Microbiota-driven gut vascular barrier disruption is a prerequisite for non-alcoholic steatohepatitis development.

BACKGROUND & AIMS Fatty liver disease, including non-alcoholic fatty liver (NAFLD) and steatohepatitis (NASH), has been associated with increased intestinal barrier permeability and translocation of bacteria or bacterial products into the blood circulation. In this study, we aimed to unravel the role of both intestinal barrier integrity and microbiota in NAFLD/NASH development. METHODS C57BL/6J mice were fed with high-fat diet (HFD) or methionine-choline-deficient diet for 1 week or longer to recapitulate aspects of NASH (steatosis, inflammation, insulin resistance). Genetic and pharmacological strategies were then used to modulate intestinal barrier integrity. RESULTS We show that disruption of the intestinal epithelial barrier and gut vascular barrier (GVB) are early events in NASH pathogenesis. Mice fed HFD for only 1 week undergo a diet-induced dysbiosis that drives GVB damage and bacterial translocation into the liver. Fecal microbiota transplantation from HFD-fed mice into specific pathogen-free recipients induces GVB damage and epididymal adipose tissue enlargement. GVB disruption depends on interference with the WNT/β-catenin signaling pathway, as shown by genetic intervention driving β-catenin activation only in endothelial cells, preventing GVB disruption and NASH development. The bile acid analogue and farnesoid X receptor agonist obeticholic acid (OCA) drives β-catenin activation in endothelial cells. Accordingly, pharmacologic intervention with OCA protects against GVB disruption, both as a preventive and therapeutic agent. Importantly, we found upregulation of the GVB leakage marker in the colon of patients with NASH. CONCLUSIONS We have identified a new player in NASH development, the GVB, whose damage leads to bacteria or bacterial product translocation into the blood circulation. Treatment aimed at restoring β-catenin activation in endothelial cells, such as administration of OCA, protects against GVB damage and NASH development. LAY SUMMARY The incidence of fatty liver disease is reaching epidemic levels in the USA, with more than 30% of adults having NAFLD (non-alcoholic fatty liver disease), which can progress to more severe non-alcoholic steatohepatitis (NASH). Herein, we show that disruption of the intestinal epithelial barrier and gut vascular barrier are early events in the development of NASH. We show that the drug obeticholic acid protects against barrier disruption and thereby prevents the development of NASH, providing further evidence for its use in the prevention or treatment of NASH

Interactions between Epithelial Cells and Dendritic Cells in Bacterial Handling

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<i>Lactobacillus paracasei</i> CBA L74 Metabolic Products and Fermented Milk for Infant Formula Have Anti-Inflammatory Activity on Dendritic Cells <i>In Vitro</i> and Protective Effects against Colitis and an Enteric Pathogen <i>In Vivo</i>

<div><p>The rapid expansion of commercially available fermented food products raises important safety issues particularly when infant food is concerned. In many cases, the activity of the microorganisms used for fermentation as well as what will be the immunological outcome of fermented food intake is not known. In this manuscript we used complex <i>in vitro</i>, <i>ex-vivo</i> and <i>in vivo</i> systems to study the immunomodulatory properties of probiotic-fermented products (culture supernatant and fermented milk without live bacteria to be used in infant formula).</p><p>We found <i>in vitro</i> and <i>ex-vivo</i> that fermented products of <i>Lactobacillus paracasei</i> CBA L74 act via the inhibition of proinflammatory cytokine release leaving anti-inflammatory cytokines either unaffected or even increased in response to <i>Salmonella</i> typhimurium. These activities are not dependent on the inactivated bacteria but to metabolic products released during the fermentation process. We also show that our <i>in vitro</i> systems are predictive of an <i>in vivo</i> efficacy by the fermented products. Indeed CBA L74 fermented products (both culture medium and fermented milk) could protect against colitis and against an enteric pathogen infection (<i>Salmonella</i> typhimurium). Hence we found that fermented products can act via the inhibition of immune cell inflammation and can protect the host from pathobionts and enteric pathogens. These results open new perspectives in infant nutrition and suggest that <i>L. paracasei</i> CBA L74 fermented formula can provide immune benefits to formula-fed infants, without carrying live bacteria that may be potentially dangerous to an immature infant immune system.</p></div

<i>Lactobacillus paracaseii</i> CBA L74 supernatant has anti-inflammatory properties on MoDCs.

