DDR2/STAT3 Positive Feedback Loop Mediates the Immunosuppressive Microenvironment by Upregulating PD-L1 and Recruiting MDSCs in Oxaliplatin-Resistant HCC.
Cellular and molecular gastroenterology and hepatology
BACKGROUND AND AIMS:Transcriptome sequencing revealed high expression of DDR2 in oxaliplatin-resistant hepatocellular carcinoma (HCC). This study aimed to explore the role of DDR2 in oxaliplatin resistance and immune evasion in HCC. METHODS:Oxaliplatin-resistant HCC cell lines were established. The interaction between DDR2 and STAT3 was investigated, along with the mechanisms involved in DDR2/STAT3-mediated PD-L1 upregulation and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) accumulation both in vitro and in vivo. RESULTS:DDR2 was found to induce the phosphorylation of STAT3, leading to its nuclear translocation. Conversely, the activation of STAT3 enhanced DDR2 expression. A positive feedback loop involving DDR2/STAT3 was identified in oxaliplatin-resistant HCC, which was associated with PD-L1 upregulation and PMN-MDSCs accumulation. Knockdown of DDR2 and STAT3 sensitized oxaliplatin-resistant HCC cells to oxaliplatin and resulted in decreased PMN-MDSCs and increased CD8 T cells in the tumor microenvironment. Enzyme-linked immunosorbent array and MDSC transwell migration assays indicated that oxaliplatin-resistant HCC cells recruited PMN-MDSCs through CCL20. Dual luciferase reporter assays demonstrated that STAT3 can directly enhance the transcription of PD-L1 and CCL20. Furthermore, treatment with a PD-L1 antibody in combination with CCL20 blockade had significant antitumor effects on oxaliplatin-resistant HCC. CONCLUSIONS:Our findings revealed a positive feedback mechanism involving DDR2 and STAT3 that mediates the immunosuppressive microenvironment and promotes oxaliplatin resistance and immune evasion via PD-L1 upregulation and PMN-MDSC recruitment. Targeting the DDR2/STAT3 pathway may be a promising therapeutic strategy to overcome immune escape and chemoresistance in HCC.
10.1016/j.jcmgh.2024.101377
Inhibition of EZH2 Reduces Aging-Related Decline in Interstitial Cells of Cajal of the Mouse Stomach.
Cellular and molecular gastroenterology and hepatology
BACKGROUND & AIMS:Restricted gastric motor functions contribute to aging-associated undernutrition, sarcopenia, and frailty. We previously identified a decline in interstitial cells of Cajal (ICC; gastrointestinal pacemaker and neuromodulator cells) and their stem cells (ICC-SC) as a key factor of gastric aging. Altered functionality of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is central to organismal aging. Here, we investigated the role of EZH2 in the aging-related loss of ICC/ICC-SC. METHODS:klotho mice, a model of accelerated aging, were treated with the most clinically advanced EZH2 inhibitor, EPZ6438 (tazemetostat; 160 mg/kg intraperitoneally twice a day for 3 weeks). Gastric ICC were analyzed by Western blotting and immunohistochemistry. ICC and ICC-SC were quantified by flow cytometry. Gastric slow wave activity was assessed by intracellular electrophysiology. Ezh2 was deactivated in ICC by treating Kit;Ezh2 mice with tamoxifen. TRP53, a key mediator of aging-related ICC loss, was induced with nutlin 3a in gastric muscle organotypic cultures and an ICC-SC line. RESULTS:In klotho mice, EPZ6438 treatment mitigated the decline in the ICC growth factor KIT ligand/stem cell factor and gastric ICC. EPZ6438 also improved gastric slow wave activity and mitigated the reduced food intake and impaired body weight gain characteristic of this strain. Conditional genomic deletion of Ezh2 in Kit-expressing cells also prevented ICC loss. In organotypic cultures and ICC-SC, EZH2 inhibition prevented the aging-like effects of TRP53 stabilization on ICC/ICC-SC. CONCLUSIONS:Inhibition of EZH2 with EPZ6438 mitigates aging-related ICC/ICC-SC loss and gastric motor dysfunction, improving slow wave activity and food intake in klotho mice.
