Heart failure (HF) affects 2% of the EU population and is an unmet clinical need due to the limited treatment options. HF is associated with alterations of the immune system. Non-Alcoholic Fatty Liver Disease (NAFLD), an emerging health problem associated with metabolic diseases, such as obesity and type 2 diabetes (T2D), is an independent cardiovascular disease risk factor. When NAFLD progresses from simple steatosis to Non-Alcoholic SteatoHepatitis (NASH) with fibrosis, the risk to develop hepatic and cardiac complications increases. However, the mechanisms linking NASH and HF are not understood. Strikingly, monocytes (Mono) and macrophages (Macro) are important contributors to cardiovascular disease and these cells also plays a key role in NASH. Moreover, our preliminary results identify these immune cells as potential link between NAFLD and cardiac dysfunction. We thus hypothesize that NASH and its metabolic alterations impact on the immune compartment, which in turn functionally contributes to inappropriate cardiac remodelling (CR), leading to HF. We propose an ambitious project combining systems biology approaches, state-of-the-art molecular technologies, original preclinical models and human translational studies to: 1. Define the role of NASH in (the aggravation of) CR in mice; 2. Characterize the immune cell alterations in NASH-induced CR with a focus on Mono and Macro; 3. Determine the functional role of thus identified immune cells in CR; 4. Translate our findings to the human pathology by analyzing the impact of NAFLD on cardiac complications (atrial fibrillation and HF) in patients undergoing aortic valve stenosis surgery. The unique combination of expertise in immunology, metabolism and cardiology within our consortium is critical to decipher how immune system alterations link liver pathology to heart disease. Our results will improve our understanding of HF pathophysiology allowing the identification of novel patient management approaches.

Despite decades of lipid lowering drug delivery, prevention strategies and efforts in research, cardiovascular diseases, mainly caused by atherosclerosis, are still the leading cause of death worldwide. New therapies are then mandatory to reduce the residual cardiovascular risk and to prevent atherothrombotic events. Atherosclerosis is a chronic inflammatory disease of the vascular wall triggered by low density lipoprotein internalisation within the subendothelial space. More than the obstruction of the arterial lumen, instability and rupture of the plaque are now recognized as the most deleterious events. Among processes triggering plaque instability, intraplaque neovascularization accelerates plaque progression, induces plaque rupture and attenuates statin benefit in human. Our preliminary data identified the nuclear receptor Rev-erb-a as a putative inhibitor of intraplaque neovascularization in human and mouse plaques in vivo as well as in vitro in endothelial cells. We then hypothesize that Rev-erb-a represents a new anti-atherogenic target that prevents intraplaque neovascularization by inhibiting the angiogenic activity of endothelial cells. This proposal will be the first to address the role of Rev-erba in angiogenesis and intraplaque neovascularization. This project will involve original available mouse models, innovative validated whole organ imaging techniques, in combination with cellular omics and mechanistic studies and a translational clinical part addressing the relevance of experimental results to human disease. We anticipate to identify Rev-erb-a as a novel therapeutic target to reduce intraplaque neovascularization and cardiovascular diseases.

Background. Aortic valve replacement is the sole therapeutic solution to improve outcomes in patients with aortic stenosis. AVR is commonly performed during on-pump cardiac surgery, whereas trans-catheter aortic valve implantation is increasingly performed in patients with high-risk for surgery complications. Whatever the technical approach, AVR is expected to improve not only survival, but also functional status as a result of cardiac reverse remodeling, i.e. regression of the left ventricular hypertrophy. No medication is currently available to specifically improve and/or accelerate reverse remodeling after AVR. We have recently demonstrated that afternoon (vs. morning) AVR results in decreased incidence of both immediate perioperative myocardial infarction and acute heart failure within 500 days after surgery. Interestingly, disruption of diurnal rhythms by modulating molecular clock genes immediately after myocardial infarction in mice impaired healing and exacerbated maladaptive cardiac remodeling. Working hypothesis. We postulate that the circadian clock plays a key role in immune cell recruitment and peri-operative myocardial injury healing and remodeling after AVR, i.e. differences in pre- and post-operative immune-inflammatory responses and subsequent cardiac repair and remodeling are responsible for the lower frequency of heart failure development months after afternoon (vs. morning) AVR. Objectives and methods. The project will be organized to determine: (i) the impact of peri-operative inflammation on surgery complications and subsequent cardiac remodeling after AVR, (ii) the impact of the time-of-day on peri-operative inflammatory responses and their role in post-operative outcomes. We will perform a bi-centric prospective randomized study at the University Hospital CHU de Lille (France) and CHU Rennes (France) in patients undergoing first AVR for severe aortic stenosis with LVEF>50% to determine whether the pre-operative leukocyte immune-phenotype and their post-operative activation display a morning-afternoon variation, and whether this activation is associated with reverse cardiac remodeling during the year following AVR. Deep immuno-phenotyping of peripheral blood leukocytes will be performed by mass cytometry (CYTOF). Second, the transcriptional pathways associated with the time-of-the-day variation and AVR-induced immune-phenotype will be explored in cells identified in previous WP by RNAseq analysis. Third, based on our previous studies, we will determine the role of the clock gene REV-ERB? in the regulation of the time-of-the-day variation of the immune-inflammatory response and its impact on cardiac remodeling. We will perform functional analysis on freshly isolated human monocyte sub-populations and study cardiac remodeling after abdominal aortic constriction (AAC) and reverse remodelling after AAC relief in mouse models deficient for REV-ERB? (cell type to be chosen based on the results from other WP). Finally, studies using novel REV-ERB? ligands will be done to validate this target as a pharmacological approach. Expected results and relevance of the project. Our translational project including patients and preclinical models will identify the contribution of the immune-inflammatory response in maladaptive cardiac healing and reverse remodeling after AVR., as well as proof-of-concept studies for feasible therapeutic strategies interfering with clock gene signaling in immune-inflammatory cells to prevent maladaptive cardiac remodeling for which virtually no drugs exist to date.

