Proteolytic bacteria expansion during colitis amplifies inflammation through cleavage of the external domain of PAR2.

Imbalances in proteolytic activity have been linked to the development of inflammatory bowel diseases (IBD) and experimental colitis. Proteases in the intestine play important roles in maintaining homeostasis, but exposure of mucosal tissues to excess proteolytic activity can promote pathology through protease-activated receptors (PARs). Previous research implicates microbial proteases in IBD, but the underlying pathways and specific interactions between microbes and PARs remain unclear. In this study, we investigated the role of microbial proteolytic activation of the external domain of PAR2 in intestinal injury using mice expressing PAR2 with a mutated N-terminal external domain that is resistant to canonical activation by proteolytic cleavage. Our findings demonstrate the key role of proteolytic cleavage of the PAR2 external domain in promoting intestinal permeability and inflammation during colitis. In wild-type mice expressing protease-sensitive PAR2, excessive inflammation leads to the expansion of bacterial taxa that cleave the external domain of PAR2, exacerbating colitis severity. In contrast, mice expressing mutated protease-resistant PAR2 exhibit attenuated colitis severity and do not experience the same proteolytic bacterial expansion. Colonization of wild-type mice with proteolytic PAR2-activating Enterococcus and Staphylococcus worsens colitis severity. Our study identifies a previously unknown interaction between proteolytic bacterial communities, which are shaped by inflammation, and the external domain of PAR2 in colitis. The findings should encourage new therapeutic developments for IBD by targeting excessive PAR2 cleavage by bacterial proteases.

Diet-induced obesity alters intestinal monocyte-derived and tissue-resident macrophages and increases intestinal permeability in female mice independent of tumor necrosis factor.

Macrophages are essential for homeostatic maintenance of the anti-inflammatory and tolerogenic intestinal environment, yet monocyte-derived macrophages can promote local inflammation. Proinflammatory macrophage accumulation within the intestines may contribute to the development of systemic chronic inflammation and immunometabolic dysfunction in obesity. Using a model of high-fat diet-induced obesity in C57BL/6J female mice, we assessed intestinal paracellular permeability by in vivo and ex vivo assays and quantitated intestinal macrophages in ileum and colon tissues by multicolor flow cytometry after short (6 wk), intermediate (12 wk), and prolonged (18 wk) diet allocation. We characterized monocyte-derived CD4-TIM4- and CD4+TIM4- macrophages, as well as tissue-resident CD4+TIM4+ macrophages. Diet-induced obesity had tissue- and time-dependent effects on intestinal permeability, as well as monocyte and macrophage numbers, surface marker phenotype, and intracellular production of the cytokines IL-10 and tumor necrosis factor (TNF). We found that obese mice had increased paracellular permeability, in particular within the ileum, but this did not elicit recruitment of monocytes nor a local proinflammatory response by monocyte-derived or tissue-resident macrophages in either the ileum or colon. Proliferation of monocyte-derived and tissue-resident macrophages was also unchanged. Wild-type and TNF-/- littermate mice had similar intestinal permeability and macrophage population characteristics in response to diet-induced obesity. These data are unique from reported effects of diet-induced obesity on macrophages in metabolic tissues, as well as outcomes of acute inflammation within the intestines. These experiments also collectively indicate that TNF does not mediate effects of diet-induced obesity on paracellular permeability or intestinal monocyte-derived and tissue-resident intestinal macrophages in young female mice.NEW & NOTEWORTHY We found that diet-induced obesity in female mice has tissue- and time-dependent effects on intestinal paracellular permeability as well as monocyte-derived and tissue-resident macrophage numbers, surface marker phenotype, and intracellular production of the cytokines IL-10 and TNF. These changes were not mediated by TNF.

Psychological stress impairs IL22-driven protective gut mucosal immunity against colonising pathobionts.