10.1016/j.jcmgh.2024.101376
Ablation of Intestinal Epithelial Sialylation Predisposes to Acute and Chronic Intestinal Inflammation in Mice.
Cellular and molecular gastroenterology and hepatology
BACKGROUND & AIMS:Addition of sialic acids (sialylation) to glycoconjugates is a common capping step of glycosylation. Our study aims to determine the roles of the overall sialylation in intestinal mucosal homeostasis. METHODS:Mice with constitutive deletion of intestinal epithelial sialylation (IEC Slc35a1 mice) and mice with inducible deletion of sialylation in intestinal epithelium (TM-IEC Slc35a1 mice) were generated, which were used to determine the roles of overall sialylation in intestinal mucosal homeostasis by ex vivo and mutiomics studies. RESULTS:IEC Slc35a1 mice developed mild spontaneous microbiota-dependent colitis. Additionally, 30% of IEC Slc35a1 mice had spontaneous tumors in the rectum greater than the age of 12 months. TM-IEC Slc35a1 mice were highly susceptible to acute inflammation induced by 1% dextran sulfate sodium versus control animals. Loss of total sialylation was associated with reduced mucus thickness on fecal sections and within colon tissues. TM-IEC Slc35a1 mice showed altered microbiota with an increase in Clostridium disporicum, which is associated a global reduction in the abundance of at least 10 unique taxa; however, metabolomic analysis did not show any significant differences in short-chain fatty acid levels. Treatment with 5-fluorouracil led to more severe small intestine mucositis in the IEC Slc35a1 mice versus wild-type littermates, which was associated with reduced Lgr5 cell representation in small intestinal crypts in IEC Slc35a1 mice. CONCLUSIONS:Loss of overall sialylation impairs mucus stability and the stem cell niche leading to microbiota-dependent spontaneous colitis and tumorigenesis.
10.1016/j.jcmgh.2024.101378
Hepatocyte-specific Epidermal Growth Factor Receptor Deletion Promotes Fibrosis but has no Effect on Steatosis in Fast-food Diet Model of Metabolic Dysfunction-associated Steatotic Liver Disease.
Cellular and molecular gastroenterology and hepatology
BACKGROUND & AIMS:Metabolic dysfunction-associated steatotic liver disease (MASLD) has become the most prevalent chronic liver disorder, with no approved treatment. Our previous work demonstrated the efficacy of a pan-ErbB inhibitor, Canertinib, in reducing steatosis and fibrosis in a murine fast-food diet (FFD) model of MASLD. The current study explores the effects of hepatocyte-specific ErbB1 (ie, epidermal growth factor receptor [EGFR]) deletion in the FFD model. METHODS:EGFR mice, treated with AAV8-TBG-CRE to delete EGFR specifically in hepatocytes (EGFR-KO), were fed either a chow-diet or FFD for 2 or 5 months. RESULTS:Hepatocyte-specific EGFR deletion reduced serum triglyceride levels but did not prevent steatosis. Surprisingly, hepatic fibrosis was increased in EGFR-KO mice in the long-term study, which correlated with activation of transforming growth factor-β/fibrosis signaling pathways. Further, nuclear levels of some of the major MASLD regulating transcription factors (SREBP1, PPARγ, PPARα, and HNF4α) were altered in FFD-fed EGFR-KO mice. Transcriptomic analysis revealed significant alteration of lipid metabolism pathways in EGFR-KO mice with changes in several relevant genes, including downregulation of fatty-acid synthase and induction of lipolysis gene, Pnpla2, without impacting overall steatosis. Interestingly, EGFR downstream signaling mediators, including AKT, remain activated in EGFR-KO mice, which correlated with increased activity pattern of other receptor tyrosine kinases, including ErbB3/MET, in transcriptomic analysis. Lastly, Canertinib treatment in EGFR-KO mice, which inhibits all ErbB receptors, successfully reduced steatosis, suggesting the compensatory roles of other ErbB receptors in supporting MASLD without EGFR. CONCLUSIONS:Hepatocyte-specific EGFR-KO did not impact steatosis, but enhanced fibrosis in the FFD model of MASLD. Gene networks associated with lipid metabolism were greatly altered in EGFR-KO, but phenotypic effects might be compensated by alternate signaling pathways.
