Osteoarthritis (OA) is the most frequent joint disease in adult. It is responsible for disability and increased mortality. There is no efficient treatment and OA leads to cartilage degradation and joint destruction. Cartilage calcification favors cartilage degradation and worsens OA disease. It is composed of calcium orthophosphate, mainly as carbonated apatite, and calcium pyrophosphate dihydrates (CPPD) crystals. These crystals stimulate the production of catabolic mediators and proteolytic enzymes by joint cells. How these crystals activate cells remains unclear, especially their interactions with cell membrane and intracellular outcomes. Moreover, their individual role in OA is unknown since current imaging techniques possess low capacity to discriminate apatite from CPPD crystals. OASIS project aims: 1/ in fundamental studies to decipher calcium crystal-cell membrane interactions, crystal internalization and intracellular crystal outcomes and ; 2/ in applied issues to optimize spectral multi-energy computerized tomography (SPCCT) acquisition and analysis procedures allowing discrimination between apatite and CPP crystals in OA patients. OASIS project involves a consortium of 5 multidisciplinary academic partners (physicians, chemists, cell biologists, physicists and computer scientists) and an industrial partner (Canon Medical System), well-renowned as CT constructor. This consortium possesses complementary skills including a unique know-how in the synthesis of pure phase apatite, CPPD crystals and fluorescent organic nanoparticles (FON), a mastery of cellular and physico-chemical analysis techniques (Raman spectroscopy, X-ray diffraction, atomic force microscopy (AFM)), deep knowledge of scanner procedures and the access of cohort of well-phenotyped patients and the last spectral CT prototype (AQUILION PRISM). We achieved for the first time to obtain stable FON-coupled calcium phosphate crystals which will allow their internalization and intracellular follow-up. In addition, first tests showed the ability of the spectral CT AQUILION PRISM to differentiate CPPD crystals contained in quadriceps muscle from apatite contained in the femur. These preliminary results guarantee the success of this project. This project will include nanoscale characterization, cellular and animal studies and end with application in patient care. FON-coupled crystals will serve to study their cell membrane interactions and intracellular outcomes using AFM and confocal microscopy in artificial membrane, macrophage and in vivo models. Blocks of apatite or CPPD crystals with different size and mass, grafted in mouse quadriceps and then embedded in resin, will permit ex-vivo identification of SPCCT optimal parameters that discriminate apatite from CPPD crystals. These parameters will be applied in OA patients who will undergo total knee joint replacement surgery. SPCCT results will be compared to ex vivo analysis performed on surgical samples. The capacity to differentiate apatite from CPPD crystals will be a major step in the understanding of OA pathogenesis and allow a better disease management. OASIS project possesses high valorization potential in fundamental and applied issues. Identification of cellular activation mechanisms will open new therapeutic targets and understanding of calcium phosphates crystal internalization mechanisms can be generalized to other inorganic particles. Similarly, the SPCCT diagnosis procedures that permit discrimination between apatite and CPPD crystals can be applied to other calcification tissues and fields including kidney stones, vessel calcification, tumor calcification, etc… These procedures will be integrated in software of future CT.