Crohn's disease is an inflammatory disease of the gastrointestinal tract characterized by an aberrant response to microbial and environmental triggers. This includes an altered microbiome dominated by Enterobacteriaceae and in particular adherent-invasive E. coli (AIEC). Clinical evidence implicates periods of psychological stress in Crohn's disease exacerbation, and disturbances in the gut microbiome might contribute to the pathogenic mechanism. Here we show that stress-exposed mice develop ileal dysbiosis, dominated by the expansion of Enterobacteriaceae. In an AIEC colonisation model, stress-induced glucocorticoids promote apoptosis of CD45+CD90+ cells that normally produce IL-22, a cytokine that is essential for the maintenance of ileal mucosal barrier integrity. Blockade of glucocorticoid signaling or administration of recombinant IL-22 restores mucosal immunity, prevents ileal dysbiosis, and blocks AIEC expansion. We conclude that psychological stress impairs IL-22-driven protective immunity in the gut, which creates a favorable niche for the expansion of pathobionts that have been implicated in Crohn's disease. Importantly, this work also shows that immunomodulation can counteract the negative effects of psychological stress on gut immunity and hence disease-associated dysbiosis.

Saccharomyces boulardii CNCM I-745 modulates the microbiota-gut-brain axis in a humanized mouse model of Irritable Bowel Syndrome.

BACKGROUND: Gnotobiotic mice colonized with microbiota from patients with irritable bowel syndrome (IBS) and comorbid anxiety (IBS+A) display gut dysfunction and anxiety-like behavior compared to mice colonized with microbiota from healthy volunteers. Using this model, we tested the therapeutic potential of the probiotic yeast Saccharomyces boulardii strain CNCM I-745 (S. bou) and investigated underlying mechanisms. METHODS: Germ-free Swiss Webster mice were colonized with fecal microbiota from an IBS+A patient or a healthy control (HC). Three weeks later, mice were gavaged daily with S. boulardii or placebo for two weeks. Anxiety-like behavior (light preference and step-down tests), gastrointestinal transit, and permeability were assessed. After sacrifice, samples were taken for gene expression by NanoString and qRT-PCR, microbiota 16S rRNA profiling, and indole quantification. KEY RESULTS: Mice colonized with IBS+A microbiota developed faster gastrointestinal transit and anxiety-like behavior (longer step-down latency) compared to mice with HC microbiota. S. bou administration normalized gastrointestinal transit and anxiety-like behavior in mice with IBS+A microbiota. Step-down latency correlated with colonic Trpv1 expression and was associated with altered microbiota profile and increased Indole-3-acetic acid (IAA) levels. CONCLUSIONS & INFERENCES: Treatment with S. bou improves gastrointestinal motility and anxiety-like behavior in mice with IBS+A microbiota. Putative mechanisms include effects on pain pathways, direct modulation of the microbiota, and indole production by commensal bacteria.

Aryl hydrocarbon receptor ligand production by the gut microbiota is decreased in celiac disease leading to intestinal inflammation.

Metabolism of tryptophan by the gut microbiota into derivatives that activate the aryl hydrocarbon receptor (AhR) contributes to intestinal homeostasis. Many chronic inflammatory conditions, including celiac disease involving a loss of tolerance to dietary gluten, are influenced by cues from the gut microbiota. We investigated whether AhR ligand production by the gut microbiota could influence gluten immunopathology in nonobese diabetic (NOD) mice expressing DQ8, a celiac disease susceptibility gene. NOD/DQ8 mice, exposed or not exposed to gluten, were subjected to three interventions directed at enhancing AhR pathway activation. These included a high-tryptophan diet, gavage with Lactobacillus reuteri that produces AhR ligands or treatment with an AhR agonist. We investigated intestinal permeability, gut microbiota composition determined by 16S rRNA gene sequencing, AhR pathway activation in intestinal contents, and small intestinal pathology and inflammatory markers. In NOD/DQ8 mice, a high-tryptophan diet modulated gut microbiota composition and enhanced AhR ligand production. AhR pathway activation by an enriched tryptophan diet, treatment with the AhR ligand producer L. reuteri, or pharmacological stimulation using 6-formylindolo (3,2-b) carbazole (Ficz) decreased immunopathology in NOD/DQ8 mice exposed to gluten. We then determined AhR ligand production by the fecal microbiota and AhR activation in patients with active celiac disease compared to nonceliac control individuals. Patients with active celiac disease demonstrated reduced AhR ligand production and lower intestinal AhR pathway activation. These results highlight gut microbiota-dependent modulation of the AhR pathway in celiac disease and suggest a new therapeutic strategy for treating this disorder.

