A detailed screen of BA receptor expression in immune cells identified platelets, produced from haematopoietic megakaryocytes, to express the BA FXR. Pharmacological studies revealed that FXR activation inhibits platelet activation in response to collagen or thrombin receptor stimulation, resulting in decreased Ca2+ signalling, fibrinogen binding, aggregation, integrin signalling, spreading and stimulation of clot retraction. Platelets from FXR-deficient (KO) mice were refractory to treatment with FXR activators. Thus, FXR may be a potential target for the prevention of atherothrombotic disease 1. As microbiota, which produce secondary BA, have an impact on the immune system, we investigated whether changes in the composition and/or function of microbiota by administration of "prebiotics" have an impact on vascular function through changes in BA. In a model of metabolically induced endothelial dysfunction (n-3 PUFA-depleted ApoE-KO mice), inulin-like fructan (ITF) supplementation reversed endothelial dysfunction in the mesenteric and carotid arteries through activation of the nitric oxide (NO) synthesis pathway. Prebiotic administration increased NO-producing bacteria, reconstituted the flora with the beneficial bacterium Akkermansia and decreased the abundance of taxa involved in secondary BA synthesis. Changes in gut and liver gene expression occur upon ITF administration, suggesting an increase in GLP-1 production and BA turnover as drivers of endothelial function preservation, highlighting the key role of BA in the regulation of endothelial function 2. The minipig is increasingly used as an animal model, especially in surgery. Furthermore, its BA pool is more similar to that of humans than rodents, being particularly enriched in hyocholic acid 3. This model was therefore used to link increased BA concentrations to the metabolic benefits of bariatric surgery, particularly Roux Y gastric bypass (RYGB). Total systemic BA concentrations increased after RYGB, due to an increase in conjugated BA. The ratio of portal to systemic conjugated BAs decreased after RYGB, indicating alterations in liver function in the changes in systemic BA concentrations during RYGB surgery due to a decrease in hepatic selective reuptake. Thus, alterations in liver function contribute to the increase in systemic BA after RYGB 4.

 

To improve the available range of BA agonists and antagonists, a medicinal chemistry programme was undertaken to identify TGR5 agonists that lacked deleterious systemic effects, but retained beneficial effects on the enteroendocrine and enteroimmune systems. A screening of TGR5 agonists with exposure primarily in the gut as measured by GLP-1 secretion from enteroendocrine L-cells identified a potent secretagogue with a small effect on gallbladder volume, but improving metabolic homeostasis in a mouse model of diet-induced obesity and insulin resistance (IR) 5. This topical intestinal TGR5 agonist is now being tested for its potential actions on the immunoinflammatory system as a tool and potential therapeutic agent. Several translational studies have been initiated to determine the role of BAs in human metabolism and pathophysiology. To delineate the contribution of BAs in the transition to steatohepatitis (NASH) in non-alcoholic fatty liver disease, we investigated whether alterations in BAs are associated with NASH independently of body weight and alterations in glucose metabolism. Plasma BA concentrations did not correlate with liver damage in NASH. In contrast, primary BA were strongly correlated with IR. Transcriptomic analyses showed that hepatic metabolism of BA was not impaired in NASH patients. Finally, the plasma FGF-19, secondary BA to primary BA and free BA to conjugated BA ratios were similar, suggesting unaltered intestinal BA metabolism and signalling. Thus, the composition of the peripheral plasma BA pool is primarily determined by the metabolic state of obese patients rather than by liver pathology.6 Further studies are underway to determine how BA signalling through its receptors modulates NASH and fibrosis and whether these actions involve direct regulation of the immune-inflammatory system in the liver. A translational study aims to determine which immune cell types are associated with the transition from simple steatosis to NASH. Transcriptional profiling of liver biopsies together with detailed immune profiling was performed in two cohorts of NASH patients and the signatures altered by lifestyle intervention (LSI) were identified. Analysis of liver biopsies revealed a liver gene signature associated with the transition from steatosis to NASH, sensitive to regression of NASH activity on LSI, independent of body weight loss and enriched in immune-related genes linked to inflammatory responses, antigen presentation and cytotoxic cells. In an independent cohort, NASH was associated with alterations in blood immune cell populations, including classical dendritic cells (cDC) type 1 and 2, and cytotoxic CD8 T cells. Lobular inflammation and bloating are associated with the accumulation of CD8 T cells in the liver. A diet-driven mouse model of NASH has been developed and used to demonstrate that progression from simple steatosis to NASH results in a comparable hepatic immune expression signature and accumulation of cDCs and intrahepatic CD8 T cells 7. This translational model will be used to investigate the role of BA signalling on these NASH-associated immune populations, which express FXR and TRG5. We have also published 3 reviews on BAs, their receptors and their modulatory roles on pathophysiology 8-10.

