Chronic inflammatory diseases (IDs) are the third cause of death in developed countries, after cancer and cardiovascular disorders, and their prevalence is growing in westernized countries. These diseases constitute a heterogeneous group of illnesses, including non-exhaustively, rheumatic diseases (rheumatoid arthritis (RA)), autoimmune systemic diseases (systemic lupus erythematosus (SLE)), and inflammatory bowel disorders (IBDs). All these diseases, which appear clinically different, share many similarities, such as common genetic background, common pathophysiological pathways and not surprisingly similar treatments. They are characterized by an autoimmune response with circulating autoantibodies secreted by B cells, which are activated by a specific subset of effector CD4+ T cells, follicular helper T cells (Tfh). Tissue lesions in these pathologies involve another subset of effector T cell, the IL17-secreting T cells (Th17). Interestingly, we recently highlighted the crucial role of CD95L in SLE pathogenesis. CD95L (FasL) belongs to the TNF family. While its receptor CD95 (Fas) is ubiquitously expressed, CD95L is mainly detected at the surface of lymphocytes and NK cells where it plays a pivotal role in the elimination of infected and transformed cells. CD95L is a transmembrane protein acting through cell-to-cell contact but it can be cleaved by metalloproteases, releasing a soluble ligand (cleaved CD95L or cl-CD95L) whose biological function remains to be defined. We observed that cl-CD95L is increased in lupus patients, and this soluble ligand aggravates inflammation in SLE by inducing non-apoptotic signaling pathways (NF-?B and PI3K). CD95 harbors an intracellular stretch designated death domain (DD). Binding of membrane-bound CD95L to CD95 leads to the recruitment of the adaptor protein FADD via the DD. FADD in turn aggregates the initiator caspase-8 and caspase-10. The CD95/FADD/caspase complex is called death-inducing signalling complex (DISC) and implements the apoptotic signaling pathway. In contrast, cl-CD95L fails to form DISC, but instead triggers the formation of a non-apoptotic complex termed motility-inducing signaling complex (MISC) inducing a Ca2+ response. Recent data from our group highlighted that this Ca2+ response occurred through the direct recruitment of PLC?1 by CD95. Indeed, in presence of cl-CD95L, the juxtamembrane region of CD95, called calcium-inducing domain (CID), binds PLC?1 to induce endothelial transmigration of Th17 cells in SLE (Immunity, 2017). Moreover, a chimeric molecule consisting of the CID conjugated to the cell-penetrating domain (designated TAT-CID) binds PLC?1 and prevents its recruitment to CD95. Strikingly, injections of TAT-CID in lupus-prone mice dampen the accumulation of Th17 cells in inflamed organs and alleviate clinical symptoms. Our preliminary data indicate that cl-CD95L also triggers endothelial transmigration of Tfh cells and this process is inhibited by TAT-CID. Furthermore, cl-CD95L favors the activation of Tfh cells and by doing so, their ability to promote the differentiation of B cells into Ig-producing plasma cells. These data urge us to investigate the molecular mechanisms by which cl-CD95L stimulates Tfh cells and decipher whether the therapeutic effect of TAT-CID in lupus-prone mice is related to a combined action on Th17 and Tfh cells. Our consortium intends to address whether i) cl-CD95L is increased not only in the sera of SLE patients but also in those of RA and IBD patients and ii) how this ligand promotes migration/differentiation in Th17 and Tfh cells. Also, using Protein-fragment complementation assay (PCA), high-throughput screening will be performed iii) to identify new inhibitors of CD95/PLC?1 interaction. In conclusion, this project will extend our observations on the role of cl-CD95L in several Th17 and/or Tfh-driven IDs and translate those results into innovative therapeutic molecules for ID patients.

The study aims to characterize how acyl-CoA synthetase 4 (ACSL4) regulates kidney function by controlling the functions of the thick ascending limb of the loop of Henle (cTAL) and the juxtaglomerular (JG) apparatus, two major structures regulating the renin-angiotensin system and salt and water conservation. ACSL4 sensitizes cells to ferroptosis, but we believe that it plays a crucial physiological role in renal function through its impact on membrane lipid composition. ACSL4 enriches membrane phospholipids with arachidonic acid (AA). We conducted lipidomic analysis of tubular segments isolated from mice and found that cTAL is predominantly enriched in phospholipids containing AA. We also have shown that ACSL4 is predominantly expressed in the cTAL segment and especially in the macula densa. We hypothesize that ACSL4 is involved in provision of the appropriate lipid environment for cTAL transporter NKCC2's activity, as well as the requisite amount of AA-containing phospholipids for prostaglandin E2 (PGE2) production and renin-angiotensin system activation. In this model, inhibiting ACSL4 would reduce NKCC2 activity or expression, leading to a decrease in NaCl reabsorption by the cTAL, and reduce AA incorporation into membrane phospholipids and PGE2 production, which would decrease activation of the renin-angiotensin system. Our aims are to: 1) demonstrate that ACSL4 enriches phospholipids with AA in cTAL and that suppression of ACSL4 disrupts cTAL phospholipid composition and affects cTAL function and JG apparatus activity in vitro and in vivo; 2) demonstrate that ACSL4 activity is involved in the progression of chronic kidney disease, and 3) screen and test novel molecules that specifically inhibit ACSL4. If we demonstrate the importance of ACSL4 in regulating glomerular hemodynamics, we will be equipped with evidence to examine small molecule inhibitors of ACSL4 with reno protective properties, allowing for new nephroprotection management options.