Lactobacilli Degrade Wheat Amylase Trypsin Inhibitors to Reduce Intestinal Dysfunction Induced by Immunogenic Wheat Proteins.

BACKGROUND & AIMS: Wheat-related disorders, a spectrum of conditions induced by the ingestion of gluten-containing cereals, have been increasing in prevalence. Patients with celiac disease have gluten-specific immune responses, but the contribution of non-gluten proteins to symptoms in patients with celiac disease or other wheat-related disorders is controversial. METHODS: C57BL/6 (control), Myd88-/-, Ticam1-/-, and Il15-/- mice were placed on diets that lacked wheat or gluten, with or without wheat amylase trypsin inhibitors (ATIs), for 1 week. Small intestine tissues were collected and intestinal intraepithelial lymphocytes (IELs) were measured; we also investigated gut permeability and intestinal transit. Control mice fed ATIs for 1 week were gavaged daily with Lactobacillus strains that had high or low ATI-degrading capacity. Nonobese diabetic/DQ8 mice were sensitized to gluten and fed an ATI diet, a gluten-containing diet or a diet with ATIs and gluten for 2 weeks. Mice were also treated with Lactobacillus strains that had high or low ATI-degrading capacity. Intestinal tissues were collected and IELs, gene expression, gut permeability and intestinal microbiota profiles were measured. RESULTS: In intestinal tissues from control mice, ATIs induced an innate immune response by activation of Toll-like receptor 4 signaling to MD2 and CD14, and caused barrier dysfunction in the absence of mucosal damage. Administration of ATIs to gluten-sensitized mice expressing HLA-DQ8 increased intestinal inflammation in response to gluten in the diet. We found ATIs to be degraded by Lactobacillus, which reduced the inflammatory effects of ATIs. CONCLUSIONS: ATIs mediate wheat-induced intestinal dysfunction in wild-type mice and exacerbate inflammation to gluten in susceptible mice. Microbiome-modulating strategies, such as administration of bacteria with ATI-degrading capacity, may be effective in patients with wheat-sensitive disorders.

Commensal microbiota induces colonic barrier structure and functions that contribute to homeostasis.

The intestinal barrier encompasses structural, permeability and immune aspects of the gut mucosa that, when disrupted, may contribute to chronic inflammation. Although gnotobiotic studies have demonstrated the effects of microbiota on mucosal and systemic immunity, as well as intestinal barrier architecture and innate immune characteristics, its impact on barrier function remains unclear. We compared germ-free and conventional mice, as well as mice colonized with human fecal microbiota that were followed for 21 days post-colonization. Colonic barrier structure was investigated by immunohistochemistry, molecular and electron microscopy techniques. Permeability was assessed in colon tissue by Ussing chambers, and by serum LPS and MDP detection using TLR4- and NOD2-NFκB reporter assays. Microbiota profile was determined by Illumina 16S rRNA gene sequencing. Low dose dextran sodium sulfate was administered to assess microbiota-induced barrier changes on resistance to colonic injury. Permeability to paracellular probes and mucus layer structure resembled that of conventional mice by day 7 post-colonization, coinciding with reduced claudin-1 expression and transient IL-18 production by intestinal epithelial cells. These post-colonization adaptations were associated with decreased systemic bacterial antigen exposure and reduced susceptibility to intestinal injury. In conclusion, commensal colonization promotes physiological barrier structural and functional adaptations that contribute to intestinal homeostasis.

