Background: Obesity currently touches 17% of the adult French population and, by leading to diabetes, cardiovascular disease and certain forms of cancer, it represents an important medical and socio-economic burden. Treating obesity can be successful only if weight loss is maintained over time and weight regain is avoided. Unfortunately, most patients with obesity who receive weight loss interventions eventually exhibit weight regain. Hence, there is an important and urgent need to understand the mechanisms underlying the failure to maintain a lower body weight in the post-obese state. To continuously adjust energy availability at the organism level, the brain hypothalamus and the adipose tissue must interact through blood-borne and nervous pathways, with the hypothalamic-sympathetic nervous system (SNS) top-down signaling being key in this context. Beyond an obvious role for hypothalamic neurons and adipocytes, recent studies have pointed to the involvement of hypothalamic microglia (the brain resident macrophages) and adipose tissue macrophages in the regulation of energy balance, as these immune cell types critically affect the functions of neurons and adipocytes, respectively, by regulating inflammatory responses among other mechanisms. Rationale and objectives: Here we hypothesize that after an episode of obesity, an altered neuro-immune crosstalk, involving hypothalamic microglia, the SNS and adipose tissue macrophages, drives a new, higher, body weight set-point by modifying use of energy substrates as well as systemic and tissue inflammatory responses. Our aims are: 1) To characterize the putative mechanisms underlying this altered neuro-immune crosstalk; 2) To establish their causality in modifying the body weight set-point and ensuing weight regain; 3) To determine their translational relevance in patients with weight regain after bariatric surgery. Methods: To reach these objectives, we will use an experimental mouse model based on diet switch to mimic weight gain/weight loss/weight regain in which behavioral and in vivo metabolic studies will be combined with histological, molecular and biochemical analyses. Some of these studies will be carried out also on adipose tissues samples of bariatric surgery patients with/without weight regain to assess translatability of the findings towards humans. Ad hoc approaches, including generation of transgenic mice, pharmacology and use of RNAseq and cell metabolomics, will then provide in depth information on the roles of hypothalamic microglia, SNS and adipose tissue macrophages in the metabolic changes that accompany the post-obese state and that favor weight regain. Originality: Our project stems from an original and timely hypothesis proposing that the body acquires a memory of past obesity episodes through changes in the neuroimmune-SNS crosstalk involving the hypothalamus and the adipose tissue, which in turn favor ensuing body weight gain and metabolic alterations. The scientific objectives that we therefore aim at reaching through MEMOBESE are novel and original, while sitting on several published and unpublished observations that overall support the hypothesis proposed and its logic. Expected results and potential benefits: MEMOBESE is expected to deliver greater understanding of the biological mechanisms at the core of the failure of weight loss maintenance. In the short term, this knowledge is expected to advance the fields of Physiology, Nutrition, Neuroscience, Immunology and Metabolism. In the medium and long term, the evidence provided will foster further studies on identified mechanisms, potentially leading to more effective treatment strategies and to the use of novel biological markers to better characterize and stratify patients for improved medical care.

Nonalcoholic fatty liver disease (NAFLD) is the most prominent form of liver disease in the western world, affecting approximately one­third of the population, and is projected to become a major clinical and economic burden worldwide within the next decade. Chronic inflammation favors the perpetuation and aggravation of NAFLD, notably through the accumulation of activated macrophages. However, in inflamed tissues, macrophages are diverse. Indeed, two types of macrophages with different developmental origins cohabite: tissue resident macrophages (rMf) that self-maintain independently from circulating monocytes by proliferating locally, and inflammatory monocyte-derived macrophages (iMf) differentiated from circulating Ly-6C+ monocytes recruited to the inflamed tissue. While iMf are believed to play a detrimental role in NALFD and its complications, there is no available information on the specific roles played by rMf in these disorders. Such lack of knowledge is largely due to the absence of tools allowing to specifically target liver rMf, known as Kupffer cells (KCs), and this had precluded their appropriate study in physiology and physiopathology in general. To fill this gap, we have developed new mouse models and methods allowing us to study how KCs impact on liver metabolic diseases outcome. We will take advantage of these tools to study how specific modulation of pro and anti-inflammatory genes in KCs modulates NAFLD and its complications. In addition, our preliminary data also suggest that self-maintenance and activities of KCs are modulated by the environmental context of these pathologies (inflammation, lipid stress, …). Indeed, contrary to what is observed during healthy homeostasis, a significant part of KCs is replaced by cells of monocytic origin under conditions of NAFLD and steatohepatitis (NASH). We propose to further study this phenomenon. More specifically, our proposal entitled TARGETKC will allow us to: 1) Determine the mechanism underlying the participation of monocytes to the Kupffer cell pool under conditions of lipid stress, as well as the physiopathological role played by monocyte-derived KCs; 2) Determine how TLR4-mediated sensing of danger signals (gut-derived LPS, lipids, fetuin A, HMGB1, …) and tonic IL-10 production by KCs alter liver function, systemic inflammation as well as liver lipid metabolism in the context of NASH; and 3) Uncover molecular and lipid pathways induced in Kupffer cells under conditions of NASH using transcriptomic and lipidomics approaches.

