L. Pastorelli

Central role of the gut epithelial barrier in the pathogenesis of chronic intestinal inflammation: lessons learned from animal models and human genetics.

The gut mucosa is constantly challenged by a bombardment of foreign antigens and environmental microorganisms. As such, the precise regulation of the intestinal barrier allows the maintenance of mucosal immune homeostasis and prevents the onset of uncontrolled inflammation. In support of this concept, emerging evidence points to defects in components of the epithelial barrier as etiologic factors in the pathogenesis of inflammatory bowel diseases (IBDs). In fact, the integrity of the intestinal barrier relies on different elements, including robust innate immune responses, epithelial paracellular permeability, epithelial cell integrity, as well as the production of mucus. The purpose of this review is to systematically evaluate how alterations in the aforementioned epithelial components can lead to the disruption of intestinal immune homeostasis, and subsequent inflammation. In this regard, the wealth of data from mouse models of intestinal inflammation and human genetics are pivotal in understanding pathogenic pathways, for example, that are initiated from the specific loss of function of a single protein leading to the onset of intestinal disease. On the other hand, several recently proposed therapeutic approaches to treat human IBD are targeted at enhancing different elements of gut barrier function, further supporting a primary role of the epithelium in the pathogenesis of chronic intestinal inflammation and emphasizing the importance of maintaining a healthy and effective intestinal barrier.

SAMP1/YitFc mouse strain: A spontaneous model of Crohn's disease‐like ileitis

The SAMP1/YitFc mouse strain represents a model of Crohns disease (CD)-like ileitis that is ideal for investigating the pathogenesis of chronic intestinal inflammation. Different from the vast majority of animal models of colitis, the ileal-specific phenotype characteristic of SAMP1/YitFc mice occurs spontaneously, without genetic, chemical, or immunological manipulation. In addition, SAMP1/YitFc mice possess remarkable similarities to the human condition with regard to disease location, histologic features, incidence of extraintestinal manifestations, and response to conventional therapies. SAMP1/YitFc mice also display a well-defined time course of a predisease state and phases of acute and chronic ileitis. As such, the SAMP1/YitFc model is particularly suitable for elucidating pathways that precede the clinical phenotype that may lead to preventive, and therefore more efficacious, intervention with the natural course of disease, or alternatively, for the development of therapeutic strategies directed against chronic, established ileitis. In this review we summarize important contributions made by our group and others that uncover potential mechanisms in the pathogenesis of CD using this unique murine model of chronic intestinal inflammation.

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.

Novel cytokine signaling pathways in inflammatory bowel disease: insight into the dichotomous functions of IL-33 during chronic intestinal inflammation:

In 2010, four independent groups almost simultaneously reported the association of the novel interleukin-1 (IL-1) family member, IL-33, with inflammatory bowel disease (IBD). The findings were remarkably consistent and demonstrated that IL-33 is markedly upregulated in, and specific to, ulcerative colitis (UC). In addition, although a variety of gut-associated immune cell subsets express IL-33, the primary source appears to be the intestinal epithelium. IL-33’s receptor, ST2, a formerly orphaned IL-1 receptor-related protein, was also found to be increased in UC patients, although the cellular source of ST2 appears to be somewhat more ambiguous. In fact, emerging evidence indicates that the IL-33/ST2 axis plays a critical role in several other chronic inflammatory and immune disorders. In the gut, IL-33 has been shown to be important in the clearance of intestinal parasites, and inducing epithelial cell hyperplasia, mucus production and mucosal eosinophilic infiltration. However, despite the established trend of increased IL-33 and ST2 expression during IBD, specifically UC, the precise pathophysiologic relevance of these findings has yet to be determined. Interestingly, IL-33 has the ability to potentiate pathogenic Th2 and Th17 responses in gut-associated lymphoid tissues, while also promoting healing of damaged mucosa following inflammatory insults. Indeed, further mechanistic studies are warranted to confirm the possible dichotomous functions of IL-33 during chronic intestinal inflammation and better define its precise role in the pathogenesis of IBD. Herein, we discuss what is currently known about IL-33/ST2 in the gut and speculate as to the potential role of the IL-33/ST2 system in IBD.

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.

