Phagocytosis is a mechanism of internalization and digestion of objects larger than 0.5 microns that relies on receptor triggering leading to actin polymerization and membrane deformation. Partners 1 and 2 have contributed to describe these mechanisms. How multiple receptors simultaneously recognize microbes, pathogens or debris both through direct binding and opsonization, leading to a complex interplay between the signaling pathways and a fine tuning of the fate of the internalised material, is still not well understood. In particular, phagocytosis by C-type lectin receptors that bind carbohydrates residues on the surface of various microorganisms has been overlooked so far. Mannose receptors for instance importantly bind glycoconjugates with terminal mannose, fucose and N-Acetylglucosamine present in bacterial and yeast walls. Partners 2 and 3 developed functionalized lipid droplets coated with tailor-made fluorescent mannolipids to study C type lectin receptors-induced phagocytosis. The complement receptor 3 (CR3) is an integrin that binds microorganisms directly or a complement opsonized-target indirectly. Partner 1 has contributed to dissect signaling associated with CR3. The integrin CR3 was reported to cooperate with Immunoglobulins receptors to ensure efficient phagocytosis, but how different receptors cooperate with each other during signaling, force generation, phagosome formation and maturation, remains to be investigated. This proposal aims at untangling critical steps of phagocytosis taking advantage of a multi-disciplinary approach and unique deformable emulsion droplets coated with fluorescent receptor-targeted ligands as targets for phagocytosis by human macrophages to : - determine by FRET receptors binding and clustering during phagocytosis - monitor directly the forces generated by the phagocyte and identify important regulators of force generation analyse the fate of the internalized material and phagosome maturation upon various receptor engagement, taking advantage of novel functional fluorescent probes. To this end, the complementary expertise of three groups, who have separately made important contributions in their fields and have already collaborated, will be brought together. They will take advantage of a new class of materials, oil-in-water emulsion droplets, developed by Partner 2, which are deformable particles that can be functionalized with biological ligands freely-diffusing at the interface. The interaction and clustering of different receptors in the contact zone between the phagocytic cell and the droplet will be investigated with high spatial resolution using FRET between fluorescent carbohydrate ligands prepared by Partner 3. The deformable droplets are unique tools to directly measure mechanical stresses. The intimate relations between MR and CR3 will be analyzed and the role of potential regulators of the CR3 previously identified by Partner 1 will be tested both on receptor clustering and force generation. To study how receptors influence the fate of the internalized material during phagocytosis, we will combine new probes developed by Partner 3 in various colors allowing multiplexing with the lipid particles. The phagocytosis assays will be performed in primary human macrophages by Partner 1. With this project, we will extend the toolkit to address unsolved questions on phagocytic receptors dynamics in relevant phagocytic cells. We will be able to monitor the underlying mechanobiology of the phagocytosis process, with a novel set of combined expertise and techniques: ligand design, particle formulation, mechanical measurements, time-lapse microscopy and force-dependent integrin partners. Importantly, the receptors of interest play a crucial role in clearance of pathogens as well as neuron pruning, and the phagocytic properties of macrophages can be hijacked in some pathological conditions, which increases the relevance of a better understanding of phagocytosis.

Anemia, defined as a decreased quantity of circulating red blood cells, is a major source of morbidity and mortality affecting a-third of the worldwide population. As a functional component of erythrocytes hemoglobin, iron is essential for oxygen storage and transport. The liver-derived peptide hepcidin is the master regulator of iron homeostasis. During anemia, the erythroid hormone erythroferrone regulates hepcidin synthesis to ensure the proper supply of iron to the bone marrow for red blood cells synthesis. However, we provided evidence that another factor may exert a similar function. We identified a previously undescribed suppressor of hepcidin that is highly induced in the liver in response to hypoxia during the recovery from anemia and in thalassemic mice. We demonstrated that this hepatokine is a potent suppressor of hepcidin in vitro and in vivo. Our preliminary data further indicated that it acts by impairing the canonical BMP-SMAD signaling cascade that governs hepcidin regulation but the molecular mechanism is unknown. Our aims are to confirm the role this hepatokine in hepcidin regulation and iron metabolism, to identify its exact mechanism, to explore its involvement in human pathologies and to investigate the therapeutic potential of its manipulation in murine models of anemia. Successful completion of this project will open new areas of investigation and provide a better understanding of the pathophysiology of anemia and hypoxia and liver physiology. Ultimately, it will lead to the development of new therapeutic strategies for the treatment of various forms of anemias for which current treatments remain largely ineffective.

