controls homeostasis of adult intestinal smooth muscle. Zacharias William J,Madison Blair B,Kretovich Katherine E,Walton Katherine D,Richards Neil,Udager Aaron M,Li Xing,Gumucio Deborah L Developmental biology The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed. 10.1016/j.ydbio.2011.04.025
    Stromal and immune cells in gut fibrosis: the myofibroblast and the scarface. Valatas Vassilis,Filidou Eirini,Drygiannakis Ioannis,Kolios George Annals of gastroenterology Post-inflammatory scarring is the end-result of excessive extracellular matrix (ECM) accumulation and tissue architectural destruction. It represents a failure to effectively remodel ECM and achieve proper reinstitution and healing during chronic relapsing inflammatory processes. Scarring may affect the functionality of any organ, and in the case of inflammatory bowel disease (IBD)-associated fibrosis leads to stricture formation and often surgery to remove the affected bowel. The activated myofibroblast is the final effector cell that overproduces ECM under the influence of various mediators generated by an intense interplay of classic and non-classic immune cells. This review focuses on how proinflammatory mediators from various sources produced in different stages of intestinal inflammation can form profibrotic pathways that eventually lead to tissue scarring through sustained activation of myofibroblasts. 10.20524/aog.2017.0146
    TNF-α potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts. Rodriguez Perez Citlali Ekaterina,Nie Wenxian,Sinnett-Smith James,Rozengurt Enrique,Yoo James American journal of physiology. Gastrointestinal and liver physiology The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Here, we show that treatment of 18Co cells, a model of human colonic MFBs, with TNF-α and lysophosphatidic acid (LPA) induced striking synergistic cyclooxygenase-2 (COX-2) protein expression and production of PGE(2). This effect was prevented by the LPA(1) receptor antagonist Ki16425, the G(iα)-specific inhibitor pertussis toxin, and by the preferential protein kinase (PK) C inhibitors GF109203X and Go6983. As a known downstream target of LPA and PKC, we tested whether PKD, recently implicated in the regulation of COX-2 expression in MFB, was involved in this response. TNF-α, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation stimulated by LPA, as measured by PKD autophosphorylation at Ser(910). LPA-induced PKD activation was also inhibited by Ki16425, pertussis toxin, GF109203X, and Go6983. Transfection of 18Co cells with short interfering RNA targeting PKD completely inhibited the synergistic increase in COX-2 protein, demonstrating a critical role of PKD in this response. Our results imply that cross talk between TNF-α and LPA results in the amplification of COX-2 protein expression via a conserved PKD-dependent signaling pathway that appears to involve the LPA(1) receptor and the G protein G(iα). PKD plays a critical role in the expression of COX-2 in human colonic MFBs and may contribute to an inflammatory microenvironment that promotes tumor growth. 10.1152/ajpgi.00381.2010
    Counteracting effect of TRPC1-associated Ca2+ influx on TNF-α-induced COX-2-dependent prostaglandin E2 production in human colonic myofibroblasts. Hai Lin,Kawarabayashi Yasuhiro,Imai Yuko,Honda Akira,Inoue Ryuji American journal of physiology. Gastrointestinal and liver physiology TNF-α-NF-κB signaling plays a central role in inflammation, apoptosis, and neoplasia. One major consequence of this signaling in the gut is increased production of prostaglandin E(2) (PGE(2)) via cyclooxygenase-2 (COX-2) induction in myofibroblasts, which has been reported to be dependent on Ca(2+). In this study, we explored a potential role of canonical transient receptor potential (TRPC) proteins in this Ca(2+)-mediated signaling using a human colonic myofibroblast cell line CCD-18Co. In CCD-18Co cell, treatment with TNF-α greatly enhanced Ca(2+) influx induced by store depletion along with increased cell-surface expression of TRPC1 protein (but not of the other TRPC isoforms) and induction of a Gd(3+)-sensitive nonselective cationic conductance. Selective inhibition of TRPC1 expression by small interfering RNA (siRNA) or functionally effective TRPC1 antibody targeting the near-pore region of TRPC1 (T1E3) antagonized the enhancement of store-dependent Ca(2+) influx by TNF-α, whereas potentiated TNF-α induced PGE(2) production. Overexpression of TRPC1 in CCD-18Co produced opposite consequences. Inhibitors of NF-κB (curcumin, SN-50) attenuated TNF-α-induced enhancement of TRPC1 expression, store-dependent Ca(2+) influx, and COX-2-dependent PGE(2) production. In contrast, inhibition of calcineurin-nuclear factor of activated T-cell proteins (NFAT) signaling by FK506 or NFAT Activation Inhibitor III enhanced the PGE(2) production without affecting TRPC1 expression and the Ca(2+) influx. Finally, the suppression of store-dependent Ca(2+) influx by T1E3 antibody or siRNA knockdown significantly facilitated TNF-α-induced NF-κB nuclear translocation. In aggregate, these results strongly suggest that, in colonic myofibroblasts, NF-κB and NFAT serve as important positive and negative transcriptional regulators of TNF-α-induced COX-2-dependent PGE(2) production, respectively, at the downstream of TRPC1-associated Ca(2+) influx. 10.1152/ajpgi.00354.2010
    Celecoxib induces proliferation and Amphiregulin production in colon subepithelial myofibroblasts, activating erk1-2 signaling in synergy with EGFR. Benelli Roberto,Venè Roberta,Minghelli Simona,Carlone Sebastiano,Gatteschi Beatrice,Ferrari Nicoletta Cancer letters The COX-2 inhibitor Celecoxib, tested in phase III trials for the prevention of sporadic colon adenomas, reduced the appearance of new adenomas, but was unable to affect the incidence of colon cancer. Moreover the 5years follow-up showed that patients discontinuing Celecoxib treatment had an increased incidence of adenomas as compared to the placebo arm. In the APC(min/+) mouse model short term treatment with Celecoxib reduced gut adenomas, but a prolonged administration of the drug induced fibroblast activation and intestinal fibrosis with a final tumor burden. The way Celecoxib could directly activate human colon myofibroblasts (MF) has not yet been investigated. We found that MF are activated by non toxic doses of Celecoxib. Celecoxib induces erk1-2 and Akt phosphorylation within 5'. This short term activation is apparently insufficient to cause phenotypic changes, but the contemporary triggering of EGFR causes an impressive synergic effect inducing MF proliferation and the neo-expression and release of Amphiregulin (AREG), a well known EGFR agonist involved in colon cancer progression. As a confirm to these observations, the erk inhibitor U0126 and the EGFR inhibitors Tyrphostin and Cetuximab were able to contrast AREG induction. Our data provide evidence that Celecoxib directly activates MF empowering EGFR signaling. According to these results the association with EGFR (or erk1-2) inhibitors could abolish the off-target activity of Celecoxib, possibly extending the potential of this drug for colon cancer prevention. 10.1016/j.canlet.2012.09.008
    Dermatan sulfate reduces monocyte chemoattractant protein 1 and TGF-β production, as well as macrophage recruitment and myofibroblast accumulation in mice with unilateral ureteral obstruction. Belmiro C L R,Gonçalves R G,Kozlowski E O,Werneck A F,Takyia C M,Leite M,Pavão M S G Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas Selectins play an essential role in most inflammatory reactions, mediating the initial leukocyte-rolling event on activated endothelium. Heparin and dermatan sulfate (DS) bind and block P- and L-selectin function in vitro. Recently, we reported that subcutaneous administration of DS inhibits colon inflammation in rats by reducing macrophage and T-cell recruitment and macrophage activation. In the present study, we examined the effect of porcine intestinal mucosa DS on renal inflammation and fibrosis in mice after unilateral ureteral obstruction (UUO). Twenty-four adult male Swiss mice weighing 20-25 g were divided into 4 groups: group C (N = 6) was not subjected to any surgical manipulation; group SH (N = 6) was subjected to surgical manipulation but without ureter ligation; group UUO (N = 6) was subjected to unilateral ureteral obstruction and received no treatment; group UUO plus DS (N = 6) was subjected to UUO and received DS (4 mg/kg) subcutaneously daily for 14 days. An immunoblot study was also performed for TGF-β. Collagen (stained area ~3700 µm(2)), MCP-1 (stained area ~1700 µm(2)), TGF-β (stained area ~13% of total area), macrophage (number of cells ~40), and myofibroblast (stained area ~1900 µm(2)) levels were significantly (P < 0.05) higher in the UUO group compared to control. DS treatment significantly (P < 0.05) reduced the content of collagen (stained area ~700 µm(2)), MCP-1 (stained area ~160 µm(2)) and TGF-β (stained area ~5% of total area), in addition to myofibroblast (stained area ~190 µm(2)) and macrophage (number of cells ~32) accumulation in the obstructed kidney. Overall, these results indicate that DS attenuates kidney inflammation by reducing macrophage recruitment, myofibroblast population and fibrosis in mice submitted to UUO. 10.1590/s0100-879x2011007500077
    Cytoglobin expression of rectal subepithelial myofibroblasts: significant alterations of cytoglobin+ stromal cells in long-standing ulcerative colitis. Okayasu Isao,Mikami Tetuo,Yoshida Tsutomu,Hana Kiyomi,Yokozawa Masashi,Sada Miwa,Fujiwara Mutsunori,Kawada Norifumi Histology and histopathology Cytoglobin/stellate cell activation-associated protein (Cygb/STAP), a hemoprotein, functions as part of an O2 reservoir with protective effects against oxidative stress in hepatic stellate cells. Heterogeneous expression of the neural cell adhesion molecule (NCAM)+ and/or α-smooth muscle actin (αSMA)+ has been noted in subepithelial myofibroblasts and interstitial cells of the same lineage in the colorectum. We have demonstrated that early genomic instability of both epithelial and stromal cells in ulcerative colitis (UC) is important for colorectal tumorigenesis, as well as for mucosal remodeling. To further clarify possible roles of stromal cells in mucosal remodeling and tumor development in UC, we here focused on Cygb expression of subepithelial myofibroblasts and interstitial cells, as well as αSMA and HSP47. Noncancerous mucosa of resected rectae from UC patients with or without colorectal neoplasia (14 and 20 cases, respectively) and of sporadic rectal cancer cases (16) was analyzed immunohistochemically, as well as by immuno-fluorescence and electron microscopy. The results, heterogeneous phenotypes of Cygb+, αSMA+ and HSP47+ subepithelial myofibroblasts and interstitial cells, corresponding to rectal stellate cells, were demonstrated. A decrease of Cygb+ subepithelial myofibroblasts and an increase of αSMA+ interstitial cells were significant in UC, as compared to normal rectal mucosa. Furthermore, a decrease of Cygb+ subepithelial myofibroblasts, correlating with αSMA+ and HSP47+ cells, was significant in long-standing UC with neoplasia. In conclusion, there are heterogeneous phenotypes of Cygb+, αSMA+ and HSP47+ subepithelial myofibroblasts and interstitial cells in the rectal mucosa. Mucosal remodeling with alterations of Cygb+ and/or αSMA+/HSP47+ stromal cells might have some relation to UC-associated tumorigenesis. 10.14670/HH-26.679
    Interleukin (IL)-1β Is a Strong Inducer of IL-36γ Expression in Human Colonic Myofibroblasts. Takahashi Kenichiro,Nishida Atsushi,Shioya Makoto,Imaeda Hirotsugu,Bamba Shigeki,Inatomi Osamu,Shimizu Tomoharu,Kitoh Katsuyuki,Andoh Akira PloS one BACKGROUNDS AND AIMS:Interleukin (IL)-36 cytokines are members of the IL-1 cytokine family. In this study, we investigated the expression of IL-36γ in human colonic myofibroblasts to explore the molecular mechanisms underlying IL-36γ induction. MATERIALS AND METHODS:IL-36 mRNA was analyzed by real-time PCR method. Secretion of IL-36γ protein was evaluated by Western blot and ELISA analyses. Molecular mechanism of IL-36γ induction was evaluated by siRNA analyses and immunofluorescence experiments. RESULTS:IL-36γ mRNA expression was scarcely detected in the cells without stimulation. IL-1β induced a marked increase of IL-36γ mRNA expression. TNF-α markedly enhanced IL-1β-induced IL-36γ mRNA expression. These responses were confirmed at the protein levels. The inhibitors for ERK1/2 (PD98059 and U0216) and a p38 MAPK (SB203580) significantly reduced the IL-1β-induced IL-36γ mRNA expression. In addition, the siRNAs specific for NF-κB p65 and AP-1 (c-Jun) significantly reduced the expression of IL-1β-induced IL-36γ mRNA. CONCLUSIONS:Colonic myofibroblasts are cellular source of IL-36γ in the intestine. IL-36γ expression was induced by the combination of IL-1β and TNF-α via activation of MAPKs and transcription factors, NF-κB and AP-1. 10.1371/journal.pone.0138423