Cellular stress induced by the abnormal accumulation of improperly folded proteins in the endoplasmic reticulum (ER) is emerging as a major actor in disease development and an appealing actionable target. ER stress levels are under constant surveillance by the unfolded protein response (UPR), a major adaptive mechanism that lies at the core of cellular homeostasis and is responsible for cellular life-or-death decisions. The Inositol-requiring enzyme 1 (IRE1), the most conserved UPR transducer, is an ER-resident transmembrane protein with a cytosolic dual kinase/RNase activity controlling pro-survival or pro-death signals. Yet, despite >20 years of investigations, the precise molecular mechanisms by which IRE1 is activated and exerts its catalytic and scaffolding functions still remain unclear and a subject of debate. Major discoveries over the past year did not completely solve the mechanisms underlying IRE1 signaling and highlighted discrepancies in vitro and in cellular models that currently raise a plethora of new questions. The INSPIRE1 project aims to decode IRE1 molecular mechanisms of activation through a unique and novel prism at the interface of chemical biology, supramolecular chemistry and structural biology to provide a timely breakthrough in addressing this knowledge gap. At the core of the project is an integrated approach relying on three synthetic nanoplatforms, each tailored to study and address specific questions in a well-controlled environment. This unique and novel strategy promises to overcome the current limitations that hinder the full comprehension of IRE1 signaling mechanisms. The functional and structural knowledge gained will have far-reaching implications for the biological understanding of IRE1-dependent disorders and for harnessing IRE1 as a potential therapeutic target.

The incidence of Non-alcoholic fatty liver disease (NAFLD) is constantly increasing, owing to the obesity epidemic. The first step in the course of NAFLD progression is hepatic steatosis. Steatosis can progress to nonalcoholic steatohepatitis (NASH), a progressive disease defined as a combination of lipid accumulation, hepatocyte death, inflammation and activation of fibrogenic pathways that may lead to liver fibrosis, cirrhosis, liver failure and/or hepatocellular carcinoma. Therefore, it is important to elucidate the molecular mechanisms underlying the steatosis-NASH transition to propose new therapeutic avenues. There is a major unmet clinical need, as no efficient pharmacologic treatment is available, yet. We hypothesized that, aberrant activation of the ER stress sensor Inositol-requiring enzyme 1 (IRE1) could be crucial in the steatosis-NASH transition. IRE1 RNase is involved in two major signaling pathways including first the non-conventional splicing of XBP1 mRNA and second, the degradation of mRNA and miRNA, an activity named Regulated IRE1 Dependent Decay of RNA (RIDD). While XBP1 has been reported to be involved in cell fate and hepatic lipid homeostasis, the RIDD is much less characterized. We identified bax-inhibitor-1 (BI-1), the endogenous inhibitor of IRE1 (PNAS, 2006, JBC 2010). The analysis of human NASH liver biopsies revealed that BI-1 gene downregulation parallels to the upregulation of IRE1 RNase signaling (CDD 2015, Hepatology, 2018). BI-1 deficient mice challenged with a diet-induced obesity (HFD) mimicked NASH with overwhelmed hepatic IRE1 RNase. Pharmacologically targeting IRE1 RNase rescues HFD-BI-1 deficient mice from NASH suggesting that, inhibiting IRE1 could be effective in NASH. Our aims are: 1) To identify the IRE1-dependent mechanisms (cell death, inflammation, fibrosis) driving-NASH. a) We will determine the cell type in which, IRE1 RNase drives the steatosis-NASH transition (hepatocytes or parenchymal cells). b) Accordingly, we will identify the XBP1 and RIDD networks in cell death, inflammation, fibrosis involved in NASH pathogenesis by using transgenic mice specifically silenced for XBP1 and RIDD respectively, in hepatocytes (or macrophages). 2) To determine the mechanisms of IRE1 activation. Lipids can directly induce IRE1a. a) We will determine a lipidomic-signature specific to NASH progression (sera from patients). b) Accordingly, we will test whether the "identified-lipid-subspecies" trigger IRE1-signaling in hepatocytes and macrophages. 3) To test therapeutic strategies inhibiting IRE1-dependent cell death and inflammasome in NASH animals. Strategies with IRE1 RNase pharmacological inhibitors or genetic probes (Adenovirus BI-1) will be tested in preclinical models of NASH. 4) To identify an hepatic IRE1 signature specific to NASH progression. Monitoring the level of IRE1 RNase in NASH patients could represent a new companion diagnostic and prognostic for NASH. The research program will bring together a complementary team of experts in ER stress and metabolic diseases (steatosis, NASH and diabetes) with advanced research skills in translational NASH research (patient samples, nutritional/transgenic NASH models), cell signaling, the UPR, omics technologies (genomics, proteomics, metabolomics). This proposal is based on strong preliminary data obtained by the 3 partners (Hepatology, 2018, Diabetologia 2018, EMBO Mol Med 2018). Overall the complementary expertise of the partners (Nature Reviews Endocrinology 2016, Nat Cell Biology 2015) will bring synergy to the outstanding realization of the research program. By means of our research program, we hope to understand the central role of IRE1 in NASH to validate the therapeutic relevance of targeting IRE1 RNase in NASH. We propose that pharmacologically targeting IRE1a RNase will be effective also in metabolic diseases linked to excessive IRE1 RNase activity such as type 2 diabetes and arteriosclerosis.