Carlo De Salvo

IL-33 activates tumor stroma to promote intestinal polyposis

Significance Colorectal cancer results from genetic lesions in epithelial cells. However, the tumor microenvironment, which is formed by nonepithelial stromal cells, also plays an important role in this disease. The influence of the microenvironment on tumorigenesis is mediated by paracrine signals between tumor epithelial cells and neighboring stromal cells. We found that expression of interleukin 33 (IL-33), an important mediator of type 2 immunity and wound repair, is induced in epithelial cells of human and mouse intestinal tumors. IL-33 promoted intestinal tumorigenesis in ApcMin/+ mice and activated two stromal cell types, subepithelial myofibroblasts and mast cells, known to mediate intestinal dysplasia. Tumor epithelial cells are proposed to coopt IL-33–mediated immune and wound-healing responses to create a microenvironment favorable to tumorigenesis. 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 ApcMin/+ 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 ApcMin/+ 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 ApcMin/+ 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.

The Role of IL-33 in Gut Mucosal Inflammation

Interleukin (IL)-33 is a recently identified cytokine belonging to the IL-1 family that is widely expressed throughout the body and has the ability to induce Th2 immune responses. In addition, IL-33 plays a key role in promoting host defenses against parasites through the expansion of a novel population of innate lymphoid cells. In recent years, a growing body of evidence has shown that the proinflammatory properties displayed by IL-33 are detrimental in several experimental models of inflammation; in others, however, IL-33 appears to have protective functions. In 2010, four different research groups consistently described the upregulation of IL-33 in patients with inflammatory bowel disease (IBD). Animal models of IBD were subsequently utilized in order to mechanistically determine the precise role of IL-33 in chronic intestinal inflammation, without, however, reaching conclusive evidence demonstrating whether IL-33 is pathogenic or protective. Indeed, data generated from these studies suggest that IL-33 may possess dichotomous functions, enhancing inflammatory responses on one hand and promoting epithelial integrity on the other. This review focuses on the available data regarding IL-33/ST2 in the physiological and inflammatory states of the gut in order to speculate on the possible roles of this novel IL-1 family member in intestinal inflammation.

IL-33 drives eosinophil infiltration and pathogenic type 2 helper T-cell immune responses leading to chronic experimental ileitis

Although a clear association has been established between IL-33 and inflammatory bowel disease, mechanistic studies to date, primarily using acute murine models of colitis, have yielded contradicting results, demonstrating both pathogenic and protective roles. We used a well-characterized, spontaneous model of inflammatory bowel disease [ie, SAMP1/YitFc (SAMP) mice] to investigate the role of IL-33 during chronic intestinal inflammation. Our results showed marked eosinophil infiltration into the gut mucosa with increased levels of eotaxins and type 2 helper T-cell (Th2) cytokines as disease progressed and became more severe, which could be reversed upon either eosinophil depletion or blockade of IL-33 signaling. Exogenous IL-33 administration recapitulated these effects in ilea of uninflamed (parental) control AKR/J mice. Human data supported these findings, showing colocalization and up-regulation of IL-33 and eosinophils in the colonic mucosa of inflammatory bowel disease patients versus noninflamed controls. Finally, colonization of commensal flora by fecal material transplantation into germ-free SAMP and the presence of the gut microbiome induced IL-33, subsequent eosinophil infiltration, and mounting of Th2 immune responses, leading to exacerbation of chronic intestinal inflammation characteristic of SAMP mice. These data demonstrate a pathogenic role for IL-33-mediated eosinophilia and activation of Th2 immunity in chronic intestinal inflammation that is dependent on the gut microbiome. Targeting IL-33 may represent a novel therapeutic approach to treat patients with inflammatory bowel disease.

Tu1950 Pathogenic Role of IL-33-Mediated Eosinophil Infiltration and Function in Experimental IBD

KO mice are not activated and may not be fully immunosuppressive. Adoptive transfer of In Vitro generated iTregs into 2-3 week old DC-TGFβRII KO mice significantly reduced the frequency of activated T cells in the spleen and MLN. The severity of colitis was also markedly reduced in iTreg transferred KO mice. However, the mice were not protected from gastritis. Conclusions: DCs from DC-TGFβRII KO mice are more proinflammatory and less tolerogenic. This altered function of DCs combined with the potential loss of function of regulatory T cells may contribute to the development of autoimmune disease in DC-TGFβRII KO mice.

Mo1779 Interleukin-33 Induces Distinct Gastric Epithelial Alterations and Plays an Important Role in the Pathogenesis of Murine Gastritis

active in CD Paneth cells. TEM high magnification observation revealed that autophagy activation was associated with a significant decrease in the number of secretory granules in CD patients. The granules appeared to be wrapped around autophagolysosomal vacuoles and abnormally degraded. Conclusion: This work describes of a novel selective autophagy pathway, called crinophagy that targets specifically secretory granules of CD Paneth cells. This process consists of the self-digestion of secretory granules by the autophago-lysosomal pathway. Crinophagy may explain the decrease of CD secretory granule observed in our study as well as the disorganization of the secretory granules and the exocytosis defect previously reported in CD.