    Inflammatory bowel disease-associated interleukin-33 is preferentially expressed in ulceration-associated myofibroblasts. Sponheim Jon,Pollheimer Jürgen,Olsen Trine,Balogh Johanna,Hammarström Clara,Loos Tamara,Kasprzycka Monika,Sørensen Dag Reidar,Nilsen Hogne Røed,Küchler Axel M,Vatn Morten H,Haraldsen Guttorm The American journal of pathology Interleukin-33 (IL-33) is a novel member of the interleukin-1 family that induces mucosal pathology in vivo and may drive fibrosis development and angiogenesis. To address its potential role in inflammatory bowel disease, we explored its tissue expression in biopsy specimens from untreated ulcerative colitis patients, observing a 2.6-fold up-regulation of IL-33 mRNA levels, compared to controls. Immunohistochemical analyses of surgical specimens showed that a prominent source of IL-33 in ulcerative colitis lesions were ulceration-associated myofibroblasts that co-expressed the fibroblast marker heat shock protein 47, platelet-derived growth factor receptor (PDGFR)β, and, in part, the myofibroblast marker α-smooth muscle actin (SMA). In contrast, IL-33-positive myofibroblasts were almost absent near the deep fissures seen in Crohn's disease. A screen of known and putative activators of IL-33 in cultured fibroblasts revealed that the Toll-like receptor-3 agonist poly (I:C) was among the strongest inducers of IL-33 and that it synergized with transforming growth factor-β, a combination also known to boost myofibroblast differentiation. Experimental wound healing in rat skin revealed that the de novo induction of IL-33 in pericytes and the possible activation of scattered, tissue-resident IL-33(+)PDGFRβ(+)αSMA(-) fibroblast-like cells were early events that preceded the later appearance of IL-33(+)PDGFRβ(+)αSMA(+) cells. In conclusion, our data point to a novel role for IL-33 in mucosal healing and wound repair and to an interesting difference between ulcerative colitis and Crohn's disease. 10.2353/ajpath.2010.100378
    TNF-α induces upregulation of EGFR expression and signaling in human colonic myofibroblasts. Yoo James,Rodriguez Perez Citlali Ekaterina,Nie Wenxian,Edwards Robert A,Sinnett-Smith James,Rozengurt Enrique American journal of physiology. Gastrointestinal and liver physiology The myofibroblast has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), which has been associated with many human cancers. Here, we show that long-term exposure of 18Co cells, a model of human colonic myofibroblasts, with TNF-α led to a striking increase in cell surface EGFR expression, an effect that was completely inhibited by cycloheximide. Subsequent EGFR binding by EGF and heparin binding (HB)-EGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a significant increase in cyclooxygenase-2 (COX-2) expression compared with 18Co cells treated with EGF and HB-EGF alone. TNF-α also increased EGFR expression and signaling in primary myofibroblasts isolated from human colon tissue. TNF-α-induced upregulation of EGFR may be a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes inflammatory bowel disease and that may contribute to a microenvironment that predisposes to colitis-associated cancer through enhanced COX-2 expression. 10.1152/ajpgi.00522.2011
    Regulation of eotaxin-3/CC chemokine ligand 26 expression by T helper type 2 cytokines in human colonic myofibroblasts. Takahashi K,Imaeda H,Fujimoto T,Ban H,Bamba S,Tsujikawa T,Sasaki M,Fujiyama Y,Andoh A Clinical and experimental immunology Eotaxins induce the trafficking of eosinophils to the sites of inflammation via CC chemokine receptor 3 (CCR3). In this study, we investigated eotaxin-3/CC chemokine ligand 26 (CCL26) expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for eotaxin-3 expression in human colonic myofibroblasts. Eotaxin-3 mRNA and protein expression was evaluated by real time-polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Eotaxin-3 mRNA expression was elevated significantly in the active lesions of ulcerative colitis (UC) patients. Significant elevations were also observed in the active lesions of Crohn's disease (CD) patients, but this was significantly lower than that detected in the active UC lesions. There were no significant increases in the inactive lesions of UC or CD patients. Colonic myofibroblasts were identified as a major source of eotaxin-3 in the colonic mucosa, and interleukin (IL)-4 and IL-13 enhanced eotaxin-3 mRNA and protein expression significantly in these cells. There was a significant positive correlation between mucosal eotaxin-3 and IL-4 mRNA expression in the active lesions of IBD patients. The IL-4- and IL-13-induced eotaxin-3 mRNA expression was regulated by the signal transducer and activator of transcription-6 (STAT-6) and suppressor of cytokine signalling (SOCS)1-mediated pathways. Interferon (IFN)-γ acts as a negative regulator on the IL-4- and IL-13-induced eotaxin-3 expression via STAT-1 activation. Eotaxin-3 expression was elevated specifically in the active lesions of IBD, in particular UC. Eotaxin-3 derived from colonic myofibroblasts may play an important role in the pathophysiology of UC. 10.1111/cei.12117
    BCL2 Regulates Differentiation of Intestinal Fibroblasts. Weder Bruce,Mamie Céline,Rogler Gerhard,Clarke Stephen,McRae Bradford,Ruiz Pedro A,Hausmann Martin Inflammatory bowel diseases BACKGROUND:Fibrosis in patients with Crohn's disease (CD) results from an imbalance toward excessive fibrous tissue formation driven by fibroblasts. Activation of fibroblasts is linked to the B-cell lymphoma 2 (BCL2) family, which is involved in the induction of apoptosis. We investigated the impact of BCL2 repression on fibrogenesis. METHODS:The model of dextran sodium sulfate (DSS)-induced chronic colitis and the heterotopic transplantation model of fibrosis were used. Following the administration of the BCL2 antagonist (ABT-737, 50 mg/kg/d), collagen layer thickness and hydroxyproline (HYP) content were determined. Fibroblasts were stimulated with the BCL2 antagonist (0.01-100 µM). BCL2, alpha smooth muscle actin (αSMA), and collagen I (COL1A1) were determined by quantitative polymerase chain reaction (qPCR), immunofluorescence microscopy (IF), and western blot (WB). mRNA expression pattern was determined by next-generation sequencing (NGS). RESULTS:Collagen layer thickness was significantly decreased in both DSS-induced chronic colitis and the transplantation model of fibrosis upon BCL2 antagonist administration compared with vehicle. Decreased HYP content confirmed the preventive effects of the BCL2 antagonist on fibrosis. In vitro, a significant increase in PI+/annexin V+ human colonic fibroblasts was determined by fluorescence-activated cell sorting upon treatment with high-dose BCL2 antagonist; at a lower dose, αSMA, COL1A1, and TGF were decreased. NGS, IF, and qPCR revealed decreased expression and nuclear translocation of GATA6 and SOX9, known for reprogramming fibroblasts. CONCLUSION:BCL2 antagonist administration partially prevented fibrogenesis in both fibrosis models. The BCL2 antagonist reduced the expression of TGFβ-induced factors involved in differentiation of myofibroblasts, and therefore might represent a potential treatment option against CD-associated fibrosis. 10.1093/ibd/izy147
    Diet in Intestinal Fibrosis: A Double-Edged Sword. Marion-Letellier Rachel,Leboutte Mathilde,Amamou Asma,Raman Maitreyi,Savoye Guillaume,Ghosh Subrata Nutrients The natural history of inflammatory bowel diseases, especially Crohn's disease, is frequently complicated by intestinal fibrosis. Because of the lack of effective treatments for intestinal fibrosis, there is an urgent need to develop new therapies. Factors promoting intestinal fibrosis are currently unclear, but diet is a potential culprit. Diet may influence predisposition to develop intestinal fibrosis or alter its natural history by modification of both the host immune response and intestinal microbial composition. Few studies have documented the effects of dietary factors in modulating IBD-induced intestinal fibrosis. As the mechanisms behind fibrogenesis in the gut are believed to be broadly similar to those from extra-intestinal organs, it may be relevant to investigate which dietary components can inhibit or promote fibrosis factors such as myofibroblasts progenitor activation in other fibrotic diseases. 10.3390/nu13093148
    TNF-α and LPA promote synergistic expression of COX-2 in human colonic myofibroblasts: role of LPA-mediated transactivation of upregulated EGFR. Yoo James,Rodriguez Perez Citlali Ekaterina,Nie Wenxian,Sinnett-Smith James,Rozengurt Enrique BMC gastroenterology BACKGROUND:Enhanced EGF receptor (EGFR) signaling is a hallmark of many human cancers, though the role of enhanced EGFR signaling within the surrounding tumor stroma has not been well studied. The myofibroblast is an important stromal cell that demonstrates enhanced EGFR expression in the setting of inflammation, though the functional relevance is not known. We recently reported that TNF-α and the G protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA) lead to synergistic cyclo-oxygenase-2 (COX-2) expression, an enzyme strongly associated with the development of colitis-associated cancer. Here, we investigate whether EGFR signaling plays a role in the synergistic COX-2 expression induced by LPA and TNF-α. METHODS:18Co cells, a model of human colonic myofibroblasts, were grown to confluence on 35 × 10 mm cell culture dishes and were used from passages 10-14. 18Co cells were treated with TNF-α (8.3 ng/ml) and LPA (10 μM). EGFR and COX-2 protein expression, Y1068 phosphorylation, and p42/44 MAPK phosphorylation were assessed by Western Blot, in the presence and absence of various inhibitors. RESULTS:Exposure of 18Co cells to either TNF-α or LPA alone had no effect on EGFR autophosphorylation at Y1068. However, chronic exposure to TNF-α led to upregulation of EGFR in association with sustained LPA-mediated EGFR phosphorylation at Y1068. TNF-α and LPA also led to sustained p42/44 MAPK phosphorylation and synergistic COX-2 expression, effects that were partially inhibited by the EGFR tyrosine kinase inhibitor AG1478. p42/44 MAPK phosphorylation and COX-2 expression were inhibited to the same degree by the MMP inhibitors GM6001 and BB-94, suggesting that LPA-mediated EGFR transactivation involved MMP-mediated release of EGFR ligands from the cell surface. The Src inhibitor SU6556 inhibited TNF-α/LPA-mediated EGFR phosphorylation at Y1068, p42/44 MAPK phosphorylation, and COX-2 expression in a dose-dependent fashion, suggesting an upstream role of Src in the transactivation of EGFR. CONCLUSION:Synergistic COX-2 expression induced by TNF-α and LPA involves Src/MMP-mediated transactivation of EGFR and downstream p42/44 MAPK activation in human colonic myofibroblasts. Enhanced EGFR expression induced by TNF-α promotes GPCR-mediated EGFR transactivation in colonic myofibroblasts, providing an important mechanism for stromal COX-2 over-expression that may predispose to the development of colitis-associated cancer. 10.1186/1471-230X-13-90
    Inducible NOS mediates CNP-induced relaxation of intestinal myofibroblasts. Chen Yishi,Chitapanarux Taned,Wu Jianfeng,Soon Russell K,Melton Andrew C,Yee Hal F American journal of physiology. Gastrointestinal and liver physiology Contraction of intestinal myofibroblasts (IMF) contributes to the development of strictures and fistulas seen in inflammatory bowel disease, but the mechanisms that regulate tension within these cells are poorly understood. In this study we investigated the role of nitric oxide (NO) signaling in C-type natriuretic peptide (CNP)-induced relaxation of IMF. We found that treatment with ODQ, a soluble guanylyl cyclase (sGC) inhibitor, or N(G)-nitro-L-arginine (L-NNA) or N(G)-monomethyl-L-arginine (L-NMMA), inhibitors of NO production, all impaired the relaxation of human and mouse IMF in response to CNP. ODQ, L-NNA, and L-NMMA also prevented CNP-induced elevations in cGMP concentrations, and L-NNA or L-NMMA blocked CNP-induced decreases in myosin light phosphorylation. IMF isolated from transgenic mice deficient in inducible nitric oxide synthase (iNOS) had reduced relaxation responses to CNP compared with IMF from control mice and were insensitive to the effects of ODQ, L-NNA, and L-NMMA on CNP treatment. Together these data indicate that stimulation of sGC though NO produced by iNOS activation is required for maximal CNP-induced relaxation in IMF. 10.1152/ajpgi.00214.2012
