We observed that Tgfb1 is upregulated at the transcription level in cells deficient for the four and a half LIM-only protein 2 (FHL2). The upregulation of Tgfb1 leads to increased liver and kidney fibrogenesis in FHL2-/- mice. We have carried out microarray analysis with mouse embryonic fibroblasts derived from FHL2-/- embryos. We would like to have help for the analysis of our microarray data to identify the transcription factors that would be responsible for the upregulation of Tgfb1 expression in FHL2-/- cells. These transcription factors should potentially bind to the murine promoter of Tgfb1.

We have recently identified around 500 long non-coding RNAs that follow a very precise expression pattern in undifferentiated mouse Embryonic Stem cells. In order to assess if any of those molecules are functionally relevant for the biology of ES cells we will perform a functional screening based on the CRISPR technology. We plan to use a modified version of the CRISPR-CAS9 technology, the CRISPRon system, in which (i) the guide-RNAs (gRNAs) will target the promoters of our candidate lncRNAs (at least 5 gRNAs per promoter) and (ii) an enzymatically-inactive CAS9 will be engineered to recrute 10 molecules of the potent VP64 transactivator. This will enable to upregulate the targeted lncRNA from its endogeneous locus.

Skeletal muscle stem cells constitute a population of cells with heterogeneous properties. Interestingly, muscle stem cells have a remarkable capacity to regenerate muscle fibres after regeneration. We are performing a molecular analysis of these stem cells.

Grâce à la plateforme PF2 à Pasteur, nous avons réalisé une étude de microarray ( Affymetrix Genechip Mouse Gene 2.0 ST Array) avec les fibroblastes de souris. Pourriez vous SVP nous aider à analyser ces données?

Gli are transcriptional regulators involved in the Shh signalling pathway. Gli3 binding sites on DNA have been defined in the mouse genome by ChIP-chip experiments (Vokes et al., 2008). We are working on the Msx transcription factor family, and have strong evidence indicative of interactions between Msx1 and Gli3 at the protein level. This might reflect in Msx1 binding to DNA in the vicinity of Gli3 binding sites. However, Msx1 binding sites are ill-defined. One stategy to confirm this hypothesis is to compare sequences around gli3 binding sites (around 4200 such sequences in published data) and look for conserved stretches that might define Msx1 binding sites.

Aim : In vitro infection of innate immune cells by L. amazonensis (L.am.) seems to be associated to an increase in resistance to cell death of infected cells. This project aims at deciphering the impact of in vitro L. amazonensis (L.am.) amastigotes infection on cell death processes including apoptosis, autophagy, pyroptosis and necroptosis in different host cells, i.e. Bone-marrow- derived macrophages (BMDMs) and dendritic cells (BMDCs) after one day of infection. Material : RNA samples were obtained from control and infected BMDMs (BALB/c mice) or BMDCs (BALB/c, C57BL/6 and DBA/2 mice) after their sorting by Fluorescence Activated- Cell sorting. For BALB/c BMDCs, amastigotes were also added to cells in presence of immune serum to trigger their opsonization. Samples were analysed a few years ago at the « Plateforme Transcriptome et Epigénome » with the Affymetrix technology. RNAs from BMDMs and BMDCs were analysed with the Affymetrix Mouse430_2 GeneChips and the Affymetrix Mouse Gene ST 1.0 arrays respectively.  

assess the faisability of the chip analysis to our samples from laser capture microdissection of the mouse intestine, of diverse quality and preparation levels

Detection of nucleic acids at the single cell level using microscopy has now reach high throughput levels which promise exciting discoveries concerning the functionning of the genomes. The HSC3D project funded by the CITECH in the Pasteur institute aims at multiplexing nucleic acid detection by Fluorescence In Situ Hybridisation (FISH). This necessitates libraries in the range of tens of thousands of  100mer DNA oligonucleotides which will be synthesised by digital lithography. Several constrains apply to these oligonucleotides which therefore require heavy duty bioinformatics for their design .

Screening for NSm function in infected mammalian cells by proteomic analysis.

After the behavioural characterisation of the Shank3 KO cohort, we extracted and dissected different region of the brain: cortex, hippocampus, striatum, cerebellum.

These regions were selected according to the expression level of Shank3 (Peça et al. 2011). Using QUIAGEN miRNAeasy with DNAse, miRNA-enriched RNA was extracted from the brain regions in order to perform RNAseq.

We will ask 3 questions:

• What is the pattern of Shank3 isoforms in different brain regions?

• What are the genes/pathways differentially expressed in wild-type and Shank3 knock-out mice?

• What are the genes/pathways associated with the severity of the self grooming behaviour ?