10.1016/j.jcmgh.2024.101380
COVID-19 Diarrhea Is Inflammatory, Caused by Direct Viral Effects Plus Major Role of Virus-induced Cytokines.
Cellular and molecular gastroenterology and hepatology
BACKGROUND & AIMS:Diarrhea occurs in up to 50% of cases of COVID-19. Nonetheless, the pathophysiologic mechanism(s) have not been determined. METHODS:This was examined using normal human enteroid monolayers exposed apically to live SARS-CoV-2 or non-replicating virus-like particles (VLPs) bearing the 4 SARS-CoV-2 structural proteins or irradiated virus, all of which bound and entered enterocytes. RESULTS:Live virus and VLPs incrieased secretion of multiple cytokines and reduced mRNAs of ACE2, NHE3, and DRA. Interleukin (IL)-6 plus IL-8 alone reduced NHE3 mRNA and protein and DRA mRNA and protein. Neither VLPs nor IL-6 plus IL-8 alone altered Cl secretion, but together they caused Cl secretion, which was Ca-dependent, CFTR-independent, blocked partially by a specific TMEM16A inhibitor, and entirely by a general TMEM16 family inhibitor. VLPs and irradiated virus, but not IL-6 plus IL-8, produced Ca waves that began within minutes of VLP exposure, lasted for at least 60 minutes, and were prevented by pretreatment with apyrase, a P2Y1 receptor antagonist, and general TMEM16 family inhibitor but not by the specific TMEM16A inhibitor. CONCLUSIONS:The pathophysiology of COVID-19 diarrhea appears to be a unique example of a calcium-dependent inflammatory diarrhea that is caused by direct viral effects plus the virus-induced intestinal epithelial cytokine secretion.
10.1016/j.jcmgh.2024.101383
Acvr1b Loss Increases Formation of Pancreatic Precancerous Lesions From Acinar and Ductal Cells of Origin.
Cellular and molecular gastroenterology and hepatology
BACKGROUND & AIMS:Pancreatic ductal adenocarcinoma can develop from precursor lesions, including pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasm (IPMN). Previous studies indicated that loss of Acvr1b accelerates the Kras-mediated development of papillary IPMN in the mouse pancreas; however, the cell type predominantly affected by these genetic changes remains unclear. METHODS:We investigated the contribution of cellular origin by inducing IPMN associated mutations (KRAS expression and Acvr1b loss) specifically in acinar (Ptf1a;Kras;Acvr1b mice) or ductal (Sox9CreER;Kras;Acvr1b mice) cells in mice. We then performed magnetic resonance imaging and a thorough histopathologic analysis of their pancreatic tissues. RESULTS:The loss of Acvr1b increased the development of pancreatic intraepithelial neoplasia and IPMN-like lesions when either acinar or ductal cells expressed a Kras mutation. Magnetic resonance imaging, immunohistochemistry, and histology revealed large IPMN-like lesions in these mice that exhibited features of flat, gastric epithelium. In addition, cyst formation in both mouse models was accompanied by chronic pancreatitis. Experimental acute pancreatitis accelerated the development of large mucinous cysts and pancreatic intraepithelial neoplasia when acinar, but not ductal, cells expressed mutant Kras and lost Acvr1b. CONCLUSIONS:These findings indicate that loss of Acvr1b in the presence of the Kras oncogene promotes the development of large and small precancerous lesions from both ductal and acinar cells. However, the IPMN-like phenotype was not equivalent to that observed when these mutations were made in all pancreatic cells during development. Our study underscores the significance of the cellular context in the initiation and progression of precursor lesions from exocrine cells.
10.1016/j.jcmgh.2024.101387
Select Gut Microbiota Impede Rotavirus Vaccine Efficacy.
