Clostridium ramosum regulates enterochromaffin cell development and serotonin release.

Peripheral serotonin (5-hydroxytryptamine: 5-HT) synthesized in the intestine by enterochromaffin cells (ECs), plays an important role in the regulation of peristaltic of the gut, epithelial secretion and promotes the development and maintenance of the enteric neurons. Recent studies showed that the indigenous gut microbiota modulates 5-HT signalling and that ECs use sensory receptors to detect dietary and microbiota-derived signals from the lumen to subsequently transduce the information to the nervous system. We hypothesized that Clostridium ramosum by increasing gut 5-HT availability consequently contributes to high-fat diet-induced obesity. Using germ-free mice and mice monoassociated with C. ramosum, intestinal cell lines and mouse organoids, we demonstrated that bacterial cell components stimulate host 5-HT secretion and program the differentiation of colonic intestinal stem progenitors toward the secretory 5-HT-producing lineage. An elevated 5-HT level regulates the expression of major proteins involved in intestinal fatty acid absorption in vitro, suggesting that the presence of C. ramosum in the gut promotes 5-HT secretion and thereby could facilitates intestinal lipid absorption and the development of obesity.

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.

Do we choose control diets wisely?

In a recent article in Cell Reports, Dalby and colleagues convincingly demonstrate that choosing an inadequate control diet in animal experiments that investigate the interaction of nutrition, gut microbiota, and obesity development may lead to the wrong conclusions. The authors systematically compared the effects of refined high- and low-fat diets (rHFD and rLFD) with those of a standard chow diet on mouse physiology, microbiota composition, cecal fermentation, and intestinal morphology. The results obtained in this study question the conclusions drawn from animal studies that compared the effects of HFDs with those of chow diets.

Glycan-Functionalized Microgels for Scavenging and Specific Binding of Lectins.

Lectins are proteins with a well-defined carbohydrate recognition domain. Many microbial proteins such as bacterial toxins possess lectin or lectin-like binding domains to interact with cell membranes that are decorated with glycan recognition motifs. We report a straightforward way to prepare monodisperse and biocompatible polyethylene glycol microgels, which carry glycan motifs for specific binding to lectins. The sugar-functionalized colloids exhibit a wide mesh size and a highly accessible volume. The microgels are prepared via drop-based microfluidics combined with radical polymerization. GSII and ECL are used as model lectins that bind specifically to the corresponding carbohydrates, namely, GlcNAc and LacNAc. LacNAc microgels bind ECL with a high capacity and high affinity (Kd ≈ 0.5 to 1 µM), suggesting multivalent binding of the lectin to the LacNAc-decorated flexible microgel network. Glycan-functionalized microgels present a useful tool for lectin scavenging in biomedical applications.

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.

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.

ARC is a novel therapeutic approach against acetaminophen-induced hepatocellular necrosis.

BACKGROUND & AIMS: Acetaminophen (AAP) overdose is the most frequent cause of drug-induced liver failure. c-Jun N-terminal kinase (JNK) is thought to play a central role in AAP-induced hepatocellular necrosis. The apoptosis repressor with caspase recruitment domain (ARC) is a death repressor that inhibits death receptor and mitochondrial apoptotic signaling. Here, we investigated ARC's therapeutic effect and molecular mechanisms on AAP-induced hepatocellular necrosis. METHODS: We tested the in vivo and in vitro effects of ARC fused with the transduction domain of HIV-1 (TAT-ARC) on murine AAP hepatotoxicity. RESULTS: Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by AAP overdose in C57BL/6 mice. AAP triggered caspase-independent necrosis, as evidenced by liver histology, elevated serum transaminases, and secreted HMGB1 that was inhibited by ARC. ARC-mediated hepatoprotection was not caused by an alteration of AAP metabolism, but resulted in reduced oxidative stress. AAP overdose led to induction of RIP-dependent signaling with subsequent JNK activation. Ectopic ARC inhibited JNK activation by specific interactions between ARC and JNK1 and JNK2. Importantly, survival of mice was even preserved when ARC therapy was initiated in a delayed manner after AAP administration. CONCLUSIONS: This work identifies for the first time ARC-JNK-binding with subsequent inhibition of JNK signaling as a specific mechanism of ARC to interfere with AAP-dependent necrosis. Our data suggests that AAP-mediated induction of RIP signaling serves as a critical switch for hepatocellular necrosis. The efficacy of TAT-ARC protein transduction in murine AAP hepatotoxicity suggests its therapeutic potential for reversing AAP intoxication also in humans.

TAT-apoptosis repressor with caspase recruitment domain protein transduction rescues mice from fulminant liver failure.

UNLABELLED: Acute liver failure (ALF) is associated with massive hepatocyte cell death and high mortality rates. Therapeutic approaches targeting hepatocyte injury in ALF are hampered by the activation of distinct stimulus-dependent pathways, mechanism of cell death, and a limited therapeutic window. The apoptosis repressor with caspase recruitment domain (ARC) is a recently discovered death repressor that inhibits both death receptor and mitochondrial apoptotic signaling. Here, we investigated the in vivo effects of ARC fused with the transduction domain of human immunodeficiency virus 1 (HIV-1) (TAT-ARC) on Fas- and tumor necrosis factor (TNF)-mediated murine models of fulminant liver failure. Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by Fas-agonistic antibody (Jo2), concanavalin A (ConA), or D-galactosamine/lipopolysaccharide (GalN/LPS) administration. Importantly, survival of mice was even preserved when TAT-ARC therapy was initiated in a delayed manner after stimulation with Jo2, ConA, or GalN/LPS. ARC blocked hepatocyte apoptosis by directly interacting with members of the death-inducing signaling complex. TNF-mediated liver damage was inhibited by two independent mechanisms: inhibition of jun kinase (JNK)-mediated TNF-α expression and prevention of hepatocyte apoptosis by inhibition of both death receptor and mitochondrial death signaling. We identified JNK as a novel target of ARC. ARC's caspase recruitment domain (CARD) directly interacts with JNK1 and JNK2, which correlates with decreased JNK activation and JNK-dependent TNF-α production. CONCLUSION: This work suggests that ARC confers hepatoprotection upstream and at the hepatocyte level. The efficacy of TAT-ARC protein transduction in multiple murine models of ALF demonstrates its therapeutic potential for reversing liver failure.