Beside data driven eperiments, we will test candidate genes/pathways such as glutamatergic receptors and <

We previously showed that humanized immune system (HIS) mice generated in Balb/c Rag2^-/-γ_c^-/- SIRP^NOD (BRGS) recipients are susceptible to HIV-1 infection (X4 and R5 isolates) and maintain circulating HIV-1 in the plasma, resulting in a dramatic depletion of human CD4⁺ T cells. We also characterized features of HIV physiopathology in this model. Human thymocyte subsets developing in the thymus of HIS mice appear phenotypically normal, but in the periphery the T cell repertoire is restricted compared with that of human peripheral blood T cells. This negatively impacts on the ability of HIS mice to generate antigen-specific human immune responses when mice are vaccinated with protein antigens or following infection with lymphotropic viruses such as HIV. One likely explanation for these functional deficiencies involves the fact that human T cells are selected intrathymically by mouse MHC molecules and that naïve T cells in peripheral lymphoid organs interact primarily with mouse DC (as human DC development in HIS mice is limited). As a first line of improvement, we recently generated a novel mouse model by crossing our BRGS mice with the HLA-A*02-HHD class I transgenic mice and the HLA-DRB1*15 class II transgenic mice, resulting in BRGS-A2DR2 mice. Following intra-hepatic injection of these mice with MHC-matched CD34⁺ stem cells we observed increased engraftment, with faster kinetics. Moreover BRGS-A2DR2 HIS mice have an increased T cell development leading to a more equilibrated B/T and CD4/CD8 phenotype. We showed that BRGS-A2DR2 HIS mice were able to sustain replication of HIV R5 virus as the BRGS hosts. Viremia was similar in a first phase and then lower in a second phase in BRGS-A2DR2 compared to BRGS HIS mice, which could be a consequence of a better quality of the immune response. However, the viremia reached a similar plateau in the last phase. We propose to study the impact of the immune res

The post-translational modification by SUMO is an essential regulatory mechanism of protein function that is involved in most challenges faced by eukaryotic cells. Gene expression is particularly regulated by sumoylation as many SUMO substrates are transcription factors and chromatin-associated proteins, including histones. The emerging paradigm for the proposed work is that sumoylation controls multiple aspects of chromatin structure and function in response to external cues. According to this view, sumoylation is expected to impact both global and specific transcriptional programs thereby affecting constitutive and inducible expression of both coding and non coding genes. Recently, we found SUMO as an integral and instructive component of chromatin in cell growth, senescence and cancer, thus establishing sumoylation as a new and paradigmatic chromatin modification. This work now paves the way for detailed understanding of the contribution of SUMO as a multifaceted modifier of chromatin.

Gene ontology analysis of RNAseq data from uninfected and Leishmania-infected mouse macrophages.  Scientific context During the course of cutaneous or visceral disease in humans or experimental animal models, the resolution of leishmanial infections or the control of parasite growth is dependent on appropriate innate and adaptive immune responses developed by the parasitized host. Leishmania largely evades and subverts these responses by its intracellular life style inside the mammalian host, where the parasites develop into amastigotes mainly within macrophages (BMDMs). We have focused our interest in the BMDM inflammasome and the way Leishmania amastigotes interfere or subvert BMDM inflammatory responses. Our recent data are in favor of an absence of stimulation, even a down-regulation of the inflammasome in BMDMs harboring replicating amastigotes at the gene and protein expression levels. To go further on this, we have performed RNAseq experiments on uninfected and infected BMDMs. This project was done at the “Transcriptome and Epigenome” platform and in close collaboration with the C3BI for normalization and statistical analysis procedures. Objective In the present proposal we will perform a deep analysis of the repartition of modulated genes between the different conditions using these RNAseq data. Using C3Bi expertise we will classify known functions of modulated genes into GO aspects i.e. molecular function, cellular component and biological process, visualize gene annotations and perform statistical analyses for the distribution of the annotated genes over the GO hierarchy for the different gene lists analyzed. Hopefully, these analyses will bring us a better understanding of the mechanisms underlying the subversion of BMDM functions in the innate and adaptive immune response to Leishmania infection which is a prerequisite to design novel anti-parasitic intervention strategies targeting the infected host cell rather than the parasite.

In order to study a differential regulation of RNA and protein expression level during Rift valley fever virus (RVFV) infection in a murine model, we want to compare transcriptomic and proteomic analysis.  

We have identified a candidate gene associated with increased resistance to a pathogen. This gene is poorly annotated in public databases. To get insight into its function we are focusing on genes that are co-expressed with this candidate gene in various datasets, including microarray collection on various mouse tissues or in a given tissue across inbred strains of mice. Indeed, coexpression is one of the central idea in gene expression analysis. The 'Guilt by association' principle states that gene coexpression might indicate shared regulatory mechanisms and roles in related biological processes. We have established lists of genes that are co-expressed in various tissues, and in collection of individuals with different genotypes. We are willing to get graphical representation giving a compact overview of genes that are coexpressed with our candidate gene.

I am interested in gut-brain axis and specifically how a bacterial metabolite, MDP, can be sensed directly by neurons.

Notre but est de trouver un test statistique capable de dire si deux matrices de nombres sont différents. Ces matrices correspondent à l’utilisation de fragments de gènes (V ou J) pour créer un gène ré-arrangé (V-J) codant pour un anticorps de spécificité particulière. Ici nous avons représenté pour chaque gène V la famille de gène J utilisé (parmi J1, J2, J3, J4). Le nombre correspondant au nombre d’occurrences que nous avons trouvé après séquençage du répertoire d’anticorps spécifiques à partir de souris immunisées contre l’un de ces antigènes. Les matrices ont été réalisées pour les deux chaines codant la spécificité d’un anticorps: la chaine lourde (VH) ou la chaine légère (VL). Le but est de comparer les deux matrices VH l’une avec l’autre, et les deux matrices VL l’une avec l’autre.

