Background : Fatty acids (FAs) appear to be essential for macrophage plasticity as they are ideally positioned at the crossroads between anabolic and catabolic pathways. Macrophages possess the ability to modulate dynamically and autonomously their intracellular FA metabolism including FA oxidation and FA synthesis. As observed for cholesterol, beyond the purely quantitative accumulation of fatty acid in the cells, it is likely that qualitative alterations of fatty acid profile and distribution may affect some biological functions of the macrophages either through the production of bioactive lipid mediators or to changes of cell membranes properties. Some recent studies support that view and have demonstrated that genetic modulation of FA pathways even though restricted to myeloid cells directly affects macrophage functions and subsequently influences atherosclerosis development. By using targeted lipidomic and transcriptomic approaches, we have characterized a tightly regulated set of genes that control membrane polyunsaturated fatty acid (PUFA) composition in macrophages. These genes are involved in the elongation of PUFAs (ELOVL5) and in their incorporation into phospholipids (LPCAT3). ELOVL5 and LPCAT3 display a strong specificity toward n-3 and n-6 PUFA with 20 carbons including arachidonic acid (AA) and eicosapentaenoic acid( EPA). Macrophages deficient for Elovl5 and/or Lpcat3 display dramatic alteration of AA and EPA metabolism. Unexpectedly, the TLR-sensitive transcription factor IRF5 (interferon regulatory factor -5) has been identified as a potential transcriptional regulator of both LPCAT3 and ELOVL5. Aims : The overall concept of this project is to demonstrate that the LCPAT3/ELOVL5 axis modulates atherosclerosis development by controlling key macrophages functions. Our research program will combine in vitro and in vivo approaches using genetically engineered mouse models and translational studies in human populations First, by using Lpcat3KOMAC and Elovl5-/- mice we will explore the role of the LPCAT3/ELOVL5 axis in macrophages in the context of atherosclerosis and we will decipher the molecular mechanisms involved. We will also test the ability of Elovl5/lpcat3 to modulate the antiatherogenic action of eicosapentaenoic acid (EPA) in a mouse model of atherosclerosis. In a second WP, we will investigate the involvement of IRF5 in the reprogramming of FA metabolism through ELOVL5 and LPCAT3 during inflammation. We will explore the interplay between IRF5, LPCAT3/ELOVL5 pathways, PUFA composition and synthesis of pro-resolving mediators following LPS activation. Additionally, we expect to characterize the atherogenic function of IRF5 in macrophages, which is to date unclear. In WP3, we will perform translational studies to validate our hypothesis and to determine whether biomarkers of LPCAT3/ELOVL5 activity are associated with CV risk in selected human populations, including Type 2 Diabetic subjects with low versus high cardiovascular risk. Finally, we will analyze whether these markers have significantly different levels in stable Vs vulnerable regions of human carotid atherosclerotic plaques. Perspectives : Our project fits into a context in which new therapies targeting macrophages and inflammation appear as potentially effective strategies for atherosclerosis treatment and prevention. On the one hand, this program may unravel new and original mechanisms associating fatty acids and macrophage activation thus paving the way for the development of therapeutic strategies. On the other hand, this project should lead to the identification of new biomarkers of CV risk, thus allowing earlier detection and improving the stratification of high-risk patients. Finally, our program should also provide new insights into the mechanisms that account for the potential atheroprotective effects of EPA at a time when the impact of omega 3 fatty acids on the cardiovascular risk is being reevaluated.

Women's fertility is linked to their oocytes quality. Indeed, before ovulation, oocytes have to resume their meiosis, then to activate the zygote upon fertilization and finally to support the embryonic development for the 5 days of tubal transit before implantation in the uterus. This represents a burst of energy, which is mostly supported by their mitochondria. But their mitochondrial activity permanently decreases with the women’s age leading to aneuploid oocytes and infertility. Furthermore, nowadays young women are increasingly delaying their first pregnancy for social reasons. Indeed, today, the age of the first delivery is at 31, compared with 23, 30 years ago. It reduces dramatically the length of their genital life, since the oocytes quality drops dramatically around 37 years old. It is a major social problem leading to procreative tourism towards countries with less care to ethical behavior and women's rights, since Assisted Reproductive Technology (ART) by itself does not improve the oocyte’s quality. Actually, before 35 years old an oocyte has 4.5% chance to give a child: at 40 years old, it takes 140 oocytes to have a baby, while the ovarian reserve falls. Many attempts have been run to solve the problem, mostly based on mitochondrial transfer. They are either banned because of heteroplasmy or are difficult to implement requiring surgical procedures and ovarian biopsies prior to ART attempt. The Fertiline molecule that we discovered (Patents No. FR0313545 and No. FR1558899) represents a real new solution. Just supplementing the medium with the molecule, it improves the gametes metabolism of energy. In mice, it increases the fertilization rates, the blastoformation by more than 20% and the percentage of healthy offsprings per embryo transferred by more than 30%. In a prospective randomized clinical trial, authorized by the “Agence de la BioMedecine” (ABM) (Clinicaltrial.gov: NCT02161861), it improved the in vitro maturation rate of immature oocytes by more than 50% and reduced the miscarriages rate, suggesting an improvement of the oocyte and embryo ploidy. A complete transcriptomic analysis of embryos will be conducted by RNA seq, CGH array and methylome analysis in three species, with particular attention being paid to imprinted genes expression. - In mice which has the advantage of quick gestation, - In bovine which, as human being, belongs to mono-ovulating species with heterogeneous follicular cohorts, - In humans, using embryos donated for research. Our project will be complemented by a prospective, randomized clinical trial at the Cochin Hospital for which the authorization is being asked to ANSM and ABM who already authorized the first clinical trial. Our hypothesis is that in humans Fertiline should significantly improve the percentage of mature oocytes and baby born by ART, even for women over 37 years old, thus answering to the Societal Challenge 4 "Life, health and well-being”. The study of the mechanism of action of the molecule will furthermore be a very significant scientific advance in the understanding of cellular mechanisms of energy regulation, and the production of transcriptomic data in three species an important step in the comparative analysis of species evolution. Our consortium combining fundamental research, exploration in several species and a clinical application will be able to ensure the success of the project. There are 140,000 attempts of ART in France, more than one million in the world for which more than 40% of women are over 35 years old. It is therefore a major issue for women’s fertility and couple’s life.