Osteoporosis, the major cause of fractures, results from an imbalance between bone formation and bone resorption. While accelerated bone resorption characterizes postmenopausal bone loss related to estrogen failure, reduced bone formation is responsible for idiopathic and age-related bone loss. The objective of TARGETBONE is to provide an integrated understanding of the cellular and molecular mechanisms that underlay the pathophysiology of osteoporosis focusing on the differentiation process of Bone Marrow Mesenchymal Stem Cells (BM-MSC) and bone marrow progenitors towards the osteoblastic lineage. The transcription factors Dlx5 and Dlx6, which play an important role in osteogenesis, are expressed by BM-MSC and are upstream regulators of Osterix (Osx) and Runx2, two master genes involved in the induction of osteoblast differentiation. Our aim is to characterize the cellular and molecular factors that promote BM-MSCs differentiation modifying directly the function of Dlx5 and Dlx6 in BM-MSCs or in more differentiated progenitors in vivo and in vitro. To this end, we will generate the conditional deletion of Dlx5/6 in mouse osteoblast progenitors by crossing our recently generated Dlx5/6Hboxlox/lox with Osx-creERT2 mice to obtain Dlx5/6Osx-creERT2 mice in which Tamoxifen injection will permit to delete both gene in Osx-expressing precursors. The bone phenotype of these mice will be studied, and BM-MSCs and more differentiated osteoblasts will be cultured and analysed. Complementary genetic, viral and siRNA strategies will be used to analyze the effect of Dlx5/6 on the differentiation potential of cultured BM-MSC from mice and from humans. The capacity of normal and ablated cells to differentiate towards the osteoblastic or adipocytic lineage will be tested using standardized molecular approaches. The interaction of Dlx5 with the adaptator protein MAGED1 is required to determine the cellular and/or transactivating activity of Dlx5 during osteoblast differentiation. We will characterize the protein complexes which include Dlx5 by analyzing through mass-spectroscopy the proteins that are coprecipitated with Dlx5 and/or MAGED1. The conformational interaction of Dlx5 and MAGED1 will be investigated through crystallography and in silico analysis with the objective to identify new protein-preotein interactions which could become targets for drug discovery. The identification of new molecular targets will prompt the design of innovative molecular tools to promote bone anabolic response. To further translate the impact of our findings to osteoporosis, we will analyze the osteogenic functions exerted by DLX5/6 in human bone and human BM-MSCs from different ages. The strength of TARGETBONE resides in the multidisciplinary nature of the consortium. The combined expertise of three complementary teams expert in molecular biology, drug discovery and osteoporosis provides a unique opportunity to transfer this new knowledge from basic science to clinical applications.

CONTEXT: T lymphocytes are critical cellular actors in the host response to various pathogens i.e. viruses, bacteria and fungi and in the control of malignant cell transformation. Importantly, the thymus ensures a continuous production of naïve T cells with a diverse TCR repertoire capable to mount effective immune responses. However, this organ undergoes severe changes upon aging in its mass and architecture, accompanied by a progressive decrease in T cell production, a process referred to as “thymic involution”. Furthermore, the thymus is highly sensitive to myeloablative treatments used to cure hematological disorders such as genetic disorders or cancer by hematopoietic stem cell transplantation. Both aging and myeloablative treatments have deleterious effects on thymic epithelial cells, leading to a drastic reduction in T cell production and consequently to increased susceptibility to opportunistic infections, autoimmunity and cancer. Nevertheless, the thymus shows a high plasticity and is prone to regenerative therapies. Although in mice, certain growth factors or cytokines protect or regenerate thymic epithelial cells and improve thymopoiesis, their efficacy in humans in preclinical trials either failed or remains to be determined. Identifying effective therapeutic molecules to regenerate thymic functions thus still constitutes an unmet clinical need. In this context, we recently showed in mice that the administration of the epithelial growth and differentiation cytokine, RANK ligand (RANKL), ameliorates the regeneration of the thymus and T-cell reconstitution upon myeloablative treatments and bone marrow transplantation (BMT) (Lopes et al. EMBO Mol Med 2017 et brevet WO/2018/154122). OBJECTIVES: The objectives of this project are to determine: (1) whether T-cell production improved by RANKL enhances protection against lethal opportunistic infections in a pre-clinical BMT mouse model, (2) to what extent RANKL is beneficial to reverse age-related thymic involution, (3) the impact of RANKL on the prevention of bone loss by bisphosphonates upon BMT and thymic involution, (4) the translational potential of using RANKL to regenerate human thymic functions. In summary, the ambition of this project is to bring the proof of concept that RANKL could be clinically pertinent to improve the recovery of T-cell functions without inducing bone loss in pathophysiological conditions in which the thymus has been severely damaged. CONSORTIUM: This project is a close collaboration between three French research teams. It brings together a fully complementary expertise on the biology of thymic function in mice (Team #1) and humans (Team #2) as well as on bone physiology (Team #3). BIOMEDICAL RELEVANCE: This project is expected to improve the immune system by acting on the production of T lymphocytes by regenerating the thymic function. We are convinced that the expected results could have a significant impact on public health, given the incidence of 20 to 30% of mortality related to infections after HSC transplantation and the demographic increase in the number of elderly people in our societies, a phenomenon also associated with a high rate of infections. This project has the potential to improve the health of many patients who incur morbidity and mortality due to T cell deficiency and reduce the associated medical costs.

