During embryo development, looping of the heart tube is the first morphological sign of left-right asymmetry. It is required for the alignment of cardiac chambers and thus the plumbing of the blood flow. Whereas the molecular cascade initiating left-right patterning has been well characterised in the early embryo, how it is later transposed in different populations of cardiac precursor cells for heart looping remains poorly understood. Our aim is to understand how left-right signalling controls the mechanism of heart looping and the alignment of cardiac chambers using cutting-edge technologies. Our preliminary results by computer modelling, mouse genetics and human genetics have validated the feasibility of the project. In the mouse model, we will dissect the role of the left determinant Nodal by conditional gene inactivation. The mechanism of heart looping, as well as the structural defects in chamber alignment that might fit with human heart diseases, will be analysed using High Resolution Episcopic Microscopy (HREM). To identify novel markers of the left and right heart fields, we will adopt a genome-wide transcriptional approach, tomo-seq. On a clinical point of view, we will focus on a rare cardiac malformation, Congenitally Corrected Transposition of the Great Arteries (CCTGA), a model of abnormal heart looping characterized by the left/right inversion of the ventricles. Taking advantage of a large cohort of patients and the associated collection of DNA samples (CARREG) and phenotypic descriptions, we aim to analyse the functional characteristics of CCTGA by multimodal imaging, as well as the genetic variations by whole genome sequencing. Selected genetic variations will be validated in the zebrafish model and also by developing novel mouse models. This will open perspectives to study the pathological mechanisms involved, with particular reference to heart looping. Combining pediatric cardiology, human genetics and developmental biology to analyse the left-right asymmetry of the heart, our innovative project is expected to impact both fundamental research for the mechanism of left-right patterning during organogenesis, as well as clinical practice for genetic counselling and prognosis of cardiac chamber misalignment, representing about 20% of congenital heart defects (CHD), the most common malformation in humans.

Cardiovascular diseases (CVD), including ischemic heart disease (IHD), are the main cause of mortality in nearly all European Union member states, accounting for almost 40% of all deaths in the region in 2011. In particular, the cardiovascular risk in people with diabetes mellitus is two to three times higher than in those without the disease and CVD. This situation clearly calls for the absolute necessity to pursue basic and translational research in order to progress beyond state-of-the-art, identify and test new strategies to limit CVD especially in patients experiencing type 2 diabetes (T2D). Emerging evidences suggest that myeloid-derived cells may provide the necessary signals to drive both cardiogenesis and tissue remodeling. In response to acute myocardial infarction (AMI), different subpopulations of myeloid cells, such as monocytes, macrophages and mast cells are recruited in the infarcted heart and participate to cardiac repair and remodeling. On T2D background, monocytes, macrophages as well as mast cells fate switches towards an inflammatory phenotype that could precipitate adverse ventricular remodeling after AMI. The classical paradigm indicates that recruited myeloid-derived cells populating the ischemic heart mainly derive from genuine hematopoietic progenitors residing in the bone marrow (BM). Remarkably, recent works have challenged the view of medullary origin of hematopoiesis and found that systemic inflammation can also stimulate extramedullary hematopoiesis in adult mice and humans. First and foremost, pioneer work from partner 1 showed that WAT-stroma vascular fraction exhibits hematopoietic regenerative potential. Such WAT hematopoietic activity relies on the presence of a large population of hematopoietic stem/progenitor cells, producing 80 to 100% of mast cells and macrophages within WAT, but also in other organs. These original results suggest that, in addition to the BM, WAT constitutes an alternative source of innate immune cells that may contribute to inter-organ dialogue. We thus hypothesize that beside metabolic and endocrine signals, WAT interacts with other organs via the production of myeloid cells, notably monocytes/macrophages and mast cells that participate to remodelling and/or regenerative processes in target organs. A dysfunction of WAT-hematopoiesis may lead to adverse effects on these processes. In parallel, WAT-hematopoietic activity is controlled by external signals from injured tissues, and in turn controls the WAT physiology itself. In line with this hypothesis, preliminary results obtained by the consortium suggest that WAT-derived myeloid cells are able to home to the cardiac tissue where they can participate to regenerative and/or reparative processes. In addition, a dysfunction of WAT-hematopoiesis is observed T2D, and plays a pivotal and causative role in the development of the disease. The main objectives of this project are to demonstrate that (i) immune cells contributing to cardiac remodeling after AMI partly derive from WAT, (ii) diabetes–induced dysfunction of WAT hematopoiesis contributes to alteration in cardiac remodeling and (iii) the WAT-hematopoietic activity depends on signals from the infarcted heart. Hence, the major conceptual breakthrough of the present project implies a switch in the conceptual archetype depicting the origin of myeloid cells and our major objective is to establish proof of concept of this hypothesis, emphasizing the intrinsic crosstalk between WAT-derived myeloid cells and the cardiac homeostasis. Gain of knowledge in this novel and unexpected function of the WAT could lead to identify innovative therapeutic targets to prevent or treat IHD associated with diabetes.

