The treatment of Inflammatory Bowel Disease (IBD) represents a major medical challenge. IBD is a highly debilitating disease, which incidence is constantly growing in developed countries. The current therapies are costly, present severe side effects, and a number of patients are resistant to any forms of treatment. Therefore, intensive research has aimed at understanding the mechanisms of those diseases, to define new potential targets. Using pre-clinical models, we have identified previously unknown anti-inflammatory properties for Elafin, a protein naturally expressed in the human gut, that is able to inhibit elastolytic activity. We then have cloned the gene of human Elafin and expressed it in recombinant bacteria that are present in daily food: the Lactococcus lactis, assuming that Elafin delivery to the gut by such a probiotic strain, would exert protective effects against colitis. Treatments with those recombinant bacteria drastically reduced inflammatory symptoms associated with IBD in animal models. However, animal models provide limited knowledge as they have an etiology different from the human disease, and very often do not implicate the same mediators. In addition, the mechanisms by which Elafin delivery to the gut would be protective are unclear. Protease inhibition is probably implicated, but potentially antimicrobial properties and inhibition of nuclear factors are also involved. The cells on which Elafin exerts its anti-inflammatory properties are also undefined. If Elafin-recombinant L. lactis has to be considered as a possible treatment for IBD in human, there is an absolute need to define the mechanisms by which such recombinant L. lactis strains protects against intestinal inflammation, in a context that would be as close as possible to the human disease and its mediators. The general objective of the project is to investigate the mechanisms by which Elafin delivery is protective against intestinal inflammation, and to determine whether Elafin delivery by lactic acid bacteria protects from the deleterious environment present in tissues from IBD patients. Specifically, we aim at determining the effects of Elafin-recombinant L. lactis on three general types of cells that are in contact with the recombinant LAB: 1/ Intestinal Epithelial Cells 2/ Mucosal Immune Cells 3/ Microbiota For all those cellular targets, we will determine whether Elafin’s effects are due to protease inhibition or to other properties, by comparing the effects of wild-type or mutated forms of Elafin recombined in L. lactis. This approach will shed definitive and unique light on the mechanisms of action of Elafin upon mucosal inflammation.

According to the World Health Organization, cardiovascular diseases are the leading cause of death worldwide, with the majority due to coronary heart diseases. It is expected that by 2030, almost 23.6 million people will die from coronary heart diseases. Understanding the underlying mechanisms thus represents a great challenge for basic scientists. Despite major progress in experimental reperfusion therapy, virtually no intervention has shown clearly protective effects against ischemia-reperfusion (IR) injury in the clinical setting, indicating that reevaluation of existing or development of new modalities to protect the heart from IR injury is urgently needed. Accumulating evidence indicates that reperfusion caused irreversible myocardial damage, possibly through a form of mitochondrial dysfunction named permeability transition. Although calcium (Ca2+) overload and excessive production of reactive oxygen species in the early minutes of reflow have been shown to trigger a nonspecific high-conductance channel (mitochondrial permeability-transition pore, PTP), its regulation is still unclear during reperfusion. Mitochondria and sarco/endoplasmic reticulum (SR/ER) have fundamental roles in energy production and Ca2+ transport, which are central in physiopathological functions of the cardiac muscle. Their involvement during myocardial IR begins to be recognized but the underlying mechanisms are not yet well understood. Recently the host lab and others demonstrated that mitochondria-SR/ER interaction sites allow mitochondria to sense SR/ER stress through local delivery of Ca2+ and other signaling entities. We have identified several proteins enriched at the SR/ER-mitochondria interface, including cyclophilin D (CypD) and glycogen synthase kinase 3 beta (GSK3ß), two potential PTP regulators, with functional and structural significance, thus highlighting the emerging role of this region within the cell. The general objective of this research program is to understand the role and mechanisms of the physical and functional Ca2+ coupling between SR/ER and mitochondria in the regulation of PTP during cardioprotection and to propose GSK3ß inhibition as a new therapeutic for patients ongoing acute myocardial infarction (AMI). Based on our recent study, we hypothesize that decreasing SR/ER-mitochondria Ca2+ transfer by targeting GSK3ß will promote cardioprotection after IR by preventing mitochondrial Ca2+ overload through the reduction of the local Ca2+ transfer from SR/ER to mitochondria. To determine the underlying mechanisms, Ca2+ imaging and proteomics will be conducted in both human and mice isolated cardiomyocytes. Thanks to the complementarity of the PI expertise with the host lab environment, we believe that this translational project might help to better understand the physiopathology and to identify new molecular targets for the treatment of cardiovascular diseases.