    Caveolin-1 Alleviates Crohn's Disease-induced Intestinal Fibrosis by Inhibiting Fibroblasts Autophagy Through Modulating Sequestosome 1. Inflammatory bowel diseases BACKGROUND:Intestinal fibrosis is a common complication of Crohn's disease (CD) and is characterized by the excessive accumulation of extracellular matrix produced by activated myofibroblasts. Caveolin-1 (CAV1) inhibits fibrosis. However, limited data show that CAV1 affects intestinal fibrosis. METHODS:Human CD tissue samples were gained from patients with CD who underwent surgical resection of the intestine and were defined as stenotic or nonstenotic areas. A dextran sodium sulfate-induced mouse model of intestinal fibrosis was established. For in vitro experiments, we purchased CCD-18Co intestinal fibrosis cells and isolated and cultured human primary colonic fibroblasts. These fibroblasts were activated by transforming growth factor β administration for 48 hours. In the functional experiments, a specific small interfering RNA or overexpression plasmid was transfected into fibroblasts. The messenger RNA levels of fibrosis markers, such as α-smooth muscle actin, fibronectin, connective tissue growth factor, and collagen I1α, were determined using quantitative polymerase chain reaction. Western blot analysis was applied to detect the expression of CAV1, SQSTM1/p62 (sequestosome 1), and other fibrosis markers. RESULTS:In human CD samples and the dextran sodium sulfate-induced mouse model of intestinal fibrosis, we observed a downregulation of CAV1 in fibrosis-activated areas. Mechanistically, CAV1 knockdown in both human primary colonic fibroblasts and CCD-18Co cells promoted fibroblast activation, while CAV1 overexpression inhibited fibroblast activation in vitro. We found that SQSTM1/p62 positively correlated with CAV1 expression levels in patients with CD and that it was indirectly modulated by CAV1 expression. Rescue experiments showed that CAV1 decreased primary human intestinal fibroblast activation by inhibiting fibroblast autophagy through the modulation of SQSTM1/p62. CONCLUSIONS:Our data demonstrate that CAV1 deficiency induces fibroblast activation by indirectly regulating SQSTM1/p62 to promote fibroblast autophagy. CAV1 or SQSTM1/p62 may be potential therapeutic targets for intestinal fibrosis. 10.1093/ibd/izab342
    Mast Cells Tryptase Promotes Intestinal Fibrosis in Natural Decellularized Intestinal Scaffolds. Tissue engineering and regenerative medicine BACKGROUND:Standard two-dimensional (2D) culture has confirmed the mechanism of mast cells (MCs) in the pathogenesis of inflammatory bowel disease (IBD), but the regulation of signaling responses of MCs may well differ in three-dimensional (3D) microenvironments. The aim of the study was to develop a 3D culture model based on decellularized intestinal scaffolds (DIS) and verify how MCs influenced fibroblasts phenotype in the 3D model. METHODS:DIS were achieved using the detergent technique and extracellular matrix (ECM) components were verified by histologic analysis, quantification and scanning electron microscope. After human colon fibroblasts recellularized into the scaffolds and activated by MCs tryptase and TGFβ1, the changes in genes and signaling pathways during fibroblasts activation in 3D were studied and compared with the changes in 2D cell culture on plastic plates. RESULTS:Decellularization process effectively removed native cell debris while retaining natural ECM components and structure. The engrafted fibroblasts could penetrate into the scaffolds and maintain its phenotype. No matter whether fibroblasts were cultured in 2D or 3D, MCs tryptase and transforming growth factor β1 (TGF-β1) could promote the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts through Akt and Smad2/3 signaling pathways. Furthermore, the pro-collagen1α1 and fibronectin synthesis of myofibroblasts in 3D was higher than in 2D culture. CONCLUSION:Our results demonstrated that the DIS can be used as a bioactive microenvironment for the study of intestinal fibrosis, providing an innovative platform for future intestinal disease modeling and screening of genes and signaling pathways. 10.1007/s13770-022-00433-9
    Mast Cell Tryptase Promotes Inflammatory Bowel Disease-Induced Intestinal Fibrosis. Liu Bin,Yang Mu-Qing,Yu Tian-Yu,Yin Yang-Yang,Liu Ying,Wang Xiao-Dong,He Zhi-Gang,Yin Lu,Chen Chun-Qiu,Li Ji-Yu Inflammatory bowel diseases BACKGROUND:Intestinal fibrosis is the final pathological outcome of chronic intestinal inflammation without specific therapeutic drugs, which leads to ileus and surgical intervention. Intestinal fibrosis is characterized by excessive deposition of extracellular matrix (ECM). The role of mast cells (MCs), which are members of the sentinel immune cell population, is unknown in intestinal fibrosis. METHODS:In this study, we analyzed changes in MCs, tryptase proteins, and ECM components in human fibrotic and control patient intestines. We constructed dextran sodium sulfate-induced intestinal fibrosis models using wild-type mice, MC-reconstituted mice, and MC-deficient mice to explore the role of MCs and tryptase in intestinal fibrosis. The roles and mechanisms of MCs and tryptase on fibroblasts were evaluated using human MCs (HMC-1 and LAD-2), commercial tryptase proteins, human colon fibroblasts (CCD-18Co fibroblasts), the tryptase inhibitor APC366, and the protease-activated receptor-2 (PAR-2) antagonist ENMD-1068. RESULTS:Regardless of whether the colon was a human colon or a mouse colon, the fibrotic intestinal tissue had increased MC infiltration and a higher expression of ECM proteins or genes than that of the control group. The dextran sodium sulfate-induced intestinal fibrosis in MC-deficient mice was alleviated compared with that in wild-type mice. After MC reconstruction in MC-deficient mice, the alleviating effect disappeared. Tryptase, as a content stored in MC granules, was released into fibrotic intestinal tissues in the form of degranulation, resulting in an increased expression of tryptase. Compared with the control group, the tryptase inhibition group (the APC366 group) had reduced intestinal fibrosis. The CCD-18Co fibroblasts, when cocultured with MCs or treated with tryptase proteins, were activated to differentiate into myofibroblasts and secrete more ECM proteins (such as collagen and fibronectin). The underlying mechanism of fibroblast activation by tryptase was the activation of the PAR-2/Akt/mTOR pathway. CONCLUSIONS:We found that MC tryptase promotes inflammatory bowel disease-induced intestinal fibrosis. The underlying mechanism is that tryptase promotes the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts by activating the PAR-2/Akt/ mTOR pathway of fibroblasts. 10.1093/ibd/izaa125
    Potential role of protease-activated receptor-2-stimulated activation of cytosolic phospholipase A(2) in intestinal myofibroblast proliferation: Implications for stricture formation in Crohn's disease. Christerson Ulrika,Keita Asa V,Söderholm Johan D,Gustafson-Svärd Christina Journal of Crohn's & colitis BACKGROUND AND AIMS:Myofibroblast hyperplasia contributes to muscularis mucosae thickening and stricture formation in Crohn's disease (CD). Protease-activated receptor-2 (PAR-2) and cytosolic phospholipase A(2) (cPLA(2)) are known regulators of cell growth, but their significance in intestinal myofibroblast proliferation remain to be elucidated. The principle aims of the present study were to investigate if PAR-2 is expressed in the expanded muscularis mucosa in ileal CD specimens, if inflammatory cytokines may stimulate PAR-2 expression in intestinal myofibroblasts, and if PAR-2 and cPLA(2) may regulate intestinal myofibroblast growth. METHODS:Immunohistochemistry was used for detection of PAR-2 in ileal CD specimens. Studies on PAR-2 expression, PLA(2) activation and cell growth were performed in a human intestinal myofibroblast cell line, CCD-18Co. PAR-2 expression was investigated by RT-PCR and immunocytochemistry. PLA(2) activity was analyzed by quantification of released (14)C-arachidonic acid ((14)C-AA). Cell growth was examined by (3)H-thymidine incorporation and cell counting. RESULTS:The thickened muscularis mucosae of the CD specimens showed strong PAR-2 expression. In cultured myofibroblasts, tumor necrosis factor-α (TNF-α) up-regulated PAR-2 mRNA and protein, and potentiated PAR-2-stimulated (14)C-AA release by two known PAR-2 activators, trypsin and SLIGRL-NH(2). The release of (14)C-AA was dependent on cPLA(2). Trypsin stimulated the proliferation of serum-starved cells, and inhibition of cPLA(2) reduced normal cell growth and abolished the growth-promoting effect of trypsin. CONCLUSIONS:The results suggest that PAR-2-mediated cPLA(2) activation might be of importance in intestinal myofibroblast proliferation. The results also point to the possibility that PAR-2 up-regulation by inflammatory cytokines, like TNF-α, may modulate this effect. 10.1016/j.crohns.2008.10.002
    Tacrolimus (FK506) suppresses TNF-α-induced CCL2 (MCP-1) and CXCL10 (IP-10) expression via the inhibition of p38 MAP kinase activation in human colonic myofibroblasts. Aomatsu Tomoki,Imaeda Hirotsugu,Takahashi Kenichiro,Fujimoto Takehide,Kasumi Eiji,Yoden Atsushi,Tamai Hiroshi,Fujiyama Yoshihide,Andoh Akira International journal of molecular medicine In order to investigate the molecular mechanisms underlying the immunosuppressive effects of tacrolimus (FK506) on intestinal inflammation, we examined whether FK506 effects cytokine/chemokine secretion in human colonic myofibroblasts. Human colonic myofibroblasts were isolated from normal human colonic tissue. The mRNA and protein expression for human CCL2 and CXCL10 were analyzed by real-time PCR and ELISA, respectively. p38 MAP kinase activation was evaluated by western blotting. Tacrolimus (1 µM) suppressed tumor necrosis factor (TNF)-α-induced CCL2 and CXCL10 mRNA expression, but did not modulate TNF-α-induced interleukin (IL)-6 or CXCL8 mRNA expression. Dose-dependent, inhibitory effects of tacrolimus on CCL2 and CXCL10 expression were observed at the mRNA and protein levels. Significant inhibitory effects of tacrolimus were observed at concentrations as low as 0.5 µM for CCL2 and 0.1 µM for CXCL10, respectively. TNF-α-induced CCL2 and CXCL10 expression depended on p38 MAP kinase activation, and tacrolimus strongly inhibited the TNF-α-induced phosphorylation of p38 MAP kinase. Tacrolimus did not affect interferon (IFN)-γ-induced signaling transducer and activator of transcription (STAT)-1 phosphorylation, nor did it modulate CXCL10 mRNA and protein expression. In conclusion, tacrolimus suppressed CCL2 and CXCL10 expression in human colonic myofibroblasts. These inhibitory effects of tacrolimus may play key roles in the therapeutic effects of colonic inflammation in inflammatory bowel disease (IBD) patients. 10.3892/ijmm.2012.1094
    TNF-α stimulates colonic myofibroblast migration via COX-2 and Hsp27. Saini Shyla,Liu Tiegang,Yoo James The Journal of surgical research BACKROUND:Crohn's disease (CD) is a chronic inflammatory enteropathy characterized by fibrotic strictures. Myofibroblasts (MFBs) are stromal cells of the gastrointestinal tract found in increased numbers in patients with CD and represent the key effector cells involved in pathologic fibrosis. MFB is a known target of tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine strongly implicated in the pathophysiology of CD. However, the precise mechanisms through which TNF-α contributes to fibrosis remain incompletely understood. Here, we demonstrate for the first time that TNF-α increases MFB migration through the cyclooxygenase 2 (COX-2) and heat-shock protein 27 (Hsp27) pathways. MATERIALS AND METHODS:The human colonic MFB cell line 18Co was grown to confluence on 35 × 10 mm cell culture dishes and used from passages 8-14. An in vitro scratch assay assessed the effect of TNF-α (10 ng/mL) on MFB migration over 24 h in the presence or absence of several inhibitors (NS398, SB203580, Hsp27 siRNA). RESULTS:TNF-α significantly increased MFB migration over 24 h. TNF-α also led to the increased expression of COX-2 and stimulated rapid phosphorylation of Hsp27 at serine 82. TNF-α-induced COX-2 expression, Hsp27 phosphorylation, and MFB migration were all significantly inhibited by the P38 MAPK inhibitor SB203580 (P < 0.05). TNF-α-induced MFB migration was also significantly inhibited by NS398 (P < 0.05), a direct inhibitor of COX-2, and by siRNA targeting Hsp27 (P < 0.05). CONCLUSIONS:TNF-α stimulates colonic MFB migration through P38 MAPK-mediated activation of COX-2 and Hsp27. Further elucidating these inflammatory signaling pathways may lead to novel therapeutic targets for the treatment of CD-related fibrosis and strictures. 10.1016/j.jss.2016.04.034
