2021-2022: RESEARCH SUBJECTS OF MASTER 2 BIOLOGY-HEALTH COURSE PRECISION HEALTH UMR1011 – 2021-2022

Team 1

Role of the intestinal epithelial nuclear receptor FXR in the complication of non-alcoholic fatty liver disease in type 2 diabetes

Supervisor : Sophie LESTAVEL

UMR 1011 INSERM (Dir. Bart STAELS) - Laboratoire J&K, Pôle Recherche Faculté de Médecine, Boulevard du Pr Jules Leclercq 59000 Lille

Email : sophie.lestavel@univ-lille.fr

Téléphone : +33 3 20 97 42 13

The importance of the gut and its interaction with the liver in the development of type 2 diabetes (T2D) and non-alcoholic fatty diseases (NAFLD) is receiving increasing attention. In particular, deregulation of intestinal endocrine and immune functions plays a central role in metabolic diseases and disruption of intestinal permeability aggravates T2D and NAFLD. FXR is a nuclear receptor expressed in metabolic tissues such as the intestine, liver, adipose tissue and pancreas. FXR regulates energy metabolism via modulation of bile acid synthesis and lipid and glucose metabolism. Our preliminary results show that FXR deficiency limited to intestinal epithelial cells has an impact on the intestinal immune system, even when mice are fed a normal diet.

We subjected C57Bl/6 mice deficient in FXR only in their intestinal epithelial cells (intFXR KO mice) and their control littermates to a control or NAFLD-inducing diet for 24 weeks.

The intFXR KO mice and their control congeners will be characterised on the chow and NAFLD-inducing diets with respect to the progression of liver pathology (body weight monitoring, plasma ASAT/ALAT by ELISA, qPCR and liver histology) in relation to intestinal health (intestinal histology and immunofluorescence) and intestinal permeability (serum IgA by ELISA). The study of the intestinal immune system and immune cell recruitment will be performed in the small intestine and colon (bioinformatics and statistical analysis of immunophenotyping data generated during sacrifice, qPCR and immunofluorescence).

This programme should strengthen the preclinical proof of concept of FXR as a potential pharmacological target for the treatment of diabetes and its complication NAFLD.

The nuclear receptor Rev-Erbα: a new player in intestinal dietary lipid metabolism?

Supervisor: Olivier BRIAND - INSERM U1011 - EGID

Laboratoire JK, Pôle recherche Faculté de médecine, boulevard du Pr Jules Leclercq 59 Lille

Téléphone : +33 3 20 97 42 11 - olivier.briand@univ-lille.fr

Obesity and diabetes are multifactorial chronic diseases whose aetiology is an imbalance between energy intake and expenditure. Significant abnormalities in gut function, including overproduction of triglyceride-rich lipoproteins, accompany these metabolic disorders and contribute to the development of atherosclerosis and fatty liver disease (NAFLD). The nuclear receptor Rev-Erbα integrates the biological clock with metabolism in major organs. Based on our current work, we hypothesise that Rev-Erbα in the gut is a key molecular player in the orchestration of dietary lipid metabolism, as well as in the control of lipoprotein production.

This M2 project focuses on the study, in ex vivo organoid (or mini-intestine) models of human and murine origin, of the mechanisms involved in the control by Rev-Erbα of the intestinal postprandial lipid response. The inhibition in enteroids of the expression of candidate target genes, coupled with the use of inhibitors of biological processes (lipophagy, vesicular trafficking...) will allow to elucidate the molecular mechanisms. The link with the clinic will be achieved through the use of pharmacological modulators of Rev-Erbα.

The approaches used are based on cell and molecular biology techniques (analysis of gene and protein expression, indirect immunofluorescence, gene invalidation and overexpression, etc.). This project is based on extensive work in cell culture and image analysis.

Characterization of the metabolic effects of TGR5 activation in the gut: a study in a mouse model using a pharmacological tool

Supervisor: Anne TAILLEUX-Inserm UMR 1011-Institut Pasteur de Lille-Université de Lille anne.tailleux[chez]univ-lille[point]fr

Background - The intestine is an organ contributing to metabolic and inflammatory homeostasis via 1/ its nutrient absorption function, 2/ its metabolically active commensal flora, 3/ its barrier function, by controlling permeability, 4/ its enteroendocrine function, by synthesising and secreting signalling molecules, and 5/ its homeostatic function, through the immune cells it contains. TGR5 is a G protein-coupled bile acid membrane receptor expressed by different cell types involved in the regulation of metabolic homeostasis, in particular the incretin glucagon-like-peptide-1 (GLP-1)-producing enteroendocrine cells, known for its beneficial effects on metabolic homeostasis. A pharmacological tool was developed, consisting of a pharmacophore conferring agonist activity on the murine TGR5 receptor and a kinetophore that reduces its intestinal absorption and targets the actions of the molecule in the distal part of the intestine when administered orally.

