Interleukin 31 mediates MAP kinase and STAT1/3 activation in intestinal epithelial cells and its expression is upregulated in inflammatory bowel disease. Dambacher Julia,Beigel Florian,Seiderer Julia,Haller Dirk,Göke Burkhard,Auernhammer Christoph J,Brand Stephan Gut BACKGROUND/AIM:Interleukin 31 (IL31), primarily expressed in activated lymphocytes, signals through a heterodimeric receptor complex consisting of the IL31 receptor alpha (IL31Ralpha) and the oncostatin M receptor (OSMR). The aim of this study was to analyse IL31 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal inflammation. METHODS:Expression studies were performed by RT-PCR, quantitative PCR, western blotting, and immunohistochemistry. Signal transduction was analysed by western blotting. Cell proliferation was measured by MTS assays, cell migration by restitution assays. RESULTS:Colorectal cancer derived intestinal epithelial cell (IEC) lines express both IL31 receptor subunits, while their expression in unstimulated primary murine IEC was low. LPS and the proinflammatory cytokines TNF-alpha, IL1beta, IFN-gamma, and sodium butyrate stimulation increased IL31, IL31Ralpha, and OSMR mRNA expression, while IL31 itself enhanced IL8 expression in IEC. IL31 mediates ERK-1/2, Akt, STAT1, and STAT3 activation in IEC resulting in enhanced IEC migration. However, at low cell density, IL31 had significant antiproliferative capacities (p<0.005). IL31 mRNA expression was not increased in the TNFDeltaARE mouse model of ileitis but in inflamed colonic lesions compared to non-inflamed tissue in patients with Crohn's disease (CD; average 2.4-fold increase) and in patients with ulcerative colitis (UC; average 2.6-fold increase) and correlated with the IL-8 expression in these lesions (r = 0.564 for CD; r = 0.650 for UC; total number of biopsies analysed: n = 88). CONCLUSION:IEC express the functional IL31 receptor complex. IL31 modulates cell proliferation and migration suggesting a role in the regulation of intestinal barrier function particularly in intestinal inflammation. 10.1136/gut.2006.118679

In Inflamed Intestinal Tissues and Epithelial Cells, Interleukin 22 Signaling Increases Expression of H19 Long Noncoding RNA, Which Promotes Mucosal Regeneration. Gastroenterology BACKGROUND & AIMS:Inflammation affects regeneration of the intestinal epithelia; long noncoding RNAs (lncRNAs) regulate cell functions, such as proliferation, differentiation, and migration. We investigated the mechanisms by which the lncRNA H19, imprinted maternally expressed transcript (H19) regulates regeneration of intestinal epithelium using cell cultures and mouse models of inflammation. METHODS:We performed RNA-sequencing transcriptome analyses of intestinal tissues from mice with lipopolysaccharide (LPS)-induced sepsis to identify lncRNAs associated with inflammation; findings were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization analyses of intestinal tissues from mice with sepsis or dextran sulfate sodium (DSS)-induced mucosal wound healing and patients with ulcerative colitis compared to healthy individuals (controls). We screened cytokines for their ability to induce expression of H19 in HT-29 cells and intestinal epithelial cells (IECs), and confirmed findings in crypt epithelial organoids derived from mouse small intestine. IECs were incubated with different signal transduction inhibitors and effects on H19 lncRNA levels were measured. We assessed intestinal epithelial proliferation or regeneration in H19 mice given LPS or DSS vs wild-type littermates (control mice). H19 was overexpressed in IECs using lentiviral vectors and cell proliferation was measured. We performed RNA antisense purification, RNA immunoprecipitation, and luciferase reporter assays to study functions of H19 in IECs. RESULTS:In RNA-sequencing transcriptome analysis of lncRNA expression in intestinal tissues from mice, we found that levels of H19 lncRNA changed significantly with LPS exposure. Levels of H19 lncRNA increased in intestinal tissues of patients with ulcerative colitis, mice with LPS-induced and polymicrobial sepsis, or mice with DSS-induced colitis, compared with controls. Increased H19 lncRNA localized to epithelial cells in the intestine, regardless of Lgr5 messenger RNA expression. Exposure of IECs to interleukin 22 (IL22) increased levels of H19 lncRNA with time and dose, which required STAT3 and protein kinase A activity. IL22 induced expression of H19 in mouse intestinal epithelial organoids within 6 hours. Exposure to IL22 increased growth of intestinal epithelial organoids derived from control mice, but not H19 mice. Overexpression of H19 in HT-29 cells increased their proliferation. Intestinal mucosa healed more slowly after withdrawal of DSS from H19 mice vs control mice. Crypt epithelial cells from H19 mice proliferated more slowly than those from control mice after exposure to LPS. H19 lncRNA bound to p53 and microRNAs that inhibit cell proliferation, including microRNA 34a and let-7; H19 lncRNA binding blocked their function, leading to increased expression of genes that promote regeneration of the epithelium. CONCLUSIONS:The level of lncRNA H19 is increased in inflamed intestinal tissues from mice and patients. The inflammatory cytokine IL22 induces expression of H19 in IECs, which is required for intestinal epithelial proliferation and mucosal healing. H19 lncRNA appears to inhibit p53 protein and microRNA 34a and let-7 to promote proliferation of IECs and epithelial regeneration. 10.1053/j.gastro.2018.03.058

