Benedetta Mattioli

Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis.

IL-33 is a novel member of the IL-1 family and ligand for the IL-1 receptor-related protein, ST2. Recent evidence suggests that the IL-33/ST2 axis plays a critical role in several autoimmune and inflammatory disorders; however, its role in inflammatory bowel disease (IBD) has not been clearly defined. We characterized IL-33 and ST2 expression and modulation after conventional anti-TNF therapy in Crohn’s disease and ulcerative colitis (UC) patients and investigated the role of IL-33 in SAMP1/YitFc (SAMP) mice, a mixed Th1/Th2 model of IBD. Our results showed a specific increase of mucosal IL-33 in active UC, localized primarily to intestinal epithelial cells (IEC) and colonic inflammatory infiltrates. Importantly, increased expression of full-length IL-33, representing the most bioactive form, was detected in UC epithelium, whereas elevated levels of cleaved IL-33 were present in IBD serum. ST2 isoforms were differentially modulated in UC epithelium, and sST2, a soluble decoy receptor with anti-inflammatory properties, was also elevated in IBD serum. Infliximab (anti-TNF) treatment of UC decreased circulating IL-33 and increased sST2, whereas stimulation of HT-29 IEC confirmed IL-33 and sST2 regulation by TNF. Similarly, IL-33 significantly increased and correlated with disease severity, and potently induced IL-5, IL-6, and IL-17 from mucosal immune cells in SAMP mice. Taken together, the IL-33/ST2 system plays an important role in IBD and experimental colitis, is modulated by anti-TNF therapy, and may represent a specific biomarker for active UC.

Probiotic Bacteria Regulate Intestinal Epithelial Permeability in Experimental Ileitis by a TNF-Dependent Mechanism

Background We previously showed that the probiotic mixture, VSL#3, prevents the onset of ileitis in SAMP/YitFc (SAMP) mice, and this effect was associated with stimulation of epithelial-derived TNF. The aim of this study was to determine the mechanism(s) of VSL#3-mediated protection on epithelial barrier function and to further investigate the “paradoxical” effects of TNF in preventing SAMP ileitis. Methods Permeability was evaluated in SAMP mice prior to the onset of inflammation and during established disease by measuring transepithelial electrical resistance (TEER) on ex vivo-cultured ilea following exposure to VSL#3 conditioned media (CM), TNF or VSL#3-CM + anti-TNF. Tight junction (TJ) proteins were assessed by qRT-PCR, Western blot, and confocal microscopy, and TNFRI/TNFRII expression measured in freshly isolated intestinal epithelial cells (IEC) from SAMP and control AKR mice. Results Culture with either VSL#3-CM or TNF resulted in decreased ileal paracellular permeability in pre-inflamed SAMP, but not SAMP with established disease, while addition of anti-TNF abrogated these effects. Modulation of the TJ proteins, claudin-2 and occludin, occurred with a significant decrease in claudin-2 and increase in occludin following stimulation with VSL#3-CM or TNF. TNF protein levels increased in supernatants of SAMP ilea incubated with VSL#3-CM compared to vehicle, while IEC-derived TNFR mRNA expression decreased in young, and was elevated in inflamed, SAMP versus AKR mice. Conclusions Our data demonstrate that the previously established efficacy of VSL#3 in preventing SAMP ileitis is due to direct innate and homeostatic effects of TNF on the gut epithelium, modulation of the TJ proteins, claudin-2 and occludin, and overall improvement of intestinal permeability.

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.