Inflammation-related differences in mucosa-associated microbiota and intestinal barrier function in colonic Crohn's disease.

Crohn's disease (CD), characterized by discontinuous intestinal injury and inflammation, has been associated with changes in luminal microbial composition and impaired barrier function. The relationships between visual features of intestinal injury, permeability, and the mucosa-associated microbiota are unclear. Individuals undergoing routine colonoscopy (controls) and patients with CD were evaluated by clinical parameters and confocal laser scanning endomicroscopic colonoscopy (CLE). Patients with CD were categorized as either CD with no injury (CD-NI) or CD with injury (CD-I). Colonic biopsies were taken from adjacent matched sites in all individuals, and CLE images from these sites were analyzed for vascular permeability. Microbial composition was evaluated by 16S rRNA gene sequencing of the V3 region, and the mycome was identified through internal transcribed spacer 2 sequencing. Subgroup analyses were performed for histology, paracellular permeability (Ussing chamber), and encroachment of bacteria (fluorescent in situ hybridization). CD-I patients showed an altered microbial community compared with both controls and CD-NI patients, with enrichment in Escherichia and a decrease in Firmicutes, including Lachnospira, Faecalibacterium, and Blautia. In CD-I patients, bacterial encroachment to host epithelial cells was greater in sites of injury than in matched biopsy sites. Biopsies from sites of injury also demonstrated greater vascular and paracellular permeability. Overall, CD-I patients showed an altered mucosal microbial community compared with CD-NI patients and controls. Matched biopsy samples in CD-I patients revealed that sites of injury, identified endoscopically, are characterized by increased encroachment of bacteria to host epithelial cells, associated with increased paracellular and vascular permeability, which may drive inflammation in CD. NEW & NOTEWORTHY Patients with Crohn's disease (CD) with areas of colonic injury have an altered microbial community compared with patients who have no endoscopic evidence of injury or active disease. Although matched biopsies from patients with colonic injury show no differences in the mucosa-associated microbiota, injured sites are associated with increased permeability and increased encroachment. Our results support the notion that dysbiotic communities within patients with colonic injury cause or permit disruption of the mucosal and endothelial layers in CD.

Age-Associated Microbial Dysbiosis Promotes Intestinal Permeability, Systemic Inflammation, and Macrophage Dysfunction.

Levels of inflammatory mediators in circulation are known to increase with age, but the underlying cause of this age-associated inflammation is debated. We find that, when maintained under germ-free conditions, mice do not display an age-related increase in circulating pro-inflammatory cytokine levels. A higher proportion of germ-free mice live to 600 days than their conventional counterparts, and macrophages derived from aged germ-free mice maintain anti-microbial activity. Co-housing germ-free mice with old, but not young, conventionally raised mice increases pro-inflammatory cytokines in the blood. In tumor necrosis factor (TNF)-deficient mice, which are protected from age-associated inflammation, age-related microbiota changes are not observed. Furthermore, age-associated microbiota changes can be reversed by reducing TNF using anti-TNF therapy. These data suggest that aging-associated microbiota promote inflammation and that reversing these age-related microbiota changes represents a potential strategy for reducing age-associated inflammation and the accompanying morbidity.

Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice.