Hypothesis - Activation of TGR5 in the gut may lead to improved metabolic homeostasis, reduced food intake and improved glucose tolerance.

Objective - In M2, our objective will be to analyse the metabolic effects and their molecular mechanisms of the selective activation of the TGR5 receptor in the gut by a selective agonist, BDM72881.

Methods - In male C57BL6 mice, the effects of acute administration of the compound BDM72881 will be assessed by measuring food intake and energy expenditure (metabolic cages), neuronal activation (immunohistochemistry), a functional assay to assess metabolic homeostasis (OGTT), as well as the determination of GLP-1 co-secreted peptides (multiplex ELISA).

This master's project could lead to a thesis project that will assess the role of TGR5 in the intestine on endocrine function more globally.

Keywords - TGR5 receptor, enteroendocrine function, incretins, gastrointestinal peptides, metabolic homeostasis, pharmacological approach, biochemical analyses, histological analyses, gene expression measurement.

Pathophysiological role of amino acids involved in one-carbon metabolism in NAFLD: analysis of the molecular mechanisms of variations observed in a patient cohort.

Tutor :Guillaume GRZYCH, AHU CHU Lille - guillaume.grzych[chez]chru-lille[point]fr

Laboratoire : UMR 1011. Institut Pasteur de Lille. 1 rue du Pr Calmette. 59019 Lille.

Background: NAFLD (Non-Alcoholic Fatty Liver Disease), a major public health issue, is the liver complication of the metabolic syndrome and is strongly associated with diabetes and obesity. NAFLD is a progressive disease characterised by liver damage due to an accumulation of triglycerides in the liver or isolated steatosis (NAFL: Non-Alcoholic Fatty Liver). In the case of associated inflammation, NAFLD develops into steatohepatitis (NASH: Non-Alcoholic Steato Hepatitis). Possible complications of NAFLD are fibrosis, cirrhosis and hepatocellular carcinoma. The mechanisms that induce NASH from NAFLD are still poorly understood. Through a cohort study, we have identified a specific plasma metabolic profile in NASH patients involving changes in amino acids involved in one-carbon metabolism (1C metabolism). This metabolism regulates methylation, which may lead to epigenetic changes. In order to understand the molecular mechanisms underlying the plasma variations observed in NASH patients, we aim to study these modulations in a mouse model in which NASH is induced by a specific diet. The literature reports that activation of the nuclear receptor PPARɑ by fenofibrate in a mouse model and in humans leads to an increase in an intermediate metabolite of one-carbon metabolism, homocysteine (Luc et al., 2004). Since PPARɑ expression is itself decreased in NASH (Francque et al., 2015), we speculate that PPARɑ might be one of the regulators of carbon metabolism in NASH.

Aim: The project aims to analyse plasma and liver amino acid variations as well as gene expression variations in a diet-induced mouse model of NASH with control of PPARɑ expression and/or activity.

Methods: The mouse models used will have diet-induced NASH with either genetic inactivation of PPARɑ (total Ko or specific Ko in hepatocytes) or pharmacological activation of PPARɑ (by fibrates). Targeted metabolomic analyses by mass spectrometry will be performed on plasma and liver samples from these models. Depending on the metabolomic results, gene expression analyses in the liver by Q-PCR or transcriptomic analysis will identify the genes responsible for the observed metabolic variations.

Collaborations: This work will be carried out in collaboration with Dr Joël Haas and Pr Anne Tailleux (team 1 of UMR1011).

Keywords: NAFLD, mouse model, PPARɑ, amino acids, metabolism, metabolomics.

FASTRIP- Targeting strategy for the inhibition of FAT10/PPAR interaction for the treatment of NASH

Supervisor : Pr Réjane Paumelle-Lestrelin.

rejane.lestrelin[chez]univ-lille[point]fr

Tel : 03 20 97 42 09

INSERM- UMR1011 " Récepteur nucléaire, maladies métaboliques et cardiovasculaires ".

Laboratoire J&K-Faculté de médecine pôle recherche-Bd Pr Julex Leclerc-Lille

Master 2 Biology and Health: pathway: Diabetes and Cardiovascular Diseases - Precision Health

Non-alcoholic fatty liver disease (NAFLD) is rapidly becoming the most common liver disease, affecting 80% of the obese population. NAFLD is initiated by the accumulation of fat in the liver (steatosis) which progresses to non-alcoholic steatohepatitis (NASH). NASH is a risk factor for disabling and fatal liver diseases, such as cirrhosis and hepatocellular carcinoma (HCC), as well as cardiovascular disease. Currently, there is no drug treatment available to limit the severity of NASH. In this context, it is important to characterise the molecular mechanisms responsible for the development and progression of this disease in order to provide preventive and/or therapeutic solutions.