Bacterial infections constitute a major problem for human health. With the increase of antibiotic resistance, there is an urgent need for academic research to better characterize the virulence determinants associated to pathogens and envision new therapeutic strategies that are based on a deep understanding of the host-pathogen interactions involved in infection. Since innate immunity constitutes the first line of defense against bacteria, the molecular interactions underlying its activation are of particular importance. During infection, bacteria are detected through the recognition of pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors. Pathogen sensing leads to the secretion of inflammatory cytokines allowing the recruitment of immune cells to sites of infection. By applying a genome wide RNAi screening strategy, C. Arrieumerlou (coordinator, Institut Cochin) et al. recently identified a new cellular pathway regulating innate immunity during infection by several gram-negative bacteria known as major pathogens for humans. They showed that the proteins ALPK1, TIFA and TRAF6 act sequentially to induce the activation of NF-KB and the secretion of inflammatory cytokines in response to the detection of D-glycero-D-manno-heptose-1,7-bisphosphate (HBP), a newly described PAMP from gram-negative bacteria. HBP is a metabolic intermediate in the lipopolysaccharide (LPS) biosynthesis pathway. In this context, the HBPsensing proposal aims at characterizing the mechanism of HBP sensing that regulates innate immunity during infection by gram-negative bacteria. As with other PAMPs, we hypothesize that HBP sensing occurs through binding of HBP to (a) receptor(s) and that this recognition triggers a signaling cascade regulating inflammation, and thereby the onset of adaptive immunity. This new challenge of innate immunity will be tackled by a consortium of four teams, three of which at Institut Pasteur, with complementary expertise in chemistry, biophysics, biochemistry, infection biology and imaging. Our first aim consists in using state-of-the-art multistep chemical synthesis to obtain HBP and several analogues thereof. HBP is not commercially available and its chemical synthesis has not been described so far. Chemically defined HBP and the generation of different analogues will be essential throughout the project to dissect the HBP sensing pathway in absence of bacterial contaminants. The second aim is the identification of the cellular receptor(s) for HBP. This will first be addressed by RNAi screens and proteomics on ALPK1 binding proteins. If these approaches are not successful, we will capture HBP binding proteins from cell lysates by making use of HBP-based probes harboring a UV-reactive cross-linking group and biotin for purification. The HBP/receptor(s) interaction will then be addressed by biophysical methods. Our third aim is the molecular characterization of the HBP sensing pathway in epithelial cells and macrophages. In particular, we will investigate the role of ALPK1, an atypical kinase that is poorly described. We will explore the ALPK1/TIFA functional interaction and the role of ALPK1 in the regulation of IL-1b expression. Finally, our fourth aim is to obtain a spatiotemporal model for the mechanism of HBP sensing during S. flexneri infection of epithelial cells. Here, we will explore how HBP is delivered into host cells and monitor the localization of the main players of the HBP sensing pathway during infection by different imaging modalities, including super-resolution microscopy and correlative light electron microscopy. The HBPsensing project will reveal a new pathway regulating innate immunity during gram-negative bacterial infections and will provide a novel set of chemical tools to facilitate research in this emerging field. Moreover, the gained knowledge should help identifying new targets for treatments aiming at modulating inflammation in bacterial infections and sepsis.

Bone and skeletal muscle are closely linked across development, growth and aging. However, the functional interactions between bone and muscle have been poorly investigated. It is clear that a better understanding of the bone-muscle cross talks will be key to challenge numerous diseases and disorders of the musculoskeletal system. Musculoskeletal disorders affect 1 in 7 people (over 70 million in Europe). These disorders can cause long-term disabilities and have a considerable impact on public health. The goal of this proposal is to elucidate the cross talks between bone and muscle tissues at the cellular and molecular levels, in the context of tissue regeneration after combined trauma to muscle and bone. Under severe trauma, musculoskeletal injuries often cause damage of both skeletal and muscle tissues. Yet most experimental approaches address the repair of bone on one hand and the repair of muscle on the other hand. Although bone and skeletal muscle injuries trigger very efficient regenerative processes, through the activation of endogenous stem cell populations and release of growth factors within theses tissues, there are many clinical situations where bone and/or muscle regeneration need to be enhanced. We hypothesize that bidirectional interactions between skeletal muscle and adjacent bone, and most specifically the periosteum, are required during musculoskeletal regeneration, via contribution of stem cell populations and regulatory molecular systems from bone and muscle. Our preliminary data strongly suggest that bone-muscle bidirectional interactions exist at the cellular and/or molecular levels. In particular, we showed that muscle is essential to induce proper activation of skeletal stem cells within periosteum, a key cellular contributor to bone repair and a central tissue involved in these bone-muscle cross talks, as it lies at the interface between bone and muscle. We propose 3 aims, which will explore complementarily aspects of the role of bone-muscle cross talks during musculoskeletal regeneration at the tissue, cellular and molecular levels. We will define the stem cell contribution of periosteum and muscle to musculoskeletal regeneration, the role of the BMP pathways in mediating these molecular interactions and finally, we will aim to identify new mediators in these molecular cross talks between bone and muscle. We will use our well-established mouse models of musculoskeletal regeneration and various genetic tools to define the role of skeletal stem cells and muscle stem cells (i.e. satellite cells) during tissue regeneration. This project will combine the complementary expertise of the two partners in muscle and bone repair. By better understanding the cellular and molecular bases of musculoskeletal regeneration and the mechanisms of endogenous stem cell recruitment from muscle and bone following injury, we will be able to propose new drug-based and cell-based strategies to promote the regeneration of these tissues.