New and/or unconventional methods
 
- The 7 original papers described above all contain novel elements, particularly with regard to measurements of BA levels or correlations between immune phenotypes and transcriptomic signatures in NASH patients
- To precisely delineate BA receptor expression of (immune) cell types, reporter mice for FXR and TGR5 were generated by inserting an IRES sequence and the genes encoding the yellow fluorescent protein Profein and tdTomato downstream of the stop codon of FXR and TGR5, respectively, such that the expression of YFP and tdTomato is driven by the endogenous promoters of FXR and TGR5, respectively. YFP and tdTomato will therefore be used as surrogate markers for receptor expression. In addition, the linker-attached OST (One STrEP Tag) and HA (haemagglutinin) tags were added before the FXR and TGR5 termination codons respectively, allowing purification of FXR- or TGR5-containing protein complexes from cell lysates using high-affinity antibodies. This approach circumvents the use of poor quality antibodies against FXR and TGR5. Homozygous FXR and TGR5 reporter mice have been obtained and the colonies are currently expanding. The first analysis of the FXR reporter mice is planned for June 2019.
- The composition of the BA pool differs significantly between mice and humans, with the presence of species-specific BAs exerting distinct activities on FXR and/or TGR5, due to the activity of CYP2C70 in the synthesis of mouse-specific muricholic acid species. Indeed, CYP2C70-deficient mice exhibit a "humanised" BA pool (F.Kuipers, personal communication). Therefore, the FXR and TGR5 reporter mouse lines will be crossed with CYP2C70-/- animals and the expression of the respective receptors will be assessed in these animals at homeostasis and on different metabolic and inflammatory challenges.
- Both FXRfl/fl and TGRfl/fl lines have been obtained and the former are currently being crossed with Mb1/CD79a-Cre trangenic mice to induce a specific FXR deletion in B cells. The impact of the B-cell specific FXR deletion will be evaluated on the susceptibility to develop immunometabolic diseases in established models of diet-induced obesity/T2D as well as diet-induced NASH.
- In addition, a novel protocol to simultaneously sort 4 different liver cell types, namely hepatocytes, Kupffer cells, sinusoidal endothelial cells and stellate cells, has been developed, allowing RNAseq analysis on purified cell populations. The impact of FXR deletion in a specific (immune) cell type on the immunometabolic response to diet-induced obesity and NASH will be assessed in further experiments. Quality control data for RNAseq experiments on these purified populations were obtained. Flow and mass cytometry immunoprobing panels were set up for liver, adipose tissue and intestine to analyse both FXR and TGR5 reporter mice as well as FXRMb1/Mb1 animals. In conclusion, in the first part of the Immunobile programme, the animal models and technologies necessary to assess the role of BA receptors in the control of immune metabolism have been developed. Using these tools, we are now carrying out the functional studies in the second part of the programme.

Interdisciplinary and cross-cutting developments

Due to its fundamental design, the whole project is interdisciplinary between endocrinology and metabolism on the one hand and immunology on the other. Some aspects are translated with data obtained in human cohorts.