The present work is to systematically investigate the role of TLRs and NODs in the host defense during C. trachomatis infection using KO animals. The inflammatory cytokines and bacterial burden will be measured using Bio-Plex ELISA kit. C3BI will provide help in the data analysis.

The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections, including Influenza, HIV and Zika viruses, by inhibiting virus-cell fusion. We showed that IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses. We also reported that amino-terminal mutants of IFITM3 preventing ubiquitination or endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. Our aim is to analyze the RNA levels of the various members of the IFITM family, in various normal or pathological human or animal tissues.

The human gene ANOS1, responsible for the X-chromosome-linked form of Kallmann syndrome (a developmental disease affecting the olfactory system), has been identified in 1991 by positional cloning. It is located on the X chromosome short arm (at Xp22.3), close to the STS gene and close to the boundary of the pseudoautosomal region (common to the X and Y sex chromosomes). Since then, orthologous genes have been identified in all animal species (including invertebrates), except in the mouse, rat, and other rodents (an orthologue in the naked mole-rat Heterocephalus glaber is however present in the GeneBank data base). The orthologous STS in the mouse has been identified in the mouse, and is located in the XY pseudoautosomal region in this species. The sequence of the mouse STS is unusually GC-rich and has markedly diverged from the human orthologous sequence, even though the amino acid sequence of the protein is highly conserved. The ANOS1 orthologous genes have not yet been identified in the mouse/rat, although we have been able to detect the encoded protein anosmin-1 in these species, with antibodies directed against the human protein (the orthologous proteins in the mouse and rat have the expected size in western blot analysis, i.e. about 690 amino-acids).

Erythromyeloid progenitors (EMPs) originate from the yolk sac during early mouse development and migrate to the fetal liver via the circulation where they undergo massive expansion and differentiation into hematopoietic lineages. These events occur prior to the intraembryonic emergence of hematopoietic stem cells (HSCs). Unlike HSCs, EMPs cannot give rise to lymphoid lineages, nor can they provide long-term repopulation. As such, they are considered a transient fetal population, yet it is EMP-derived hematopoiesis that supports the growth and survival of the embryo prior to the establishment of long-term hematopoitic stem cells (HSCs). Hematopoietic cell differentiation occurs along a hierarchy of progenitors with either lymphoid or myeloid fates. Common myeloid progenitors (CMPs) give rise to further restricted granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs). This hierarchy has been well documented in adult hematopoiesis, which occurs solely from HSCs. However, fetal hematopoiesis encompasses dual origins of myeloid lineages that can originate from either EMPs or HSCs. Using genetic pulse chase labeling, we are able to distinguish these two ontologies by positively labelling EMPs and their progeny. Currently, fetal liver progenitors have been characterised by direct comparison to markers and expression profiles that are established for adult hematopoiesis. Yet, fetal hematopoietic markers may not be regulated in the same manner as their adult counterparts. Furthermore, distinguishing EMP- versus HSC-derived progeny is technically challenging and has not been properly addressed with respect to fetal liver myelopoiesis. Therefore, using our genetic pulse chase labeling approach, we would like to rebuild the differentiation tree among myeloid fetal liver progenitors. We are using high parameter flow cytometry to re-evaluate progenitor sub-populations with an expanded repertoire of markers. Since heterogeneity among progenitors (in terms of gene expression and differentiation potential) can be misrepresented and difficult to characterize on the population level, we want to investigate this on the single cell level using MARS-Seq in combination with index sorting.

Tissue resident stromal cells form the scaffold of all organs. In addition, they provide signals for proper positioning, survival and interaction of a number of other cell types, such as immune cells. Following tissue damage, the stromal microenvironment play an esssential role to organize inflammation and tissue repair. The stromal vascular fraction (SVF) of most organs is composed of immune cells, endothelial cells, mesenchymal stromal cells and tissue stem cells. Our lab investigates the impact of specific subsets of mesenchymal stromal cells (MSCs) on the different components of the SVF, at homeostasis and during the repair process. We have previoulsy identified specific subsets of MSCs that have an essential role in inflammation, tissue fibrosis or maintenance of the intestinal stem cell niche (Peduto et al., 2009; Dulauroy et al., 2012; Stzepourginski et al., 2017). This project aims at further deciphering the heterogeneity of the stromal compartment in different organs at homeostasis or following injury, and establish its transcriptional signature.

To study the neuronal mechanisms underlying the generation of distinct memories, it is necessary to perform experiments in which the sensory elements of the environment are under the precise control of the experimenter. To achieve this goal, we have developed a virtual-reality environment for rodents that we combine with imaging and electrophysiological methods to record the activity of individual neurons and populations of neurons while the animals are navigating in virtual reality. This system allows us to probe the neuronal mechanisms underlying spatial navigation and memory at the level of individual neurons and neuronal circuits. The goal of this project is to develop a modular recording and analysis pipeline that is both flexible and accessible to all experimental users, building on our existing software tools.