Emerging drugs for the treatment of ulcerative colitis

Background: Ulcerative colitis (UC) is a chronic, relapsing inflammatory disorder of the colon for which the etiology is currently unknown. At present, strategies to treat UC are primarily targeted to control inflammation during active phases of disease as well as maintain remission during quiescence. As such, several unmet needs in the treatment of UC still remain. In recent years, basic research has led to the recognition of several key factors in the pathogenesis of UC, translating into the development of several novel therapeutic agents. Objective: The aim of this study is to review emerging therapies that may advance the treatment and improve the overall care of UC patients. Methods: An extensive literature search on published manuscripts and meeting proceedings has been performed to provide a comprehensive review of future drug therapies to treat UC. Results/conclusion: The translational application of new discoveries in the basic understanding of UC pathogenesis is continuing and critical for the development of novel treatment strategies. Design of novel biologic therapies to treat UC has the challenge of addressing potential safety issues, while more traditional drugs should be further developed to facilitate patient compliance to treat this chronic, debilitating disease.

Spontaneous, Immune-Mediated Gastric Inflammation in SAMP1/YitFc Mice, a Model of Crohn's-Like Gastritis

BACKGROUND & AIMS Crohns disease (CD) can develop in any region of the gastrointestinal tract, including the stomach. The etiology and pathogenesis of Crohns gastritis are poorly understood, treatment approaches are limited, and there are not many suitable animal models for study. We characterized the features and mechanisms of chronic gastritis in SAMP1/YitFc (SAMP) mice, a spontaneous model of CD-like ileitis, along with possible therapeutic approaches. METHODS Stomachs from specific pathogen-free and germ-free SAMP and AKR mice (controls) were evaluated histologically; the presence of Helicobacter spp was tested in fecal pellets by polymerase chain reaction analysis. In vivo gastric permeability was quantified by fractional excretion of sucrose, and epithelial tight junction protein expression was measured by quantitative reverse-transcription polymerase chain reaction analysis. The effects of a proton pump inhibitor (PPI) or corticosteroids were measured, and the ability of pathogenic immune cells to mediate gastritis was assessed in adoptive transfer experiments. RESULTS SAMP mice developed Helicobacter-negative gastritis, characterized by aggregates of mononuclear cells, diffuse accumulation of neutrophils, and disruption of epithelial architecture; SAMP mice also had increased gastric permeability compared with controls, without alterations in expression of tight junction proteins. The gastritis and associated permeability defect observed in SAMP mice were independent of bacterial colonization and reduced by administration of corticosteroids but not a PPI. CD4(+) T cells isolated from draining mesenteric lymph nodes of SAMP mice were sufficient to induce gastritis in recipient SCID mice. CONCLUSIONS In SAMP mice, gastritis develops spontaneously and has many features of CD-like ileitis. These mice are a useful model to study Helicobacter-negative, immune-mediated Crohns gastritis.

IL-33 promotes recovery from acute colitis by inducing miR-320 to stimulate epithelial restitution and repair

Significance We clarify that the normal, inherent function of IL-33 following acute, resolving colitis is protection, inducing proliferation and restitution of ST2L-bearing intestinal epithelial cells (IECs). Importantly, this response occurs in otherwise healthy, immunocompetent C57BL/6J (B6) mice and may be different in other models possessing genetic and/or immunologic abnormalities that predispose to colitis, similar to patients with inflammatory bowel disease. Mechanistically, although the molecular processes responsible for control of microRNA (miR) biogenesis in response to challenge remain largely unknown, we report that IL-33 augments epithelial miR-320, which increases IEC proliferation and wound closure that is significantly diminished upon specific miR-320 inhibition. This study provides the rationale for the potential therapeutic use of either IL-33 or miR-320A to obtain optimal gut mucosal healing and the resolution of inflammation. Defective and/or delayed wound healing has been implicated in the pathogenesis of several chronic inflammatory disorders, including inflammatory bowel disease (IBD). The resolution of inflammation is particularly important in mucosal organs, such as the gut, where restoration of epithelial barrier function is critical to reestablish homeostasis with the interfacing microenvironment. Although IL-33 and its receptor ST2/ILRL1 are known to be increased and associated with IBD, studies using animal models of colitis to address the mechanism have yielded ambiguous results, suggesting both pathogenic and protective functions. Unlike those previously published studies, we focused on the functional role of IL-33/ST2 during an extended (2-wk) recovery period after initial challenge in dextran sodium sulfate (DSS)-induced colitic mice. Our results show that during acute, resolving colitis the normal function of endogenous IL-33 is protection, and the lack of either IL-33 or ST2 impedes the overall recovery process, while exogenous IL-33 administration during recovery dramatically accelerates epithelial restitution and repair, with concomitant improvement of colonic inflammation. Mechanistically, we show that IL-33 stimulates the expression of a network of microRNAs (miRs) in the Caco2 colonic intestinal epithelial cell (IEC) line, especially miR-320, which is increased by >16-fold in IECs isolated from IL-33–treated vs. vehicle-treated DSS colitic mice. Finally, IL-33–dependent in vitro proliferation and wound closure of Caco-2 IECs is significantly abrogated after specific inhibition of miR-320A. Together, our data indicate that during acute, resolving colitis, IL-33/ST2 plays a crucial role in gut mucosal healing by inducing epithelial-derived miR-320 that promotes epithelial repair/restitution and the resolution of inflammation.