    Progastrin-induced secretion of insulin-like growth factor 2 from colonic myofibroblasts stimulates colonic epithelial proliferation in mice. Duckworth Carrie A,Clyde Daniel,Worthley Daniel L,Wang Timothy C,Varro Andrea,Pritchard D Mark Gastroenterology BACKGROUND & AIMS:Many colon cancers produce the hormone progastrin, which signals via autocrine and paracrine pathways to promote tumor growth. Transgenic mice that produce high circulating levels of progastrin (hGAS) have increased proliferation of colonic epithelial cells and are more susceptible to colon carcinogenesis than control mice. We investigated whether progastrin affects signaling between colonic epithelial and myofibroblast compartments to regulate tissue homeostasis and cancer susceptibility. METHODS:Colonic myofibroblast numbers were assessed in hGAS and C57BL/6 mice by immunohistochemistry. Human CCD18Co myofibroblasts were incubated with recombinant human progastrin (rhPG)(1-80) for 18 hours, and proliferation was assessed in the presence of pharmacologic inhibitors. The proliferation of human HT29 colonic epithelial cells was assessed after addition of conditioned media from CCD18Co cells incubated with progastrin. The effects of the insulin-like growth factor (IGF)-I receptor antagonist AG1024 were investigated in cultured HT29 cells and on the colonic epithelium of hGAS mice compared with mice that did not express transgenic progastrin (controls). RESULTS:The colonic mucosa of hGAS mice contained greater numbers of myofibroblasts that expressed α-smooth muscle actin and vimentin than controls. Incubation of CCD18Co myofibroblasts with 0.1 nmol/L rhPG(1-80) increased their proliferation, which required activation of protein kinase C and phosphatidylinositol-3 kinase. CCD18Co cells secreted IGF-II in response to rhPG(1-80), and conditioned media from CCD18Co cells that had been incubated with rhPG(1-80) increased the proliferation of HT29 cells. The colonic epithelial phenotype of hGAS mice (crypt hyperplasia, increased proliferation, and altered proportions of goblet and enteroendocrine cells) was inhibited by AG1024. CONCLUSIONS:Progastrin stimulates colonic myofibroblasts to release IGF-II, which increases proliferation of colonic epithelial cells. Progastrin might therefore alter colonic epithelial cells via indirect mechanisms to promote neoplasia. 10.1053/j.gastro.2013.03.012
    Rapamycin ameliorates kidney fibrosis by inhibiting the activation of mTOR signaling in interstitial macrophages and myofibroblasts. Chen Guochun,Chen Huihui,Wang Chang,Peng Youming,Sun Lin,Liu Hong,Liu Fuyou PloS one Interstitial fibrosis is an inevitable outcome of all kinds of progressive chronic kidney disease (CKD). Emerging data indicate that rapamycin can ameliorate kidney fibrosis by reducing the interstitial infiltrates and accumulation of extra cellular matrix (ECM). However, the cellular mechanism that regulates those changes has not been well understood yet. In this study, we revealed the persistent activation of mammalian target of rapamycin (mTOR) signaling in the interstitial macrophages and myofibroblasts, but rarely in injured proximal epithelial cells, CD4+ T cells, neutrophils, or endothelial cells, during the development of kidney fibrosis. Administration of rapamycin to unilateral ureteral obstruction (UUO) mice significantly suppressed the immunoreactivity of mTOR signaling, which decreased the inflammatory responses and ECM accumulation in the obstructed kidneys. Isolated macrophages from rapamycin-treated obstructed kidneys presented less inflammatory activity than vehicle groups. In vitro study confirmed that rapamycin significantly inhibited the fibrogenic activation of cultured fibroblasts (NIH3T3 cells), which was induced by the stimulation of TGF-β(1). Further experiment revealed that rapamycin did not directly inhibit the fibrogenesis of HK2 cells with aristolochic acid treatment. Our findings clarified that rapamycin can ameliorate kidney fibrosis by blocking the mTOR signaling in interstitial macrophages and myofibroblasts. 10.1371/journal.pone.0033626
    Prostaglandin E2 promotes wound-induced migration of intestinal subepithelial myofibroblasts via EP2, EP3, and EP4 prostanoid receptor activation. Iwanaga Koichi,Okada Muneyoshi,Murata Takahisa,Hori Masatoshi,Ozaki Hiroshi The Journal of pharmacology and experimental therapeutics Intestinal subepithelial myofibroblasts (ISMFs) are mesenchymal cells that reside in the subepithelial region throughout the intestine. When the intestine is damaged, the migratory and mitotic responses of ISMFs are crucial for wound closure. However, their mechanism of action remains unknown. We have investigated the role of cyclooxygenase (COX) and its metabolite prostaglandin E(2) (PGE(2)) in the wound repair process of bovine ISMFs. The action of a mechanical scratch in a layer of ISMFs in cell culture elevated the levels of both COX-2 mRNA expression and PGE(2) secretion 1 and 6 h after the event. After 24 h ISMFs had migrated to and reduced the wounded area around the site of the scratch. Treatment with the COX-1/2 inhibitor indomethacin, the COX-2 inhibitor 3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole (CAY10404), or E prostanoid receptor 2 to 4 (EP2-EP4) antagonists significantly inhibited wound repair. Conversely, inhibition of wound closure by indomethicin was reversed by treatment with PGE(2) or agonists of the receptors EP2, EP3, or EP4 but not of EP1. Although EP2 to EP4 stimulation did not influence ISMF proliferation, it did stimulate ISMF migration in the transwell cell migration assay. It is noteworthy that cell migration stimulated by EP2 and EP4 was inhibited by the tyrosine kinase receptor inhibitor genistein and also by (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionic acid (SU6668). However, cell migration stimulated by EP3 was unaffected. Reverse transcription-polymerase chain reaction showed EP2 or EP4 stimulation elevated the level of mRNA expression for fibroblast growth factor-2, which stimulates ISMF migration. Collectively, COX-2-dependent PGE(2) secretion promotes wound healing by ISMFs. PGE(2)-EP3 signaling may directly stimulate ISMF migration. PGE(2)-EP2/4 signaling indirectly stimulates ISMF migration by elevating the level of growth factor secretion. 10.1124/jpet.111.189845
    GED-0507 attenuates lung fibrosis by counteracting myofibroblast transdifferentiation in vivo and in vitro. Speca Silvia,Dubuquoy Caroline,Rousseaux Christel,Chavatte Philippe,Desreumaux Pierre,Spagnolo Paolo PloS one The development of more effective, better tolerated drug treatments for progressive pulmonary fibrosis (of which idiopathic pulmonary fibrosis is the most common and severe form) is a research priority. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is a key regulator of inflammation and fibrosis and therefore represents a potential therapeutic target. However, the use of synthetic PPAR-γ agonists may be limited by their potentially severe adverse effects. In a mouse model of bleomycin (BLM)-induced pulmonary fibrosis, we have demonstrated that the non-racemic selective PPAR-γ modulator GED-0507 is able to reduce body weight loss, ameliorate clinical and histological features of pulmonary fibrosis, and increase survival rate without any safety concerns. Here, we focused on the biomolecular effects of GED-0507 on various inflammatory/fibrotic pathways. We demonstrated that preventive and therapeutic administration of GED-0507 reduced the BLM-induced mRNA expression of several markers of fibrosis, including transforming growth factor (TGF)-β, alpha-smooth muscle actin, collagen and fibronectin as well as epithelial-to-mesenchymal transition (EMT) and expression of mucin 5B. The beneficial effect of GED-0507 on pulmonary fibrosis was confirmed in vitro by its ability to control TGFβ-induced myofibroblast activation in the A549 human alveolar epithelial cell line, the MRC-5 lung fibroblast line, and primary human lung fibroblasts. Compared with the US Food and Drug Administration-approved antifibrotic drugs pirfenidone and nintedanib, GED-0507 displayed greater antifibrotic activity by controlling alveolar epithelial cell dysfunction, EMT, and extracellular matrix remodeling. In conclusion, GED-0507 demonstrated potent antifibrotic properties and might be a promising drug candidate for the treatment of pulmonary fibrosis. 10.1371/journal.pone.0257281
    Role of intestinal myofibroblasts in HIV-associated intestinal collagen deposition and immune reconstitution following combination antiretroviral therapy. Asmuth David M,Pinchuk Irina V,Wu Jian,Vargas Gracie,Chen Xiaoli,Mann Surinder,Albanese Anthony,Ma Zhong-Min,Saroufeem Ramez,Melcher Gregory P,Troia-Cancio Paolo,Torok Natalie J,Miller Christopher J,Powell Don W AIDS (London, England) OBJECTIVE:To investigate the potential role of mucosal intestinal myofibroblasts (IMFs) in HIV and associated fibrosis in gut-associated lymphoid tissue. DESIGN:Profibrotic changes within the secondary lymphoid organs and mucosa have been implicated in failed immune reconstitution following effective combination antiretroviral therapy (cART). Microbial translocation is believed to be sustaining these systemic inflammatory pathways. IMFs are nonprofessional antigen-presenting cells with both immunoregulatory and mesenchymal functions that are ideally positioned to respond to translocating microbial antigen. METHODS:Duodenal biopsies, obtained from patients naive to cART, underwent trichrome staining and were examined for tissue growth factor-beta (TGF-β) expression. Combined immunostaining and second harmonic generation analysis were used to determine IMF activation and collagen deposition. Confocal microscopy was performed to examine IMF activation and Toll-like receptor (TLR)4 expression. Finally, primary IMF cultures were stimulated with lipopolysaccharide to demonstrate the expression of the inflammatory biomarkers. RESULTS:The expression of the fibrosis-promoting molecule, TGF-β1, is significantly increased in duodenal biopsies from HIV patients naïve to cART, and negatively correlated with subsequent peripheral CD4(+) recovery. The increase in TGF-β1 coincided with an increase in collagen deposition in the duodenal mucosa in the tissue area adjacent to the IMFs. We also observed that IMFs expressed TLR4 and had an activated phenotype since they were positive for fibroblast activation protein. Finally, stimulation of IMFs from HIV patients with TLR4 resulted in significantly increased expression of profibrotic molecules, TGF-β1, and interleukin-6. CONCLUSION:Our data support the hypothesis that activated IMFs may be among the major cells contributing to the profibrotic changes, and thus, the establishment and maintenance of systemic inflammation interfering with immune reconstitution in HIV patients. 10.1097/QAD.0000000000000636
    TAK1 is a key modulator of the profibrogenic phenotype of human ileal myofibroblasts in Crohn's disease. Grillo Alessia Rosaria,Scarpa Melania,D'Incà Renata,Brun Paola,Scarpa Marco,Porzionato Andrea,De Caro Raffaele,Martines Diego,Buda Andrea,Angriman Imerio,Palù Giorgio,Sturniolo Giacomo Carlo,Castagliuolo Ignazio American journal of physiology. Gastrointestinal and liver physiology Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) signaling can mediate inflammatory responses as well as tissue remodeling. Intestinal mucosal myofibroblast (IMF) activation drives gut fibrosis in Crohn's disease (CD); however, the molecular pathways involved are largely unknown. Thus we investigated the yet-unknown expression and function of TAK1 in human CD-associated fibrosis. Ileal surgical specimens, ileal biopsies, and IMF isolated from controls and CD patients were analyzed for TAK1 and its active phosphorylated form (pTAK1) by Western blotting, immunohistochemistry, and real-time quantitative PCR. TAK1 pharmacological inhibition and silencing were used to assess its role in collagen and inflammatory cytokine synthesis in IMF. TAK1 and pTAK1 levels increased in ileum specimens from CD patients compared with controls and correlated to tissue fibrosis. Similarly, TAK1 mRNA in ileal biopsies of CD patients correlated with fibrogenic marker expression but not inflammatory cytokines. CD-derived IMF showed higher TAK1 and pTAK1 expression associated with increased collagen1(α)1 mRNA levels compared with control IMF. TGF-β1 promoted pTAK1 nuclear translocation and collagen synthesis. TAK1 inhibition or silencing significantly reduced TGF-β1-stimulated collagen production and normalized the profibrogenic phenotype of CD-derived IMF. Taken together, these data suggest that TAK1 activation and nuclear translocation induce and maintain a fibrogenic phenotype in the IMF. Thus the TAK1 signaling pathway may represent a suitable target to design new, antifibrotic therapies. 10.1152/ajpgi.00400.2014