Tu1736 IL-33/ST2 Axis Modulates Epithelial Repair and Gut Mucosal Wound Healing in DSS-Colitic Mice

Background. Increasing evidence confirms that IL-33 and its receptor, ST2, are important factors in the pathogenesis of IBD. However, animal studies have yielded ambiguous results, reporting both pathogenic as well as protective functions. The aim of our study was to characterize and functionally evaluate the precise role of the IL-33/ST2 axis following acute epithelial injury and mucosal repair in dextran sodium sulphate (DSS)-induced colitic mice. Methods. 3% DSS was administered for 5d to C57/BL6 wild-type (WT), IL-33 KO and ST2 KO mice to induce colitis. DSS was then replaced with regular drinking water for 2 wks (recovery period). Another group of WT mice received DSS for 5d and IL-33 (33ug/kg, i.p.) or vehicle (VEH) every other day during the recovery period. Mice were sacrificed either after DSS challenge or after 1 or 2 wks of recovery; control mice (CT) not exposed to DSS were sacrificed at similar time points, at which time colons were harvested. Body weight, occult blood test, and stool consistency were measured daily to calculate Disease Activity Index (DAI), and endoscopic and histological evaluation of colons were performed using established scoring systems. IHC, qPCR and Western blots were done on full-thickness colons for IL-33 and ST2 localization, mRNA expression, and evaluation of protein isoforms, respectively. Results. DSS administered to WT mice resulted in increased body weight loss and DAI. More severe colitis was observed following DSS+1wk recovery vs. after 5d of DSS, which decreased after DSS+2wks recovery. IL-33 mRNA transcripts were dramatically elevated after DSS, and even more so following DSS+recovery vs. CT, but similar to CT after DSS+2wks recovery. ST2 mRNA expression was also increased after DSS+recovery vs. CT, while no difference was found between 5d of DSS challenge and CT and after DSS+2wks recovery. Full-length, bioactive IL33 (31 kDa), ST2L (76 kDa) and sST2 (52 kDa) were expressed in all experimental groups; the cleaved, less active form of IL33 (24 kDa) was increased in only DSS exposed mice vs. CT. IHC showed intense IL-33 and ST2 staining within the inflamed and ulceratedmucosa of DSS-treatedmice. ST2 staining was more evident during the recovery phase following DSS, notably localized to subepithelial myofibroblasts in close proximity to areas of re-epithelialization. Remarkably, both IL-33 and ST2 KO mice showed increased colonic inflammation after 2 wks recovery compared to after 5d DSS and vs also WT, suggesting the importance of IL-33/ST2 axis in mucosal healing. Likewise, IL33 treatment of WT mice resulted in increased body weight, reduced DAI, and decreased colonic inflammation after 2 wks recovery vs. VEH. Conclusions. Our results suggest that activation of the IL-33/ST2 axis promotes epithelial repair and mucosal healing following acute epithelial injury during DSS-induced colitis.

Sa1847 Emerging Role of the Interleukin (IL)-33/ST2 Axis in Gut Mucosal Wound Healing and Epithelial Repair

BACKGROUND: Danger signals have been postulated as regulators of gut mucosal immunity. During intestinal inflammation, the epithelium is compromised and signals, alerting adjacent cells of tissue damage, are released. Hence, we were interested in metallothioneins (MTs), small proteins which have been identified at inflammation sites.We explored triggers releasing MTs from colon epithelial cells and identified their role as extracellular danger signal during experimental colitis. METHODS: HT29 cells were subjected to the following treatments: 200ng/ml LPS, 200 microM H2O2, hypoxia (~1% oxygen), 100ng/ml TNF-alpha + 300ng/ ml IFN-gamma to induce apoptosis, and repeated freeze/thaw cycles to mimic necrosis. Supernatant was analysed for MT levels using western blot and for lactate dehydrogenase activity (LDH). A Boyden transwell migration assay with blood leukocytes was applied to evaluate the chemotactic potential of extracellular MT and the capacity of monoclonal antiMT antibody (100μg/ml UC1MT) to abolish this. The role of MT as chemo-attractant was further explored using dextran sulphate sodium (DSS)-induced colitis in MT knockout (MT-/-), transgenic (MT+/+) and wild type mice (WT). The therapeutical use of monoclonal therapy was tested in DSSand 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Inflammatory cell infiltrate was evaluated in all experiments together with standard inflammation markers. RESULTS: Necrosis and TNF-induced apoptosis resulted in detectable MT levels in supernatant of HT29 cells, which was not the case for LPS, H2O2 nor hypoxia treatment. LDH activity was not increased after stimulation with TNF, ruling out an uncontrolled release of MT from TNF-treated cells. Increased leukocyte migration towards this MT-containing supernatant was detected, whereas the addition of UC1MT was able to overcome this chemo-attraction (p,0.05). Significantly less neutrophil infiltration was observed in MT-/mice compared to MT+/+ and WT mice in DSS colitis (p,0.05). Complementary, i.p. UC1MT treatment reduced the number of F4/80-positive macrophages in DSSand TNBS-induced colitis (p,0.05). Less inflammatory infiltrate was associated with reduced histological inflammation in all three colitis experiments. CONCLUSION: We characterized metallothionein as danger signal released from HT29 cells after necrotic and TNF-induced apoptotic cell death. Inhibiting MT function by monoclonal therapy reduces leukocyte infiltration and represents a novel therapy dampening experimental colitis.