 

The three most significant achievements:

- Legry V., Francque S., Haas J.T., Verrijken A., Caron S., Chavez-Talavera O., Vallez E., Vonghia L., Dirinck E., Verhaegen A.,Kouach M., Lestavel S., Lefebvre P., Van Gaal L., Tailleux A., Paumelle R., Staels B.: Bile acid alterations are associated with insulin resistance, but not with NASH in obese subjects. J. Clin. Endocrinol. Metab. 2017; 102, 3783

- Haas, J.T., Vonghia, L., Mogilenko, D., Verrijken, A., Fleury, S., Deprince, A., Nikitin, A., Woitrain, E., Durocq-Geoffroy, L., Pic, S., et al. (2019). Presence and activity of NASH correlate with a hepatic immune-related transcriptomic signature and increased cytotoxic CD8 T lymphocytes. Nat Metab in press

- Lasalle, M., Hoguet, V., Hennuyer, N., Leroux, F., Piveteau, C., Belloy, L., Lestavel, S., Vallez, E., Dorchies, E., Duplan, I., et al. (2017). Topical Intestinal Aminoimidazole Agonists of G-Protein-Coupled Bile Acid Receptor 1 Promote Glucagon Like Peptide-1 Secretion and Improve Glucose Tolerance. J Med Chem 60, 4185

 Dissemination and results

 

Publications

 

1             Moraes, L. A. et al. Farnesoid X Receptor and Its Ligands Inhibit the Function of Platelets. Arterioscler Thromb Vasc Biol 36, 2324-2333, doi:10.1161/ATVBAHA.116.308093 (2016).

2             Catry, E. et al. Targeting the gut microbiota with inulin-type fructans: preclinical demonstration of a novel approach in the management of endothelial dysfunction. Gut 67, 271-283, doi:10.1136/gutjnl-2016-313316 (2018).

3             Spinelli, V. et al. Influence of Roux-en-Y gastric bypass on plasma bile acid profiles: a comparative study between rats, pigs and humans. Int J Obes (Lond) 40, 1260-1267, doi:10.1038/ijo.2016.46 (2016).

4             Chavez-Talavera, O. et al. Roux-en-Y gastric bypass increases systemic but not portal bile acid concentrations by decreasing hepatic bile acid uptake in minipigs. Int J Obes (Lond) 41, 664-668, doi:10.1038/ijo.2017.7 (2017).

5             Lasalle, M. et al. Topical Intestinal Aminoimidazole Agonists of G-Protein-Coupled Bile Acid Receptor 1 Promote Glucagon Like Peptide-1 Secretion and Improve Glucose Tolerance. J Med Chem 60, 4185-4211, doi:10.1021/acs.jmedchem.6b01873 (2017).

6             Legry, V. et al. Bile Acid Alterations Are Associated With Insulin Resistance, but Not With NASH, in Obese Subjects. J Clin Endocrinol Metab 102, 3783-3794, doi:10.1210/jc.2017-01397 (2017).

7             Haas, J. T. et al. Presence and activity of NASH correlate with a hepatic immune-related transcriptomic signature and increased cytotoxic CD8 T lymphocytes. Nat Metab in press (2019).

8             Trabelsi, M. S., Lestavel, S., Staels, B. & Collet, X. Intestinal bile acid receptors are key regulators of glucose homeostasis. Proc Nutr Soc 76, 192-202, doi:10.1017/S0029665116002834 (2017).

9             Chavez-Talavera, O., Tailleux, A., Lefebvre, P. & Staels, B. Bile acid control of metabolism and inflammation in obesity, type 2 diabetes, dyslipidemia and NAFLD. Gastroenterology, doi:10.1053/j.gastro.2017.01.055 (2017).

10           Chavez-Talavera, O., Haas, J., Grzych, G., Tailleux, A. & Staels, B. Bile acid alterations in nonalcoholic fatty liver disease, obesity, insulin resistance and type 2 diabetes: what do the human studies tell? Curr Opin Lipidol 30, 244-254, doi:10.1097/MOL.0000000000000597 (2019).

 

Keywords:


Bile acids, Nuclear receptors, Immune cells, Metabolism