At the back of the eye lies a monolayer of cells essential for vision: the retinal pigment epithelium (RPE). Apical microvilli from these polarized cells make close contact with the photosensitive photoreceptor outer segments (POS). POS are constantly renewed to fight the high levels of oxidative damage they are subjected to, and one of RPE cell main roles to maintain lifelong vision is the daily elimination of used POS tips by phagocytosis. Absence or failure to complete this task leads to the development of blinding diseases for which no treatment exists, such as early-onset retinal dystrophies or age-related macular degeneration (AMD). An important feature of RPE phagocytosis is its rhythmic activity. We showed previously that the alphavbeta5 integrin receptor controls the rhythmic activation of RPE phagocytosis. Subsequently, an intracellular signaling cascade activates the Mer tyrosine kinase (MerTK) internalization receptor. Our recent studies suggest that MerTK also controls the amounts of POS that can be tethered by RPE cells, including via the extracellular cleavage of MerTK. Interestingly, the known machinery for RPE phagocytosis is close to the clearance of apoptotic cells by macrophages. In macrophages, many molecules intervene in such processes, suggesting that similar intricate protein networks could operate in RPE cells. However, tissue specificity exists, including the permanent contact between photoreceptors and RPE cells, thus regulation of the machinery has to be controlled very tightly via several mechanisms to launch and stop phagocytosis at the proper time. Our recent data on the tissue-specific opposite role of MerTK ligands reinforce this idea. Moreover, several other receptors have been shown to be expressed by RPE cells, but their participation in POS phagocytosis has not been investigated yet. So far, studies on the phagocytic machinery have been performed on nocturnal rodent species using mostly rod photoreceptors sensitive to dim light. However, central vision in humans is mostly due to cones that give us details resolution and color vision. Rod and cone POS membrane structures are different and they are used for vision almost in exclusion of each other, either at night or during the day, respectively. Therefore, our hypothesis is that the elimination of used cone POS could take place at a different timing and with a different molecular machinery than for rod POS. For these reasons, with the REPHAGO project we plan on identifying the contribution of new membrane receptors in controlling the daily activation of rod (Aim 1) and cone (Aim 2) POS phagocytosis by RPE cells. Candidate receptors for rod POS phagocytosis will be validated in vitro and then in vivo for interesting candidates according to their implication during the phagocytic process. We will characterize cone-specific molecules for POS clearance using transcriptome studies associated with functional validation assays. For this project, we will use multiple state-of the art approaches (functional phagocytosis assays, in vivo phagocytosis assessment, RNAseq, visual animal phenotyping…) as well as new and innovative animal models (RPE-specific knockout mouse models for rod receptor candidates, cone-rich diurnal rodent model). Identified receptors will be then explored in other phagocytic cells. Understanding the complexity and specificity of protein networks and interactions to complete daily this crucial task will enlighten us both on normal retinal function and on the consequences of phagocytic defects. This will help us consider new avenues for therapies and therapeutic targets for these pathologies for which no treatment exists. As well, the sequential activation of the RPE machinery can help us decipher molecular pathways that are used in other phagocytic cells, and in particular macrophages. Thus, our results could contribute to the understanding of phagocytic processes occurring in other tissues or pathologies such as atherosclerosis.