Osteoporosis is a common age-related disorder characterized by low bone mass and deterioration in bone microarchitecture, leading to increased skeletal fragility and fracture risk. Age-related osteoporosis fractures are increasing in link with the proportion of elderly population is increasing, thus resulting in human burden for the health system. Beside the efficacy of antiresorbing drugs at stopping bone loss, the major question arising now is how is it possible to restore lost bone? For this aim, there is a crucial need for identifying new targets especially on bone forming cells (osteoblasts). Our project will focus on the lysophosphatidic acid (LPA)-producing enzyme Autotaxin (ATX) because LPA has an anabolic action on bone by activating osteoblasts. However, LPA also promotes osteoclastic bone resorption. Thus, specific therapeutic blocking of LPA’s catabolic activity could promote its anabolic action on bone tissue. ATX is the main producer of LPA in the organism. Remarkably, global deletion of ATX gene (Enpp2) is lethal at the embryonic stage making impossible the use of mice for bone study. Nevertheless, the choice of ATX was supported because of our recent observations in MC3T3 cell line and calvaria primary osteoblasts that express high levels of ATX making osteoblasts a potent source of LPA at the bone site. The first objective of the project is to elucidate the role of ATX/LPA axis during bone mass acquisition in youth and bone loss in aging and to determine its impact on osteoblast function and bone quality. For this aim we will analyze ATXdeltaOb mice already generated by crossing Enpp2flox/flox mice with Osx:GFP-Cre/+ animals allowing specific invalidation of ATX expression in osteoblast progenitors and hypertrophic chondrocytes. These animals present a remarkable low bone mass phenotype. Animal bone phenotype will be characterized based on technics used in routine in partners’ laboratories such as microCT, histology, immunohistochemistry, fluorescence imaging. The second objective of the project is to characterize novel signaling pathways that control the anabolic activity of osteoblasts and that connect osteoblast to osteoclast functions. Our project will decipher in primary mouse and human osteoblasts the molecular connections between Wnt/beta-catenin and ATX/LPA signaling pathways that have recently been revealed in malignant cells allowing the identification of potential new therapeutic targets. The third objective of the project is to develop new therapeutic tools promoting bone gain. To this aim we will characterize at molecular levels domains of ATX that bind to cell surface of osteoblasts and osteoclasts because of ATX new role emerging as a docking molecule required for the proper presentation of LPA to the cell surface, leading to the activation of specific LPA receptors. We and other have shown that ATX binds to beta-1, beta-3 integrins and heparan sulfate proteoglycans. We have demonstrated that ATX controls osteoclastic bone erosion in inflammatory conditions. Because of the paramount role of beta-3 integrin in osteoclast function, we will develop a unique strategy in the field by performing in silico proteochemometrics studies based on artificial intelligence followed by co-crystallography and biochemical analyses of ATX with identified beta-3 integrins interactants that will be validated both in osteoclast activity and osteoblast/osteoclast coculture assays. Functionally active peptides will be PEGylated to increase stability in vivo and used in our preclinical animal models of osteoporosis as a proof of concept in the development of new therapies against bone loss. Altogether, based on animal whole body bone characterization, histology, osteoblast and osteoclast cell biology, in silico proteochemometrics and crystallography analyses, the project will fully characterize the molecular of actions of ATX on bone cells and will develop new tools dedicated to bone regeneration therapies.