Cellular and molecular gastroenterology and hepatology
BACKGROUND & AIMS:The protection provided by rotavirus (RV) vaccines is highly heterogeneous among individuals. We hypothesized that microbiota composition might influence RV vaccine efficacy. METHODS:First, we examined the potential of segmented filamentous bacteria (SFB) colonization to influence RV vaccine efficacy in mice. Next, we probed the influence of human microbiomes on RV vaccination via administering mice fecal microbial transplants (FMTs) from children with robust or minimal RV vaccine responsiveness. Post-FMT, mice were subjected to RV vaccination followed by RV challenge. RESULTS:SFB colonization induced a phenotype that was reminiscent of RV vaccine failure (ie, failure to generate RV antigens and, consequently, anti-RV antibodies following RV vaccination resulting in proneness to RV challenge after SFB levels diminished). FMTs from children to mice recapitulated donor vaccination phenotype. Specifically, mice receiving FMTs from high-responsive vaccinees copiously shed RV antigens and robustly generated anti-RV antibodies following RV vaccination. Concomitantly, such mice were impervious to RV challenge. In contrast, mice receiving FMTs from children who had not responded to RV vaccination exhibited only modest responses to RV vaccination and, concomitantly, remained prone to RV challenge. Microbiome analysis ruled out a role for SFB but suggested involvement of Clostridium perfringens. Oral administration of cultured C. perfringens to gnotobiotic mice partially recapitulated the RV vaccine non-responder phenotype. Analysis of published microbiome data found C. perfringens abundance in children modestly associated with RV vaccine failure. CONCLUSION:Microbiota composition influences RV vaccine efficacy with C. perfringens being one, perhaps of many, potential contributing taxa.
10.1016/j.jcmgh.2024.101393
Quantifying Forms and Functions of Enterohepatic Bile Acid Pools in Mice.
Cellular and molecular gastroenterology and hepatology
BACKGROUNDS & AIMS:Bile acids (BAs) are core gastrointestinal metabolites with dual functions in lipid absorption and cell signaling. BAs circulate between the liver and distal small intestine (i.e., ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with host intestinal cells in vivo remain poorly understood. Because ileal absorption is rate-limiting in determining which BAs in the intestinal lumen gain access to host intestinal cells and receptors, and at what concentrations, we hypothesized that defining the rates and routes of ileal BA absorption in vivo would yield novel insights into the physiological forms and functions of mouse enterohepatic BA pools. METHODS:Using ex vivo mass spectrometry, we quantified 88 BA species and metabolites in the intestinal lumen and superior mesenteric vein of individual wild-type mice, and cage-mates lacking the ileal BA transporter, Asbt/Slc10a2. RESULTS:Using these data, we calculated that the pool of BAs circulating through ileal tissue (i.e., the ileal BA pool) in fasting C57BL/6J female mice is ∼0.3 μmol/g. Asbt-mediated transport accounted for ∼80% of this pool and amplified size. Passive permeability explained the remaining ∼20% and generated diversity. Compared with wild-type mice, the ileal BA pool in Asbt-deficient mice was ∼5-fold smaller, enriched in secondary BA species and metabolites normally found in the colon, and elicited unique transcriptional responses on addition to exvivo-cultured ileal explants. CONCLUSIONS:This study defines quantitative traits of the mouse enterohepatic BA pool and reveals how aberrant BA metabolism can impinge directly on host intestinal physiology.
10.1016/j.jcmgh.2024.101392
Wall of Resilience: How the Intestinal Epithelium Prevents Inflammatory Onslaught in the Gut.