Loss of estrogen-mediated immunoprotection underlies female gender bias in experimental Crohn’s-like ileitis

The incidence and severity of Crohn’s disease (CD) are increased in female patients. Using SAMP1/YitFc (SAMP) mice, a spontaneous model of chronic intestinal inflammation that displays histologic and pathogenic similarities to human CD, we investigated the potential mechanism(s) contributing to sex differences observed in CD. Similar to gender differences observed in CD patients, SAMP female (SAMP-F) mice displayed an earlier onset and more severe ileitis compared with SAMP male (SAMP-M) mice. Furthermore, T-regulatory cells (Tregs) from gut-associated lymphoid tissue (GALT) of SAMP-F mice were reduced in frequency and impaired in their in vitro and in vivo suppressive functions compared with that of SAMP-M mice. Given the interaction between sex hormones and Treg function, we investigated the possible role of estrogen (E2) in SAMP ileitis. SAMP-M mice responded to exogenous E2 administration by expanding Treg frequency and reducing ileal inflammation, whereas SAMP-F mice were resistant. Conventional T cells and Tregs responded differentially to estrogen signaling, leading to distinct immunoprotective effects mediated by distinct estrogen receptor (ER) isoforms. These mechanisms were impaired in T cells from SAMP-F mice. Thus, hormone signaling influences the expansion and function of GALT Tregs in an ER-dependent manner and contributes to gender-based differences in experimental CD.

P002 The potential role of IL-33 in inflammatory-associated gut fibrosis: Induction of pro-fibrogenic gene expression and myofibroblast hypertrophy

Background: The pathogenesis of human ulcerative colitis (UC) is characterized by the presence of IL-13-producing Natural Killer T cells (NKT cells). One major complication of UC is the development of colitis-associated colorectal cancer. In these studies we determined the role of alternatively activated “M2 macrophages” in a new mouse model of colitis-associated carcinogenesis. Further, we verified these results through immunological analysis of surgical specimens obtained from patients with UC-associated colon cancer. Methods: Studies were performed using a mouse model for colitis-associated carcinogenesis based on chronic Oxazolonecolitis in combination with an initial i.p. azoxymethane (AOM) injection in female BALB/c mice. We observed chronic intestinal inflammation lasting for 12 weeks in mice repeatedly administered Oxazolone. Surgical specimens with UC-associated colon cancer were analyzed by flow cytometry and immunohistochemistry. Results: We could show that Oxazolone-colitis was mediated by colonic alpha-galactosyl-ceramide+CD1+ NKT-cells producing IL-13 and therefore resembles the immunological characteristics of human UC. Colon cancer development in this model was dependent on F4/80+CD11bhighGr1low macrophages producing IL-6 and EGF. Those cells inherited phenotypic characteristics of IL-13-stimulated alternativelyactivated “M2 macrophages”. To elucidate the importance of these “M2 macrophages”, we conducted bone marrow chimera studies and demonstrated that innate immune signaling through MyD88 in “M2 macrophages” is the key event for the production of tumor supporting factors like IL-6 and EGF. In surgical specimens obtained from patients with UC-associated colon cancer we verified those experimental findings. We consistently found a dense accumulation of IL-13-producing NKT cells inside the tumors. In addition, in this IL-13-dominated tumor micromilieu CD14+ and CD68+ antigen-presenting cells accumulated intensively and those antigen-presenting cells demonstrated an alternatively activated “M2 phenotype” by the expression of CD163 and CD205. Conclusions: We could demonstrate that in an experimental UC model, as well as in human UC, pathogenic IL-13producing NKT cells accumulate in the tumor micromilieu of UC-associated colon cancers. This immunologic situation inside the tumor microenvironment of mice and men induces tumor promoting M2 macrophages. Therefore M2 macrophages represent a potential target for future therapeutic strategies.