Our goal is to have a bioinformatic tool that performs the 2 x 2 comparison of matrices of numbers in matrix batches. Each matrix corresponds to the use of gene fragments (V or J) to create a re-arranged gene (VJ) encoding a specific antibody, identified by droplet microfluidic or flow cytometry techniques followed by high throughput sequencing (NGS). ). The matrices are made for the two chains encoding the specificity of an antibody: the heavy chain (VH) or the light chain (VL). The goal is to compare VH matrices one against another, and VL matrices one against another for antibody gene rearrangements in mice, llama, and humans.

The development of the mammary gland occurs in five distinct phases: embryogenesis, puberty, pregnancy, lactation, and involution. Due to its extraordinary regenerative capacity, the mammary epithelium is a fantastic system to study the physiological regulation of cellular plasticity in vivo. It comprises two major cellular lineages, the outer myoepithelial (also called basal) and inner luminal cell layers. Although the existence of post-natal bipotent mammary stem cells (MaSCs) remains debatable, it is well accepted that there are subtypes of epithelial cells (progenitors, unipotent and/or bipotent stem cells) that are responsible for the remodeling and renewal of the mammary gland. Moreover, these cells have also been suggested to be the cell of breast cancer origin. Therefore, it is crucial to further understand how mammary gland maintains its proper cellular plasticity, especially during the cycles of pregnancy and involution, which might shed new lights on the initiation, progression, and metastasis of breast cancer.

The three HP1 proteins (Heterochromatin Protein 1 alpha, -beta, -gamma) are epigenetic markers of heterochromatin, the condensed, repressed form of chromatin. They are typically known to associate to the di-/tri-methylated lysine 9 of histone H3 (H3-K9me2/3), a repressive histone mark, HP1s are therefore linked to chromatin silencing. But on the other hand, HP1g has been shown to localize within the body of coding genes in correlation with their transcriptional activity. We have identified functional links between HP1g and chromatin-associated RNA on gene models, but we are now dissecting these links on a genome-wide basis.

Stromal cells are essential during organ morphogenesis and for the maintenance of tissue homeostasis. In addition, increasing evidence indicates that stromal cells play a role in certain type of chronic diseases, such as cancer. In this project, we will investigate stromal cell heterogeneity at the single cell level in specific organs at homeostasis and during pathology.

The project aims at developing (1) a database dedicated to the storage and statistical analysis of Collaborative Cross based data and (2) implement genetic analysis tools based on rqtl2 for the identification of QTL on this data.

Disruption of GABAergic inhibitory circuits is one of the common alteration responsible for several psychiatric developmental disorders. Gephyrin (GPHN) is the common and main molecular organizer of inhibitory synapses. It acts as a hub under the postsynaptic membrane for the multiple protein-protein interactions. Intriguingly, inhibitory synapses are highly heterogeneous, bearing various inhibitory postsynaptic potential (IPSP) properties and also specific subcellular localization on their target neuron. The molecular mechanism responsible of this diversity is still unknown although it could result, in part, of alternative splicing regulation that will produce specific GPHN isoforms carrying versatile properties. Interestingly, exons alternatively included in Gphn transcripts are proposed to change the binding of GPHN protein with inhibitory receptor as well as its oligomerization. Thus, alternative splicing regulation of Gphn expression intuitively provides a potential molecular mechanism to finely regulate several aspect of inhibitory synapse development, however this regulation step is still largely unexplored. In collaboration with Fabrice Ango (IGF-Montpellier), we have designed an experimental approach to sequence GPHN transcripts using the technologies from Pacific Bioscience and Oxford Nanopore. It was applied to samples prepared from mouse and human tissues. To date, we got sequences from Pacific Bioscience sequencing and our interaction with E. Kornobis allow us to get preliminary data that have revealed a high level of complexity in alternative transcripts expressed by GPHN in Mouse brain samples. However, we are fighting to cluster these sequences and pulling together alternative GPHN transcripts with a bioinformatic pipeline able to decipher properly between light variation of sequences and sequencing errors associated to long read sequencing. Results obtained from currently available solutions, such as PacBio IsoSeq3 analysis pipeline, led us to believe that a more suitable software solution is needed, especially to properly characterize Gephyrin splicing diversity. We propose to build a new bioinformatic pipeline to analyze our data and usable to long read sequencing obtained with Pacific Bioscience and Oxford Nanopore technologies.