Non-alcoholic fatty liver disease (NAFLD) is a major global health problem for which there are no effective treatments. Early stages of NAFLD are described by isolated hepatic lipid accumulation, which, over time, can lead to inflammation and fibrosis, called Non-alcoholic steatohepatitis (NASH). NAFLD and especially NASH are closely associated to dysfunction in hepatic glucose and lipid homeostasis. However, the mechanisms driving activation of the hepatic immune system and progression from steatosis to NASH, remain poorly understood. We recently identified hepatic dendritic cells (DC) as a key immune population that is associated with NASH in humans. We hypothesize that the altered metabolic environment, including excess circulating lipids, alters the hepatic DC population triggering progression to NASH. The present proposal aims to dissect the key intracellular metabolic pathways of hepatic DC that are affected by activation during NASH pathogenesis.

CONTEXT. Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in western countries. It represents a continuum encompassing stages ranging from isolated steatosis to non-alcoholic steatohepatitis (NASH), characterized by lobular inflammation and ballooning, with or without fibrosis. Some NASH will progress toward the hepatocellular carcinoma (HCC). The liver is a frontline immunological organ containing numerous innate and adaptive immune cells. An imbalance of pro- and anti-inflammatory responses involving both innate and adaptive mechanisms affects the induction and progression of NASH. Type 1 innate lymphoid cell (ILC1) and NK cells are the most abundant hepatic subsets of innate lymphocytes. They promptly sense changes in liver homeostasis. ILC1 are resident in the liver and specifically display cytotoxic capacity directly through expression of Granzyme B and indirectly through IFN? production. Recent studies have suggested a role for ILC in immune-mediated liver diseases. HYPOTHESIS. We suspect that ILC subsets participate to the damaging inflammatory process through their alteration resulting from, or their involvement in, gut-liver axis dysregulation. PRELIMINARY RESULTS. Using seatosis-inducing MCD diet and a new NASH diet that we validated, we showed early alterations in ILC1 and NK populations in gut and liver with impairment of IFNg production and increased gut permeability as well late alterations in CD8 T cells. In a clinical study, we also showed that circulating NK, cDC2 and IFN? - or Perforin-expressing CD8 T cell and liver cytotoxic CD8 T cells correlate with NASH-associated inflammation, ballooning and with distinct sets of associated genes in the liver, including cytotoxic markers, MHCII antigen presentation for CD8 T cells, and inflammatory cytokines for NK cells. AIM. We will determine the contribution of ILC, in part through IFN? production, to the different stages of steatosis, NASH and HCC development and their interactions with CD8 T cells and Kupffer cells. WORKPLAN & METHODS. We will determine whether: 1. ILC1 directly contribute to the liver pathology in NASH. Using new mouse lines for inducible depletion of all IFN? -secreting cells or only functional NK/ILC1, we will delete these populations at different time points after diet initiation and evaluate both immune and metabolic parameters of NASH. We will evaluate NASH outcome in IFN? and IFN?R-deficient animals. 2. ILC indirectly contribute to NASH through modulation of intestinal functions. Using a new ILC3 fate mapping mouse line, we will monitor a) their in situ differentiation from precursors, b) their gut localization, c) their plasticity towards ILC1 subset. 3. ILC1 interact with CD8+ T cells and/or KC. We will isolate ILC from circulation, liver, adipose tissue and intestine from NASH or chow diet-fed mice and perform single cell RNAseq analysis. Contribution of dysregulated molecules and pathways in liver ILC1, identified by RNAseq, to a direct and indirect crosstalk with CD8+ T cells and KC will be analysed. 4. Specific components of NASH-inducing diet impact on immune populations and intracellular metabolism. We will mimic the effect of a NASH diet in vitro to elucidate the impact of the steatogenic compounds on ILC and CD8+ T cells immune functions. They will be sorted and cultured in the presence of palmitate, high glucose concentrations or cholesterol and their phenotype and functional changes investigated. IMPACT. Beyond the understanding of the role of ILC in NASH, we will establish a rational basis for new therapies targeting these innate cell subsets to prevent NAFLD progression toward irreversible stages of fibrosis and HCC.