Justin McCarville

Intestinal Microbiota Modulates Gluten-Induced Immunopathology in Humanized Mice

Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. The recent increase in CD incidence suggests that additional environmental factors, such as intestinal microbiota alterations, are involved in its pathogenesis. However, there is no direct evidence of modulation of gluten-induced immunopathology by the microbiota. We investigated whether specific microbiota compositions influence immune responses to gluten in mice expressing the human DQ8 gene, which confers moderate CD genetic susceptibility. Germ-free mice, clean specific-pathogen-free (SPF) mice colonized with a microbiota devoid of opportunistic pathogens and Proteobacteria, and conventional SPF mice that harbor a complex microbiota that includes opportunistic pathogens were used. Clean SPF mice had attenuated responses to gluten compared to germ-free and conventional SPF mice. Germ-free mice developed increased intraepithelial lymphocytes, markers of intraepithelial lymphocyte cytotoxicity, gliadin-specific antibodies, and a proinflammatory gliadin-specific T-cell response. Antibiotic treatment, leading to Proteobacteria expansion, further enhanced gluten-induced immunopathology in conventional SPF mice. Protection against gluten-induced immunopathology in clean SPF mice was reversed after supplementation with a member of the Proteobacteria phylum, an enteroadherent Escherichia coli isolated from a CD patient. The intestinal microbiota can both positively and negatively modulate gluten-induced immunopathology in mice. In subjects with moderate genetic susceptibility, intestinal microbiota changes may be a factor that increases CD risk.

Novel perspectives on therapeutic modulation of the gut microbiota

The gut microbiota contributes to the maintenance of health and, when disrupted, may drive gastrointestinal and extragastrointestinal disease. This can occur through direct pathways such as interaction with the epithelial barrier and mucosal immune system or indirectly via production of metabolites. There is no current curative therapy for chronic inflammatory conditions such as inflammatory bowel disease, which are complex multifactorial disorders involving genetic predisposition, and environmental triggers. Therapies are directed to suppress inflammation rather than the driver, and these approaches are not devoid of adverse effects. Therefore, there is great interest in modulation of the gut microbiota to provide protection from disease. Interventions that modulate the microbiota include diet, probiotics and more recently the emergence of experimental therapies such as fecal microbiota transplant or phage therapy. Emerging data indicate that certain bacteria can induce protective immune responses and enhance intestinal barrier function, which could be potential therapeutic targets. However, mechanistic links and specific therapeutic recommendations are still lacking. Here we provide a pathophysiological overview of potential therapeutic applications of the gut microbiota.

Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease

Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1.

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.

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.

SHP-2 Phosphatase Prevents Colonic Inflammation by Controlling Secretory Cell Differentiation and Maintaining Host-Microbiota Homeostasis.

Polymorphisms in the PTPN11 gene encoding for the tyrosine phosphatase SHP‐2 were described in patients with ulcerative colitis. We have recently demonstrated that mice with an intestinal epithelial cell‐specific deletion of SHP‐2 (SHP‐2IEC‐KO) develop severe colitis 1 month after birth. However, the mechanisms by which SHP‐2 deletion induces colonic inflammation remain to be elucidated. We generated SHP‐2IEC‐KO mice lacking Myd88 exclusively in the intestinal epithelium. The colonic phenotype was histologically analyzed and cell differentiation was determined by electron microscopy and lysozyme or Alcian blue staining. Microbiota composition was analyzed by 16S sequencing. Results show that innate defense genes including those specific to Paneth cells were strongly up‐regulated in SHP‐2‐deficient colons. Expansion of intermediate cells (common progenitors of the Goblet and Paneth cell lineages) was found in the colon of SHP‐2IEC‐KO mice whereas Goblet cell number was clearly diminished. These alterations in Goblet/intermediate cell ratio were noticed 2 weeks after birth, before the onset of inflammation and were associated with significant alterations in microbiota composition. Indeed, an increase in Enterobacteriaceae and a decrease in Firmicutes were observed in the colon of these mice, indicating that dysbiosis also occurred prior to inflammation. Importantly, loss of epithelial Myd88 expression inhibited colitis development in SHP‐2IEC‐KO mice, rescued Goblet/intermediate cell ratio, and prevented NFκB hyperactivation and inflammation. These data indicate that SHP‐2 is functionally important for the maintenance of appropriate barrier function and host‐microbiota homeostasis in the large intestine. J. Cell. Physiol. 231: 2529–2540, 2016.

Pharmacological approaches in celiac disease

Celiac disease is an autoimmune enteropathy triggered by the ingestion of gluten, characterized by immune responses toward gluten constituents and the autoantigen transglutaminase 2. The only current treatment available for celiac disease is a gluten-free diet, however there are a plethora of therapies in development for the treatment of celiac disease (e.g. vaccine), management of symptoms while consuming gluten (e.g. Necator americanus) or adjuvant therapies in conjunction with the gluten-free diet (e.g. larazotide acetate). Current approaches in development target barrier function, immune responses, detoxifying gluten or sequestering gluten. Developing therapies include those targeting environmental factors, such as the microbiota or proteases.