Complex chronic diseases are caused by the accumulation of genetic, microbial and lifestyle factors. The number and complexity of such factors makes prediction of pathogenesis and therapy particularly difficult. Although a single factor is rarely sufficient to trigger pathology, genetic and environmental factors have so far been studied in isolation. Nevertheless, a substantial number of genetic variants have been associated with disease risk and the concomitant lifestyle shift and excessive hygiene are thought to contribute to the increased incidence in inflammatory diseases in industrialized countries. Moreover, clinical and experimental observations suggest a strong impact of gut microbiota on susceptibility to inflammatory diseases. The aim of 3D PATH is to explore the multiplicity and complexity of genetic, microbial and lifestyle factors associated with vulnerability to inflammatory pathology, using mice of the Collaborative Cross (CC), that model human genetic variability. Quantitative trait loci analyses, as well as integrative data analyses on metabolic and inflammatory outcomes will reveal new genetic variants and combinations of variants associated with disease susceptibility, and whether alterations of the gut microbiota in genetically susceptible mouse strains can trigger the phenotype. Moreover, the longitudinal design of experiments will allow us to identify early biomarkers that predict the pathology later in life. In a second step, validation of the identified risk factor combinations and exploration of the underlying molecular mechanisms will be performed taking advantage of the mouse model.

Erythromyeloid progenitors (EMPs) originate from the yolk sac during early mouse development and migrate to the fetal liver via the circulation where they undergo massive expansion and differentiation into hematopoietic lineages. These events occur prior to the intraembryonic emergence of hematopoietic stem cells (HSCs). Unlike HSCs, EMPs cannot give rise to lymphoid lineages, nor can they provide long-term repopulation. As such, they are considered a transient fetal population, yet it is EMP-derived hematopoiesis that supports the growth and survival of the embryo independently of HSCs. HSC differentiation respects a hierarchy of progenitors with either lymphoid or myeloid fates and has been well documented in adult hematopoiesis. However, fetal hematopoiesis ensues from erythroid and myeloid progenitors with dual origins from either EMPs or HSCs. Using a genetic pulse chase labeling approach to distinguish these two ontogenies, we would like to explore heterogeneity on the single cell level and to build the differentiation tree among fetal liver myeloid progenitors using MARS-Seq.

Dengue virus (DENV) induces strong T and B cell responses upon infection. However, there is currently neither vaccine nor specific treatment against DENV, which is spreading worldwide causing 400 million new infections every year, of which 100 million cases are symptomatic, ranging from a self-limiting febrile illness named dengue fever (DF) to more severe life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). One of the major obstacles of dengue vaccine development is the cross-reactivity among antibodies against the different DENV serotypes (designed as DENV1-4) that are 67-75% identical at the amino acid level. Indeed, while a primary infection by one DENV serotype can induce a lifelong immunity against re-infection by the same serotype, subsequent infections by heterologous serotypes increase the risk of developing severe dengue, a phenomenon due to non-neutralizing or sub-neutralizing antibodies and called antibody-dependent enhancement (ADE). To avoid the induction of such enhancing antibodies, and given the identification of CD4+ and CD8+ T cell epitopes from previously dengue virus (DENV)-infected donors, we have designed a minimal DENV antigen (called DENV1-NS, patent EP14305984.8 filled on June 23 2014), which is enriched in conserved and highly antigenic epitopes. Using this minimal DENV1-NS antigen, we have first established in vivo its immunogenicity in transgenic mice expressing HLA class II and class I alleles (with the activation of DENV-specific CD4 and CD8 T cells). We have also shown that a prime-boost DNA immunization of these HLA transgenic mice induces a strong T cell immunity, with a significant protection against DENV1 infection, in the absence of neutralizing or sub-neutralizing anti-DENV antibodies (Roth et al., accepted for publication). Our proposal aims, therefore, to identify early transcriptional signatures correlated with the development of memory CD4 and CD8 T cells in vaccinated animals, which promote enhanced anti-dengue immunity.

This projects aims at characterizing at the single cell level the stromal microenvironment of the skeletal muscle, to get a better insight into the heterogeneity and function of different cell populations involved in skeletal muscle homeostasis and regeneration.

The therapeutic anti-IgE antibody Omalizumab is used for the treatment of severe asthma, and is known to trigger anaphylaxis in some patients. Since Omalizumab is a humanized IgG1, so we hypothesized that Omalizumab could trigger anaphylaxis through the alternative FcgR-mediated pathway. Indeed, we observed that Omalizumab can trigger anaphylaxis in genetically modified mice expressing human FcgRs. We further produced a mutant version of Omalizumab which can still block IgE but cannot bind FcgRs, and demonstrated that this mutant anti-IgE does not induce FcgR-mediated anaphylaxis.

Gene-modified T cells expressing a chimeric antigen receptor (CAR) targeting the CD19 molecule have demonstrated promising clinical efficacy in the treatment of B cell malignancies. However, the frequent relapses and toxic adverse events such as cytokine release syndrome represent hurdles to the success of CAR T cell therapies. In most clinical settings, CAR T cells are generated from a mixture of autologous CD4+ and CD8+ T cells before being infused into patients. This inter-patient heterogeneity within the composition of CAR T cell products renders the large variety of response efficacy and toxicity difficult to interpret. Using a model of B cell aggressive lymphoma developing in the bone marrow, we investigate the differential contributions of CD4+ and CD8+ anti-CD19 CAR T cells to tumor outcome and changes in the tumor microenvironment. Our first in vivo imaging and flow cytometry results suggest that CD4+ CAR T cells have poor cytotoxic potential compared to CD8+ CAR T cell. On the other hand, CD4+ CAR T cells were largely responsible for the cytokine release syndrome and have a unique role in boosting the accumulation of NK cells at the tumor site. Using single cell RNAseq, we aim to identify the changes in the bone marrow tumor microenvironment induced by CD4+ and CD8+ CAR T cells, focusing on the recruitment of host specific immune cell populations and their activation status. Identifying specific contributions of the CD4+ and CD8+ CAR T cells to immune cell recruitment and tumor outcome would help designing optimal CAR T cell products, with important clinical implications.