P.05.2 PATHOGENIC ROLE OF IL-33-MEDIATED EOS INFILTRATION AND FUNCTION IN EXPERIMENTAL INFLAMMATORY BOWEL DISEASE

Background and aim: IL-33, a new member of the IL-1 family, is linked to several immune and inflammatory disorders, such as IBD. Eosinophils (EOS) are found in active inflammatory lesions of IBD patients and can secrete proinflammatory molecules, alter epithelial barrier function and initiate mucosal immune responses leading to chronic gut inflammation. Recent evidence show that IL-33 can regulate EOS infiltration and function. The aim of this study is to determine IL-33-mediated EOS activation and ileal inflammation in the SAMP1/YitFc (SAMP) murine model of chronic enteritis. Material and methods: EOS were detected by IHC for major basic protein (MBP). IL-33 and EOS-associated cytokines, IL-5 and eotaxin-1,-2,-3, were measured by qPCR in a timecourse study of SAMP and AKR (parental control) ilea. Th2 cytokines were measured by ELISA in mesenteric lymph nodes (MLN) of SAMP treated daily with rIL-33 (33 ug/kg, ip for 1wk). EOS depletion and IL-33 neutralization studies were performed by ip administration (2X/wk for 6 wks) of Abs against IL-5/CCR3 and anti-ST2 (all 5mg/kg), respectively, on inflamed SAMP, and ileitis and cytokine expression evaluated. Results: Ileal IL-33, IL-5, and eotaxin-1 & -2 mRNA transcripts increased according to disease severity in SAMP (4vs. 20-wk-old, p<0.01) and were elevated vs. age-matched AKR controls (p<0.05). EOS infiltration showed a similar trend with more abundant and intense MBP staining in cells morphologically resembling EOS during disease progression in SAMP, and was virtually absent in AKR. Administration of rIL-33 induced an increase in the Th2 cytokines, IL-4, -5, &-10, levels in MLN of treated vs. vehicle-treated mice (p<0.05). Lastly, administration of either anti-IL-5/anti-CCR3 or antiST2 to inflamed SAMP resulted in a marked decrease in ileal inflammation (p<0.01), EOS infiltration, and Th2 cytokine/eotaxin-1&-2 mRNA expression (p<0.01) vs. IgG control-treated mice. Conclusions: Taken together, these data demonstrate a pathogenic role of IL33-mediated EOS infiltration and function in chronic intestinal inflammation, and that blockade of IL-33 and/or downstream EOS activation may represent a novel therapeutic modality to treat patients with IBD.

M1770 Interaction Between Sex Hormones/Sex Hormone Receptors With the Intestinal Epithelium Modulates Barrier Function and Subsequent Inflammation in Experimental IBD

Background & Aims: Inflammatory bowel diseases (IBD) are characterized by chronic intestinal inflammation due to the loss of immune tolerance againstmucosal antigens. Mesenchymal stromal cells were recently shown to possess immunomodulatory action with beneficial therapeutic effects in immune mediated disorders. We investigated the potential therapeutic action of subcutaneous adipose tissue (AT-MSCs) or bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a model of IBD. Methods: Male Wistar rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis were treated with AT-MSCs or BM-MSCs through intraperitoneal injection after colonoscopic detection of inflammation, at day 4. Colonoscopic and histologic scores were evaluated. Inflammatory response was determined by measuring the levels of inflammatory cytokines in cultures of colon samples, by ELISA. Collagen fibers were stained with picric acid dye and the density of collagen deposition was evaluated using a computerized image analysis system. MSCs were labeled with Tc-99m and administered contralateral to the site of inflammation. Scans were taken 30 minutes and 2 hours after injection, and isolated colon scans were obtained after euthanasia. Results: Intra-peritoneal injection of AT-MSCs or BM-MSCs significantly reduced the endoscopic (p<0.01) and histopathologic (p<0.02) severity of colitis . The therapeutic effect was mediated by downregulating the levels of TNF-alpha (p<0.01), and interleukin-1 beta (p<0.03). The high baseline levels of VEGF-a in TNBS-colitis did not decrease significantly after MSCs-therapy. A significant reduction in collagen deposition was observed in MSC-treated animals (p<0.01). Effects observed with AT-MSCs were significantly greater than the ones obtained with BMMSCs. Scintigraphy showed 99mTc-MSCs uptake in TNBS-induced animals but no uptake in controls. Labeled MSCs clearly migrated towards the sites of inflammation, and the uptake increased from 30 min to 2h. Conclusions: Intra-peritoneally injected MSCs are effective in the treatment of experimental colitis, probably due to a local paracrine anti-inflammatory action. Because MSCs are easily and safely obtained from the abundant subcutaneous fat tissue, AT-MSCs emerge as a promising therapy for IBD.