High salt diet exacerbates colitis in mice by decreasing Lactobacillus levels and butyrate production

BackgroundChanges in hygiene and dietary habits, including increased consumption of foods high in fat, simple sugars, and salt that are known to impact the composition and function of the intestinal microbiota, may explain the increase in prevalence of chronic inflammatory diseases. High salt consumption has been shown to worsen autoimmune encephalomyelitis and colitis in mouse models through p38/MAPK signaling pathway. However, the effect of high salt diet (HSD) on gut microbiota and on intestinal immune homeostasis, and their roles in determining vulnerability to intestinal inflammatory stimuli are unknown. Here, we investigate the role of gut microbiota alterations induced by HSD on the severity of murine experimental colitis.ResultsCompared to control diet, HSD altered fecal microbiota composition and function, reducing Lactobacillus sp. relative abundance and butyrate production. Moreover, HSD affected the colonic, and to a lesser extent small intestine mucosal immunity by enhancing the expression of pro-inflammatory genes such as Rac1, Map2k1, Map2k6, Atf2, while suppressing many cytokine and chemokine genes, such as Ccl3, Ccl4, Cxcl2, Cxcr4, Ccr7. Conventionally raised mice fed with HSD developed more severe DSS- (dextran sodium sulfate) and DNBS- (dinitrobenzene sulfonic acid) induced colitis compared to mice on control diet, and this effect was absent in germ-free mice. Transfer experiments into germ-free mice indicated that the HSD-associated microbiota profile is critically dependent on continued exposure to dietary salt.ConclusionsOur results indicate that the exacerbation of colitis induced by HSD is associated with reduction in Lactobacillus sp. and protective short-chain fatty acid production, as well as changes in host immune status. We hypothesize that these changes alter gut immune homeostasis and lead to increased vulnerability to inflammatory insults.

Tu1749 Gluten-Induced Responses in NOD/DQ8 Mice Are Influenced by Bacterial Colonization

Introduction: Disturbances of the intestinal microbiota due to enhanced hygienic conditions or antibiotic use have been linked to the increasing prevalence of obesity, allergy, autoimmunity, and functional and inflammatory disorders of the gut, including celiac disease (CD). It is unknown whether perturbation of normal gut colonization plays a causal role in subsequent development of immune responses to gluten. Thus, our aim was to determine and characterize whether absence of gut commensals modifies the response to gluten using germ-free (GF), specific pathogen free (SPF) and altered Schaedler flora (ASF) colonized NOD/DQ8 mice. Methods: GF, SPF, and ASF 8-10 week old male and female NOD/DQ8 mice were mucosally sensitized with peptic tryptic (PT) gliadin (500μg) and cholera toxin (CT; 25μg) by oral gavage once a week for three weeks. Control mice were sensitized with CT alone. Following sensitization, mice were challenged three times a week for two weeks with 2mg of gluten by oral gavage. All materials for GF experiments were aseptically prepared and administered to mice housed in gnotobiotic isolators. Twenty-four hours following the final gluten challenge enteropathy was evaluated by intraepithelial lymphocyte (IEL) counts and villus/crypt (V/C) ratios. Anti-gliadin IgA antibodies (AGA) in intestinal washes were determined as markers of sensitization and specificity towards gliadin was tested by Western blot. Gliadin-specific T cell responses were tested using in vitro proliferation assays. Results: V/C ratios in GF NOD/DQ8 mice decreased by 52% following sensitization, which was significantly greater than SPF (40%) andASF (38%) gliadin-sensitizedmice (p<0.01). Gliadinsensitized GF mice also had the greatest increase in IELs compared to gliadin-sensitized SPF and ASF mice (p<0.01). No control GF, SPF or ASF mouse had a positive AGA result. In gliadin-sensitized mice, positive AGA was detected in 75% of GF mice, 33% of SPF mice and no ASF mice. The specificity of AGA towards gliadin was confirmed by Western blot. T cells isolated from mesenteric lymph nodes of GF gliadin-sensitized mice responded to PT-gliadin stimulation in vitro, but not to PT-zein stimulation. T cells from non-sensitized mice did not respond to PT-gliadin stimulation. Conclusions: Gluten-induced enteropathy and development of gliadin-specific AGA was enhanced under germ-free conditions in NOD/ DQ8 mice and T cell responses are gliadin-specific. These results suggest that the commensal intestinal microbiota may have a protective effect during gliadin-sensitization and challenge in NOD/DQ8mice. Themechanisms leading to this protective effect are under current investigation.

A Commensal Bifidobacterium longum Strain Prevents Gluten-Related Immunopathology in Mice through Expression of a Serine Protease Inhibitor

ABSTRACT Microbiota-modulating strategies, including probiotic administration, have been tested for the treatment of chronic gastrointestinal diseases despite limited information regarding their mechanisms of action. We previously demonstrated that patients with active celiac disease have decreased duodenal expression of elafin, a human serine protease inhibitor, and supplementation of elafin by a recombinant Lactococcus lactis strain prevents gliadin-induced immunopathology in the NOD/DQ8 mouse model of gluten sensitivity. The commensal probiotic strain Bifidobacterium longum NCC2705 produces a serine protease inhibitor (Srp) that exhibits immune-modulating properties. Here, we demonstrate that B. longum NCC2705, but not a srp knockout mutant, attenuates gliadin-induced immunopathology and impacts intestinal microbial composition in NOD/DQ8 mice. Our results highlight the beneficial effects of a serine protease inhibitor produced by commensal B. longum strains. IMPORTANCE Probiotic therapies have been widely used to treat gastrointestinal disorders with variable success and poor mechanistic insight. Delivery of specific anti-inflammatory molecules has been limited to the use of genetically modified organisms, which has raised some public and regulatory concerns. By examining a specific microbial product naturally expressed by a commensal bacterial strain, we provide insight into a mechanistic basis for the use of B. longum NCC2705 to help treat gluten-related disorders.