Intestinal Microbiota Protects against MCD Diet-Induced Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in western countries, with a continuously rising incidence. Gut-liver communication and microbiota composition have been identified as critical drivers of the NAFLD progression. Hence, it has been shown that microbiota depletion can ameliorate high-fat diet or western-diet induced experimental Non-alcoholic steatohepatitis (NASH). However, its functional implications in the methionine-choline dietary model, remain incompletely understood. Here, we investigated the physiological relevance of gut microbiota in methionine-choline deficient (MCD) diet induced NASH. Experimental liver disease was induced by 8 weeks of MCD feeding in wild-type (WT) mice, either with or without commensal microbiota depletion, by continuous broad-spectrum antibiotic (AB) treatment. MCD diet induced steatohepatitis was accompanied by a reduced gut microbiota diversity, indicating intestinal dysbiosis. MCD treatment prompted macroscopic shortening of the intestine, as well as intestinal villi in histology. However, gut microbiota composition of MCD-treated mice, neither resembled human NASH, nor did it augment the intestinal barrier integrity or intestinal inflammation. In the MCD model, AB treatment resulted in increased steatohepatitis activity, compared to microbiota proficient control mice. This phenotype was driven by pronounced neutrophil infiltration, while AB treatment only slightly increased monocyte-derived macrophages (MoMF) abundance. Our data demonstrated the differential role of gut microbiota, during steatohepatitis development. In the context of MCD induced steatohepatitis, commensal microbiota was found to be hepatoprotective.

Anti-food and anti-microbial IgG subclass antibodies in inflammatory bowel disease.

OBJECTIVES: Inflammatory bowel disease (IBD), particularly Crohn's disease (CD), is associated with increased microbial-specific IgG and IgA antibodies, whereas alterations of anti-food antibodies are still disputed. The knowledge about IgG subclass antibodies in IBD is limited. In this study we analysed IgG subclass antibodies specific for nutritional and commensal antigens in IBD patients and controls. METHODS: Serum IgG1, IgG2, IgG3 and IgG4 specific for wheat and milk extracts, purified ovalbumin, Escherichia coli and Bacteroides fragilis lysates and mannan from Saccharomyces cerevisiae were analysed by ELISA in patients with CD (n = 56), ulcerative colitis (UC; n = 29), acute gastroenteritis/colitis (n = 12) as well as non-inflammatory controls (n = 62). RESULTS: Anti-Saccharomyces cerevisiae antibodies (ASCA) of all IgG subclasses and anti-B. fragilis IgG1 levels were increased in CD patients compared to UC patients and controls. The discriminant validity of ASCA IgG2 and IgG4 was comparable with that of ASCA pan-IgG and IgA, whereas it was inferior for ASCA IgG1/IgG3 and anti-B. fragilis IgG1. Complicated CD defined by the presence of perianal, stricturing or penetrating disease phenotypes was associated with increased ASCA IgG1/IgG3/IgG4, anti-B. fragilis IgG1 and anti-E. coli IgG1 levels. Anti-food IgG subclass levels were not different between IBD patients and controls and did not correlate with food intolerance. In contrast to anti-microbial Abs, food-specific IgG responses were predominately of the IgG4 isotype and all food-specific IgG subclass levels correlated negatively with age. CONCLUSION: Our study supports the notion that the adaptive immune recognition of food and commensal antigens are differentially regulated.

Subcellular antigen localization in commensal E. coli is critical for T cell activation and induction of specific tolerance.

Oral tolerance to soluble antigens is critically important for the maintenance of immunological homeostasis in the gut. The mechanisms of tolerance induction to antigens of the gut microbiota are still less well understood. Here, we investigate whether the subcellular localization of antigens within non-pathogenic E. coli has a role for its ability to induce antigen-specific tolerance. E. coli that express an ovalbumin (OVA) peptide in the cytoplasm, at the outer membrane or as secreted protein were generated. Intestinal colonization of mice with non-pathogenic E. coli expressing OVA at the membrane induced the expansion of antigen-specific Foxp3+ Tregs and mediated systemic immune tolerance. In contrast, cytoplasmic OVA was ignored by antigen-specific CD4+ T cells and failed to induce tolerance. In vitro experiments revealed that surface-displayed OVA of viable E. coli was about two times of magnitude more efficient to activate antigen-specific CD4+ T cells than soluble antigens, surface-displayed antigens of heat-killed E. coli or cytoplasmic antigen of viable or heat-killed E. coli. This effect was independent of the antigen uptake efficiency in dendritic cells. In summary, our results show that subcellular antigen localization in viable E. coli strongly influences antigen-specific CD4+ cell expansion and tolerance induction upon intestinal colonization.

Distinct patterns of IgG and IgA against food and microbial antigens in serum and feces of patients with inflammatory bowel diseases.

BACKGROUND: Inflammatory bowel disease (IBD) is associated with a defective intestinal barrier and enhanced adaptive immune responses against commensal microbiota. Immune responses against food antigens in IBD patients remain poorly defined. METHODS: IgG and IgA specific for food and microfloral antigens (wheat and milk extracts; purified ovalbumin; Escherichia coli and Bacteroides fragilis lysates; mannan from Saccharomyces cerevisiae) were analyzed by ELISA in the serum and feces of patients with Crohn's disease (CD; n = 52 for serum and n = 20 for feces), ulcerative colitis (UC; n = 29; n = 17), acute gastroenteritis/colitis (AGE; n = 12; n = 9) as well as non-inflammatory controls (n = 61; n = 39). RESULTS: Serum anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-B. fragilis IgG and IgA levels were increased in CD patients whereas antibody (Ab) levels against E. coli and food antigens were not significantly different within the patient groups and controls. Subgroup analysis revealed that CD patients with severe diseases defined by stricturing and penetrating lesions have slightly higher anti-food and anti-microbial IgA levels whereas CD and UC patients with arthropathy have decreased anti-food IgG levels. Treatment with anti-TNF-α Abs in CD patients was associated with significantly decreased ASCA IgG and IgA and anti-E. coli IgG. In the feces specific IgG levels against all antigens were higher in CD and AGE patients while specific IgA levels were higher in non-IBD patients. Anti-food IgG and IgA levels did not correlate with food intolerance. SUMMARY: In contrast to anti-microbial Abs, we found only minor changes in serum anti-food Ab levels in specific subgroups of IBD patients. Fecal Ab levels towards microbial and food antigens show distinct patterns in controls, CD and UC patients.

TRIM27 negatively regulates NOD2 by ubiquitination and proteasomal degradation.

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohn's disease. We found that TRIM27 expression is increased in Crohn's disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus.We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.