Type I interferons (IFNs) have long been recognised as key immune mediators, in particular in anti-viral defence. Due to their potent and broad effect the IFN response operates along a spectrum, and must be balanced between protection against infection versus risk of inflammatory disease and immunopathogenesis. As such, the induction, transmission, and resolution of IFN responses are tightly regulated. Type I interferonopathies and related non-monogenic phenotypes represent examples of a disturbance of the homeostatic control of this complex system. The identification of such diseases is of clinical importance as ‘anti-IFN’ treatments are developed based on an understanding of pathology. Furthermore, these diseases provide an unprecedented opportunity to define the role of type I IFNs in human health and disease. Despite their key roles in immune defence and human pathology, no routine laboratory test exists in medical practice for the assessment of type I IFN signalling. We will take advantage of a breakthrough technology, digital ELISA that permits for the first time the direct quantification of type I IFN proteins in human samples. We emphasize here the true novelty of this approach, which is based on ex vivo analysis in a human context to dissect out the regulation and function of IFNs in a range of diverse disease settings. We present compelling preliminary data demonstrating the potential use of this revolutionary platform, and highlight the exceptional patient cohorts that we have established which will enable us to pursue these studies. Importantly, this proposal will provide new information about the role and cellular sources of different IFNs, how IFN-stimulated signals are transduced, and the engagement of feedback loops during therapeutic intervention. Previous studies have addressed some of these questions with transcriptomic approaches, but an assessment at the functional proteomic level has not been possible until now. We believe that our proposed approach using the Simoa platform represents a technological step-change, which has major significance for the future diagnosis and treatment of patients with disease related to an upregulation of type I IFN signalling, and in the study of underlying mechanisms relevant to these disorders. With a focus on diagnostics, stratification and target identification, we consider that our application has remarkable potential for translation into near-term clinical benefit.

We recently reported a novel combined immunodeficiency in patients with loss of function mutations in the CTPS1 gene and characterized by high susceptibility to viral infection due to inability of T cell to proliferate and control infections. The project aims at acquiring fundamental knowledge on the biology of CTPS1, a key enzyme for the de-novo synthesis of CTP, including its role in embryonic development and in the immune system. Cellular and mouse models (including conditional KO and inducible KO) will be used to study the function and the regulation of CTPS1 under normal and pathological settings in the immune system. These studies will also help to determine whether CTPS1 could be a target for drugs to specifically dampen lymphocytes proliferation in autoimmune and inflammatory disorders. Chemical inhibitors of CTPS1 recently developed will be tested. The preliminary data already obtained by the team guarantee the workability of the project. This project relies on the complementary expertise of the four partners of the consortium.

SCIENTIFIC BACKGROUND AND OBJECTIVES Retroelements (retroviruses and long terminal repeats (LTR)-retrotransposons) replicate by reverse transcription of their RNA genome into a cDNA that is next integrated into host-cell chromosomal DNA, by the retroelement-encoded integrase. To accomplish a productive replication, the cDNA must traffic through the cytoplasm, cross the nuclear membrane barrier with the integrase, and access a site of integration that allows active transcription of the viral genes. Whereas important advances have been made on the traffic of viral particles within the cytoplasm, the events occurring at the interface between the cytoplasm and the nucleus, until their integration into the host DNA remain largely unknown. This question is fundamental since cDNA integration is an essential step to achieve productive infection and long-term viral persistence. The goal of this project is to address the fundamental question of the interplay between the replication of retroelements and the nuclear architecture, from viral nuclear import to its genomic integration. DESCRIPTION OF THE PROJECT/METHODOLOGY LTR-retrotransposons are ubiquitous in eukaryotic genomes, and important understandings on retroviral biology have been gained by their study in yeast. Therefore, we will use yeast Ty1 LTR-retrotransposon, as working model. Integration does not occur randomly throughout the host-cell genome in vivo, revealing a retroelement-specific pattern of preferred sites, which depends in part on transcription determinants. Our first goal is to study the influence of transcription on Ty1 integration site selection. We will: - Set up advanced in vitro integration systems coupling Ty1 integration to an active transcription; - Develop innovative strategies to identify in vivo, new Ty1 co-factors involved in nuclear import and integration, and characterize their impact on integration site selection in genome-wide studies. In the nucleus, chromosomes and individual loci display dynamic but non-random spatial positional preferences, which appear to influence biological process linked to DNA metabolism. Our second goal is to determine the impact of chromosome architecture on retroviral integration, as well as the influence of retroviral insertion on 3D nuclear organization. We will use the most modern tools to localize loci, follow them in living cells, and statistically map their position in two dimensions in the nuclear space, in order to: - study the localization of distinct Ty1 genomic loci in the nuclear space; - compare the position of different loci before and after Ty1 integration. On their nuclear import journey, cDNA and viral proteins of several retroviruses accumulate at the centrosome but the nature of events occurring at this organelle and their impact on nuclear import and integration is unknown. Our third goal is to decipher the contribution of the Spindle Pole Body (the yeast centrosome) and the microtubule network in Ty1 replication. We will: - analyze the localization of Ty1 and SPB proteins by immunofluorescence microscopy and at super resolution; - determine whether a fully functional SPB is required for Ty1 retrotransposition, using genetic and genomic approaches. EXPECTED RESULTS The outcome of this project will be to: - identify new Ty1 cellular partners; - decipher at the molecular level the connection between the transcriptional machinery and the preintegration complex, and its role in the integration process; - determine the impact of the chromosomes spatial organization on the integration process; - get insight into the contribution of the SPB in Ty1 lifecycle. Furthermore, this project should open new directions to explore the interplay between cellular machineries and mammal retroviruses, which should have valuable implications in clinical therapy for the treatment of retroviral infections and for the development of novel retroviruses-based vectors for gene therapy.