Irritable bowel syndrome (IBS) is a common disorder characterized by altered gut function and often is accompanied by comorbid anxiety. Although changes in the gut microbiota have been documented, their relevance to the clinical expression of IBS is unknown. To evaluate a functional role for commensal gut bacteria in IBS, we colonized germ-free mice with the fecal microbiota from healthy control individuals or IBS patients with diarrhea (IBS-D), with or without anxiety, and monitored gut function and behavior in the transplanted mice. Microbiota profiles in recipient mice clustered according to the microbiota profiles of the human donors. Mice receiving the IBS-D fecal microbiota showed a taxonomically similar microbial composition to that of mice receiving the healthy control fecal microbiota. However, IBS-D mice showed different serum metabolomic profiles. Mice receiving the IBS-D fecal microbiota, but not the healthy control fecal microbiota, exhibited faster gastrointestinal transit, intestinal barrier dysfunction, innate immune activation, and anxiety-like behavior. These results indicate the potential of the gut microbiota to contribute to both intestinal and behavioral manifestations of IBS-D and suggest the potential value of microbiota-directed therapies in IBS patients.

Duodenal Bacteria From Patients With Celiac Disease and Healthy Subjects Distinctly Affect Gluten Breakdown and Immunogenicity.

BACKGROUND & AIMS: Partially degraded gluten peptides from cereals trigger celiac disease (CD), an autoimmune enteropathy occurring in genetically susceptible persons. Susceptibility genes are necessary but not sufficient to induce CD, and additional environmental factors related to unfavorable alterations in the microbiota have been proposed. We investigated gluten metabolism by opportunistic pathogens and commensal duodenal bacteria and characterized the capacity of the produced peptides to activate gluten-specific T-cells from CD patients. METHODS: We colonized germ-free C57BL/6 mice with bacteria isolated from the small intestine of CD patients or healthy controls, selected for their in vitro gluten-degrading capacity. After gluten gavage, gliadin amount and proteolytic activities were measured in intestinal contents. Peptides produced by bacteria used in mouse colonizations from the immunogenic 33-mer gluten peptide were characterized by liquid chromatography tandem mass spectrometry and their immunogenic potential was evaluated using peripheral blood mononuclear cells from celiac patients after receiving a 3-day gluten challenge. RESULTS: Bacterial colonizations produced distinct gluten-degradation patterns in the mouse small intestine. Pseudomonas aeruginosa, an opportunistic pathogen from CD patients, exhibited elastase activity and produced peptides that better translocated the mouse intestinal barrier. P aeruginosa-modified gluten peptides activated gluten-specific T-cells from CD patients. In contrast, Lactobacillus spp. from the duodenum of non-CD controls degraded gluten peptides produced by human and P aeruginosa proteases, reducing their immunogenicity. CONCLUSIONS: Small intestinal bacteria exhibit distinct gluten metabolic patterns in vivo, increasing or reducing gluten peptide immunogenicity. This microbe-gluten-host interaction may modulate autoimmune risk in genetically susceptible persons and may underlie the reported association of dysbiosis and CD.

Mechanisms of innate immune activation by gluten peptide p31-43 in mice.

Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. Innate immunity contributes to the pathogenesis of CD, but the mechanisms remain poorly understood. Although previous in vitro work suggests that gliadin peptide p31-43 acts as an innate immune trigger, the underlying pathways are unclear and have not been explored in vivo. Here we show that intraluminal delivery of p31-43 induces morphological changes in the small intestinal mucosa of normal mice consistent with those seen in CD, including increased cell death and expression of inflammatory mediators. The effects of p31-43 were dependent on MyD88 and type I IFNs, but not Toll-like receptor 4 (TLR4), and were enhanced by coadministration of the TLR3 agonist polyinosinic:polycytidylic acid. Together, these results indicate that gliadin peptide p31-43 activates the innate immune pathways in vivo, such as IFN-dependent inflammation, relevant to CD. Our findings also suggest a common mechanism for the potential interaction between dietary gluten and viral infections in the pathogenesis of CD.

Ecobiotherapy Rich in Firmicutes Decreases Susceptibility to Colitis in a Humanized Gnotobiotic Mouse Model.

