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1. CD32+CD4+ T Cells Sharing B Cell Properties Increase With Simian Immunodeficiency Virus Replication in Lymphoid Tissues

Author

Huot, Nicolas, Rascle, Philippe, Planchais, Cyril, Contreras, Vanessa, Passaes, Caroline, Le Grand, Roger, Beignon, Anne-Sophie, Kornobis, Etienne, Legendre, Rachel, Varet, Hugo, Saez-Cirion, Asier, Mouquet, Hugo, Jacquelin, Beatrice, Müller-Trutwin, Michaela, HIV, Inflammation et persistance - HIV, Inflammation and Persistence, Institut Pasteur [Paris] (IP), Université Paris Diderot, Sorbonne Paris Cité, Paris, France, Université Paris Diderot - Paris 7 (UPD7), Immunologie humorale - Humoral Immunology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Pôle Biomics (C2RT), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris] (IP)-Institut Pasteur [Paris] (IP), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), For sequencing and analysis support, we thank the Biomics Platform, C2RT, Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09-09) and IBISA. NH was supported by the Fondation Jacquelin Beytout and Institut Pasteur. PR was recipient of a PhD fellowship from the University Paris Diderot, Sorbonne Paris Cité and also supportedby the NIH (R01AI143457). CP was the recipient of a PhD fellowship from the ANRS. HM received core grants from the Institut Pasteur, the INSERM and the Milieu Intérieur Program(ANR-10-LABX-69-01). We would like to acknowledge funding support from ANRS, MSDAvenir and Institut Pasteur to MM-Tand AS-C. We gratefully acknowledge the support to IDMIT from the French government: Investments for the Future program for infrastructures (PIA) through the ANR-11-INBS-0008 grant as well as from the PIA grant ANR-10-EQPX-02-01 to the FlowCyTech facility at IDMIT., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), ANR-11-INBS-0008,IDMIT,Infrastructure nationale pour la modélisation des maladies infectieuses humaines(2011), ANR-10-EQPX-0002,FlowCyTech,Plateforme de phénotypage en Mass cytométrie pour l'analyse multiparamétrique de biomarqueurs complexes de l'efficacité de nouvelles stratégies thérapeutiques ou vaccinales(2010), HIV, Inflammation et persistance, Institut Pasteur [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Institut Pasteur [Paris], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Varet, Hugo, Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID, Laboratoires d'excellence - GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE - - MILIEU INTERIEUR2010 - ANR-10-LABX-0069 - LABX - VALID, Infrastructures - Infrastructure nationale pour la modélisation des maladies infectieuses humaines - - IDMIT2011 - ANR-11-INBS-0008 - INBS - VALID, and Equipements d'excellence - Plateforme de phénotypage en Mass cytométrie pour l'analyse multiparamétrique de biomarqueurs complexes de l'efficacité de nouvelles stratégies thérapeutiques ou vaccinales - - FlowCyTech2010 - ANR-10-EQPX-0002 - EQPX - VALID

Subjects
[SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM] Life Sciences [q-bio]/Immunology, [SDV]Life Sciences [q-bio], Immunology, HIV, CD4, LN, [SDV] Life Sciences [q-bio], SIV, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, natural host, Immunology and Allergy, CD32, [SDV.IMM]Life Sciences [q-bio]/Immunology, CD20, intestine
Abstract

International audience; CD4 T cell responses constitute an important component of adaptive immunity and are critical regulators of anti-microbial protection. CD4 + T cells expressing CD32a have been identified as a target for HIV. CD32a is an Fcγ receptor known to be expressed on myeloid cells, granulocytes, B cells and NK cells. Little is known about the biology of CD32 + CD4 + T cells. Our goal was to understand the dynamics of CD32 + CD4 + T cells in tissues. We analyzed these cells in the blood, lymph nodes, spleen, ileum, jejunum and liver of two nonhuman primate models frequently used in biomedical research: African green monkeys (AGM) and macaques. We studied them in healthy animals and during viral (SIV) infection. We performed phenotypic and transcriptomic analysis at different stages of infection. In addition, we compared CD32+CD4+ T cells in tissues with well-controlled (spleen) and not efficiently controlled (jejunum) SIV replication in AGM. The CD32 + CD4 + T cells more frequently expressed markers associated with T cell activation and HIV infection (CCR5, PD-1, CXCR5, CXCR3) and had higher levels of actively transcribed SIV RNA than CD32 - CD4 + T cells. Furthermore, CD32 + CD4 + T cells from lymphoid tissues strongly expressed B-cell-related transcriptomic signatures, and displayed B cell markers at the cell surface, including immunoglobulins CD32+CD4+ T cells were rare in healthy animals and blood but increased strongly in tissues with ongoing viral replication. CD32 + CD4 + T cell levels in tissues correlated with viremia. Our results suggest that the tissue environment induced by SIV replication drives the accumulation of these unusual cells with enhanced susceptibility to viral infection.