<p>MoDCs were infected with either <i>Salmonella</i> SL1344 (MOI 10∶1) or <i>Lactobacillus</i> CBA L74 (MOI 10∶1) for 1 hour in medium without antibiotics or in the presence of 2% LB, MRS, FB62 supernatant (Sn FB62) or CBA L74 supernatant in MRS (Sn CBA L74). After washing and 24 h incubations in medium with antibiotics cytokine abundance was evaluated. A. Concentrations of IL-12p70 and IL-10 as determined by ELISA and CBA assays respectively. B. The % of response relative to FB62 infected MoDCs is shown after grouping six experiments together. C. Anti-inflammatory index calculated as ratio of IL-10/IL12p70 concentrations. * p<0,05; *** p<0,001.</p

CBA L74 fermented milk reduces inflammatory mediators expression.

<p>At the end of the experiment described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087615#pone-0087615-g005" target="_blank">Figure 5</a> RNA was extracted from colons and expression levels of Tbet, Gata3, Inf-γ, Il-17a, Il-6, Il-22, Il-33, Il-1β, Kc, Ccl2, Cox2, iNos, Ahr, Indo, Zo-1, Reg3β, Reg3γ were assessed by RT-qPCR. Expression levels are normalized to the housekeeping mRNA Ralp32. Dots represent individual mice measurements, lines represent the average value.</p

Oral administration of fermented milk preparation protects against colitis.

<p>Mice (n = 5) were orally administered on a daily basis for 14 consecutive days (days −2 to 11) with vehicle (water), non-fermented milk (NFM) or fermented milk (FM). Acute colitis was induced by dissolving 3% DSS in drinking water for 9 days (days 0–9). A, Single mouse body weight curves, Kaplan-Meier survival and colon length for vehicle, NFM and FM. B. Body weight curves (mean ± SEM) for two pooled experiments. * p<0,05; ** p<0,01.</p

Fermented milk preparation has anti-inflammatory properties on MoDCs.

<p>MoDCs were infected with <i>Salmonella</i> FB62 (MOI 10:1) for 1 hour in medium without antibiotics or in different infant formulas: non-fermented milk (NFM) and fermented milk (FM) obtained through fermentation of Lactobacillus CBA L74 alone (CBA L74), fermentation in the presence <i>S. thermophilus</i> TH3 (S. th) or double fermentation with CBA L74 and <i>S. thermophilus</i> TH3 (CBA L74+S. th). After washing and 24 h incubations in medium with antibiotics cytokine abundance was evaluated. Panels show % of response of IL-12p70 and IL-10 relative to MoDCs infected with SL1334 in medium without antibiotics. *** p<0,001.</p

IL10 Secretion Endows Intestinal Human iNKT Cells with Regulatory Functions Towards Pathogenic T Lymphocytes

Background and aims: Invariant natural killer T [iNKT] cells perform pleiotropic functions in different tissues by secreting a vast array of pro-inflammatory and cytotoxic molecules. However, the presence and function of human intestinal iNKT cells capable of secreting immunomodulatory molecules such as IL-10 has never been reported so far. Here we describe for the first time the presence of IL10-producing iNKT cells [NKT10 cells] in the intestinal lamina propria of healthy individuals and of Crohn's disease [CD] patients. Methods: Frequency and phenotype of NKT10 cells were analysed ex vivo from intestinal specimens of Crohn's disease [n = 17] and controls [n = 7]. Stable CD-derived intestinal NKT10 cell lines were used to perform in vitro suppression assays and co-cultures with patient-derived mucosa-associated microbiota. Experimental colitis models were performed by adoptive cell transfer of splenic naïve CD4+ T cells in the presence or absence of IL10-sufficient or -deficient iNKT cells. In vivo induction of NKT10 cells was performed by administration of short chain fatty acids [SCFA] by oral gavage. Results: Patient-derived intestinal NKT10 cells demonstrated suppressive capabilities towards pathogenic CD4+ T cells. The presence of increased proportions of mucosal NKT10 cells associated with better clinical outcomes in CD patients. Moreover, an intestinal microbial community enriched in SCFA-producing bacteria sustained the production of IL10 by iNKT cells. Finally, IL10-deficient iNKT cells failed to control the pathogenic activity of adoptively transferred CD4+ T cells in an experimental colitis model. Conclusions: These results describe an unprecedentd IL10-mediated immunoregulatory role of intestinal iNKT cells in controlling the pathogenic functions of mucosal T helper subsets and in maintaining the intestinal immune homeostasis