Interleukin-37: A Peacekeeper at the Intestinal Borders

The interleukin (IL)-1 family of cytokines encompasses a wide spectrum of soluble mediators and receptor antagonists. Their expression, present in nearly all organ systems, is of particular importance at mucosal barriers interfacing the external microenvironment, such as the skin, the respiratory tract, and the gastrointestinal (GI) system. IL-1 and IL-1-related cytokines are key factors in the promotion, enhancement, and regulation of host innate immunity and in the orchestration of their interplay with the adaptive immune system. Interestingly, several IL-1 family members, including IL-1a/IL-1b, IL-18, and IL-33, display dichotomous, opposing functions in the pathogenesis of chronic intestinal inflammation, by both inducing inflammation and promoting epithelial restitution/repair and mucosal healing [1]. Emerging evidence, however, suggests that the IL-1 family member 7 (IL-1F7), also termed IL-37, possesses predominant and seemingly exclusive anti-inflammatory activities, particularly in the setting of inflammatory bowel disease (IBD) and other colitic conditions. Discovery and Characterization of IL-37

DOP006 IFI16 is dysregulated in inflammatory bowel diseases and its epithelial expression induced by pro-inflammatory cytokines

Background: c-Jun N-terminal kinases (JNK), members of the mitogen-activated protein kinase family, regulate several cellular functions such as proliferation, differentiation, apoptosis as well as the production of cytokines and antimicrobial peptides. In this study we analyzed the role of JNK 1 and 2 isoforms in the regulation of the gastrointestinal barrier. Methods: JNK1flox mice were crossed with Villin-Cre mice to create an intestinal epithelial-cell specific knockout (KO) of JNK1 (JNK1 DIEC). Furthermore, constitutive JNK2 knockout (JNK2 / ) and JNK1 DIEC/JNK2 / double-KO mice were used. Bone marrow transplantation was performed to create chimeric mice with isolated JNK2-deletions in the hematopoietic or nonhematopoietic compartment. Mice were investigated under normal conditions and after induced colitis with 3% dextran sodium sulfate (DSS) for 7 days. Disease activity was monitored by the disease activity index (DAI) for DSS-induced colitis (based on weight loss, stool consistency, and bleeding), endoscopy, histology and functional testing of the intestinal barrier. Molecular analyses were performed by immmuhistochemistry, PCR, western blot and enzymatic assays. Results: JNK1 and JNK2 were equally expressed in the intestinal epithelium and phosphorylation was induced in both isoforms upon DSS colitis. Non-treated JNK1 DIEC, JNK2 / , and JNK1 DIEC/JNK2 / developed normally, did not show any phenotype up to 45 weeks. Upon induction of DSS colitis, disease activity was unchanged in JNK1 DIEC compared to WT animals, whereas JNK2 / and JNK1 DIEC/JNK2 / mice showed significantly higher disease activity with enhanced mucosa destruction, increased barrier dysfunction and higher inflammation as indicated by tissue myeloperoxidase levels, CD11b immunostaining, and mRNA expression of proinflammatory cytokine. The analysis of chimeric mice revealed that the loss of JNK2 in the non-hematopoietic compartment was mainly responsible for the enhanced disease phenotype. JNK2-deficient mice showed augmented caspase 3-dependent epithelial cell apoptosis and increased expression of genes related to endoplasmic reticulum stress. Cell culture experiments with the colon carcinoma cell line HT-29 showed enhanced apoptosis induction upon oxidative stress after treatment with the JNK inhibitor SP600125. Conclusions: Our data indicate that JNK2, but not JNK1, protects against stress induced apoptosis in intestinal epithelial cells and therefore improves barrier function and secondary inflammation.