Published
2021

2. Transcriptional adaptation of Mycobacterium ulcerans in an original mouse model: New insights into the regulation of mycolactone

Author

Robbe-Saule, Marie, Foulon, Mélanie, Poncin, Isabelle, Esnault, Lucille, Varet, Hugo, Legendre, Rachel, Besnard, Alban, Grzegorzewicz, Anna, Jackson, Mary, Canaan, Stéphane, Marsollier, Laurent, Marion, Estelle, Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), Aix Marseille Université (AMU), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Pôle Biomics (C2RT), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris] (IP)-Institut Pasteur [Paris] (IP), Mycobacteria Research Laboratories [Fort Collins, CO, États-Unis], Department of Microbiology, Immunology and Pathology [Fort Collins, CO, États-Unis], Colorado State University [Fort Collins] (CSU)-Colorado State University [Fort Collins] (CSU), This work was supported by the Agence Nationale de la Recherche [Grant ANR-15-IFEC-0006], Agence Nationale de la Recherche [Grant ANR- 16-CE12-0023], Fondation pour la Recherche Médicale [Equipe FRM], Region Pays de la Loire [Grant Starter], Fondation Raoul Follereau, INSERM., ANR-15-IFEC-0006,BU_SPONT_HEAL,Transcriptome Analysis of Animal Models of Spontaneous Healing of Buruli Ulcer(2015), ANR-16-CE12-0023,MYCOPARADOX,Dissection de l'architecture génétique des réactions paradoxales dans la Lèpre et l'Ulcère de Buruli(2016), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Institut Pasteur [Paris], ATOMycA (CRCINA-ÉQUIPE 6), Centre hospitalier universitaire de Nantes (CHU Nantes)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes (UN)-Université d'Angers (UA)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes (UN)-Université d'Angers (UA), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Institut Pasteur [Paris], Colorado State University [Fort Collins] (CSU), Varet, Hugo, Transcriptome Analysis of Animal Models of Spontaneous Healing of Buruli Ulcer - - BU_SPONT_HEAL2015 - ANR-15-IFEC-0006 - Infect-ERA - VALID, and Dissection de l'architecture génétique des réactions paradoxales dans la Lèpre et l'Ulcère de Buruli - - MYCOPARADOX2016 - ANR-16-CE12-0023 - AAPG2016 - VALID

Subjects
Mycobacterium Infections, Mycobacterium ulcerans, [SDV]Life Sciences [q-bio], Adaptation, Biological, mycolactone, Infectious and parasitic diseases, RC109-216, Gene Expression Regulation, Bacterial, [SDV] Life Sciences [q-bio], Mice, host-bacterium interaction, Animals, Humans, rna-sequencing, Macrolides, Buruli Ulcer, metabolism, Research Article, Research Paper, rnasequencing
Abstract

We thank all members of the animal SCAHU platform.; International audience; Mycobacterium ulcerans is the causal agent of Buruli ulcer, a chronic infectious disease and the third most common mycobacterial disease worldwide. Without early treatment, M. ulcerans provokes massive skin ulcers, caused by the mycolactone toxin, its main virulence factor. However, spontaneous healing may occur in Buruli ulcer patients several months or years after the disease onset. We have shown, in an original mouse model, that bacterial load remains high and viable in spontaneously healed tissues, with a switch of M. ulcerans to low levels of mycolactone production, adapting its strategy to survive in such a hostile environment. This original model offers the possibility to investigate the regulation of mycolactone production, by using an RNA-seq strategy to study bacterial adaptation during mouse infection. Pathway analysis and characterization of the tissue environment showed that the bacillus adapted to its new environment by modifying its metabolic activity and switching nutrient sources. Thus, M. ulcerans ensures its survival in healing tissues by reducing its secondary metabolism, leading to an inhibition of mycolactone synthesis. These findings shed new light on mycolactone regulation and pave the way for new therapeutic strategies.