Epidemiological data report an association between obesity and inflammatory bowel disease (IBD). Furthermore, animal models demonstrate that maternal high fat diet (HFD) and maternal obesity increase susceptibility to IBD in offsprings. However, the mechanisms that translate maternal obesity and HFD into increased susceptibility to IBD later in life remain unknown. Here we report that excess calorie intake by neonatal mice, as a consequence of maternal HFD, forced feeding of neonates or low litter competition, leads to multiple causally-linked perturbations in the intestine that imprint the individual with long term susceptibility to IBD.

The global aim of the project is to characterize and explore the role of dermal macrophage populations in the control of vascular integrity in health and diseases. Currently, we are limited by the lack of population-specific markers that would allow us to study them down to the molecular level. We thus propose to perform a single cell gene expression analysis on pan leukocytes (CD45+) isolated from mouse skin dermis and epidermis in order to identify and define the genetic signature of the steady-state dermal macrophage subpopulations.

Cellular senescence is a stable cell cycle arrest that can be triggered by various biological stresses. Importantly, senescent cells remain metabolically active and secrete numerous molecules, such as cytokines, chemokines, proteases and growth factors. This secretome is called SASP (senescence associated secretory phenotype). Senescence plays a role in several processes, most notably in cancer, where it acts as an intrinsic tumor suppressor mechanism by inhibiting cell growth of premalignant cells. More recently, senescence has been shown to be involved in other biological responses, notably in tissue repair and aging. We recently showed that senescence upon tissue damage was promoting cellular reprogramming and cellular plasticity, notably via SASP. More precisely, we showed that IL-6 was an important mediator of SASP effect. Blocking IL-6 abolished beneficial effect of the SASP on reprogramming. Nonetheless, we speculate that other factors may be important for reprogramming. Indeed SASP factors have been previously shown to play redundant roles, notably in mediating senescence. Therefore we performed mass spectrometry analysis to identify other SASP factors in collaboration with proteomic platform of Institut Pasteur. We already identified promising candidates but we would also like to have a better global understanding of SASP complexity and which pathways it could activate in recipient cells. Thus, to investigate SASP effects more in details, we collaborate with C3BI platform of Institut Pasteur. Finally, understanding how SASP impact cellular plasticity in the context to tissue regeneration is essential for devising new strategies based on in vivo reprogramming.

The transcriptome signature during rabies infection will be analyzed both in animal model (mouse) and in human, at the central nervous system level, to identify the main canonical pathways involved during the infection.

Streptococcus gallolyticus sous espèce gallolyticus, autrefois dénommée Streptococcus bovis biotype I, est une bactérie de la flore intestinale qui constitue une cause émergente de septicémies et d’endocardites chez les personnes âgées. Depuis plus de 50 ans, les études épidémiologiques indiquent l’existence d’une forte association entre les infections invasives à S. gallolyticus et le CCR (cancer colorectal) (Pasquereau-Kotula et al., 2018). Néanmoins, le rôle exact de S.gallolyticus dans le développement du cancer colorectal n'est toujours pas déterminé. L’objectif principal de ce projet de recherche est de mieux comprendre le rôle de S. gallolyticus dans les CCR. Pour vérifier le rôle du S. gallolyticus dans le dévalement du CCR on a utilisé un modèle de cancer colorectal chez la souris (A/J + AOM). Grace à ce model o a peux démonter que S. gallolyticus accélère la tumorigenèse. Pour essayer de trouver un mechanism responsable de ce effect nous avons réalisé un protéome et un phosphoprotéome complets sur des tissus de souris colonisés par bactérie contrôle ou S. gallolyticus. L'analyse des changements dans le total et le phospho protéome aidera à mettre en évidence le mécanisme pertinent de la cancérogenèse induite par le S. gallolyticus.

Left-right asymmetry of the heart is essential for establishing the double blood circulation. Impairment of left-right embryo patterning is associated with severe congenital heart defects. During embryonic development, the heart initially forms as a straight tube. While it elongates, it acquires the shape of a rightward helix, a process referred to as heart looping. Previous work suggests that dynamic signaling in the heart precursor cells are essential for heart looping completion, but so far only a few genes have been identified as asymmetrically expressed. In order to investigate the dynamic spatiotemporal patterning of heart precursors, and screen for novel players in asymmetric heart morphogenesis, we aim to perform several transcriptomic analyses.

This project aims at characterizing the transcriptional signature of skeletal muscle during regeneration.