    AXL Is a Potential Target for the Treatment of Intestinal Fibrosis. Inflammatory bowel diseases BACKGROUND:Fibrosis is the final common pathway to intestinal failure in Crohn's disease, but no medical therapies exist to treat intestinal fibrosis. Activated myofibroblasts are key effector cells of fibrosis in multiple organ systems, including the intestine. AXL is a receptor tyrosine kinase that has been implicated in fibrogenic pathways involving myofibroblast activation. We aimed to investigate the AXL pathway as a potential target for the treatment of intestinal fibrosis. METHODS:To establish proof of concept, we first analyzed AXL gene expression in 2 in vivo models of intestinal fibrosis and 3 in vitro models of intestinal fibrosis. We then tested whether pharmacological inhibition of AXL signaling could reduce fibrogenesis in 3 in vitro models of intestinal fibrosis. In vitro testing included 2 distinct cell culture models of intestinal fibrosis (matrix stiffness and TGF-β1 treatment) and a human intestinal organoid model using TGF-β1 cytokine stimulation. RESULTS:Our findings suggest that the AXL pathway is induced in models of intestinal fibrosis. We demonstrate that inhibition of AXL signaling with the small molecule inhibitor BGB324 abrogates both matrix-stiffness and transforming growth factor beta (TGF-β1)-induced fibrogenesis in human colonic myofibroblasts. AXL inhibition with BGB324 sensitizes myofibroblasts to apoptosis. Finally, AXL inhibition with BGB324 blocks TGF-β1-induced fibrogenic gene and protein expression in human intestinal organoids. CONCLUSIONS:The AXL pathway is active in multiple models of intestinal fibrosis. In vitro experiments suggest that inhibiting AXL signaling could represent a novel approach to antifibrotic therapy for intestinal fibrosis such as in Crohn's disease. 10.1093/ibd/izaa169
    Inhibition of Fibroblast Activation Protein Restores a Balanced Extracellular Matrix and Reduces Fibrosis in Crohn's Disease Strictures Ex Vivo. Truffi Marta,Sorrentino Luca,Monieri Matteo,Fociani Paolo,Mazzucchelli Serena,Bonzini Matteo,Zerbi Pietro,Sampietro Gianluca M,Di Sabatino Antonio,Corsi Fabio Inflammatory bowel diseases Background:Crohn's disease (CD) is a chronic bowel inflammation that ultimately leads to fibrosis, for which medical therapy is currently unavailable. Fibrotic strictures in CD are characterized by excessive extracellular matrix (ECM) deposition, altered balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), and overexpression of fibroblast activation protein (FAP), a marker of active fibroblasts. Here we investigated the role of FAP-targeted therapy in ECM remodeling in CD strictures ex vivo. Methods:Bowel specimens were obtained from stenotic and nonstenotic ileal segments from 30 patients with fibrostenotic CD undergoing surgery. FAP expression was evaluated in isolated mucosal myofibroblasts by immunoblotting and flow cytometry. Bowel tissue cultures were treated with anti-FAP antibody, and soluble collagen, TIMP-1, and MMPs were measured in tissue culture supernatants by immunoblotting. Anti-FAP-treated myofibroblasts were analyzed for TIMP-1 expression by immunoblotting, for migratory potential by wound healing assay, and for apoptosis by Annexin V staining. Results:Myofibroblasts from stenotic CD mucosa showed upregulation of FAP expression when compared with nonstenotic mucosa. Treatment of stenotic tissues with anti-FAP antibody induced a dose-dependent decrease in collagen production, particularly affecting type I collagen. The treatment also reduced TIMP-1 production in CD strictures, without altering MMP-3 and MMP-12 secretion. Accordingly, anti-FAP treatment inhibited TIMP-1 expression in stenotic CD myofibroblasts and enhanced myofibroblast migration without affecting survival. Conclusions:FAP inhibition reduced type I collagen and TIMP-1 production by CD strictures ex vivo without compromising uninvolved bowel areas. These results suggest that targeting FAP could reconstitute ECM homeostasis in fibrostenotic CD. 10.1093/ibd/izx008
    Oxidative state and IL-6 production in intestinal myofibroblasts of Crohn's disease patients. Catarzi Serena,Favilli Fabio,Romagnoli Cecilia,Marcucci Tommaso,Picariello Lucia,Tonelli Francesco,Vincenzini Maria Teresa,Iantomasi Teresa Inflammatory bowel diseases BACKGROUND:Intestinal subepithelial myofibroblasts (ISEMFs) produce inflammatory cytokines in response to certain stimuli. In the intestine of patients with Crohn's disease (CD), cytokine synthesis is modified and an increased number of myofibroblasts has been observed. The intracellular redox state influences cytokine production and oxidative stress is present in the intestinal mucosa of CD patients. METHODS:This study was performed in ISEMFs isolated from the colon of patients with active CD and in a myofibroblast cell line derived from human colonic mucosa: 18Co cells. Cellular glutathione (GSH) levels were modulated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, or N-acetylcysteine, a GSH precursor. GSH and oxidized glutathione (GSSG) levels were measured by high-performance liquid chromatography (HPLC) methods. Interleukin (IL)-6 production was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS:ISEMFs of CD patients exhibited an increased oxidative state due to a decrease in the GSH/GSSG ratio, which is related to an increase in basal IL-6 production or is stimulated by tumor necrosis factor alpha (TNFα) or bacterial products. This relationship was also confirmed in 18Co cells. Phosphorylation and activation of ERK1/2 and p38 MAPK, which are signaling factors involved in the IL-6 synthesis, were also increased when there is oxidative stress in ISEMFs. CONCLUSIONS:This study shows for the first time in ISEMFs of CD patients an increased production of IL-6 synthesis related to the decrease in the GSH/GSSH ratio, suggesting redox regulation with the involvement of specific kinase activation. The present data shed light on the pathogenesis of inflammatory chronic processes and relapses that occur in this pathology. 10.1002/ibd.21552
    Activation of Myofibroblast TRPA1 by Steroids and Pirfenidone Ameliorates Fibrosis in Experimental Crohn's Disease. Kurahara Lin Hai,Hiraishi Keizo,Hu Yaopeng,Koga Kaori,Onitsuka Miki,Doi Mayumi,Aoyagi Kunihiko,Takedatsu Hidetoshi,Kojima Daibo,Fujihara Yoshitaka,Jian Yuwen,Inoue Ryuji Cellular and molecular gastroenterology and hepatology BACKGROUND & AIMS:The transient receptor potential ankyrin 1 (TRPA1) channel is highly expressed in the intestinal lamina propria, but its contribution to gut physiology/pathophysiology is unclear. Here, we evaluated the function of myofibroblast TRPA1 channels in intestinal remodeling. METHODS:An intestinal myofibroblast cell line (InMyoFibs) was stimulated by transforming growth factor-β1 to induce in vitro fibrosis. knockout mice were generated using the Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. A murine chronic colitis model was established by weekly intrarectal trinitrobenzene sulfonic acid (TNBS) administration. Samples from the intestines of Crohn's disease (CD) patients were used for pathologic staining and quantitative analyses. RESULTS:In InMyoFibs, TRPA1 showed the highest expression among TRP family members. In TNBS chronic colitis model mice, the extents of inflammation and fibrotic changes were more prominent in TRPA1 knockout than in wild-type mice. One-week enema administration of prednisolone suppressed fibrotic lesions in wild-type mice, but not in TRPA1 knockout mice. Steroids and pirfenidone induced Ca influx in InMyoFibs, which was antagonized by the selective TRPA1 channel blocker HC-030031. Steroids and pirfenidone counteracted transforming growth factor-β1-induced expression of heat shock protein 47, type 1 collagen, and α-smooth muscle actin, and reduced Smad-2 phosphorylation and myocardin expression in InMyoFibs. In stenotic intestinal regions of CD patients, TRPA1 expression was increased significantly. TRPA1/heat shock protein 47 double-positive cells accumulated in the stenotic intestinal regions of both CD patients and TNBS-treated mice. CONCLUSIONS:TRPA1, in addition to its anti-inflammatory actions, may protect against intestinal fibrosis, thus being a novel therapeutic target for highly incurable inflammatory/fibrotic disorders. 10.1016/j.jcmgh.2017.12.005
    Treatment of Intestinal Fibrosis in Experimental Inflammatory Bowel Disease by the Pleiotropic Actions of a Local Rho Kinase Inhibitor. Holvoet Tom,Devriese Sarah,Castermans Karolien,Boland Sandro,Leysen Dirk,Vandewynckel Yves-Paul,Devisscher Lindsey,Van den Bossche Lien,Van Welden Sophie,Dullaers Melissa,Vandenbroucke Roosmarijn E,De Rycke Riet,Geboes Karel,Bourin Arnaud,Defert Olivier,Hindryckx Pieter,De Vos Martine,Laukens Debby Gastroenterology BACKGROUND:Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFβ-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. METHODS:Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. RESULTS:ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFβ1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. CONCLUSIONS:Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD. 10.1053/j.gastro.2017.06.013
    Thrombin Induced Apoptosis through Calcium-Mediated Activation of Cytosolic Phospholipase A in Intestinal Myofibroblasts. Biomolecules & therapeutics Thrombin is a serine protease that participates in a variety of biological signaling through protease-activated receptors. Intestinal myofibroblasts play central roles in maintaining intestinal homeostasis. In this study, we found that thrombin-induced apoptosis is mediated by the calcium-mediated activation of cytosolic phospholipase A in the CCD-18Co cell. Thrombin reduced cell viability by inducing apoptosis and proteinase-activated receptor-1 antagonist attenuated thrombin-induced cell death. Endogenous ceramide did not affect the cell viability itself, but a ceramide-mediated pathway was involved in thrombin-induced cell death. Thrombin increased intracellular calcium levels and cytosolic phospholipase A activity. The ceramide synthase inhibitor Fumonisin B, intracellular calcium chelator BAPTA-AM, and cytosolic phospholipase A inhibitor AACOCF inhibited thrombin-induced cell death. Thrombin stimulated arachidonic acid release and reactive oxygen species generation, which was blocked by AACOCF, BAPTA-AM, and the antioxidant reagent Trolox. Taken together, thrombin triggered apoptosis through calcium-mediated activation of cytosolic phospholipase A in intestinal myofibroblasts. 10.4062/biomolther.2022.043
    Transforming growth factor β1-induced collagen production in myofibroblasts is mediated by reactive oxygen species derived from NADPH oxidase 4. Hotta Yuma,Uchiyama Kazuhiko,Takagi Tomohisa,Kashiwagi Saori,Nakano Takahiro,Mukai Rieko,Toyokawa Yuki,Yasuda Tomoyo,Ueda Tomohiro,Suyama Yosuke,Murakami Takaaki,Tanaka Makoto,Majima Atsushi,Doi Toshifumi,Hirai Yasuko,Mizushima Katsura,Morita Mayuko,Higashimura Yasuki,Inoue Ken,Fukui Akifumi,Okayama Tetsuya,Katada Kazuhiro,Kamada Kazuhiro,Handa Osamu,Ishikawa Takeshi,Naito Yuji,Itoh Yoshito Biochemical and biophysical research communications Intestinal fibrosis with stricture formation is a severe complication of Crohn's disease (CD). Though new therapeutic targets to enable the prevention or treatment of intestinal fibrosis are needed, markers of this condition and the basic mechanisms responsible have not been established. NADPH oxidase (NOX) 4 has already been reported to play a key role in models of fibrogenesis, including that of the lung. However, its importance in intestinal fibrogenesis remains unclear. In this study, we examined the role of NOX4 in collagen production by intestinal myofibroblasts stimulated with transforming growth factor (TGF)-β1. Using LmcMF cells, an intestinal subepithelial myofibroblast (ISEMF) line, we first examined the induction of collagen production by TGF-β1. Subsequently, we investigated the role of NOX4 in TGF-β1-induced collagen I production in these cells using SB525334 (an SMAD2/3 inhibitor), diphenyleneiodonium (an NOX inhibitor), and Nox4 small interfering RNA (siRNA). Production of collagen was assessed with Sirius red staining, and Nox4 expression was measured by quantitative real-time PCR. Reactive oxygen species (ROS) production was determined using DCFDA and fluorescent microscopy. We observed that TGF-β1 induced collagen production via NOX4 activation and ROS generation in LmcMF cells. Nox4 siRNA and inhibitors of TGF-β1 receptor and NOX significantly reduced TGF-β1-induced ROS and collagen production. Thus, in the present study, we revealed that collagen production in ISEMFs is induced via an NOX4-dependent pathway. This work supports a function for NOX4 in intestinal fibrogenesis and identifies it as a potential therapeutic target in recalcitrant fibrotic disorders of CD patients. 10.1016/j.bbrc.2018.10.116