Published
2021

3. SIV-induced terminally differentiated adaptive NK cells in lymph nodes associated with enhanced MHC-E restricted activity

Author

Huot, Nicolas, Rascle, Philippe, Petitdemange, Caroline, Contreras, Vanessa, Stürzel, Christina M., Baquero, Eduard, Harper, Justin L., Passaes, Caroline, Legendre, Rachel, Varet, Hugo, Madec, Yoann, Sauermann, Ulrike, Stahl-Hennig, Christiane, Nattermann, Jacob, Saez-Cirion, Asier, Le Grand, Roger, Keith Reeves, R., Paiardini, Mirko, Kirchhoff, Frank, Jacquelin, Beatrice, Müller-Trutwin, Michaela, Varet, Hugo, Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID, Infrastructures - Infrastructure nationale pour la modélisation des maladies infectieuses humaines - - IDMIT2011 - ANR-11-INBS-0008 - INBS - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, HIV, Inflammation et persistance - HIV, Inflammation and Persistence, Institut Pasteur [Paris] (IP), Immunologie des maladies virales, auto-immunes, hématologiques et bactériennes (IMVA-HB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, University of Ulm (UUlm), Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Yerkes National Primate Research Center [Lawrenceville, GA], Emory University [Atlanta, GA], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Pôle Biomics (C2RT), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris] (IP)-Institut Pasteur [Paris] (IP), Epidémiologie des Maladies Emergentes - Emerging Diseases Epidemiology, Pasteur-Cnam Risques infectieux et émergents (PACRI), Institut Pasteur [Paris] (IP)-Conservatoire National des Arts et Métiers [CNAM] (CNAM), HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-Institut Pasteur [Paris] (IP)-Conservatoire National des Arts et Métiers [CNAM] (CNAM), HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM), German Primate Center - Deutsches Primatenzentrum -- Leibniz Insitute for Primate Research -- [Göttingen, Allemagne] (GPC - DPZ), Universitätsklinikum Bonn (UKB), Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Harvard Medical School [Boston] (HMS), Emory University School of Medicine, The Biomics Platform (C2RT, Institut Pasteur) is supported by France Génomique (ANR-10-INBS-09-09) and IBISA NH was supported by the VRI Labex, the Fondation Jacquelin Beytout and Institut Pasteur, and PR was recipient of a PhD fellowship from the University Paris Diderot, Sorbonne Paris Cité. C.P. was supported by a MSDAvenir Postdoctoral Fellowship Grant. FK was supported by the DFG (CRC 1279 and SPP 1923. We would like to acknowledge grant support from ANRS, the Fondation Jacqueline Beytout, and the Fondation Les Ailes to M.M.T., from the NIH (R01AI143457) to M.M.T. and R.K.R. and from MSDAvenir to A.S.C. and M.M.T., J.L.H. and M.P. were supported by NIH grant R01AI116379 to M.P. and by ORIP/OD award P51OD011132 to Yerkes National Primate Research Center. J.N. was supported by the DFG (SPP 1937, SFB TR57), the Hector Foundation, and the DZIF (TTU-HIV). We gratefully acknowledge the support to IDMIT from the French government: Investments for the Future program for infrastructures (PIA) through the ANR-11-INBS-0008 grant as well as from the PIA grant ANR-10-EQPX-02-01 to the FlowCyTech facility at IDMIT. We equally acknowledge the Investments for Future grant ANR-10–INSB–04 to support the UtechS Photonic BioImaging (Imagopole) and C2RT facilities at Institut Pasteur., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), ANR-11-INBS-0008,IDMIT,Infrastructure nationale pour la modélisation des maladies infectieuses humaines(2011), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), HIV, Inflammation et persistance, Institut Pasteur [Paris], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Institut Pasteur [Paris], Institut Pasteur [Paris]-Conservatoire National des Arts et Métiers [CNAM] (CNAM)-Institut Pasteur [Paris]-Conservatoire National des Arts et Métiers [CNAM] (CNAM), and Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
CD4-Positive T-Lymphocytes, Male, MESH: Killer Cells, Natural, MESH: Cell Differentiation, Lymphoid Tissue, Science, animal diseases, [SDV]Life Sciences [q-bio], MESH: Simian Immunodeficiency Virus, Simian Acquired Immunodeficiency Syndrome, Fluorescent Antibody Technique, MESH: Algorithms, MESH: Lymph Nodes, MESH: Flow Cytometry, NK cells, Article, MESH: Chlorocebus aethiops, Chlorocebus aethiops, Animals, Humans, MESH: Animals, MESH: Fluorescent Antibody Technique, MESH: K562 Cells, Retrovirus, MESH: Humans, MESH: Transcriptome, MESH: Macaca, MESH: CD4-Positive T-Lymphocytes, Cell Differentiation, Flow Cytometry, MESH: Male, Killer Cells, Natural, [SDV] Life Sciences [q-bio], MESH: NK Cell Lectin-Like Receptor Subfamily C, Viral infection, MESH: Lymphoid Tissue, Macaca, Lymph node, Female, Simian Immunodeficiency Virus, Lymph Nodes, MESH: Simian Acquired Immunodeficiency Syndrome, K562 Cells, NK Cell Lectin-Like Receptor Subfamily C, Transcriptome, MESH: Female, Algorithms
Abstract