BACKGROUND: Alterations in the intestinal microbiota, characterized by depletion of anti-inflammatory bacteria, such as Firmicutes, in patients with ulcerative colitis (UC) have prompted interest in microbiota-modulating strategies for this condition. The aim of this study was to evaluate the role of fecal and synthetic human microbial ecosystems, low or enriched in Firmicutes, on colitis susceptibility and host immune responses. METHODS: The microbiota of selected healthy and UC human donors was characterized by culture method and 16S rRNA-based sequencing. Germ-free mice were colonized with fecal or a synthetic ecosystem enriched (healthy donors) or low (UC donors) in Firmicutes. Experimental colitis was induced using dextran sodium sulfate. Colon transcriptome and colon lamina propria cells were evaluated in mice postcolonization by RNA-seq and flow cytometry, respectively, and T helper (TH) 17 differentiation was assessed in vitro. RESULTS: Mice colonized with microbiota from patients with UC low in Firmicutes had increased sensitivity to colitis compared with mice colonized with fecal or synthetic ecosystems rich in Firmicutes. Microbiota low in Firmicutes increased expression of TH17-related genes and expansion of interleukin-17A-expressing CD4 cells in vivo. Supplementation with bacterial isolates belonging to the Firmicutes phylum abrogated the heightened TH17 responses in vitro. CONCLUSIONS: A microbiota rich in Firmicutes derived from fecal samples of a healthy human donor, or assembled synthetically, downregulated colonic inflammation and TH17 pathways in mice. The results support the use of ecobiotherapy strategies, enriched in Firmicutes, for the prevention or treatment of UC.

Faecalibacterium prausnitzii prevents physiological damages in a chronic low-grade inflammation murine model.

BACKGROUND: The human gut houses one of the most complex and abundant ecosystems composed of up to 10(13)-10(14) microorganisms. The importance of this intestinal microbiota is highlighted when a disruption of the intestinal ecosystem equilibrium appears (a phenomenon called dysbiosis) leading to an illness status, such as inflammatory bowel diseases (IBD). Indeed, the reduction of the commensal bacterium Faecalibacterium prausnitzii (one of the most prevalent intestinal bacterial species in healthy adults) has been correlated with several diseases, including IBD, and most importantly, it has been shown that this bacterium has anti-inflammatory and protective effects in pre-clinical models of colitis. Some dysbiosis disorders are characterized by functional and physiological alterations. Here, we report the beneficial effects of F. prausnitzii in the physiological changes induced by a chronic low-grade inflammation in a murine model. Chronic low-grade inflammation and gut dysfunction were induced in mice by two episodes of dinitro-benzene sulfonic acid (DNBS) instillations. Markers of inflammation, gut permeability, colonic serotonin and cytokine levels were studied. The effects of F. prausnitzii strain A2-165 and its culture supernatant (SN) were then investigated. RESULTS: No significant differences were observed in classical inflammation markers confirming that inflammation was subclinical. However, gut permeability, colonic serotonin levels and the colonic levels of the cytokines IL-6, INF-γ, IL-4 and IL-22 were higher in DNBS-treated than in untreated mice. Importantly, mice treated with either F. prausnitzii or its SN exhibited significant decreases in intestinal permeability, tissue cytokines and serotonin levels. CONCLUSIONS: Our results show that F. prausnitzii and its SN had beneficial effects on intestinal epithelial barrier impairment in a chronic low-grade inflammation model. These observations confirm the potential of this bacterium as a novel probiotic treatment in the management of gut dysfunction and low-grade inflammation.

BL-7010 demonstrates specific binding to gliadin and reduces gluten-associated pathology in a chronic mouse model of gliadin sensitivity.

