Kristen O. Arseneau

Probiotics promote gut health through stimulation of epithelial innate immunity.

Probiotic formulations are widely available and have a variety of proposed beneficial effects, including promotion of gut health. The mechanisms of action of probiotic bacteria in the intestine are still unclear but are generally attributed to an antiinflammatory effect. Here, we demonstrate that the multiple probiotic formulation VSL#3 prevents the onset of intestinal inflammation by local stimulation of epithelial innate immune responses (i.e., increased production of epithelial-derived TNF-α and restoration of epithelial barrier function in vivo). We also demonstrate that probiotic bacteria stimulate epithelial production of TNF-α and activate NF-κB in vitro. Our results support the hypothesis that probiotics promote gut health through stimulation, rather than suppression, of the innate immune system. Furthermore, our findings provide the perspective that defects in innate immunity may play a critical role in the pathogenesis and progression of intestinal disorders, such as inflammatory bowel disease.

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.

The dual role of nod-like receptors in mucosal innate immunity and chronic intestinal inflammation

Nucleotide-binding and oligomerization domain NOD-like receptors (NLRs) are highly conserved cytosolic pattern recognition receptors that play, in combination with toll-like receptors, a critical role in innate immunity and inflammation. These proteins are characterized by a central oligomerization domain termed nucleotide-binding domain, and a protein interaction domain containing leucine-rich repeats. Some NLRs, including NOD1 and NOD2, sense the cytosolic presence of conserved bacterial molecular signatures and drive the activation of mitogen-activated protein kinase and the transcription factor NF-κB. A different set of NLRs induces caspase-1 activation through the assembly of large protein complexes known as inflammasomes. Activation of NLR proteins results in secretion of pro-inflammatory cytokines and subsequent inflammatory responses. The critical role of NLRs in innate immunity is underscored by the fact that polymorphisms within their genes are implicated in the development of several immune-mediated diseases, including inflammatory bowel disease. Over the past few years, the role of NLRs in intestinal homeostasis has been highlighted, however the mechanism by which dysfunction in these proteins leads to aberrant inflammation is still the focus of much investigation. The purpose of this review is to systematically evaluate the function of NLRs in mucosal innate immunity and understand how genetic or functional alterations in these components can lead to the disruption of intestinal homeostasis, and the subsequent development of chronic inflammation.

Tregs are Dysfunctional in Vivo in a Spontaneous Murine Model of Crohn’s Disease

Although regulatory T cells (Tregs) have been implicated in inflammatory bowel disease, Tregs from Crohns disease (CD) patients are increased in number and function normally in vitro. To clarify this disparity, we studied Treg function in vivo using a spontaneous model of CD-like ileitis. We first administered anti-CD25-depleting antibodies to SAMP1/YitFc (SAMP) mice to assess ileitis; mesenteric lymph node cells were then transferred into SCID (severe combined immunodeficient) recipients to induce colitis. CD25 depletion increased the severity of both spontaneous ileitis and adoptively transferred colitis. Interestingly, a second transfer of CD4+CD25+ cells from untreated AKR control mice was able to ameliorate the induced colitis, whereas CD4+CD25+ cells from untreated SAMP mice were not, suggesting a functional abnormality in SAMP Tregs. Anti-CD25 treatment in SAMP mice also induced proliferation of CD25−Foxp3+ Tregs, which had a proinflammatory intestinal T helper type 1/ T helper type 2 (Th1/Th2) effector phenotype. These studies demonstrate Treg dysfunction in a spontaneous model of CD-like ileitis.

Cytokines and mucosal immunity.

Purpose of review Cytokines are integral mediators for maintaining intestinal mucosal homeostasis, as well as prominent effector molecules during chronic gut inflammatory diseases. This review focuses on recent studies of the role of specific cytokines in mucosal immunity. Recent findings Dichotomous, or even opposing, functions have been described for several cytokines involved in intestinal innate immunity (most notably for members of the interleukin-1 family), which depend on the specific inflammatory conditions within the intestinal mucosa. For example, both interleukin-1&agr; and interleukin-33 exhibit ‘alarmin’-type properties that can signal tissue or cell damage, which further add to their well described proinflammatory roles. Costimulatory molecules of the tumor necrosis factor/tumor necrosis factor receptor superfamily, such as TNF-like cytokine 1A and LIGHT, are actively involved in mucosal proinflammatory pathways, but also may exert protection against infectious agents to facilitate recovery from acute inflammation. Finally, innate lymphoid cells are increasingly recognized as important cellular sources of pivotal mucosal cytokines, including the interleukin-23/T helper 17 cytokine, interleukin-22. Summary Elucidating the complexity of cytokine signaling within the normal mucosa and during acute and chronic inflammation will be a pivotal step toward understanding the pathogenesis of immune-mediated gut diseases and developing effective therapies to treat them.