Because of ageing, various life trauma and bone diseases, bone grafts and bone substitutes are being used to replace missing or no longer viable bone tissue. The number of bone grafting procedures will in the next decade reach 500 000 in 2020 in Europe and the sales volumes of high performance synthetic bone substitute grafts with the attractive price tag could reach 4 million cm3 by 2020. Among the synthetic bone substitutes, silicate-based bioactive glasses have clinically proved to be as efficient as autograft in the long term but lack short term performances which are not yet optimal for spinal surgery, fracture consolidation and alveolar ridge augmentation in dental implantology, for which the bone tissue regeneration is needed within four months. The PyVerres project intends to develop (from fundamental to applied aspects), new pyrophosphate-based glasses (PP-glasses) to regenerate bone tissues in orthopedics and dental applications. These biomaterials, recently patented by CIRIMAT, are composed of pyro- and orthophosphate entities and calcium ions. Their elaboration by soft chemistry (40°C maximum, including the drying steps) and their structure have never been described in the literature before. The nature of these materials and their synthesis process are in line with the need to reduce the manufacturing costs, i.e. cheaper raw materials, low temperature and solvent-free elaboration processes. From the biological point of view, their novelty is mainly associated to the pyrophosphate entities which can be hydrolyzed into orthophosphate ones thanks to chemical and enzymatic (phosphatase enzymes naturally produced by osteoblasts) activities. Their expected tunable properties by biochemical control of the resorption rate (chemical and enzymatic hydrolysis triggered by the pyro/ortho-phosphate ratio) open the way to the design of materials able to induce a specific biological response, and act as biologically active inorganic glasses enhancing the bone healing process. Most importantly, this project aims at assessing the PP-glasses technology and transferring the low-temperature solvent-free synthesis process and characterization of these novel tunable bioactive glasses to a SME company, Noraker which has successful experiences in technology assessment and has already brought to the market several innovative bioactive glass based products from academic collaborations. To ensure to reach these main R&D objectives, fundamental chemical, physico-chemical and biological studies need to be carried out to establish a link between the elaboration process, the glass compositions and their biological properties. Therefore a consortium has been formed with 5 academic and 1 industrial partners, providing complementary domains of knowledge and expertise for the good development of this inter- and multidisciplinary PRCE project. The consortium includes two Carnot Institutes, CIRIMAT in Toulouse and Chimie-Balard in Montpellier, the LCMCP in Paris, BioTis U1026 in Bordeaux, B2OA in Paris and Noraker company in Villeurbanne. The work program includes a 1st step dedicated to the elaboration of glasses by screening synthesis conditions such as composition, pH, temperature, followed by the technology transfer of the low temperature synthesis process to Noraker and, fine structural, morphological and thermal characterizations and modelling. The in vitro study of these glasses reactivity in biological fluids is the 2nd important step, necessary to anticipate the physico-chemical phenomena occurring at the surface or within of the glass in presence or absence of enzymes. Then, biological tests will be conducted: i) in vitro to study the biological response of cells and also of phosphatase enzymes depending on the composition of the glass (pyro/ortho-phosphate ratio) and, ii) in vivo through the animal implantation of new glasses to study their biocompatibility, osteoconductivity and resorbability kinetics compared to commercial bioglass.