Celiac disease (CD) is an autoimmune disorder in individuals that carry DQ2 or DQ8 MHC class II haplotypes, triggered by the ingestion of gluten. There is no current treatment other than a gluten-free diet (GFD). We have previously shown that the BL-7010 copolymer poly(hydroxyethyl methacrylate-co-styrene sulfonate) (P(HEMA-co-SS)) binds with higher efficiency to gliadin than to other proteins present in the small intestine, ameliorating gliadin-induced pathology in the HLA-HCD4/DQ8 model of gluten sensitivity. The aim of this study was to investigate the efficiency of two batches of BL-7010 to interact with gliadin, essential vitamins and digestive enzymes not previously tested, and to assess the ability of the copolymer to reduce gluten-associated pathology using the NOD-DQ8 mouse model, which exhibits more significant small intestinal damage when challenged with gluten than HCD4/DQ8 mice. In addition, the safety and systemic exposure of BL-7010 was evaluated in vivo (in rats) and in vitro (genetic toxicity studies). In vitro binding data showed that BL-7010 interacted with high affinity with gliadin and that BL-7010 had no interaction with the tested vitamins and digestive enzymes. BL-7010 was effective at preventing gluten-induced decreases in villus-to-crypt ratios, intraepithelial lymphocytosis and alterations in paracellular permeability and putative anion transporter-1 mRNA expression in the small intestine. In rats, BL-7010 was well-tolerated and safe following 14 days of daily repeated administration of 3000 mg/kg. BL-7010 did not exhibit any mutagenic effect in the genetic toxicity studies. Using complementary animal models and chronic gluten exposure the results demonstrate that administration of BL-7010 is effective and safe and that it is able to decrease pathology associated with gliadin sensitization warranting the progression to Phase I trials in humans.

Intraluminal administration of poly I:C causes an enteropathy that is exacerbated by administration of oral dietary antigen.

Systemic administration of polyinosinic:polycytidylic acid (poly I:C), mimics virally-induced activation of TLR3 signalling causing acute small intestine damage, but whether and how mucosal administration of poly I:C causes enteropathy is less clear. Our aim was to investigate the inflammatory pathways elicited after intraluminal administration of poly I:C and determine acute and delayed consequences of this locally induced immune activation. Intraluminal poly I:C induced rapid mucosal immune activation in C57BL/6 mice involving IFNß and the CXCL10/CXCR3 axis, that may drive inflammation towards a Th1 profile. Intraluminal poly I:C also caused enteropathy and gut dysfunction in gliadin-sensitive NOD-DQ8 mice, and this was prolonged by concomitant oral administration of gliadin. Our results indicate that small intestine pathology can be induced in mice by intraluminal administration of poly I:C and that this is exacerbated by subsequent oral delivery of a relevant dietary antigen.

Novel role of the serine protease inhibitor elafin in gluten-related disorders.

OBJECTIVES: Elafin, an endogenous serine protease inhibitor, modulates colonic inflammation. We investigated the role of elafin in celiac disease (CD) using human small intestinal tissues and in vitro assays of gliadin deamidation. We also investigated the potential beneficial effects of elafin in a mouse model of gluten sensitivity. METHODS: Epithelial elafin expression in the small intestine of patients with active CD, treated CD, and controls without CD was determined by immunofluorescence. Interaction of elafin with human tissue transglutaminase-2 (TG-2) was investigated in vitro. The 33-mer peptide, a highly immunogenic gliadin peptide, was incubated with TG-2 and elafin at different concentrations. The degree of deamidation of the 33-mer peptide was analyzed by liquid chromatography-mass spectrometry. Elafin was delivered to the intestine of gluten-sensitive mice using a recombinant Lactococcus lactis vector. Small intestinal barrier function, inflammation, proteolytic activity, and zonula occludens-1 (ZO-1) expression were assessed. RESULTS: Elafin expression in the small intestinal epithelium was lower in patients with active CD compared with control patients. In vitro, elafin significantly slowed the kinetics of the deamidation of the 33-mer peptide to its more immunogenic form. Treatment of gluten-sensitive mice with elafin delivered by the L. lactis vector normalized inflammation, improved permeability, and maintained ZO-1 expression. CONCLUSIONS: The decreased elafin expression in the small intestine of patients with active CD, the reduction of 33-mer peptide deamidation by elafin, coupled to the barrier enhancing and anti-inflammatory effects observed in gluten-sensitive mice, suggest that this molecule may have pathophysiological and therapeutic importance in gluten-related disorders.