    Ogerin mediated inhibition of TGF-β(1) induced myofibroblast differentiation is potentiated by acidic pH. PloS one Transforming growth factor beta (TGF-β) induced myofibroblast differentiation is central to the pathological scarring observed in Idiopathic Pulmonary Fibrosis (IPF) and other fibrotic diseases. Our lab has recently identified expression of GPR68 (Ovarian Cancer Gene Receptor 1, OGR1), a pH sensing G-protein coupled receptor, as a negative regulator of TGF-β induced profibrotic effects in primary human lung fibroblasts (PHLFs). We therefore hypothesized that small molecule activators of GPR68 would inhibit myofibroblast differentiation. Ogerin is a positive allosteric modulator (PAM) of GPR68, inducing a leftward shift of the dose response curve to proton induced signaling. Using PHLFs derived from patients with both non-fibrotic and IPF diagnoses, we show that Ogerin inhibits, and partially reverses TGF-β induced myofibroblast differentiation in a dose dependent manner. This occurs at the transcriptional level without inhibition of canonical TGF-β induced SMAD signaling. Ogerin induces PKA dependent CREB phosphorylation, a marker of Gαs pathway activation. The ability of Ogerin to inhibit both basal and TGF-β induced collagen gene transcription, and induction of Gαs signaling is enhanced at an acidic pH (pH 6.8). Similar findings were also found using fibroblasts derived from dermal, intestinal, and orbital tissue. The biological role of GPR68 in different tissues, cell types, and disease states is an evolving and emerging field. This work adds to the understanding of Gαs coupled GPCRs in fibrotic lung disease, the ability to harness the pH sensing properties of GPR68, and conserved mechanisms of fibrosis across different organ systems. 10.1371/journal.pone.0271608
    Role of N-acetylcysteine and GSH redox system on total and active MMP-2 in intestinal myofibroblasts of Crohn's disease patients. Romagnoli Cecilia,Marcucci Tommaso,Picariello Lucia,Tonelli Francesco,Vincenzini Maria Teresa,Iantomasi Teresa International journal of colorectal disease PURPOSE:Intestinal subepithelial myofibroblasts (ISEMFs)(1) are the predominant source of matrix metalloproteinase-2 (MMP-2) in gut, and a decrease in glutathione/oxidized glutathione (GSH/GSSG) ratio, intracellular redox state index, occurs in the ISEMFs of patients with Crohn's disease (CD). The aim of this study is to demonstrate a relationship between MMP-2 secretion and activation and changes of GSH/GSSG ratio in ISEMFs stimulated or not with tumor necrosis factor alpha (TNFα). METHODS:ISEMFs were isolated from ill and healthy colon mucosa of patients with active CD. Buthionine sulfoximine, GSH synthesis inhibitor, and N-acetylcysteine (NAC), precursor of GSH synthesis, were used to modulate GSH/GSSG ratio. GSH and GSSG were measured by HPLC and MMP-2 by ELISA Kit. RESULTS:In cells, stimulated or not with TNFα, a significant increase in MMP-2 secretion and activation, related to increased oxidative stress, due to low GSH/GSSG ratio, was detected. NAC treatment, increasing this ratio, reduced MMP-2 secretion and exhibited a direct effect on the secreted MMP-2 activity. In NAC-treated and TNFα-stimulated ISEMFs of CD patients' MMP-2 activity were restored to physiological value. The involvement of c-Jun N-terminal kinase pathway on redox regulation of MMP-2 secretion has been demonstrated. CONCLUSION:For the first time, in CD patient ISEMFs, a redox regulation of MMP-2 secretion and activation related to GSH/GSSG ratio and inflammatory state have been demonstrated. This study suggests that compounds able to maintain GSH/GSSG ratio to physiological values can be useful to restore normal MMP-2 levels reducing in CD patient intestine the dysfunction of epithelial barrier. 10.1007/s00384-012-1632-2
    Myofibroblast activation in colorectal cancer lymph node metastases. Yeung T M,Buskens C,Wang L M,Mortensen N J,Bodmer W F British journal of cancer BACKGROUND:Myofibroblasts have an important role in regulating the normal colorectal stem cell niche. While the activation of myofibroblasts in primary colorectal cancers has been previously described, myofibroblast activation in lymph node metastases has not been described before. METHODS:Paraffin-embedded lymph node sections from patients with macrometastases, micrometastases and isolated tumour cells were stained to identify myofibroblasts and to characterise the distribution of different cell types in tumour-containing lymph nodes. The extent of myofibroblast presence was quantified and compared with the size of the metastasis and degree of proliferation and differentiation of the cancer cells. RESULTS:We show substantial activation of myofibroblasts in the presence of colorectal metastases in lymph nodes, which is intimately associated with glandular structures, both in micro- and macrometastases. The degree of activation is positively associated with the size of the metastases and the proportion of Ki67+ve cancer cells, and negatively associated with the degree of enterocyte differentiation as measured by CK20 expression. CONCLUSION:The substantial activation of myofibroblasts in tumour-containing lymph nodes strongly suggests that these metastatic cancer cells are still significantly dependent on their microenvironment. Further understanding of these epithelial-mesenchymal interactions could lead to the development of new therapies in metastatic disease. 10.1038/bjc.2013.209
    Dietary salt exacerbates intestinal fibrosis in chronic TNBS colitis via fibroblasts activation. Amamou Asma,Rouland Matthieu,Yaker Linda,Goichon Alexis,Guérin Charlène,Aziz Moutaz,Savoye Guillaume,Marion-Letellier Rachel Scientific reports Intestinal fibrosis is a frequent complication in inflammatory bowel diseases (IBD). It is a challenge to identify environmental factors such as diet that may be driving this risk. Intestinal fibrosis result from accumulation of extracellular matrix (ECM) proteins secreted by myofibroblasts. Factors promoting intestinal fibrosis are unknown, but diet appears to be a critical component in its development. Consumption of salt above nutritional recommendations can exacerbate chronic inflammation. So far, high salt diet (HSD) have not been thoroughly investigated in the context of intestinal fibrosis associated to IBD. In the present study, we analyze the role of dietary salt in TNBS chronic colitis induced in rat, an intestinal fibrosis model, or in human colon fibroblast cells. Here, we have shown that high-salt diet exacerbates undernutrition and promoted ECM-associated proteins in fibroblasts. Taken together, our results suggested that dietary salt can activate intestinal fibroblasts, thereby contributing to exacerbation of intestinal fibrosis. Dietary salt may be considered as a putative environmental factor that drives intestinal fibrosis risk. 10.1038/s41598-021-94280-8
    MyD88 in myofibroblasts enhances colitis-associated tumorigenesis via promoting macrophage M2 polarization. Yuan Qi,Gu Jianchun,Zhang Jie,Liu Shi,Wang Qinchuan,Tian Tian,Chen Zhinan,Zhang Jinhua Cell reports The signal adaptor MyD88, an essential component of TLR signaling, plays an important role in gut-microbiome interactions. However, its contribution to colitis-associated cancer (CAC) is still controversial. Far less is known about the specific effects of MyD88 signaling in myofibroblasts in CAC development. Here, we used a CAC mouse model in which MyD88 was selectively depleted in myofibroblasts. Myofibroblast MyD88-deficient mice are resistant to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced tumorigenesis, as evidenced by the decrease in the number and sizes of tumors. MyD88 deficiency in myofibroblasts attenuates intestinal epithelial cell (IEC) proliferation after acute DSS-induced colitis. Furthermore, MyD88 signaling in myofibroblasts increases the secretion of osteopontin (OPN), which promotes macrophage M2 polarization through binding to αβ and CD44, leading to activation of the STAT3/PPARγ pathway. Thus, MyD88 signaling in myofibroblasts crucially contributes to colorectal cancer development and provides a promising therapeutic target for the prevention of colitis-associated carcinogenesis. 10.1016/j.celrep.2021.108724
    Tpl2 regulates intestinal myofibroblast HGF release to suppress colitis-associated tumorigenesis. Koliaraki Vasiliki,Roulis Manolis,Kollias George The Journal of clinical investigation The tumor microenvironment plays a significant role in colitis-associated cancer (CAC). Intestinal myofibroblasts (IMFs) are cells in the intestinal lamina propria secreting factors that are known to modulate carcinogenesis; however, the physiological role of IMFs and signaling pathways influencing CAC have remained unknown. Tumor progression locus 2 (Tpl2) is a MAPK that regulates inflammatory and oncogenic pathways. In this study we addressed the role of Tpl2 in CAC using complete and tissue-specific ablation of Tpl2 in mutant mice. Tpl2-deficient mice did not exhibit significant differences in inflammatory burdens following azoxymethane (AOM)/dextran sodium sulfate (DSS) administration compared with wild-type mice; however, the mutant mice developed significantly increased numbers and sizes of tumors, associated with enhanced epithelial proliferation and decreased apoptosis. Cell-specific ablation of Tpl2 in IMFs, but not in intestinal epithelial or myeloid cells, conferred a similar susceptibility to adenocarcinoma formation. Tpl2-deficient IMFs upregulated HGF production and became less sensitive to the negative regulation of HGF by TGF-β3. In vivo inhibition of HGF-mediated c-Met activation blocked early, enhanced colon dysplasia in Tpl2-deficient mice, indicating that Tpl2 normally suppresses the HGF/c-Met pathway. These findings establish a mesenchyme-specific role for Tpl2 in the regulation of HGF production and suppression of epithelial tumorigenesis. 10.1172/JCI63917
    Bradykinin stimulates protein kinase D-mediated colonic myofibroblast migration via cyclooxygenase-2 and heat shock protein 27. Chu Eric,Saini Shyla,Liu Tiegang,Yoo James The Journal of surgical research BACKGROUND:Inflammatory bowel disease is characterized by episodic intestinal injury and repair. Myofibroblasts are gastrointestinal tract stromal cells that regulate the reparative process and are known targets of inflammatory mediators including bradykinin (BK). However, the mechanisms through which inflammation regulates myofibroblast-induced wound healing remain incompletely understood. Here, we demonstrate, for the first time, that BK stimulates myofibroblast migration through protein kinase D (PKD)-mediated activation of the cyclooxygenase-2 (COX-2) and heat shock protein 27 (Hsp27) pathways. MATERIALS AND METHODS:CCD-18Co is a human colonic myofibroblast cell line used from passages 8 to 14. An in vitro scratch assay assessed the effect of BK (100 nM) on myofibroblast migration over 24 h in the presence or absence of several inhibitors (CID755673 [10 μM] and NS398 [10 μM]). Hsp27 small interfering RNA evaluated the effect of Hsp27 on colonic myofibroblast migration. Antibodies to pPKD, pHsp27, and COX-2 evaluated expression levels by Western blot. RESULTS:BK stimulated myofibroblast migration over 24 h. BK also led to rapid and sustained phosphorylation of PKD at Ser-916, rapid phosphorylation of Hsp27 at Ser-82, and increased COX-2 expression over 4 h. BK-mediated COX-2 expression and Hsp27 phosphorylation were both inhibited by the PKD inhibitor CID755673. Similarly, BK-induced myofibroblast migration was significantly inhibited by CID755673 (P < 0.05), by the direct COX-2 inhibitor NS398 (P < 0.05), and by Hsp27 small interfering RNA (P < 0.05). CONCLUSIONS:BK stimulates myofibroblast migration through PKD-mediated activation of COX-2 and Hsp27. PKD, COX-2, and Hsp27 all appear to regulate myofibroblast cell migration, a stromal population that may play an important role in mucosal healing in the setting of inflammation. 10.1016/j.jss.2016.10.014