Cellular and molecular gastroenterology and hepatology
The intestinal epithelium forms the boundary between the intestinal immune system in the lamina propria and the outside world, the intestinal lumen, which contains a diverse array of microbial and environmental antigens. Composed of specialized cells, this epithelial monolayer has an exceptional turnover rate. Differentiated epithelial cells are released into the intestinal lumen within a few days, at the villus tip, a process that requires strict regulation. Dysfunction of the epithelial barrier increases the intestinal permeability and paves the way for luminal antigens to pass into the intestinal serosa. Stem cells at the bottom of Lieberkühn crypts provide a constant supply of mature epithelial cells. Differentiated intestinal epithelial cells exhibit a diverse array of mechanisms that enable communication with surrounding cells, fortification against microorganisms, and orchestration of nutrient absorption and hormonal balance. Furthermore, tight junctions regulate paracellular permeability properties, and their disruption can lead to an impairment of the intestinal barrier, allowing inflammation to develop or further progress. Intestinal epithelial cells provide a communication platform through which they maintain homeostasis with a spectrum of entities including immune cells, neuronal cells, and connective tissue cells. This homeostasis can be disrupted in disease, such as inflammatory bowel disease. Patients suffering from inflammatory bowel disease show an impaired gut barrier, dysregulated cellular communication, and aberrant proliferation and demise of cells. This review summarizes the individual cellular and molecular mechanisms pivotal for upholding the integrity of the intestinal epithelial barrier and shows how these can be disrupted in diseases, such as inflammatory bowel disease.
10.1016/j.jcmgh.2024.101423
Early Onset Colorectal Cancer: Molecular Underpinnings Accelerating Occurrence.
Cellular and molecular gastroenterology and hepatology
The onset of colorectal cancer (CRC) in patients younger than 50 continues to rapidly increase. This study highlights the epidemiologic changes, risk factors, clinical characteristics, and molecular profiles prevalent in early onset CRC patients, and identifies key areas for future research. It has been noted that only a small fraction of early onset CRC cases is attributed to known hereditary mutations and fit the canonical pathway of late-onset colorectal cancer development. To highlight this, we review the genetic and epigenetic modifications specific to early onset CRC. We also discuss the synergetic effect of single-nucleotide polymorphisms and environmental factors on the early onset of CRC. Additionally, we discuss the potential of noninvasive biomarker assays to enhance early detection, screening, diagnosis, and prognostic outcome predictions.
10.1016/j.jcmgh.2024.101425
Normalization of Cystic Fibrosis Immune System Reverses Intestinal Neutrophilic Inflammation and Significantly Improves the Survival of Cystic Fibrosis Mice.
Cellular and molecular gastroenterology and hepatology
BACKGROUND & AIMS:Cystic fibrosis (CF) is an autosomal recessive genetic disorder, affecting multiple organ systems. CF intestinal disease develops early, manifesting as intestinal bacterial overgrowth/dysbiosis, neutrophilic inflammation, and obstruction. As unresolvable infection and inflammation reflect host immune deficiency, we sought to determine if the CF-affected immune system plays any significant role in CF intestinal disease pathogenesis. METHODS:CF and sibling wild-type (WT) mice underwent reciprocal bone marrow transplantation. After immune reconstitution, their mortality, intestinal transit, fecal inflammatory markers, and mucosal immune cell composition were assessed. Moreover, reciprocal neutrophil transfusion was conducted to determine if neutrophil function affects intestinal movement. Furthermore, expression of induced nitric oxide synthase (iNOS) and production of nitric oxide (NO) in CF and WT neutrophils were compared. Lastly, specific iNOS inhibitor 1400W was tested to prevent CF intestinal obstruction. RESULTS:Immune restoration in CF mice reversed the intestinal neutrophilic inflammation, improved the intestinal dysmotility, and rescued the mice from mortality. Transfusion of WT neutrophils into CF mice ameliorated the retarded bowel movement. CF neutrophils expressed significantly more iNOS and produced significantly more NO. Pharmaceutical blocking of iNOS significantly improved intestinal transit and survival of CF mice. CONCLUSIONS:CF immune defect plays a critical role in CF intestinal disease development. Activation of iNOS in inflammatory cells produces excessive NO, slows the bowel movement, and facilitates intestinal paralysis and obstruction in CF. Thus, normalization of the CF immune system may offer a novel therapy to treat CF intestinal disease.
10.1016/j.jcmgh.2024.101424
PKMζ, a Brain-specific PKCζ Isoform, is Required for Glycolysis and Myofibroblastic Activation of Hepatic Stellate Cells.