Our proposal aims to unravel the molecular mechanisms through which membrane cholesterol and lipid transporters (ABCA1 and/or ABCG1) shape adaptive immune response. First, this question will be investigated in T lymphocytes by the phenotypic and mechanistic analysis of conditional deficiencies of ABCA1 and/or ABCG1 in mouse T cells. We will determine the impact of the lipid transporters deficiency on the distribution and membrane dynamics of the T-cell receptor (TCR) at steady state and following stimulation. We expect that changes in lipid storage could modify intracellular trafficking and induce epigenetic reprogramming which would lead to specific cell fates. In second exploratory phase, multiomic exploration of T cells from Tangier patients and ABCA1/G1 deficient mouse will enable us to decipher how the absence each transporter modifies in T cells the lipidome, the phosphoproteome and the TCR signalosome. This axis will be extended to autoimmune pathologies and metabolic disorders.

Ten percent of the European population is on long-term statin therapy. Statins reduce major vascular events and vascular mortality in a large number of individuals. However, the efficacy and safety of this therapy is uncertain with elderly people, who often present co-morbidities and potential drug interactions. In addition, statin responses, both in terms of LDL cholesterol reduction and side effects, are patient-dependant and very variable, with good or poor responders to treatment and some being intolerant to it. The therapeutic effects of statins include immunomodulatory and anticancer effects on host cells, suggesting their potential for broader applications. In particular, some statins exhibit bactericidal activities that influence the growth and virulence of bacterial strains in the intestinal microbiota. Given the growing evidence linking the gut microbiota to immunity and to the pharmacokinetics-pharmacodynamics of drugs, our project aims to determine the role of statins in the gut microbiota-mediated Tc1 (CD8+ interferon-g+ T cells) response in chronic inflammatory diseases and associated phenotypes in human and mouse models. Our recent works directly related to the project revealed (i) the role of the intestinal microbiota in the regulation of host cholesterol homeostasis; the pleiotropic effect of statins on (ii) Human enterotypes and (iii) metabolotypes in human microbiome recipient mice ; (iv) the role of bile acids in the control of bacterial proliferation and hepatoprotection, and (v) alcoholic hepatitis; (vi) the metabolomic profiling as a tool for clustering patient subtypes. Our observations suggest that the heterogeneity of statins responses originate from their properties to remodel intestinal microbiota and this way impact immunity and associated chronic inflammatory diseases. Our preliminary data revealed that microbiota composition modulates statin impact on disease progression in mouse models of chronic inflammatory diseases (atherosclerosis, grafted colorectal cancer). Pathophysiological responses (harmful or protective) to statins are associated with a significant alteration Tc1 cells response conditioned by functional remodeling of the basal intestinal microbiota. Based on these data, our objectives are: - determination of the impact of statins on bacterial metabolites and taxa, associated with Tc1 responses, first in murine pathological models (task 1) and then in a cohort of dyslipidemic patients (task 2); - to transfer and validate in vivo “pathological phenotypes” in recipient models of atherosclerosis combined with spontaneous colorectal cancer on the basis of selection of good and poor human Tc1 cell responders, (task 3); - to identify biomarkers (metabolites and/or bacterial strains) that predict effects on the progression of both diseases (task 4). The research will include metabolomic/lipidomics analyses, metagenomics, and immune profiling of plasma, feces, immune and inflammatory tissues (blood cells, mesenteric and para-aortic nodes, lamina propria, aorta, and tumors). In Task 1, we will complete our analysis on dyslipidemic mice (Ldlr-/-) and on a model of grafted colorectal lineage (MC38) with a particular interest in Tc1 cells in lesional and lymphoid tissues. In Task 2, we are halfway through the recruitment of 100 dyslipidemic patients as a basis for the selection of extreme phenotypes. In Task 3, we have already collected data demonstrating the feasibility of human fecal microbiota transfer in the Ldlr-/- recipient mouse model and in mice at risk to spontaneous colorectal cancer (APCmin/J). Omics data generated in this project and in the clinical project will be integrated to propose prognostic signatures of statin responses paving the way for personalized therapy.