Natural killer (NK) cells play a critical understudied role during HIV infection in tissues. In a natural host of SIV, the African green monkey (AGM), NK cells mediate a strong control of SIVagm infection in secondary lymphoid tissues. We demonstrate that SIVagm infection induces the expansion of terminally differentiated NKG2alow NK cells in secondary lymphoid organs displaying an adaptive transcriptional profile and increased MHC-E-restricted cytotoxicity in response to SIV Env peptides while expressing little IFN-γ. Such NK cell differentiation was lacking in SIVmac-infected macaques. Adaptive NK cells displayed no increased NKG2C expression. This study reveals a previously unknown profile of NK cell adaptation to a viral infection, thus accelerating strategies toward NK-cell directed therapies and viral control in tissues., NK cells control SIV infection in secondary lymphoid tissues in the natural host that typically doesn’t progress toward disease. Here the authors show that this control is associated with terminal NK cell differentiation and improved MHC-E-dependent activity lacking in pathogenic SIV infection.

Published
2021

4. Post-transcriptional regulation of Leishmania fitness gain

Author

PIEL, Laura, SHANMUGHA RAJAN, K., BUSSOTTI, Giovanni, VARET, Hugo, LEGENDRE, Rachel, PROUX, Caroline, DOUCHÉ, Thibaut, GIAI-GIANETTO, Quentin, CHAZE, Thibault, VOJTKOVA, Barbora, GORDON-BAR, Nadav, DONIGER, Tirza, COHEN-CHALAMISH, Smadar, RENGARAJ, Praveenkumar, BESSE, Céline, BOLAND, Anne, SADLOVA, Jovana, DELEUZE, Jean-François, MATONDO, Mariette, UNGER, Ron, VOLF, Petr, MICHAELI, Shulamit, PESCHER, Pascale, SPÄTH, Gerald, Parasitologie moléculaire et Signalisation / Molecular Parasitology and Signaling, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Paris (UP), Bar-Ilan University [Israël], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Biomics (plateforme technologique), Institut Pasteur [Paris], Plateforme de Protéomique / Proteomics platform, Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Charles University [Prague] (CU), Centre National de Recherche en Génomique Humaine (CNRGH), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Paris-Saclay, This work was supported by the Fondation de la Recherche Médicale contract FDT201805005619(LP), the Agence Nationale pour la Recherche Labex ‘Integrative Biology of Emerging InfectiousDiseases’ contract ANR-10-LABX-62-IBEID and Labex ‘French Alliance for Parasitology and HealthCare’ contract ANR-11-LABX-0024 (GFS, LP, PP, GB), the Campus France Franco-Israeli ProgrammeHubert Curien Maimonide 2018 (GFS and SM), the seeding grant from the Institut Pasteur International Department to the LeiSHield Consortium and the EU H2020 project LeiSHield-MATI - REP-778298-1 (GB), the France Génomique National infrastructure, funded as part of the « Investissements d’Avenir » program managed by the Agence Nationale pour la Recherche contract ANR-10-INBS-09 (HV, RL, CP), and the ERD Funds, project CePaViP (CZ.02.1.01/0.0/0.0/ 16_019/0000759) (BV, JS, PV). We thank the CEA-CNRGH for its contribution to the sequencing costs and all the CEA-CNRGH staff who contributed to sample preparation and sequencing for their excellent technical assistance., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), Bussotti, Giovanni, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID, Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV] Life Sciences [q-bio], Leishmania, transcriptomics, posttranscriptional adaptation, [SDV]Life Sciences [q-bio], genomics, [INFO]Computer Science [cs], experimental evolution, [INFO] Computer Science [cs], small nucleolar RNAs, fitness
Abstract