Cellular and molecular gastroenterology and hepatology
BACKGROUND & AIMS:Transforming growth factor (TGF)β1 induces plasma membrane (PM) accumulation of glucose transporter 1 (Glut1) required for glycolysis of hepatic stellate cells (HSCs) and HSC activation. This study aimed to understand how Glut1 is anchored/docked onto the PM of HSCs. METHODS:HSC expression of protein kinase M zeta isoform (PKMζ) was detected by reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. PKMζ level was manipulated by short hairpin RNA (shRNA) or overexpression; HSC activation was assessed by cell expression of activation markers; PM Glut1, glucose uptake, and glycolysis of HSCs were analyzed by biotinylation, 2-NBDG-based assay, and Seahorse Glycolysis Stress Test. Phospho-mutants of vasodilator-stimulated phosphorylated protein (VASP) were created by site-directed mutagenesis. TGFβ transcriptome was obtained by RNA sequencing. Single-cell RNA sequencing datasets and immunofluorescence were leveraged to analyze PKMζ expression in cancer-associated fibroblasts (CAFs) of colorectal liver metastases. Function of HSC PKMζ was determined by tumor/HSC co-implantation study. RESULTS:Primary human and murine HSCs express PKMζ, but not full-length PKCζ. PKMζ knockdown suppresses, whereas PKMζ overexpression potentiates PM accumulation of Glut1, glycolysis, and HSC activation induced by TGFβ1. Mechanistically, PKMζ binds to and induces VASP phosphorylation at serines 157 and 239 facilitating anchoring/docking of Glut1 onto the PM of HSCs. PKMζ expression is increased in the CAFs of murine and patient colorectal liver metastases compared with quiescent HSC. Targeting PKMζ suppresses transcriptome, CAF activation of HSCs, and colorectal tumor growth in mice. CONCLUSIONS:Because HSCs are also a major contributor of liver fibrosis, our data highlight PKMζ and VASP as targets to inhibit metabolic reprogramming, HSC activation, liver fibrosis, and the pro-metastatic microenvironment of the liver.
10.1016/j.jcmgh.2024.101429
Mouse Models for Pancreatic Ductal Adenocarcinoma are Affected by the cre-driver Used to Promote KRAS Activation.
Cellular and molecular gastroenterology and hepatology
BACKGROUND & AIMS:The fundamental biology of pancreatic ductal adenocarcinoma has been greatly impacted by the characterization of genetically engineered mouse models that allow temporal and spatial activation of oncogenic KRAS (KRAS). One of the most commonly used models involves targeted insertion of a cre-recombinase into the Ptf1a gene. However, this approach disrupts the Ptf1a gene, resulting in haploinsufficiency that likely affects sensitivity to oncogenic KRAS (KRAS). This study aims to determine if Ptf1a haploinsufficiency affected the acinar cell response to KRAS before and after induction of pancreatic injury. METHODS:We performed morphological and molecular analysis of 3 genetically engineered mouse models that express a tamoxifen-inducible cre-recombinase to activate Kras in acinar cells of the pancreas. The cre-recombinase was targeted to the acinar-specific transcription factor genes, Ptf1a or Mist1/Bhlha15, or expressed within a BAC-derived Elastase transgene. Histological and RNA-seq analyses were used to delineate differences between the models. RESULTS:Up to 2 months after tamoxifen induction of KRAS, morphological changes were negligible. However, induction of pancreatic injury by cerulein resulted in widespread PanIN lesions in Ptf1a pancreata within 7 days and maintained for at least 5 weeks post-injury, which was not seen in the models with 2 functional Ptf1a alleles. RNA-sequencing analysis prior to injury induction suggested Ptf1a and Mist1 mice have unique profiles of gene expression that predict a differential response to injury. Multiplex analysis of pancreatic tissue confirmed different inflammatory responses between the models. CONCLUSIONS:These findings suggest Ptf1a haploinsufficiency in Ptf1a mouse models promotes KRAS priming of genes for promotion of pancreatic ductal adenocarcinoma.
10.1016/j.jcmgh.2024.101428