The results of the UMR1011 laboratory revealed an involvement of FAT10/UBD in the progression of NASH. FAT10 is a member of the eukaryotic ubiquitin family of proteins that is not or only slightly expressed in normal tissues and whose expression is increased in inflammatory contexts. FAT10 contains two UBL domains allowing covalent interaction (FATylation) via ligases (USE1 and UBA6), or non-covalent interaction, leading its substrates to proteasomal or lysosomal degradation. Transcriptomic analysis showed a strong increase in FAT10 expression in the liver of NASH patients, which is positively correlated with steatosis, fibrosis and bloating scores, and also with NASH severity. Interestingly, FAT10 overexpression was associated with decreased expression of peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor controlling lipid metabolism and inflammation, as well as with down-regulation of the PPARα signalling pathway. Our results suggest that FAT10 can modulate NASH progression by interacting with PPARα and promoting its deactivation, highlighting the FAT10/ PPARα interaction as a potential new target to treat NASH.

In this context, the objective of this project is to :

(1) to properly characterise the type of physical interaction between FAT10 and PPARα.

(2) to develop a cell-based assay to analyse the impact of FAT10/PPARα interaction on PPARα activity.

(3) transfer the assay to the U1177 screening platform for miniaturisation and automation to increase throughput.

(4) Screen 30,000 compounds from the U1177 library to identify molecules that inhibit FAT10/PPARα interaction.

The Master 2 project will be involved in parts 1) and 2) of this project. We hope to identify new molecules disrupting the FAT10/PPARα interaction that could enter a drug discovery programme to lead to an optimised candidate for proof of concept in the mouse model of NASH developed in the UMR1011 laboratory. The results should reveal additional mechanisms of NASH development and lead to the identification of new therapeutic tools for this disease.

Characterisation of cardiac remodelling during the development of NAFLD (Non Alcoholic Fatty Liver Disease): focus on the role of myocardial immune cells.

Dr Laura BUTRUILLE, INSERM U1011 Récepteurs nucléaires, maladies métaboliques et cardiovasculaires laura.butruille[chez]pasteur-lille[point]fr

Pr David MONTAIGNE, INSERM U1011 Récepteurs nucléaires, maladies métaboliques et cardiovasculaires david.montaigne[chez]chru-lille[point]fr, 03 20 44 52 30

NAFLD (Non Alcoholic Fatty Liver Diseases) are liver manifestations of the metabolic syndrome associated with obesity and type 2 diabetes. The term NAFLD refers to the progressive succession of the NAFL (Non Alcoholic Fatty Liver) stage characterised by the presence of hepatic steatosis, followed by the NASH (Non Alcoholic Steato-Hepatitis) stage characterised by the presence of hepatic steatosis and inflammation. In the long term, NASH can lead to liver fibrosis, eventually leading to cirrhosis or cancer. These chronic liver diseases are currently untreated and many epidemiological studies report an increased risk of developing cardiovascular diseases such as atherosclerosis, heart failure or arrhythmia in these patients. However, the mechanisms linking NAFLD to cardiac pathologies are imperfectly understood. Our initial results suggest a deregulation of innate immunity induced by NAFLD, promoting the development of intracardiac inflammatory foci and fibrosis in the atria and ventricles. The project consists of characterising these mechanisms in a pre-clinical mouse model. The description of the hepatic and cardiac phenotypes will be based on macroscopic observations, functional cardiac stimulation tests, histological analyses, gene and protein expression analyses and complete immunophenotyping. These data will reinforce clinical studies performed on myocardial biopsies obtained during cardiac surgery and the postoperative follow-up of patients from a Lille cohort generated at the Heart-Lung Institute (POMI-AF: NCT03376165).

Impact of time of day on perioperative immunoinflammatory response and myocardial remodelling after cardiac surgery.

Pr David MONTAIGNE, INSERM U1011 Récepteurs nucléaires, maladies métaboliques et cardiovasculaires

03 20 44 52 30, david.montaigne[chez]chru-lille[point]fr

Dr Laura BUTRUILLE, INSERM U1011 Récepteurs nucléaires, maladies métaboliques et cardiovasculaires laura.butruille[chez]pasteur-lille[point]fr

Aortic valve stenosis is the most common heart valve disease. This valve disease is responsible for a chronic obstacle to left ventricular (LV) ejection. Faced with this high afterload, the LV adapts by increasing its muscle mass. This initially adaptive remodelling contributes to the development of intramyocardial fibrosis and altered LV relaxation and compliance. Myocardial remodelling then becomes maladaptive and leads to heart failure. Aortic valve replacement (AVR) is the only treatment option.

