Protective and aggressive bacterial subsets and metabolites modify hepatobiliary inflammation and fibrosis in a murine model of PSC.

OBJECTIVE: Conflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. GOAL: define the function of complex resident microbes and their association relevant to PSC patients by studying germ-free (GF) and antibiotic-treated specific pathogen-free (SPF) multidrug-resistant 2 deficient (mdr2-/- ) mice and microbial profiles in PSC patient cohorts. DESIGN: We measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, faecal 16S rRNA gene profiling and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2-/- mice and targeted metagenomic analysis in PSC patients. RESULTS: GF mdr2-/- mice had 100% mortality by 8 weeks with increasing hepatic bile acid (BA) accumulation and cholestasis. Early SPF autologous stool transplantation rescued liver-related mortality. Inhibition of ileal BA transport attenuated antibiotic-accelerated liver disease and decreased total serum and hepatic BAs. Depletion of vancomycin-sensitive microbiota exaggerated hepatobiliary disease. Vancomycin selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased Enterococcus faecalis and Escherichia coli liver translocation. Colonisation of GF mdr2-/- mice with translocated E. faecalis and E. coli strains accelerated hepatobiliary inflammation and mortality. Lachnospiraceae colonisation of antibiotic pretreated mdr2-/- mice reduced liver fibrosis, inflammation and translocation of pathobionts, and SCFA-producing Lachnospiraceae and purified SCFA decreased fibrosis. Faecal Lachnospiraceae negatively associated, and E. faecalis/ Enterobacteriaceae positively associated, with PSC patients' clinical severity by Mayo risk scores. CONCLUSIONS: We identified novel functionally protective and detrimental resident bacterial species in mdr2-/- mice and PSC patients with associated clinical risk score. These insights may guide personalised targeted therapeutic interventions in PSC patients.

The Emulsifier Carboxymethylcellulose Induces More Aggressive Colitis in Humanized Mice with Inflammatory Bowel Disease Microbiota Than Polysorbate-80.

Commonly used synthetic dietary emulsifiers, including carboxymethylcellulose (CMC) and polysorbate-80 (P80), promote intestinal inflammation. We compared abilities of CMC vs. P80 to potentiate colitis and impact human microbiota in an inflammatory environment using a novel colitis model of ex-germ-free (GF) IL10-/- mice colonized by pooled fecal transplant from three patients with active inflammatory bowel diseases. After three days, mice received 1% CMC or P80 in drinking water or water alone for four weeks. Inflammation was quantified by serial fecal lipocalin 2 (Lcn-2) and after four weeks by blinded colonic histologic scores and colonic inflammatory cytokine gene expression. Microbiota profiles in cecal contents were determined by shotgun metagenomic sequencing. CMC treatment significantly increased fecal Lcn-2 levels compared to P80 and water treatment by one week and throughout the experiment. Likewise, CMC treatment increased histologic inflammatory scores and colonic inflammatory cytokine gene expression compared with P80 and water controls. The two emulsifiers differentially affected specific intestinal microbiota. CMC did not impact bacterial composition but significantly decreased Caudoviricetes (bacteriophages), while P80 exposure non-significantly increased the abundance of both Actinobacteria and Proteobacteria. Commonly used dietary emulsifiers have different abilities to induce colitis in humanized mice. CMC promotes more aggressive inflammation without changing bacterial composition.

Targeting the pregnane X receptor using microbial metabolite mimicry.

The human PXR (pregnane X receptor), a master regulator of drug metabolism, has essential roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off-target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows exploiting previously unexplored parts of chemical space. Here, we report functionalized indole derivatives as first-in-class non-cytotoxic PXR agonists as a proof of concept for microbial metabolite mimicry. The lead compound, FKK6 (Felix Kopp Kortagere 6), binds directly to PXR protein in solution, induces PXR-specific target gene expression in cells, human organoids, and mice. FKK6 significantly represses pro-inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to underexploited regions of chemical space.

Manipulating resident microbiota to enhance regulatory immune function to treat inflammatory bowel diseases.

Altered intestinal microbial composition (dysbiosis) and metabolic products activate aggressive mucosal immune responses that mediate inflammatory bowel diseases (IBD). This dysbiosis impairs the function of regulatory immune cells, which normally promote mucosal homeostasis. Normalizing and maintaining regulatory immune cell function by correcting dysbiosis provides a promising approach to treat IBD patients. However, existing microbe-targeted therapies, including antibiotics, prebiotics, probiotics, and fecal microbial transplantation, provide variable outcomes that are not optimal for current clinical application. This review discusses recent progress in understanding the dysbiosis of IBD and the basis for therapeutic restoration of homeostatic immune function by manipulating an individual patient's microbiota composition and function. We believe that identifying more precise therapeutic targets and developing appropriate rapid diagnostic tools will guide more effective and safer microbe-based induction and maintenance treatments for IBD patients that can be applied in a personalized manner.

Phosphatidylinositol 3-kinase-γ signaling promotes Campylobacter jejuni-induced colitis through neutrophil recruitment in mice.

Crypt abscesses caused by excessive neutrophil accumulation are prominent features of human campylobacteriosis and its associated pathology. The molecular and cellular events responsible for this pathological situation are currently unknown. We investigated the contribution of PI3K-γ signaling in Campylobacter jejuni-induced neutrophil accumulation and intestinal inflammation. Germ-free and specific pathogen-free Il10(-/-) and germ-free Il10(-/-);Rag2(-/-) mice were infected with C. jejuni (10(9) CFU/mouse). PI3K-γ signaling was manipulated using either the pharmacological PI3K-γ inhibitor AS252424 (i.p. 10 mg/kg daily) or genetically using Pi3k-γ(-/-) mice. After up to 14 d, inflammation was assessed histologically and by measuring levels of colonic Il1ß, Cxcl2, and Il17a mRNA. Neutrophils were depleted using anti-Gr1 Ab (i.p. 0.5 mg/mouse/every 3 d). Using germ-free Il10(-/-);Rag2(-/-) mice, we observed that innate immune cells are the main cellular compartment responsible for campylobacteriosis. Pharmacological blockade of PI3K-γ signaling diminished C. jejuni-induced intestinal inflammation, neutrophil accumulation, and NF-κB activity, which correlated with reduced Il1ß (77%), Cxcl2 (73%), and Il17a (72%) mRNA accumulation. Moreover, Pi3k-γ(-/-) mice pretreated with anti-IL-10R were resistant to C. jejuni-induced intestinal inflammation compared with Wt mice. This improvement was accompanied by a reduction of C. jejuni translocation into the colon and extraintestinal tissues and by attenuation of neutrophil migratory capacity. Furthermore, neutrophil depletion attenuated C. jejuni-induced crypt abscesses and intestinal inflammation. Our findings indicate that C. jejuni-induced PI3K-γ signaling mediates neutrophil recruitment and intestinal inflammation in Il10(-/-) mice. Selective pharmacological inhibition of PI3K-γ may represent a novel means to alleviate severe cases of campylobacteriosis, especially in antibiotic-resistant strains.