In recent years the concept that only adaptive immunity mediates memory responses has been challenged by several examples of innate immune memory, as shown for macrophages and NK cells. NK cells are innate lymphoid cells, with well-established roles in targeting cancer and virally infected cells, and in addition, are crucial players in antibacterial immunity. Similarly to what has been shown in response to virus, our work is revealing that NK cells maintain a memory of bacterial infection, as seen by a faster and more tailored response to subsequent infections. In response to viral infection, it has been shown that a certain subpopulation of NK cells become “memory” cells, which can expand and contract according to necessity. Similar studies in search of a memory population to bacterial infection have not been shown. Indeed, a major setback in such experiments is the lack of established markers for memory cells. Without a known receptor-ligand interaction, like what has been described for viruses, specific memory subpopulations are very difficult to identify by classical approaches. Single cell transcriptomics represents a novel approach that has been used to discover multiple immune subtypes and will provide a method to identify new sub-populations of memory cells within the NK cell pool.

Establishment of a left-right asymmetry is essential for the function of the heart, which is to ensure a double blood circulation. This asymmetry is initiated during an embryonic process, in which the heart tube acquires a loop shape. The molecular cascade at the origin of left-right asymmetry is well established and involved in the severe heart defects of the heterotaxy syndrome. However, another 20% of heart malformations display cardiac chamber misalignment and their origin is poorly understood. Using a mutant mouse model and expertise in heart morphogenesis, we want to provide novel insight into malformations displaying an abnormal alignment of ventricles.

Complex chronic diseases such as type 1 and 2 diabetes are caused by the accumulation of genetic, microbial and lifestyle factors. The number and complexity of such factors makes prediction of pathogenesis and therapy particularly difficult. Although a single factor is rarely sufficient to trigger pathology, genetic and environmental factors have so far been studied in isolation. Nevertheless, a substantial number of genetic variants have been associated with disease risk and the concomitant lifestyle shift and excessive hygiene are thought to contribute to the increased incidence in inflammatory diseases in industrialized countries. Moreover, clinical and experimental observations suggest a strong impact of gut microbiota on susceptibility to inflammatory diseases. The aim of 3DPATH is to explore the multiplicity and complexity of genetic, microbial and lifestyle factors associated with vulnerability to inflammatory pathology, with a particular focus on type 1 and 2 diabetes, using mice of the Collaborative Cross (CC), that model human genetic variability. Quantitative trait loci analyses, as well as integrative data analyses on metabolic and inflammatory outcomes will reveal new genetic variants and combinations of variants associated with disease susceptibility, and whether alterations of the gut microbiota in genetically susceptible mouse strains can trigger the phenotype. Moreover, the longitudinal design of experiments will allow us to identify early biomarkers that predict the pathology later in life. In a second step, validation of the identified risk factor combinations and exploration of the underlying molecular mechanisms will be performed taking advantage of the mouse model.

Our project aims to study the consequences of the systemic inflammation on muscle stem cells (MuSCs). To address this question, we use two in vivo mouse models associated with high systemic inflammation, an acute viral respiratory infection, with the influenza virus, and a solid tumor growth. Both paradigms are associated with muscle catabolism, named cachexia, which is intensively studied in the cancer field. However, most studies are focusing on the muscle fibres, the contractile apparatus of the muscle. Our project is mainly focused on the study of the MuSCs, cells which give rise to skeletal muscle. We aim to characterize the molecular and functional modifications occurring in MuSCs when exposed to high inflammatory stress. RNAseq was performed for both paradigms to gain information on gene expression.

There are papain treated and papain+CPG treated group. From mouse lung tissue, I'd like to find out the target cell and their key molecule through scRNAseq and receptor-ligand analysis. When I get the target cell, I want to do ATACseq (or scATACseq) of target cell to see if memory of previous exposure to inflammatory stimuli represented by chromatin accessibility and which has something to do with 2nd exposure of same allergen.

We seek to make the appropriate statistical comparisons of oxygen consumption and mitochondrial membrane potential measurements in paired, high-resolution respirometry measurements between control and experimental samples

New mouse genetic reference populations obtained from 8 distinct founder strains, including 3 wild-derived strains, have been developped since the 2000s and are now available. These populations captured around 95% of the existing murine genetic diversity and constitute a better model than standard inbred laboratory mouse strains. These populations constitute tools to identify new phenotypes and to map the causative genetic variants (QTL mapping) behind these phenotypes. These populations include : i) Collaborative Cross (CC) inbred strains; ii) Recombinant inbred between CCs (CC-RIXs) and iii) Diversity Outbred. These populations are specifically used in the Mouse Genetics Laboratory to identify new variants involved in host susceptiblity differences to pathogens.

Muscle stem cells in different anatomical locations are programmed with distinct upstream regulators prior to acquiring myogenic identity. This intriguing observation is most evident between cranial and trunk/limb muscles, and it correlates with the fact that cranial muscle stem cells are functionally more robust in terms of expansion capacity, resistance to stress, and engraftment efficiency, when compared to those in the limb. In this project we are using genomic approaches (sn-RNAseq and sc-ATACseq) and mouse models to study how the unique properties of cranial muscle groups are defined, and how they impact on myopathies where only subsets of skeletal muscles succumb to the disease process.