Preclinical studies link circadian rhythm, inflammation and cardiac remodelling. We have recently shown that aortic valve replacement (AVR) is associated with fewer complications when performed in the afternoon compared to the morning. We hypothesise that the biological clock modulates intraoperative immune cell recruitment and thus the healing process and reverse myocardial remodelling after AVR. We aim to complement our clinical observations with a preclinical study in a mouse model. We will perform a transcriptome and epigenome on circulating leukocytes after surgery in the morning versus the afternoon to determine the impact of circadian rhythm on the systemic inflammatory response. We will analyse intramyocardial inflammatory cell populations using comprehensive immunophenotyping. Finally, we will define the impact of the nuclear receptor REV-ERBɑ and clock gene signalling on leukocyte epigenetic signatures and cardiac remodelling in this animal model.

TEAM 3

Characterization of dendritic cell (DC) and T cell (T) interactions in non-alcoholic steatohepatitis (NASH)

Supervisor. David DOMBROWICZ, DR Inserm - david.dombrowicz[chez]pasteur-lille[point]fr

Laboratoire : UMR 1011. Institut Pasteur de Lille. 1 rue du Pr Calmette. 59019 Lille. 0320877967

Background. NASH or 'fatty liver disease' is a condition that has increased dramatically in incidence worldwide and for which there is currently no pharmacological treatment. We have shown (Haas et al. Nat. Metab. 2019), in humans and in an animal model, that this metabolic pathology is associated with profound alterations of the immune system affecting in particular subpopulations of DCs (cDC1 and cDC2) and cytotoxic CD8 LTs. These alterations are potentially causative in the disease but the functional mechanisms involved, including the interactions between DC and LT, remain unknown and these populations have not been precisely characterised.

Objective. The aim of the Master 2 is to analyse the phenotypic and functional characterisation of CD8 (and CD4) T cell populations.

Methods. In a mouse model of diet-induced NASH, hepatic CD8 and CD4 T cell populations will be characterised by different techniques: flow cytometry, single-cell RNA-seq (with a particular analysis of metabolic pathways and the T cell antigen receptor -TCR-), and intracellular metabolism measurements (Seahorse). Depending on the results obtained, the interactions with DCs could be evaluated in functional tests.

Collaboration. This work will be performed in collaboration with Dr Joël Haas (UMR1011 Team 1).

Keywords: NASH, T lymphocytes, DCs, metabolism, scRNA-seq, bioinformatics, functional test.

TEAM 4

Transcriptional control of phenotypic plasticity in liver cells

Jérôme Eeckhoute - INSERM U1011 Récepteurs nucléaires, maladies cardiovasculaires et diabète Faculté de Médecine de Lille - Pôle Recherche
Boulevard du Professeur Leclerc, Bâtiment J&K
59045 Lille cedex - Tel :03.20.97.42.19

jerome.eeckhoute[chez]inserm[point]fr

u1011.pasteur-lille.fr/lunite/theme-4-analyse-transcriptionnelle-integree-des-maladies-hepatiques/

The liver performs instrumental metabolic functions that must be constantly adjusted to the nutritional and energetic status of the body. We have recently identified that the liver transcriptome is controlled by a functional interaction between chromatin structure, the epigenome and several transcriptional regulatory complexes. In this context, we aim to identify how transcriptional regulators interact with chromatin to ensure specificity and appropriate levels of expression of hepatic genes. We are also interested in the disruption of these mechanisms in the context of liver dysfunction. We propose a Master 2 internship on this topic for a student interested in elucidating the molecular mechanisms of transcriptional regulation.

TEAM 5

Brown adipose tissue from embryo to adult: implications in metabolic diseases

SupervisOR : Dr. Alicia Mayeuf-Louchart

Institut Pasteur de Lille, Université de Lille, Unité 1011-Pr B. Staels, Equipe 5-Dr. H. Duez)

03.20.87.77.75

alicia.mayeuf-louchart[chez]inserm[point]fr

Brown adipose tissue represents a new therapeutic target for the treatment of metabolic diseases. When activated by different stimuli such as exposure to cold, brown adipose tissue metabolises about 20% of the daily energy intake. Some of the glucose absorbed by brown adipocytes is stored as glycogen. However, little is known about the role of glycogen and its fate in brown adipocytes. We have recently published a paper showing that glycogen dynamics (formation and degradation) are essential for lipid droplet formation during brown adipocyte differentiation.

The objective of this project is to study glycogen metabolism in brown and beige adipocytes, in order to determine whether its metabolism may represent a potential target for improving brown adipose tissue activity in the context of metabolic diseases.

This study will be based on experiments in the mouse model, during development and in adults under different pathophysiological conditions. During the M2R, the student will perform experiments in histology (tissue sections, staining, immunoshistochemistry, microscopy), cell biology (cell culture, immunohistofluorescence, confocal microscopy), molecular biology (RNA extraction, RTqPCR) and metabolism (biochemical tests). This project aims to initiate a thesis project.