A large number of RNA viruses are neurotropic and can cause a variety of neurological disorders. The outcome of neurotropic viral infection depends on the virus and the infected neurons. In neurons, many viruses undergo a switch to a persistent infection, which is often accompanied by a drastic reduction in production of cell-free viruses. However, despite reduction of extracellular viruses, many neurotropic viruses can efficiently, disseminate within the Central Nervous System (CNS) suggesting a cell-to-cell transmission pathway that differs from the canonical spread in the periphery. The overall goal of this application is to elucidate how neurotropic RNA viruses can spread within the CNS. We will focus on two human pathogens, measles (MV) and Nipah (NiV) virus that belong to closely related single stranded RNA virus Paramyxoviridae family, with a common transcription and replication strategy. MV is restricted to humans leading in most of cases to mild, although sometimes severe and deadly disease. NiV has very high pathogenicity in humans (lethality up to 90%), can infect different animal species and requires BSL4 facility for the analysis. While Nipah virus is a new recently emerged virus, measles is re-emerging with regular outbreaks around the world, including industrialized countries. Both viruses could infect neuronal cells and cause lethal encephalitis as well as persistent brain infection, giving a relapse several months or years after primary infection. However, while CNS infection in measles is a rare event, in Nipah it is very frequent. Viral neuro-pathogenesis remains in both cases poorly understood. In the proposed studies, a combination of brain organotypic and neuronal cell culture system from transgenic mouse models will be used to analyze the spreading of these two neurotropic RNA viruses in the CNS with the expectation that comparison of these two viral infections will bring further insights in each of them. I aim at answering the five related questions (i) Do the viruses use known or alternative cellular receptor(s) to enter and spread throughout the CNS? (ii) Which glycoprotein(s) is(are) necessary/involved in entry/cell-cell spreading? (iii) How the viral core is transported from virus entry to the virus exit site assuming the cell-cell spreading? (iv) Are viral protein constituents properly assembled to give rise to virus particles responsible for the cell-cell spreading? (v) What governs the possible selectivity of virus propagation through limited neuron network(s)? In vitro and ex vivo MV and NiV infection studies will benefit from the following tools: (i) primary cultures and brain explant cultures from transgenic murine models expressing or not known viral entry receptors, (ii) molecular tools blocking the viral entry/fusion machinery and spread (iii) recombinant viruses either expressing glycoproteins blind for their receptors and/or lacking protein constituents the envelop. In addition, to identify the neuron subsets targeted by these viruses, we will build recombinant viruses expressing two fluorescent proteins with one made sensitive to the silencing by brain specific miRNA(s). The results from the proposed studies will ultimately be used to determine how acute and persistent RNA viral infections evolve to induce CNS disease. Elucidating the state of the virus during infection and mechanism of its interneuronal spread will hopefully pave the way for the design of therapies able to interfere with these processes and thus prevent fatal neurologic disorders induced by these viruses.

Liver is a vital organ, ensuring hundreds of metabolic functions and thus finely specialized for its metabolism, which consequently need from birth to adulthood to acquire its metabolic zonation and gradually enter into quiescence. Nevertheless, in response to liver damages, hepatocytes have the capacities to proliferate to maintain the hepatic mass and function, in cooperation with hepatic stem cells. This adaptative response driven by hepatocytes could favor the development of a chronic disease in case of persistent injuries. Chronic liver diseases emerge these recent years as a major health problem regarding to change in our eating habits, but also to abuse in alcohol consumption and infections by hepatitis viruses. Liver disease initiates with a single steatosis which could deteriorate into steatohepatitis (steatosis with inflammation, fibrosis and necrosis), and, finally, to cirrhosis and cancer. These last years, cumulative evidence showed that various microRNAs (miRNAs) extensively contribute to liver pathogenesis, from liver disease to cancer, and thus should constitute potent biomarkers and therapeutic targets for these pathologies. In particular, a cluster of 54 miRNAs originated from the imprinted DLK1/DIO3 locus appear as an attractive player for liver disease establishment since it is crucial both for stem cell and hepatocyte fate in mouse liver following activation of the WNT/ß-catenin signaling, a master regulator pathway for liver zonation and stem cell maintenance. Using deep-sequencing approaches in our mouse models, we importantly demonstrated that ß-catenin orchestrates a specific transcriptional program, resulting in a metabolic reprogramming and an exit from quiescence of hepatocytes, which could favor the establishment of a cancerous switch – an event observed in a third of hepatocellular carcinoma. In particular, we observed that ß-catenin promptly induced the expression of all the constituents of the locus, either expressed from the maternal or the paternal strand, dependently on methylation process and more importantly through direct binding of ß-catenin on its promoter region. In the next three years, using our mouse models, we will unveil how ß-catenin induces a loss of imprinting in the DLK1/DIO3 locus through epigenetic modifications and/or chromatin remodeling, and which factors are recruited in space and time by ß-catenin to modify the locus expression during liver pathogenesis. We will also address the functional consequences of the silencing of this locus by genome editing on hepatocyte metabolic and proliferative properties in different liver pathological contexts. This project deciphering the crosstalk between ß-catenin and epigenetic events (miRNAs, imprinting and chromatin remodeling) for gene expression should highlight new promising targets for regenerative medicine but also for different diseases associated to WNT/ß-catenin signaling such as metabolic syndromes or cancers.