Kdm6b is a histone demethylase that specifically demethylates tri-methylated histone3 lysine27 (H3K27me3), a repressive mark responsible for gene silencing. Its removal by Kdm6b enables the transcription of specific target genes. Kdm6b is expressed in many cell types such as neurons, hematopoietic stem cells, leukocytes or fibroblasts. Type 2 Innate Lymphoid cells (ILC2) are the innate counterpart of Th2 lymphocytes enriched at the barrier surfaces where they play a role in tissue repair and anti-helminth defense. Previous work from our team showed that Kdm6b expression is upregulated in IL-33-activated ILC2s. Using a IL7RCre-Kdm6bflox mouse model that specifically deletes Kdm6b in all IL7R-expressing cells including ILC2, we observed decreased ILC2 numbers in bone marrow (BM) as well as in peripheral organs. qRT-PCR on sorted Kdm6b-deficient BM ILC2 precursors revealed decreased expression of several transcription factors as well as St2, the IL-33 receptor, all involved in ILC2 maturation and activation. Stimulation via intravenous (i.v) injections of IL-33 showed alteration of cytokine production in Kdm6b-deficient ILC2s compared to WT ILC2s, in BM as well as in the periphery. This observation was confirmed using an IL-33-dependent asthma model, the papain model, where Kdm6b-deficient lung ILC2s displayed decreased secretion of inflammatory cytokines. Altogether, our results suggest a role for Kdm6b in ILC2 maturation and Th2 pathway maintenance through the IL-33/ST2 axis.

The weaning reaction is a robust intestinal immune response that is induced by the expansion of the symbiotic microbiota during food diversification from milk to solid food. This weaning reaction is critical in setting the reactivity of the immune system in the long term to confer protection against inflammatory pathology in adulthood. With the aim to identify the key players in mediating this protective immunological imprinting, this project focuses on elucidating the role of myeloid cells and aims to decipher a potential protective signature at the transcriptomic and epigenetic level in the myeloid branch, notably the hematopoietic stem and myeloid progenitor cells, which are most likely to carry the long-term healthy imprinting.

Mitochondria are dynamic organelles that undergo constant morphological changes, resulting from fusion and fission events. Mitochondrial fission is crucial for mitochondrial function, apoptosis, mitophagy, and mitochondrial segregation during mitosis. While core mitochondrial fission factors have been elucidated and characterized, it is unclear if additional molecules participate or are main players of the fission process. To solve this question, we setup a genome wide, high content imaging (HCI) screening to identify suppressors of mitochondrial fragmentation in Opa1-/- cells. The principle is that using cells deficient for mitochondrial fusion (ablated for the core fusion protein Opa1), we may identify fission factors by screening for genes for which the loss of function is able to complement Opa1-/- phenotype. This was validated in preliminary experiments of silencing of the core fission protein Drp1, where confocal and electron microscopy confirmed that ablation of Drp1 resulted in mitochondrial elongation in Opa1-/- cells without causing mitochondrial fusion in a classic polyethylene glycol fusion assay. Following miniaturization of the assay, we set up an efficient pipeline to perform an automated HCI screening in Opa1-/- MEFs transfected with a pooled siRNAs library targeting >19,000 genes. Automated imaging and high content image quantification allowed us to generate a list of potential hits, that we aim to process in collaboration with the lab of Timothy Wai and the C3BI HUB, in order to identify promising genes that will be validated and investigated in the future.

Nod2 is expressed on brain neurons, however the downstream effects of its activation by its ligand MDP are not know. We used RNAseq strategy to answer this question

We are in the process of publishing a paper showing that IRF8, a transcription factor, regulates MS4A1 expression. Indeed, we showed IRF8 was necessary for MS4A1 expression by knocking-out the gene. A reviewer is asking us to directly assess IRF8 binding on MS4A1 gene using already published CHIP-seq data (PMID:31000603).

Characterization of recombination events which occured within the immunoglobulins heavy chain locus of murine B lymphocytes via sequencing of PCR products, using the Pacbio technology.

In the past few years, evidence that Natural Killer cells can acquire memory properties has challenged the dogma that only adaptive immunity mediates memory responses. This new property of NK cells is defined by a specific enhanced response upon re-exposure linked to substantial chromatin remodeling during NK clonal expansion, and to the acquisition of stable transcriptional changes. However, NK cell memory is poorly understood at the molecular level and exclusively described in response to viral infection. Whether NK cells could acquire memory during bacterial infections and what chromatin remodeling mechanisms are involved remained uninvestigated. However, in the lab, we have shown that following LPS-induced endotoxemia, NK cells acquire long-lasting memory properties associated with specific epigenetic modifications. In this project, using S pneumo infection as a model, we will perform single cell ATAC-seq coupled with transcriptomics (Multiomics) to identify the transcriptional program of the emerging subpopulations of memory NK cells and define its epigenomic landscape. With these data, we aim to identify chromatin rearrangements and gene expression patterns that are induced and maintained in the sub-population of memory NK cells. We believe that this project will enlighten our understanding of how NK cell memory identity and function is encoded at the chromatin level, a wide-open field of study.