Intestinal-Specific TNFα Overexpression Induces Crohn's-Like Ileitis in Mice

Background and Aim Human and animal studies have clearly established tumor necrosis factor (TNF)α as an important mediator of Crohn’s disease pathogenesis. However, whether systemic or only local TNFα overproduction is required for the development of chronic intestinal inflammation and Crohn’s disease remains unclear. The aim of this study was to assess the contribution of intestinal epithelial-derived TNFα to the development of murine Crohn’s-like ileitis. Methods We adapted the well-established TNF∆ARE/+ mouse model of Crohn’s disease (which systemically overexpresses TNFα) to generate a homozygous mutant strain that overexpress TNFα only within the intestinal epithelium. Intestinal-specific TNFi∆ARE/i∆ARE mice were examined for histopathological signs of gut inflammation and extraintestinal manifestations of Crohn’s disease. The mucosal immune phenotype was characterized, and the contribution of specific lymphocyte populations to the pathogenesis of TNFi∆ARE/i∆ARE ileitis was assessed. Results TNFi∆ARE/i∆ARE mice had increased mucosal and systemic TNFα levels compared to wild-type controls (P<0.001), as well as severe chronic ileitis with increased neutrophil infiltration and villous distortion, but no extraintestinal manifestations (P<0.001 vs. wild-type controls). The gut mucosal lymphocytic compartment was also expanded in TNFi∆ARE/i∆ARE mice (P<0.05), consisting of activated CD69+ and CD4+CD62L- lymphocytes (P<0.05). FasL expression was significantly elevated in the mesenteric lymph nodes of TNFi∆ARE/i∆ARE mice (P<0.05). Adoptive transfer of mucosal TNFi∆ARE/i∆ARE lymphocytes resulted in ileitis in immunologically naïve severe combined immunodeficiency recipients (P<0.05 vs. wild-type controls), indicating an effector phenotype that was associated with increased production of both Th1 (IFNγ) and Th2 (IL-5, IL-13) cytokines. Conclusion Intestinal epithelial-derived TNFα is sufficient for the induction of Crohn’s-like ileitis, but not for the occurrence of extraintestinal manifestations, in TNFi∆ARE/i∆ARE mice. These effects were associated with generation of effector lymphocytes within the intestinal mucosa and dysregulated apoptosis. Thus, targeted intestinal blockade of TNFα may provide an effective means to neutralize gut-derived TNFα with reduced side effects.

A Novel Role for TL1A/DR3 in Protection against Intestinal Injury and Infection

TNF-like cytokine 1A (TL1A) is expressed on APCs and provides costimulatory signals to activated lymphocytes that bear its functional receptor, death receptor 3 (DR3). TL1A/DR3 signaling is involved in the pathogenesis of human and experimental inflammatory bowel disease. In the current study, we investigated the role of this cytokine/receptor pair in acute intestinal injury/repair pathways. We demonstrate that intact DR3 signaling protected mice from acute dextran sodium sulfate colitis because DR3−/− mice showed more severe mucosal inflammation and increased mortality. DR3−/− mice were compromised in their ability to maintain adequate numbers of CD4+CD25+Foxp3+ regulatory T cells in response to acute mucosal damage. This defect in immune regulation led to a nonspecific upregulation of effector proinflammatory pathways, which was most prominent for the Th17 immunophenotype. TL1A−/− mice were similarly more susceptible to dextran sodium sulfate colitis, although without mortality and with delayed kinetics compared with DR3−/− mice, and also displayed significantly reduced numbers of regulatory T cells. Infection of DR3−/− mice with Salmonella typhimurium was associated with defective microbial clearance and elevated bacterial load. Taken together, our findings indicate a novel protective role for the TL1A/DR3 axis in the regulation of mucosal homeostasis during acute intestinal injury/repair, which contrasts with its known pathogenic function during chronic intestinal inflammation.

Functional defects in NOD2 signaling in experimental and human Crohn disease

Increasing evidence suggests that a deficit in innate immunity may play a causative role in the pathogenesis of inflammatory bowel disease. The most compelling support for this hypothesis comes from the genetic association of Crohn disease (CD) with carriage of polymorphisms within the NOD2 gene, which represent the most frequent genetic defect in CD. Our findings suggest that SAMP1/YitFc mice, which develop CD-like ileitis in the absence of NOD2 genetic mutations, fail to respond to MDP administration by displaying decreased innate cytokine production and impaired bacterial clearance before the onset of disease. This provides evidence that dysregulated NOD2 signaling, genetic or functional in nature, predisposes to chronic intestinal inflammation, and supports a new paradigm that CD may occur from a deficit in innate immunity as opposed to an overly aggressive immune response. This new paradigm could lead to potential development of new preventative or therapeutic modalities for patients with CD.

Vercirnon for the treatment of Crohn's disease

Introduction: CCR9 antagonism is a promising new therapeutic approach for the treatment of Crohns disease. CCR9 is expressed on the cell surface of memory/effector CD4+ T cells and selectively binds to the small intestinal lymphocyte chemoattractant CCL25 (TECK). Blockade of the CCR9/CCL25 interaction inhibits lymphocyte homing to the intestinal mucosa, thereby limiting inflammation and disease at this site. Areas covered: This review details the current research on CCR9 antagonism and summarizes available clinical trial data for vercirnon, a selective CCR9 antagonist currently under development. Expert opinion: If the results of ongoing large-scale clinical trials of vercirnon are in line with preliminary reports, CCR9 antagonism may have comparable efficacy to anti-TNF therapies and a potentially superior safety profile, making it the latest addition to the growing arsenal of immunomodulatory drug therapies available to combat Crohns disease. Moreover, since vercirnon is an oral drug, its associated costs will likely be much lower than expensive infusion-based anti-TNF therapies, providing further economic benefits.