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network driving parasite fitness, with genome instability causing highly reproducible, gene dosage-dependent changes in protein linked to post-transcriptional regulation. These in turn were associated with a gene dosage-independent reduction in flagellar transcripts and a coordinated increase in abundance of coding and non-coding RNAs known to regulate ribosomal biogenesis and protein translation. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain, where differential regulation of mRNA stability and the generation of fitness-adapted ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania non-coding small RNome as a potential novel source for biomarker discovery.

Published
2021

5. The gut environment regulates bacterial gene expression which modulates susceptibility to bacteriophage infection

Author

Marta Lourenço, Lorenzo Chaffringeon, Quentin Lamy-Besnier, Marie Titécat, Thierry Pédron, Odile Sismeiro, Rachel Legendre, Hugo Varet, Jean-Yves Coppée, Marion Bérard, Luisa De Sordi, Laurent Debarbieux, Bactériophage, bactérie, hôte - Bacteriophage, bacterium, host, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Collège Doctoral, Sorbonne Université (SU), Centre de Recherche Saint-Antoine (CRSA), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Paris Center for Microbiome Medicine (FHU PaCeMM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institute for Translational Research in Inflammation - U 1286 (INFINITE (Ex-Liric)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Biomics (plateforme technologique), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Transcriptome et Epigénome (PF2), Institut Pasteur [Paris] (IP), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Animalerie Centrale (plateforme) - Central Animal Facility (platform) (C2RA), M.L. is funded as part of the Pasteur-Paris University (PPU) International PhD Program. M.L. is funded by Institut Carnot Pasteur Maladies Infectieuses (ANR 11-CARN 017-01). L.D.S. is funded by a Roux-Cantarini Fellowship from the Institut Pasteur (Paris, France). L.C. is funded by a PhD Fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche, France, École Doctorale 394. Q.L.-B. is funded by École Doctorale FIRE - Programme Bettencourt. M.T. received a fellowship from Fondation DigestScience (Lille, France). The transcriptome and epigenome Platform is part of the France Génomique consortium (ANR10-NBS-09-08)., We thank Jorge Moura de Sousa for writing the customized R script for the pairwise comparisons between lists of genes from the different samples and for critically reading the manuscript. We thank Dwayne Roach and Anne Chevallereau for valuable discussions. We thank the Genetics of Biofilms team (Jean-Marc Ghigo) of Institut Pasteur and in particular Anne-Aurélie Lopes for training on the biofilm assays and Christophe Beloin for access to the ASKA plasmids collection. We thank the members of the Centre for Gnotobiology Platform of the Institut Pasteur (Thierry Angélique, Eddie Maranghi, Martine Jacob, Jérome Toutain, and Marisa Gabriela Lopez Dieguez) for their help with the animal work., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), Varet, Hugo, and Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID

Subjects
intestinal microbiota, MESH: Gene Expression, [SDV]Life Sciences [q-bio], Gene Expression, enteric pathogens, Microbiology, auto-inducer, flagellum, Mice, transcriptomics, Virology, Escherichia coli, Animals, Bacteriophages, MESH: Animals, MESH: Bacteriophages, MESH: Mice, Bacteria, MESH: Escherichia coli, lipopolysaccharide, [SDV] Life Sciences [q-bio], MESH: Bacteria, motility, Genes, Bacterial, Parasitology, biofilms, MESH: Genes, Bacterial, gene regulation
Abstract

International audience; Abundance and diversity of bacteria and their viral predators, bacteriophages (phages), in the digestive tract are associated with human health. Particularly intriguing is the long-term coexistence of these two antagonistic populations. We performed genome-wide RNA sequencing on a human enteroaggregative Escherichia coli isolate to identify genes differentially expressed between in vitro conditions and in murine intestines. We experimentally demonstrated that four of these differentially expressed genes modified the interactions between E. coli and three virulent phages by either increasing or decreasing its susceptibility/resistance pattern and also by interfering with biofilm formation. Therefore, the regulation of bacterial genes expression during the colonization of the digestive tract influences the coexistence of phages and bacteria, highlighting the intricacy of tripartite relationships between phages, bacteria, and the animal host in intestinal homeostasis.

