Epilepsy is brain disorder characterized by hyper-excitable neurons and tissular remodelling as neuronal death, neuro-inflammation and vascular remodelling. The Blood Brain Barrier (BBB) disruption as well as chronic neuroinflammation are known to contribute to neuronal hyperexcitability. Neuro-inflammation in epilepsy could be acquired from systemic inflammation via the migration of peripheral leucocytes, highly activated in blood of patient, through the BBB disruption. Neutrophils (PMNs), key cells of the innate immune system, act as a first line of host defense and are also important mediators of inflammation-induced vascular and tissue injury. PMNs granules present a high level of metalloproteinase 9 that is correlated with BBB disruption in epileptic patients. IL-8, a strong chemoattractant and activator of PMNs, is released by activated microglia 4 hours after epileptic seizures in human tissue. Moreover, in rodent model, PMN infiltration in parenchyma is observed around vasogenic oedema after seizures and depleting PMN reduce the severity of seizures. Despite their critical role in innate immune system and some evidence of their possible impact on neuropathology little attention has been focused on the role of PMNs in epilepsy and more particularly on their impact on BBB disruption. This translational project in collaboration with clinicians of pitié-salpétriere hospital, will characterize the phenotype and functions of PMNs and their impact on BBB integrity in the pathophysiology of epilepsy. This project combines clinical studies, mouse model of epilepsy and in vitro experiments. First of all, we will characterize subtype and function of peripheral PMNs by flow cytometry in blood of epileptic patient and of epileptic mouse. We will take the advantage of rodent model of epilepsy to follow PMNs phenotype during epileptogenesis and seizure severity. Secondly, their impact on BBB disruption will be studied by depleting PMNs in epileptic mice. Human endothelial cells (hCMEC/D3) co-cultured with isolated PMNs from epileptic patient, will allow us to understand the mechanisms of PMNs on the loss of endothelial cell integrity. This project will gain knowledge on the activation, subtype and functions of peripheral PMNs. It will be the first study to evaluate peripheral PMNs in relation to seizures and may suggest new markers of seizure severity and frequency. This project will also be a pivotal study in the contributions of PMNs in BBB disruption during epileptogenesis. This would enhance the understanding of epileptogenesis. As still 30% of epilepsy are pharmacoresistant, our data should thus open new perspectives in the development of innovative immunotherapy strategies by modulating PMNs response.

Biliary diseases are characterized by the development of fibrosis that arises following the activation of portal fibroblasts (PFs). In primary sclerosing cholangitis (PSC), a biliary disease frequently associated with inflammatory bowel disease, the specific involvement, molecular signals and mechanisms activating PFs are still largely unknown. To address these questions, we will use mice expressing the fluorescent protein Tomato under the control of the promoter of Gli1, a marker of a subpopulation of PFs able to activate into profibrogenic cells. First, Tomato+ PFs will be traced in vivo in mouse models of biliary fibrosis. Cell depletion experiments will also be conducted to evaluate the specific impact of PFs on biliary fibrosis. Secondly, we will perform a comprehensive RNAseq analysis of Gli1+ and Gli- cells in liver from healthy and fibrotic mice to identify markers of Gli1+ derived activated PFs. Identified markers will be explored in liver specimens from PSC patients to evaluate potential predictive value on disease progression and severity. Lastly, we will identify molecular signals arising from the gut that modulate aPFs activation. Correlations between gut inflammation, activated PFs levels, and gut-derived molecular signals will be explored in PSC patients. Upon completion of this project, we will be able to establish the specific involvement of Gli1+ derived activated PFs in biliary fibrosis. This research project also holds the promise of addressing an unmet medical need in PSC by developing novel therapies to mitigate liver fibrosis.