    Tumor Necrosis Factor-Alpha Up-Regulates ICAM-1 Expression and Release in Intestinal Myofibroblasts by Redox-Dependent and -Independent Mechanisms. Fontani Filippo,Domazetovic Vladana,Marcucci Tommaso,Vincenzini Maria Teresa,Iantomasi Teresa Journal of cellular biochemistry Intercellular adhesion molecule-1 (ICAM-1) is distributed and expressed on cell surface and is present in circulation as soluble form (sICAM-1). Tumor necrosis factor-alpha (TNFα) and radical oxygen species (ROS) up-regulate the expression of ICAM-1. This study demonstrates for the first time in 18 Co cells, a myofibroblast cell line derived from human colonic mucosa, an up-regulation of ICAM-1 expression and sICAM-1 release induced by oxidative stress and TNFα stimulation. The intracellular redox state was modulated by L-buthionine-S,R-sulfoximine (BSO) or N-acetylcysteine (NAC), inhibitor and precursor respectively of GSH synthesis. ROS production increases in cells treated with BSO or TNFα, and this has been related to an up-regulation of ICAM-1 expression and sICAM-1 release. The involvement of metalloproteinases in ICAM-1 release has been demonstrated. Moreover, also expression and activation of A disintegrin and metalloproteinase 17, a membrane-bound enzyme known as TNFα-converting enzyme (TACE), have been related to ROS levels. This suggests the possible involvement of TACE in the cleavage of ICAM-1 on cell surface in condition of oxidative stress. NAC down-regulates the expression and release of ICAM-1 as well as the expression and activation of TACE. However, in TNFα stimulated cells NAC treatment reduces only in part ICAM-1 expression and sICAM-1 release. Given this TNFα may also act on these events by a redox-independent mechanism. 10.1002/jcb.25279
    Probiotic Lactobacillus acidophilus bacteria or synthetic TLR2 agonist boost the growth of chicken embryo intestinal organoids in cultures comprising epithelial cells and myofibroblasts. Pierzchalska Malgorzata,Panek Malgorzata,Czyrnek Malgorzata,Gielicz Anna,Mickowska Barbara,Grabacka Maja Comparative immunology, microbiology and infectious diseases The intestinal epithelial cells reside in close proximity to myofibroblasts and microbiota, which are supposed to have an impact on intestinal stem cells fate and to influence processes of tissue maturation and regeneration. Mechanism underlying these phenomena and their diversity among vertebrates can be studied in 3D organoid cultures. We investigated the growth of chicken embryo intestinal epithelial organoids in Matrigel with and without Toll-like receptors (TLRs) stimulation. The organoid cultures contained also some myofibroblasts with potential to promote intestinal stem cell survival. Organoid cells, expressing TLR4, TLR2 type 1 and TLR2 type 2 were incubated with their agonists (lipopolysaccharide - LPS and Pam3CSK4) or co-cultured with Lactobacillus acidophilus bacteria (LA-5). Pam3CSK4 and LA-5 promoted organoid growth, which was demonstrated by comparing the morphological parameters (mean number and area of organoids). The profile of prostaglandins (PG), known to promote intestinal regeneration, in supernatants from organoid and fibroblast cultures were evaluated. Both PGE and PGD were detected. As compared to unstimulated controls, supernatants from the Pam3CSK4-stimulated organoids contained twice as much of PGE and PGD. The changes in production of prostaglandins and the support of epithelial cell growth by myofibroblasts are factors potentially responsible for stimulatory effect of TLR2 activation. 10.1016/j.cimid.2017.06.002
    Glucagon-like petide-2 acts on colon cancer myofibroblasts to stimulate proliferation, migration and invasion of both myofibroblasts and cancer cells via the IGF pathway. Shawe-Taylor Marianne,Kumar J Dinesh,Holden Whitney,Dodd Steven,Varga Akos,Giger Olivier,Varro Andrea,Dockray Graham J Peptides Glucagon-like peptide (GLP)-2 stimulates intestinal epithelial proliferation by acting, in part, via IGF release from sub-epithelial myofibroblasts. The response of myofibroblasts to GLP-2 remains incompletely understood. We studied the action of GLP-2 on myofibroblasts from colon cancer and adjacent tissue, and the effects of conditioned medium from these cells on epithelial cell proliferation, migration and invasion. GLP-2 stimulated proliferation, migration and invasion of myofibroblasts and the proliferative and invasive responses of cancer-associated myofibroblasts were greater than those of myofibroblasts from adjacent tissue. The responses were inhibited by an IGF receptor inhibitor, AG1024. Conditioned medium from GLP-2 treated myofibroblasts increased proliferation, migration and invasion of SW480, HT29, LoVo epithelial cells and these responses were inhibited by AG1024; GLP-2 alone had no effect on these cells. In addition, when myofibroblasts and epithelial cells were co-cultured in Ibidi chambers there was mutual stimulation of migration in response to GLP-2. The latter increased both IGF-1 and IGF-2 transcript abundance in myofibroblasts. Moreover, a number of IGF binding proteins (IGFBP-4, -5, -7) were identified in myofibroblast medium; in the presence of GLP-2 there was increased abundance of the cleavage products of IGBBP-4 and IGFBP-5 suggesting activation of a degradation mechanism that might increase IGF bioavailability. The data suggest that GLP-2 stimulates cancer myofibroblast proliferation, migration and invasion; GLP-2 acts indirectly on epithelial cells partly via increased IGF expression in myofibroblasts and partly, perhaps, by increased bioavailability through degradation of IGFBPs. 10.1016/j.peptides.2017.03.008
    Regulation of intestinal myofibroblasts by KRas-mutated colorectal cancer cells through heparin-binding epidermal growth factor-like growth factor. Kawasaki Hideyoshi,Saotome Takuya,Usui Tatsuya,Ohama Takashi,Sato Koichi Oncology reports In colorectal cancer, gain-of-function mutations in KRas play a critical role in malignant transformation. Tumor growth in colorectal cancer is known to be promoted by the intestinal myofibroblasts (IMFs) that localize adjacent to the cancer cells, but the mechanisms of interaction between KRas-mutated cancer cells and the myofibroblasts remain unclear. Here, we investigated the effects of KRas-mutated cells on the behavior of myofibroblasts by using mouse primary IMFs and cells of an IMF cell line (LmcMF) and a mouse colon epithelial cell line (aMoC1). Conditioned medium (CM) was collected from aMoC1 cells overexpressing a control vector or KRasV12 vector (KRasV12-CM), and the effects of KRasV12-CM on IMFs were analyzed by performing proliferation assays, wound-healing assays, Boyden chamber assays, and western blotting. Whereas KRasV12-CM exerted little effect on the differentiation and proliferation of primary IMFs, the CM promoted migration of both primary IMFs and LmcMF cells. In KRasV12-overexpressing aMoC1 cells, mRNA expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was higher than in mock-transfected aMoC1 cells, and HB-EGF promoted the migration of primary IMFs and LmcMF cells. Moreover, KRasV12-CM-induced IMF migration was suppressed by dacomitinib, an inhibitor of HB-EGF receptors. Notably, in LmcMF cells, both KRasV12-CM and HB-EGF activated extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK), whereas KRasV12-CM-induced migration of IMFs was suppressed following treatment with either an ERK inhibitor (FR180204) or a JNK inhibitor (SP600125). These results suggest that HB-EGF secreted from KRas-mutated colorectal cancer cells promotes IMF migration through ERK and JNK activation, which, in turn, could support cancer progression. 10.3892/or.2017.5520
    Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis. Roulis Manolis,Nikolaou Christoforos,Kotsaki Elena,Kaffe Eleanna,Karagianni Niki,Koliaraki Vasiliki,Salpea Klelia,Ragoussis Jiannis,Aidinis Vassilis,Martini Eva,Becker Christoph,Herschman Harvey R,Vetrano Stefania,Danese Silvio,Kollias George Proceedings of the National Academy of Sciences of the United States of America Tumor progression locus-2 (Tpl2) kinase is a major inflammatory mediator in immune cell types recently found to be genetically associated with inflammatory bowel diseases (IBDs). Here we show that Tpl2 may exert a dominant homeostatic rather than inflammatory function in the intestine mediated specifically by subepithelial intestinal myofibroblasts (IMFs). Mice with complete or IMF-specific Tpl2 ablation are highly susceptible to epithelial injury-induced colitis showing impaired compensatory proliferation in crypts and extensive ulcerations without significant changes in inflammatory responses. Following epithelial injury, IMFs sense innate or inflammatory signals and activate, via Tpl2, the cyclooxygenase-2 (Cox-2)-prostaglandin E2 (PGE2) pathway, which we show here to be essential for the epithelial homeostatic response. Exogenous PGE2 administration rescues mice with complete or IMF-specific Tpl2 ablation from defects in crypt function and susceptibility to colitis. We also show that Tpl2 expression is decreased in IMFs isolated from the inflamed ileum of IBD patients indicating that Tpl2 function in IMFs may be highly relevant to human disease. The IMF-mediated mechanism we propose also involves the IBD-associated genes IL1R1, MAPK1, and the PGE2 receptor-encoding PTGER4. Our results establish a previously unidentified myofibroblast-specific innate pathway that regulates intestinal homeostasis and may underlie IBD susceptibility in humans. 10.1073/pnas.1415762111
    Th17 cell-derived amphiregulin promotes colitis-associated intestinal fibrosis through activation of mTOR and MEK in intestinal myofibroblasts. Gastroenterology BACKGROUND & AIMS:Intestinal fibrosis is a significant complication of Crohn's disease (CD). Gut microbiota reactive Th17 cells are crucial in the pathogenesis of CD; however, how Th17 cells induce intestinal fibrosis is still not completely understood. METHODS:In this study, T cell transfer model with wild-type (WT) and Areg Th17 cells and dextran sulfate sodium (DSS)-induced chronic colitis model in WT and Areg mice were used. CD4 T cell expression of Areg was determined by qRT-PCR and ELISA. The effect of Areg on proliferation/migration/collagen expression in human intestinal myofibroblasts was determined. Areg expression was assessed in healthy controls and CD patients with or without intestinal fibrosis. RESULTS:Although Th1 and Th17 cells induced intestinal inflammation at similar levels when transferred into Tcrβxδ mice, Th17 cells induced more severe intestinal fibrosis. Th17 cells expressed higher levels of Areg than Th1 cells. Areg mice developed less severe intestinal fibrosis compared with WT mice upon DSS insults. Transfer of Areg Th17 cells induced less severe fibrosis in Tcrβxδ mice compared with WT Th17 cells. IL-6 and IL-21 promoted Areg expression in Th17 cells by activating Stat3. Stat3 inhibitor suppressed Th17-induced intestinal fibrosis. Areg promoted human intestinal myofibroblast proliferation, motility, and collagen I expression, which was mediated by activating mTOR and MEK. Areg expression was increased in intestinal CD4 T cells in fibrotic sites compared with non-fibrotic sites from CD patients. CONCLUSIONS:These findings reveal that Th17-derived Areg promotes intestinal fibrotic responses in experimental colitis and human CD patients. Thereby, Areg might, serve as a potential therapeutic target for fibrosis in CD. 10.1053/j.gastro.2022.09.006
    Water intake accelerates ATP release from myofibroblast cells in rats: ATP-mediated podoplanin-dependent control for physiological function and immunity. Hayashi Moyuru,Watanabe-Asaka Tomomi,Nagashio Sachiho,Kaidoh Maki,Yokoyama Yumiko,Maejima Daisuke,Kajihara Ryo,Amari Kei,Arai Nariaki,Kawai Yoshiko,Ohhashi Toshio American journal of physiology. Gastrointestinal and liver physiology We previously demonstrated that water intake increased mesenteric lymph flow and the total flux of IL-22 in rat jejunum. The drained water and the higher permeability of albumin in the jejunal microcirculation contributed to increase the lymph flow and IL-22 transport via the activation of great bulk flow in the jejunal villi. To address the effects of water intake-mediated great bulk flow-dependent mechanical force on jejunal physiological function and immunological regulation of innate lymphoid cells (ILC)-3, we examined the effects of shear stress stimulation on cultured rat myofibroblast cells. Next, we investigated the effects of water intake on podoplanin and IL-22 expressions in cultured human intestinal epithelial cells and rat in vivo jejunal preparations, respectively. Shear stress stimulation of the myofibroblast cells induced ATP release via an activation of cell surface F/F ATP synthase. ATP produced podoplanin expression in the intestinal epithelial cells. Water intake accelerated immunohistochemical expressions of podoplanin and IL-22 in the interepithelial layers and lamina propria of the jejunum. ATP dose-dependently increased IL-22 mRNA expression in ILC-3, which are housed in the lamina propria. Water intake also increased immunohistochemical and mRNA expressions of ecto-nucleoside triphosphate diphosphohydrolases 2 and 5 in jejunal villi. In conclusion, water intake-mediated shear stress stimulation-dependent ATP release from myofibroblast cells maintains higher tissue colloid osmotic pressure in the jejunal microcirculation through podoplanin upregulation in the interepithelial layers. ATP induces IL-22 mRNA expression in ILC-3 in jejunal villi, which may contribute to regulation of mucosal immunity in small intestine. We investigated effects of shear stress stimulation on cultured myofibroblast cells and water intake on podoplanin and IL-22 expressions in rat jejunal villi. The stimulation induced ATP release from the cells. Water intake accelerated podoplanin and IL-22 expression levels. ATP increased IL-22 mRNA expression in innate lymphoid cells (ILC)-3. Hence, water intake maintains higher osmotic pressure in the jejunal villi through ATP release and podoplanin upregulation. Water intake may regulate the mucosal immunity. 10.1152/ajpgi.00303.2020
