Periodic Reporting for period 4 - ImmunoBile (Bile acid, immune-metabolism, lipid and glucose homeostasis) Reporting period: 2021-03-01 to 2022-02-28 Summary of the context and overall objectives of the project The role of chronic inflammation in obesity, metabolic and cardiovascular diseases is increasingly recognized. Bile acids (BA), synthesized in the liver and modified by the gut flora, facilitate lipid absorption in the intestine acting as soaps. BA also modulate lipid and glucose homeostasis by activating the nuclear receptor FXR and the GPCR TGR5. Peripheral BA concentrations are elevated in type 2 diabetes (T2D) and FXR mediates the beneficial metabolic response to gastric bypass in mice. Since the immune system plays an important role in the cross-talk with metabolic tissues, such as liver, intestine and adipose tissues, we aimed to determine whether BA modulate immune cell function. Our results identify FXR and TGR5 expression in lymphoid cells, which prompted us to study their role in the regulation of glucose and lipid metabolism through immune cell modulation. Using reporter mice and specific ligands, we are characterizing the immune cells expressing active FXR and TGR5. We will determine their role in metabolism and inflammation by immune cell-specific gene inactivation in models of obesity, T2D and non-alcoholic fatty liver disease (NAFLD). Application of mass cytometry, cell sorting and single cell transcriptomic analysis allow the identification of gene networks regulated by BA and their receptors. As microbiota generate biologically active secondary BA, we also assess the impact of microbiota depletion and subsequent BA acid pool modifications on immune cell populations. Translational studies will be performed in humans with altered BA metabolism and pharmacological treatment to assess the impact of alterations in BA metabolism and signaling on immune functions. Our project aims to identify an hitherto unexplored role of BA through modulation of the immune system on T2D, NAFLD and dyslipidemia by applying an integrative approach, possible thanks to our unique multidisciplinary expertise in basic and translational biology. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In a screening for BA receptor expression in immune cells, we found that platelets express FXR. FXR activation with pharmacological ligands inhibits platelet activation upon stimulation of collagen or thrombin receptors, resulting in diminished calcium mobilization, secretion, fibrinogen binding, aggregation. integrin signaling, spreading and ability to stimulate clot retraction. FXR function was associated with modulation of cGMP levels and downstream inhibitory signaling. FXR-deficient platelets were refractory to treatment with FXR activators. The study identifies a role for FXR in platelet activation. FXR may be a target for the prevention of athero-thrombosis (Moraes et al., 2016).- As secondary BA are produced by microbiota which impact on the immune system, we determined whether alterations in microbiota composition and/or function through the administration of “prebiotics” impacts through BA changes on vascular function. In a mouse model of metabolically-induced endothelial dysfunction, inulin-type fructan (ITF) supplementation reversed endothelial dysfunction in arteries via activation of the nitric oxide (NO) synthase/NO pathway. Prebiotic administration increased NO-producing bacteria, replenishned flora with the beneficial bacterium Akkermansia and decreased abundance in taxa involved in secondary BA synthesis. Changes in gut and liver gene expression also occur upon ITF administration suggesting increased GLP-1 production and BA turnover as drivers of endothelial function preservation, emphasizing the key role of BA in the regulation of endothelial function (Catry et al., 2018).Minipig is increasingly used as a model in particular in the field of surgery. We assessed the link between the increase of BA concentrations and the metabolic benefits of bariatric surgery, foremost Roux en Y gastric bypass (RYGB). We determined BA composition in pig, which was found more similar to human, than to mouse BA pool, yet displaying differences, mainly due to enrichment with hyocholic acid species (Spinelli et al., 2016). Systemic total BA concentrations increased after RYGB, due to an increase in conjugated BAs. The ratio of portal:systemic conjugated BAs decreased after RYGB. Our results show that the increase in systemic BAs after surgery is due to decreased selective hepatic recapture. Thus, alterations in hepatic function contribute to the increase in systemic BAs after RYGB (Chavez-Talavera et al., 2017a).To improve BA receptor agonists and antagonists, a medicinal chemistry program was undertaken to identify TGR5 agonists devoid of deleterious systemic on-target effects, but retaining beneficial effects on the entero-endocrine and entero-immune systems. A screen for TGR5 agonists with limited exposure in the intestine was performed by measuring GLP-1 secretion by enteroendocrine L-cells. A potent GLP-1 secretagogue with low effect on gallbladder volume, was identified to improve glucose homeostasis in a murine model of diet-induced obesity and IR (Lasalle et al., 2017).Translational studies were initiated to determine the role of BAs in human metabolism and pathophysiology. To delineate the contribution of BA in the transition from simple steatosis to non-alcoholic steatohepatitis (NASH), we assessed whether BA alterations are associated with NASH independent of body mass and glucose metabolism alterations. Plasma BA concentrations did not correlate with the hepatic NASH lesions. By contrast, primary BAs strongly correlated with insulin resistance. Transcriptomic analyses showed unaltered hepatic BA metabolism in NASH patients. No sign of hepatic BA accumulation or activation of the BA receptor FXR, PXR, and VDR was observed. Plasma FGF-19, secondary-to-primary BA, and free-to-conjugated BA ratios were similar, suggesting unaltered intestinal BA metabolism and signaling. Thus, peripheral plasma BA pool composition is foremost determined by the metabolic status of obese patients rather than liver pathology (Legry et al., 2017).We determined which immune cell types are associated to the transition from steatosis to NASH. Transcriptional profiling of liver biopsies and immune profiling was performed in two cohorts of NASH patients and signatures altered by lifestyle intervention (LSI) identified. Microarray analysis revealed an hepatic gene signature associated with the transition from steatosis to NASH, sensitive to regression of NASH activity upon LSI independent of body weight loss and enriched in immune-associated genes linked to inflammatory responses, antigen presentation and cytotoxic cells. In an independent cohort, NASH was also associated with alterations in blood immune populations, including DC and cytotoxic CD8 T cells. Lobular inflammation and ballooning are associated with the accumulation of liver CD8 T cells. A mouse model of diet-driven NASH demonstrated that progression from steatosis to NASH results in a comparable immune-related hepatic expression signature and the accumulation of intra-hepatic cDC and CD8 T cells (Haas et al., 2019).We also published reviews on BAs, their receptors and modulatory roles on pathophysiology (Chavez-Talavera et al., 2019; Chavez-Talavera et al., 2017b; Trabelsi et al., 2017). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In the first part of the Immunobile program, the required animal models and technologies to assess the role of BA receptors in the control of immune-metabolism have been developed. Using these tools, we are now performing the functional studies in the second part of the program. Project Summary