Differential induction of antimicrobial REGIII by the intestinal microbiota and Bifidobacterium breve NCC2950.

The intestinal microbiota is a key determinant of gut homeostasis, which is achieved, in part, through regulation of antimicrobial peptide secretion. The aim of this study was to determine the efficiency by which members of the intestinal microbiota induce the antimicrobial peptide REGIII and to elucidate the underlying pathways. We showed that germfree mice have low levels of REGIII-γ in their ileum and colon compared to mice with different intestinal microbiota backgrounds. Colonization with a microbiota of low diversity (altered Schaedler flora) did not induce the expression of REGIII-γ as effectively as a complex community (specific pathogen free). Monocolonization with the probiotic Bifidobacterium breve, but not with the nonprobiotic commensal Escherichia coli JM83, upregulated REGIII-γ expression. Induction of REGIII-γ by B. breve was abrogated in mice lacking MyD88 and Ticam1 signaling. Both live and heat-inactivated B. breve but not spent culture medium from B. breve induced the expression of REGIII-α, the human ortholog and homolog of REGIII-γ, in human colonic epithelial cells (Caco-2). Taken together, the results suggest that REGIII-γ expression in the intestine correlates with the richness of microbiota composition. Also, specific bacteria such as Bifidobacterium breve NCC2950 effectively induce REGIII production in the intestine via the MyD88-Ticam1 pathway. Treatment with this probiotic may enhance the mucosal barrier and protect the host from infection and inflammation.

Commensal and probiotic bacteria influence intestinal barrier function and susceptibility to colitis in Nod1-/-; Nod2-/- mice.

BACKGROUND: The intestinal microbiota regulates key host functions. It is unknown whether modulation of the microbiota can affect a genetically determined host phenotype. Polymorphisms in the Nucleotide oligomerization domain (Nod)-like receptor family confer genetic risk for inflammatory bowel disease (IBD). We investigated whether the intestinal microbiota and the probiotic strain Bifidobacterium breve NCC2950 affect intestinal barrier function and responses to intestinal injury in Nod1(-/-); Nod2(-/-) mice. METHODS: Specific pathogen-free (SPF) Nod1(-/-); Nod2(-/-) mice and mice gnotobiotically derived with altered Schaedler flora (ASF) biota were used. SPF Nod1(+/-); Nod2(+/-) littermates (generated by crossing SPF Nod1(-/-); Nod2(-/-) and germ-free C57BL/6 mice) and ASF Nod1(+/-); Nod2(+/-) mice were used as controls. SPF mice were gavaged daily with 10(9) -CFU B. breve for 14 days before colitis induction. Denaturing gradient gel electrophoresis (DGGE) and real-time polymerase chain reaction (PCR) were used to assess microbiota composition. Intestinal permeability was assessed by in vitro and in vivo techniques. Expressions of epithelial apical junction proteins, mucin, and antimicrobial proteins were assessed by quantitative reverse-transcription PCR (qRT-PCR) and immunofluorescence. Responses to intestinal injury were investigated using an acute experimental model of colitis. RESULTS: Under SPF conditions, Nod1(-/-); Nod2(-/-) mice had increased paracellular permeability, decreased E-cadherin, and lower colonic antimicrobial RegIII-γ expression compared to Nod1(+/-); Nod2(+/-) littermate controls. These changes were associated with increased susceptibility to colitis. ASF colonization or B. breve supplementation normalized RegIII-γ expression and decreased susceptibility to dextran sodium sulfate (DSS) colitis in Nod1(-/-); Nod2(-/-) mice. CONCLUSIONS: The intestinal microbiota influences colitis severity in Nod1(-/-); Nod2(-/-) mice. The results suggest that colonization strategies with defined commensals or exogenous specific probiotic therapy may prevent intestinal inflammation in a genetically predisposed host.