    Inflammatory and Immune Activation in Intestinal Myofibroblasts Is Developmentally Regulated. Zawahir Sharmila,Li Guanghui,Banerjee Aditi,Shiu Jessica,Blanchard Thomas G,Okogbule-Wonodi Adora C Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research We previously demonstrated that intestinal myofibroblasts from immature tissue produce excessive IL-8 in response to Escherichia coli lipopolysaccharide (LPS) compared to cells from mature tissue. However, it is unknown whether other cytokines and TLR agonists contribute to this developmentally regulated response. The aim of this study was to further characterize differences in inflammatory signaling in human primary intestinal fibroblasts from fetal (FIF) and infant (IIF) tissue and examine their potential to activate the adaptive immune response in vitro. Cytokine profiles of LPS-stimulated FIF and IIF were assessed by cytokine profile array. IL-8, IL-6, and IL-10 production in response to TLR2, TLR2/6, TLR4, and TLR5 agonists was determined by quantitative ELISA. The potential of activated myofibroblasts to activate adaptive immunity was determined by measuring surface class II MHC expression using flow cytometry. LPS-stimulated FIF produced a distinct proinflammatory cytokine profile consisting of MCP-1, GRO-alpha, IL-6, and IL-8 expression. FIF produced significant IL-8 and IL-6 in response to TLR4 agonist. IIF produced significant levels of IL-8 and IL-6 in the presence of TLR5 and TLR2 agonists. IFN-γ-treated FIF expressed greater HLA-DR levels compared to unstimulated controls and IFN-γ- and LPS-treated IIF. Activated FIF produce a more diverse inflammatory cytokine profile and greater levels of IL-8 and IL-6 in response to TLR4 stimulation compared to IIF. FIF express class II MHC proteins associated with activation of the adaptive immune response. These data suggest that FIF may contribute to bacterial-associated gut inflammation in the immature intestine. 10.1089/jir.2014.0071
    Resveratrol decreases TNFα-induced ICAM-1 expression and release by Sirt-1-independent mechanism in intestinal myofibroblasts. Domazetovic Vladana,Bonanomi Andrea Giovanni,Stio Maria,Vincenzini Maria Teresa,Iantomasi Teresa Experimental cell research Up-regulation of intercellular adhesion molecule-1 (ICAM-1) and its soluble form are involved in the chronic inflammation. For the first time, we demonstrated that resveratrol (RE), a natural polyphenol with antioxidant and anti-inflammatory properties, reduces the increase of expression and release of ICAM-1, due to TNFα-induced oxidative stress, in a myofibroblast cell line derived from human colonic (18Co cells). RE is scavenger of radical oxygen species (ROS) and modulates signaling pathways in which Sirt-1 and NF-κB are involved. Effectively, in TNFα-stimulated 18Co cells RE decreases ROS production and increases Sirt-1 expression and activity, but it reduces TNFα-induced ICAM-1 up-regulation by a Sirt-1-independent mechanism, as demonstrated by EX527 and Sirt-1 siRNA treatments. RE inhibits TNFα-induced activation of NF-κB by reducing both ROS and the degradation of IκB-α, an endogenous inhibitor of NF-κB, with consequent decrease of NF-κB nuclear translocation. This study also shows that NF-κB is not the only factor involved in the TNFα-induced ICAM-1 up-regulation and confirms our previous evidence according to which TNFα increases ICAM-1 levels by redox- and non-redox-regulated mechanisms. RE can represent good and useful support in therapies for intestinal inflammatory diseases in which TNFα plays a crucial role in the increase of adhesion molecule expression. 10.1016/j.yexcr.2019.06.024
    Daikenchuto (Da-Jian-Zhong-Tang) ameliorates intestinal fibrosis by activating myofibroblast transient receptor potential ankyrin 1 channel. Hiraishi Keizo,Kurahara Lin-Hai,Sumiyoshi Miho,Hu Yao-Peng,Koga Kaori,Onitsuka Miki,Kojima Daibo,Yue Lixia,Takedatsu Hidetoshi,Jian Yu-Wen,Inoue Ryuji World journal of gastroenterology AIM:To investigate the anti-fibrotic effects of the traditional oriental herbal medicine Daikenchuto (DKT) associated with transient receptor potential ankyrin 1 (TRPA1) channels in intestinal myofibroblasts. METHODS:Inflammatory and fibrotic changes were detected in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) chronic colitis model of wild-type and TRPA1-knockout (TRPA1-KO) mice pathological staining and immunoblotting analysis. Ca imaging experiments examined the effects of DKT and its components/ingredients on intestinal myofibroblast (InMyoFib) cell TRPA1 channel function. Pro-fibrotic factors and transforming growth factor (TGF)-β1-associated signaling were tested in an InMyoFib cell line by qPCR and immunoblotting experiments. Samples from non-stenotic and stenotic regions of the intestines of patients with Crohn's disease (CD) were used for pathological analysis. RESULTS:Chronic treatment with TNBS caused more severe inflammation and fibrotic changes in TRPA1-KO than in wild-type mice. A one-week enema administration of DKT reduced fibrotic lesions in wild-type but not in TRPA1-KO mice. The active ingredients of DKT, ., hydroxy α-sanshool and 6-shogaol, induced Ca influxes in InMyoFib, and this was antagonized by co-treatment with a selective TRPA1 channel blocker, HC-030031. DKT counteracted TGF-β1-induced expression of Type I collagen and α-smooth muscle actin (α-SMA), which were accompanied by a reduction in the phosphorylation of Smad-2 and p38-mitogen-activated protein kinase (p38-MAPK) and the expression of myocardin. Importantly, 24-h incubation with a DKT active component Japanese Pepper increased the mRNA and protein expression levels of TRPA1 in InMyoFibs, which in turn negatively regulated collagen synthesis. In the stenotic regions of the intestines of CD patients, TRPA1 expression was significantly enhanced. CONCLUSION:The effects of DKT on the expression and activation of the TRPA1 channel could be advantageous for suppressing intestinal fibrosis, and benefit inflammatory bowel disease treatment. 10.3748/wjg.v24.i35.4036
    Angiogenin regulates PKD activation and COX-2 expression induced by TNF-α and bradykinin in the colonic myofibroblast. Plummer Robert,Hu Guo-Fu,Liu Tiegang,Yoo James Biochemical and biophysical research communications INTRODUCTION:The myofibroblast is a gastrointestinal stromal cell that is a target of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine strongly implicated in colitis-associated cancer. Crosstalk between TNF-α and other pro-inflammatory mediators amplify inflammatory signaling but the mechanism is unknown. Angiogenin (ANG) is a 14-kDa angiogenesis protein that is regulated in patients with inflammatory bowel disease. However, the role of ANG on inflammatory mediator crosstalk in the myofibroblast is unknown. METHODS:The human colonic myofibroblast cell line 18Co, as well as primary mouse and human colonic myofibroblasts, were exposed to TNF-α (10 ng/ml) and bradykinin (BK, 100 nM). ANG was quantified by ELISA. The expression of cyclo-oxygenase-2 (COX-2) and phosphorylation of PKD was assessed by Western Blot. RESULTS:Primary mouse and human colonic myofibroblasts exposed to TNF-α/BK led to enhanced PKD phosphorylation and synergistic COX-2 expression. 18Co cells secrete high levels of ANG (24h, 265 ± 5 pg/ml). The monoclonal antibody 26-2F, which neutralizes ANG, inhibited TNF-α/BK-mediated PKD phosphorylation and synergistic COX-2 expression in primary human myofibroblasts. Likewise, in primary mouse myofibroblasts that do not express ANG (ANG-KO), TNF-α/BK failed to enhance PKD phosphorylation and COX-2 expression. CONCLUSIONS:TNF-α/BK enhance PKD phosphorylation and COX-2 expression in primary mouse and human colonic myofibroblasts. Angiogenin is produced by the myofibroblast, and inhibition of ANG signaling, either by its absence (ANG-KO) or by pharmacologic inhibition, blocks enhanced PKD phosphorylation and synergistic COX-2 expression induced by TNF-α/BK. ANG mediates crosstalk signaling between TNF-α/BK in the regulation of stroma-derived COX-2 and may be a novel therapeutic target for the management of colitis-associated cancer. 10.1016/j.bbrc.2020.02.169
    The fate of myofibroblasts during the development of fibrosis in Crohn's disease. Li Chao,Kuemmerle John F Journal of digestive diseases Intestinal fibrosis is a devastating complication in patients with inflammatory bowel disease. Its characteristics include the loss of regular peristalsis and nutrition absorption, excessive deposition of extracellular matrix (ECM) components, thickness of intestinal lumen due to the formation of strictures and of scar tissue. As a major cell type involved in fibrogenesis, the myofibroblasts have already been shown to have a plastic and heterogeneous function in producing abundant collagen, fibronectin and connective tissue growth factor. The primary sources of ECM-producing and vimentin-positive myofibroblasts come from different precursor cells, including bone marrow-derived mesenchymal cells, fibrocytes, pericytes, epithelial to mesenchymal transition and endothelial to mesenchymal transition. Recent immunological research findings suggest that numerous cytokines and chemokines made from macrophages, in addition to T cells and other myeloid cell types, are also important drivers of myofibroblast differentiation and hence of the activation of myofibroblast-mediated transforming growth factor and collagen production. In this review we discuss the origins, roles and cell signaling of myofibroblasts during the development of fibrosis in different organs, particularly in Crohn's disease. Finally, we suggest that the epigenetic and immunological regulation of myofibroblast differentiation may provide a novel antifibrotic strategy in the near future. 10.1111/1751-2980.12852
    Umbilical cord/placenta-derived mesenchymal stem cells inhibit fibrogenic activation in human intestinal myofibroblasts via inhibition of myocardin-related transcription factor A. Choi Yoon Jeong,Koo Jun Bon,Kim Hee Yeon,Seo Jin Won,Lee Eun Jeong,Kim Woo Ram,Cho Joo Young,Hahm Ki Baik,Hong Sung Pyo,Kim Duk Hwan,Yoo Jun-Hwan Stem cell research & therapy BACKGROUND:The lack of anti-fibrotic agents targeting intestinal fibrosis is a large unmet need in inflammatory bowel diseases, including Crohn's disease and ulcerative colitis. Previous studies have found that perinatal tissue (umbilical cord, UC; placenta, PL)-derived mesenchymal stem cells (MSCs) reduce fibrosis in several organs. However, their effects on human intestinal fibrosis are poorly understood. This study investigated the anti-fibrogenic properties and mechanisms of MSCs derived from UC and PL (UC/PL-MSCs) on human primary intestinal myofibroblasts (HIMFs). METHODS:The HIMFs were treated with TGF-β1 and co-cultured with UC/PL-MSCs. We used a small molecular inhibitor CCG-100602 to examine whether serum response factor (SRF) and its transcriptional cofactor myocardin-related transcription factor A (MRTF-A) are involved in TGF-β1-induced fibrogenic activation in HIMFs. The anti-fibrogenic mechanism of UC/PL-MSCs on HIMFs was analyzed by detecting the expression of RhoA, MRTF-A, and SRF in HIMFs. RESULTS:UC/PL-MSCs reduced TGF-β1-induced procollagen1A1, fibronectin, and α-smooth muscle actin expression in HIMFs. This anti-fibrogenic effect was more apparent in the UC-MSCs. TGF-β1 stimulation increased the expressions of RhoA, MRTF-A, and SRF in the HIMFs. TGF-β1 induced the synthesis of procollagen1A1, fibronectin, and α-smooth muscle actin through a MRTF-A/SRF-dependent mechanism. Co-culture with the UC/PL-MSCs downregulated fibrogenesis by inhibition of RhoA, MRTF-A, and SRF expression. CONCLUSIONS:UC/PL-MSCs suppress TGF-β1-induced fibrogenic activation in HIMFs by blocking the Rho/MRTF/SRF pathway and could be considered as a novel candidate for stem cell-based therapy of intestinal fibrosis. 10.1186/s13287-019-1385-8