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.

Many molecules dedicated to therapy are nowadays lately removed from the pipeline of drug discovery, in the clinical phases, because their toxicity or lack of efficacy has not been evidenced in preclinical studies. Researchers and pharmaceutical companies outline the critical need in the development of advanced in vitro trials, looking for human cells based organoids able to improve screening’s efficiency and to reduce the number of animal trials, following the 3R rule. Recent progresses in bioengineering and microtechnology pave the way to “organ-on-chip” devices ensuring 3D cell culture in conditions close to physiology. However, such models are still simplistic and not strongly validated. Based on this analysis, MimLiveronChip proposes a bioinspired approach to mimic the mandatory steps allowing the study of xenobiotic’s toxicity and metabolism in the liver. The original choice is to focus on and to reproduce two key events in series: not only the biotransformation by hepatocytes of substances transported by the blood flow, but also (and upfront), their transfer (hindered or not) across the monolayer of endothelial cells, that are very specific in the liver: they are fenestrated in healthy conditions, and lose these properties in early stages of the pathology (steatosis, fibrosis, …). In this project, we will investigate the relevance of several hypotheses regarding the effect of the mechanical and biochemical micro-environment on this fenestrated status, so as to maintain it or in contrast alter it to mimic pathological cases. Our project also addresses technological issues. As end users, we know that microfluidic devices might appear complex compared to classical 2D culture. Therefore, we propose to develop a fully integrated platform, equipped with flow and pressure controllers, as well as sensors to monitor the cell culture and allow mid throughput assays for drug screening. The newly developed tools will be benchmarked with up-to-date technology employed in CRO or pharma companies. Our goal is to position the organ-on-chip platform in the pipeline of preclinical trials, combining toxicity and metabolism evaluation. This multi-disciplinary project relies on a close collaboration among four teams bringing complementary expertise: hepatic tissue engineering at different scales (UMR UTC-CNRS Biomechanics and Bioengineering); microsystems and biology of endothelial cells (SMMIL-E UMI CNRS), as far as academia is concerned, to which can be added : a SME leader in devices for microfluidic experiments (Fluigent) and a startup dedicated to the development of advanced in vitro trials based on high throughput imaging (HCS Pharma). Preliminary common studies have demonstrated the relevance and the feasibility of the proposed methodology. Regarding socio-economical features, the work performed in our project should allow the reduction of attrition rate for drug candidates in the late clinical stages. This will thus decrease the development costs and duration in the pharmaceutical industry, but also for chemicals, in the framework of REACh regulation, for the assessment of the effect of pesticides, and in agro-industry (nutraceutics). Mid throughput cell culture devices developed in MimLiveronChip can also be used for more fundamental research (system biology, regeneration studies), as they better mimic liver structure and functions. Both companies involved in the project will benefit from direct positive feedback since their portfolio will be enriched with new equipment and screening methods, respectively. Finally, demonstrating the benefit of coupling the endothelial hepatic barrier with the 3D hepatocyte transformation unit will offer many innovative strategy integrating other organs on chip, developed in the academic labs involved in the project.