Beginning in December 2019, a novel coronavirus, designated SARS-CoV-2, has caused an international outbreak of respiratory illness termed Covid-19, requiring for a small proportion of patients, admission to Intensive Care Unit (ICU). However, despite the urgent needs of therapeutic guidelines, little is known about the exact immune and inflammatory response of patients with COVID-19 and no robust, sensitive and accurate biomarkers are available. The main objective of the present COVID-METAFLAM project is to characterize the kinetic of metabolomic inflammatory profiles associated with the inflammatory-immune response during COVID-19 pneumonia in ICU patients. We also plan to help for the prediction of the successive phases (acute phase, resolution phase) of the inflammatory-immune response; to search for an association between metabolomic profiles and clinical outcome (morbidity, mortality); to focus on metabolomic profiles in patients receiving immunomodulatory, chloroquine and/or anti-inflammatory treatments. We hypothesize that the integration and machine learning modeling of inflammatory targeted metabolomics in addition to cytokine profiles and bio-clinical data should demonstrated that some metabolites highly correlated with the pathogenic phase of the infection, whereas other are associated with the resolution phase. Our project is thus aimed at stratifying COVID-19 patients according to their inflammatory and metabolic status to help the management of this patients and to provide guidelines for patient treatment. This project will benefit from combined expertise of teams of physicians, virologists, medical biologists and biostatistician working on intensive care and metabolomics/lipidomics. We will also pay a particular attention to the ongoing therapeutic assays using anti-viral, steroids, immunomodulators and other drugs such as hydroxychloroquine. The project will last 12 months and will be organized into five tasks: (i) Recruitment of patients and collection usual demographic, anamnestic, clinical, radiological, biological and microbiological data (based on the COVID-ICU Study, REVA); (ii) Collection and transport of biological samples (based on the AP-HP COVID-Biobank); (iii) Biological explorations; (iv) Analysis of results; (v) Project management. It will be supported by existing networks: COVID-ICU Study, a French prospective multicenter (>70 ICUs, 10 ICUs in the Paris area) performed by the REVA network and the AP-HP COVID-Biobank. Over the 3-month period of inclusion, we estimate that 900 COVID-19 pneumonia patients will be admitted to the 10 participating ICUs (30 patients/month/center). We estimated that 80% of these 900 patients will be included in the COVID-ICU Study. Among them, we estimate that 50% will be included in the AP-HP COVID-Biobank. Altogether, we estimate that 360 patients will be included in the COVID-METAFLAM Study. A total of more than 200 molecular species will be quantified using Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and/or Gas Chromatography-Mass Spectrometry (GC-MS) in our certified department of clinical metabolomic at Saint Antoine hospital (Paris). We will combine data a wide variety of origins, clinical and imagery data (qualitative or quantitative data), biological results and metabolomic to provide a statistical model with prognostic and predictive values, as previously made in our laboratory for specific pathologies and anti-doping research. The project coordination will be ensured by URC –EST, the Clinical Research Center (CRC-EST), the Certified BioBank Research Center (CRB) of Sorbonne University, and the Clinical Research Unit. Our project is ambitious and innovative: (i) this is the first European project which propose a metabolomic approach to monitor kinetics of inflammatory mediators in patients with COVID-19 pneumonia; (ii) the short delay to get results will guaranty a short-term impact, with a quick translation into clinical practice and therapeutics.

Liver transplantation is the only therapeutic option for patients with end-stage liver disease. However, the rising prevalence of chronic liver diseases, such as non-alcoholic fatty liver disease (NAFLD), has increased the number of patients requiring liver transplantation, and the duration on waiting lists. The presence of steatosis in more than 30% of hepatocytes is a major cause of immediate liver graft dysfunction. Among 400 liver grafts discarded each year in France, 25% are not used because of steatosis, representing 10% of liver transplantations. The use of fatty liver grafts for transplantation would significantly increase the pool of donors and therefore, has become a topic of great interest. In this project, we will use ex situ normothermic organ perfusion to rescue fatty liver grafts by depleting them in fat and protecting them from ischemic injury during perfusion. We will target the necroptosis signaling pathway implicated both in fat storage and ischemia.

The intestinal tract is home to a densely populated microbial community, or microbiota. A growing body of evidence suggests that this commensal relationship strongly defines the health state of humans. Within this community, the richness and diversity of bacteria are shaped by the intestinal physiology (metabolism, immunity...) and by external factors (nutrients, drugs…) and are often described as crucial for maintaining a healthy state, while deviating from such situation may result in transient or chronic disorders. But the gut microbiota is composed of other microbial fractions, notably of bacteriophages (phages), the specific viruses of bacteria. Viromic studies have identified that gut phages are highly diverse and stable over time as well as very specific to single individuals. Metagenomic signatures of phage abundance and diversity were reported as associated to several diseases, such as inflammatory bowel disease (IBD). Also, increasing data suggest that the transfer of intestinal phages is crucial for the success of faecal material transplant (FMT) in resolving the recurrence of Clostridioides difficile infections. Although of invaluable significance, these studies remain largely descriptive and limited in resolving the nature and extent of the interactions taking place between the intestinal organ and its microbiota. Thus, many are the standing questions regarding their putative role in determining or contributing to a healthy or a disease state. VENTRIS is a research project aimed at characterising the ecological and molecular mechanisms governing the tripartite interaction between phages, bacteria and the intestinal tract during healthy and inflammatory conditions. This objective will be achieved by understanding (i) the impact of phages on the intestinal barrier and its inflammatory state, using in vitro and ex vivo model, including clinical samples; (ii) the mechanisms of long term phage-bacteria stable coexistence in a murine model of controlled microbiota in the presence and absence of intestinal inflammation; (iii) the potential of gut phages in controlling or reverting intestinal inflammation by modulating the gut microbiota. This project is set to have an impact on the understanding of the dynamics regulating the homeostasis of the gut microbiota. Such knowledge will open the door to deciphering the metagenomics data associated to healthy and diseased states and to designing the targeted preservation and restoration of gut health using bacteriophage-based therapeutic options.