The goal of this project is to elucidate the mechanisms responsible for the increase in spontaneous mutagenesis, which is dependent on the Mfd protein in Escherichia coli cells growing without exogenous stressors. Our hypothesis posits that the most of Mfd-dependent mutations arise from the Mfd-mediated exposure of stretches of single-stranded DNA (ssDNA) situated between the elongating RNA polymerase (RNAP) and Mfd. We propose that this occurs after Mfd restarts RNAPs stalled due to obstacles other than DNA lesions, and while it remains connected to the elongating RNAP and the DNA. The rationale behind this hypothesis is that because ssDNA is significantly less chemically stable than double-stranded DNA, it is more susceptible to premutagenic chemical modifications, such as depurination, depyrimidination, deamination and oxidation. Importantly, these modifications are not well-recognized by the NER system, and they do not block the DNA replication process. Identifying the molecular mechanisms that regulate mutation rates carries significant implications for a wide array of scientific fields, including genetics, evolution, medicine, biotechnology, and environmental science. In this context, the contribution of Mfd, an evolutionarily conserved enzyme, to spontaneous mutation rates holds particular significance. This importance stems from the fact that the inactivation of the gene encoding Mfd can be categorized as an "antimutator mutation." In other words, it reduces the rate of mutations, which can be especially relevant in the context of drug resistance and disease prevention.

The adrenal cortex is responsible for the synthesis of steroid hormones. Functional and structural changes occur during aging. The risk of adrenal tumors also increases with age and may be accompanied by alterations in steroid secretion. Animal models show that the senescence of steroid cells is accompanied by recruitment of immune cells, which play a major role in the homeostasis of the adrenal cortex. This balance is disrupted during aging and tumorigenesis. This project aims to study the impact of senescence and inflammation in human adrenal cortex. We will use single-cell transcriptomics/epigenomics and spatial transcriptomics approaches to characterize these mechanisms at the cell level during aging and through different models of tumorigenesis. These approaches will allow to better understand the pathophysiology of adrenal cortex and could lead to the discovery of new targets for treating hormone disorders and tumors.

Mucosal Associated Invariant T cells (MAIT) represent a key immune cell population because of their abundance in humans, their ability to provide immediate responses upon stimulation, and their potential involvement in many pathologies. MAIT cells recognize microbiota-derived metabolites presented by the MHC-related molecule MR1. Both MR1 and the TCR of MAIT cells are conserved in evolution, indicating non-redundant functions linked to antigen recognition. MAIT ligands produced by the microbiota control MAIT cell development. However, mechanisms underlying microbiota production of MAIT antigens remain undefined. Upon thymic egress, MAIT cells migrate to mucosal and liver tissues where they accumulate over time and reach high frequencies in humans (1-10% of T cells in the intestine, 20-45% in liver). MAIT cells are further recruited and activated in the intestinal lesions of patients with inflammatory bowel diseases, suggesting a role in these pathologies. Whether MAIT cell accumulation and function in intestinal tissues rely on the recognition of microbiota-derived antigens is unknown, and the role played by MAIT cells in the gut also remains unclear. The MicrobMAIT proposal aims at addressing the role played by microbiota-derived antigens in the biology of MAIT cells. We will take advantage of innovative genetic tools and reagents to: 1. Explore rules governing MAIT antigen production by the microbiota, in mice and humans 2. Determine the dynamics of MAIT antigens and identify antigen-presenting cells in vivo 3. Define the influence of the microbiota on MAIT cell maintenance and function during intestinal inflammation Based on our sound preliminary data, we expect to identify a new MAIT-dependent mechanism of immune surveillance of the intestinal microbiota. Future manipulation of this interaction may offer innovative therapeutic opportunities against intestinal inflammation as well as colonic carcinogenesis.