Published
2022

6. PDGFRα-induced stromal maturation is required to restrain postnatal intestinal epithelial stemness and promote defense mechanisms

Author

Jean-Marie Jacob, Selene E. Di Carlo, Igor Stzepourginski, Anthony Lepelletier, Papa Diogop Ndiaye, Hugo Varet, Rachel Legendre, Etienne Kornobis, Adam Benabid, Giulia Nigro, Lucie Peduto, Stroma, inflammation et réparation tissulaire - Stroma, Inflammation and Tissue Repair, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Biomics (plateforme technologique), This work has received funding from the Institut Pasteur, INSERM and the European Union's Horizon 2020 research and innovation program (ERC Consolidator grant No. 648428) to L.P., European Project: 648428,H2020,ERC-2014-CoG,PERIF(2015), Varet, Hugo, and Perivascular cells at the crossroads of inflammation, regeneration and fibrosis - PERIF - - H20202015-11-01 - 2020-10-31 - 648428 - VALID

Subjects
IESC, perivascular, stromal niches, Receptor, Platelet-Derived Growth Factor alpha, rsubepithelial fibroblasts, [SDV]Life Sciences [q-bio], Stem Cells, Cell Differentiation, Cell Biology, epithelial differentiation, [SDV] Life Sciences [q-bio], Intestines, intestinal barrier, intestinal repair, Mice, inflammation, Lymphotoxin beta Receptor, postnatal intestinal maturation, Genetics, Molecular Medicine, Animals, Intestinal Mucosa, intestinal epithelial stem cells, Defense Mechanisms
Abstract

International audience; After birth, the intestine undergoes major changes to shift from an immature proliferative state to a functional intestinal barrier. By combining inducible lineage tracing and transcriptomics in mouse models, we identify a prodifferentiation PDGFRαHigh intestinal stromal lineage originating from postnatal LTβR+ perivascular stromal progenitors. The genetic blockage of this lineage increased the intestinal stem cell pool while decreasing epithelial and immune maturation at weaning age, leading to reduced postnatal growth and dysregulated repair responses. Ablating PDGFRα in the LTBR stromal lineage demonstrates that PDGFRα has a major impact on the lineage fate and function, inducing a transcriptomic switch from prostemness genes, such as Rspo3 and Grem1, to prodifferentiation factors, including BMPs, retinoic acid, and laminins, and on spatial organization within the crypt-villus and repair responses. Our results show that the PDGFRα-induced transcriptomic switch in intestinal stromal cells is required in the first weeks after birth to coordinate postnatal intestinal maturation and function.

Published
2021

7. Queuine Is a Nutritional Regulator of Entamoeba histolytica Response to Oxidative Stress and a Virulence Attenuator

Author

Hajime Hisaeda, Shruti Nagaraja, Chikako Shimokawa, Samudrala Gourinath, Mohit Mazumdar, Hugo Varet, Rachel Legendre, Nancy Guillén, Maggi W. Cai, Serge Ankri, Thomas J. Begley, Peter C. Dedon, Jean-Yves Coppée, Lotem Sarid, Yana Shaulov, Yumiko Saito-Nakano, Jingjing Sun, Meirav Trebicz-Geffen, Technion - Israel Institute of Technology [Haifa], National University of Singapore (NUS), Singapore-MIT Alliance for Research and Technology (SMART), Massachusetts Institute of Technology (MIT), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris], Jawaharlal Nehru University (JNU), Biologie des Interactions Hôte-Parasite - Biology of Host-Parasite Interactions, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), National Institute of Infectious Diseases, Shinjuku-ku, The work was supported by the Israel Ministry of Health within the framework European Research Area NETwork Infect-ERA (031L0004, AMOEBAC project), the Israel Science Foundation (260/16), the ISF-NRF program (3208/19), the National Research Foundation of Singapore through the Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance IRG, the Rappaport Institute, the US–Israel Binational Science Foundation (2015211), and the Niedersachsen program., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Varet, Hugo