ARTS mediates apoptosis and regeneration of the intestinal stem cell niche. Koren Elle,Yosefzon Yahav,Ankawa Roi,Soteriou Despina,Jacob Avi,Nevelsky Alexander,Ben-Yosef Rahamim,Bar-Sela Gil,Fuchs Yaron Nature communications Stem cells (SCs) play a pivotal role in fueling homeostasis and regeneration. While much focus has been given to self-renewal and differentiation pathways regulating SC fate, little is known regarding the specific mechanisms utilized for their elimination. Here, we report that the pro-apoptotic protein ARTS (a Septin4 isoform) is highly expressed in cells comprising the intestinal SC niche and that its deletion protects Lgr5 and Paneth cells from undergoing apoptotic cell death. As a result, the Sept4/ARTS crypt displays augmented proliferation and, in culture, generates massive cystic-like organoids due to enhanced Wnt/β-catenin signaling. Importantly, Sept4/ARTS mice exhibit resistance against intestinal damage in a manner dependent upon Lgr5 SCs. Finally, we show that ARTS interacts with XIAP in intestinal crypt cells and that deletion of XIAP can abrogate Sept4/ARTS-dependent phenotypes. Our results indicate that intestinal SCs utilize specific apoptotic proteins for their elimination, representing a unique target for regenerative medicine. 10.1038/s41467-018-06941-4

TNFAIP8 controls murine intestinal stem cell homeostasis and regeneration by regulating microbiome-induced Akt signaling. Goldsmith Jason R,Spitofsky Nina,Zamani Ali,Hood Ryan,Boggs Amanda,Li Xinyuan,Li Mingyue,Reiner Elizabeth,Ayyaz Arshad,Etwebi Zienab,Lu Ling,Rivera Guzman Javier,Bou-Dargham Mayassa J,Cathoupolis Terry,Hakonarson Hakon,Sun Honghong,Wrana Jeffrey L,Gonzalez Michael V,Chen Youhai H Nature communications The intestine is a highly dynamic environment that requires tight control of the various inputs to maintain homeostasis and allow for proper responses to injury. It was recently found that the stem cell niche and epithelium is regenerated after injury by de-differentiated adult cells, through a process that gives rise to Sca1+ fetal-like cells and is driven by a transient population of Clu revival stem cells (revSCs). However, the molecular mechanisms that regulate this dynamic process have not been fully defined. Here we show that TNFAIP8 (also known as TIPE0) is a regulator of intestinal homeostasis that is vital for proper regeneration. TIPE0 functions through inhibiting basal Akt activation by the commensal microbiota via modulating membrane phospholipid abundance. Loss of TIPE0 in mice results in injury-resistant enterocytes, that are hyperproliferative, yet have regenerative deficits and are shifted towards a de-differentiated state. Tipe0 enterocytes show basal induction of the Clu regenerative program and a fetal gene expression signature marked by Sca1, but upon injury are unable to generate Sca-1/Clu revSCs and could not regenerate the epithelium. This work demonstrates the role of TIPE0 in regulating the dynamic signaling that determines the injury response and enables intestinal epithelial cell regenerative plasticity. 10.1038/s41467-020-16379-2

MET Signaling Mediates Intestinal Crypt-Villus Development, Regeneration, and Adenoma Formation and Is Promoted by Stem Cell CD44 Isoforms. Joosten Sander P J,Zeilstra Jurrit,van Andel Harmen,Mijnals R Clinton,Zaunbrecher Joost,Duivenvoorden Annet A M,van de Wetering Marc,Clevers Hans,Spaargaren Marcel,Pals Steven T Gastroenterology BACKGROUND & AIMS:Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice. METHODS:We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (Ah/Met/LacZ) or ISC-specific disruption of MET (Lgr5/Met/LacZ) and control mice (Ah/Met/LacZ, Lgr5/Met/LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5/Met/Apc and Lgr5/Met/Apc mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in Ah/Met/Apc mice compared with Ah/Met/Apc control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44, Cd44, Cd44, or Cd44 mice). RESULTS:Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in Ah/Met/LacZ mice. Lgr5/Met/LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5/Met/Apc mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (Ah/Met/Apc mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44 mice did not expand to the same extent as crypts from Cd44 mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10. CONCLUSIONS:In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors. 10.1053/j.gastro.2017.07.008