Subjects
TRNA modification, Guanine, [SDV]Life Sciences [q-bio], Queuosine, Virulence, Microbiology, Methylation, 03 medical and health sciences, chemistry.chemical_compound, Entamoeba histolytica, Mice, fluids and secretions, RNA, Transfer, Virology, parasitic diseases, Animals, Humans, Gene, 030304 developmental biology, 0303 health sciences, Mice, Inbred BALB C, biology, 030302 biochemistry & molecular biology, Queuine, Translation (biology), biology.organism_classification, QR1-502, digestive system diseases, 3. Good health, [SDV] Life Sciences [q-bio], virulence, Oxidative Stress, Biochemistry, chemistry, Transfer RNA, Female, tRNA modification, Research Article, HeLa Cells
Abstract

Entamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota., Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His, and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here, we describe the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in tRNAGUCAsp. Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in the OS response, such as heat shock protein 70 (Hsp70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in tRNAGUCAsp, parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.

Published
2021

8. Leishmania amazonensis Subverts the Transcription Factor Landscape in Dendritic Cells to Avoid Inflammasome Activation and Stall Maturation

Author

Hervé Lecoeur, Thibault Rosazza, Kossiwa Kokou, Hugo Varet, Jean-Yves Coppée, Arezou Lari, Pierre-Henri Commère, Robert Weil, Guangxun Meng, Genevieve Milon, Gerald F. Späth, Eric Prina, Key Laboratory of Molecular Virology and Immunology [Shanghai, China], Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Parasitologie moléculaire et Signalisation / Molecular Parasitology and Signaling, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Pasteur International Mixed Unit 'Inflammation and Leishmania infection' (IMU-InflaLeish), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Innate Immunity [China], Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Leishmania Lab [Corée du sud], Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Transcriptome et Epigénome (PF2), Institut Pasteur [Paris], Institut Pasteur d'Iran, Cytométrie (Plate-forme), Centre d'Immunologie et de Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Immunophysiologie et Parasitisme, This project was supported by a fund of the Institut Pasteur International Direction to the Pasteur International Unit Inflammation and Leishmania infection and by the Fonds Dèdié Sanofi-Aventis/Ministère de la Recherche et de l'Enseignement Supérieur and Combattre les Maladies Parasitaires. The Institut Pasteur Biomics Platform was supported by France Génomique (ANR-10-INBS-09-09) and IBISA., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), Varet, Hugo, Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Innate Immunity [China], Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), and Centre d'Immunologie et des Maladies Infectieuses (CIMI)

Subjects
0301 basic medicine, lcsh:Immunologic diseases. Allergy, dendritic cell, [SDV]Life Sciences [q-bio], Antigen presentation, Immunology, Biology, NF-κB, 03 medical and health sciences, 0302 clinical medicine, Immune system, NLRP3, Gene expression, MHC class I, medicine, Immunology and Allergy, Amastigote, cell sorting, transcription factor, Original Research, Inflammasome, Dendritic cell, Phenotype, amastigote, Cell biology, [SDV] Life Sciences [q-bio], 030104 developmental biology, biology.protein, lcsh:RC581-607, transcriptome, 030215 immunology, medicine.drug, Leishmania amazonensis
Abstract

International audience; Leishmania parasites are the causative agents of human leishmaniases. They infect professional phagocytes of their mammalian hosts, including dendritic cells (DCs) that are essential for the initiation of adaptive immune responses. These immune functions strictly depend on the DC's capacity to differentiate from immature, antigen-capturing cells to mature, antigen-presenting cells-a process accompanied by profound changes in cellular phenotype and expression profile. Only little is known on how intracellular Leishmania affects this important process and DC transcriptional regulation. Here, we investigate these important open questions analyzing phenotypic, cytokine profile and transcriptomic changes in murine, immature bone marrow-derived DCs (iBMDCs) infected with antibody-opsonized and non-opsonized Leishmania amazonensis (L.am) amastigotes. DCs infected by non-opsonized amastigotes remained phenotypically immature whereas those infected by opsonized parasites displayed a semi-mature phenotype. The low frequency of infected DCs in culture led us to use DsRed2-transgenic parasites allowing for the enrichment of infected BMDCs by FACS. Sorted infected DCs were then subjected to transcriptomic analyses using Affymetrix GeneChip technology. Independent of parasite opsonization, Leishmania infection induced expression of genes related to key DC processes involved in MHC Class I-restricted antigen presentation and alternative NF-κB activation. DCs infected by non-opsonized parasites maintained an immature phenotype and showed a small but significant down-regulation of gene expression related to pro-inflammatory TLR signaling, the canonical NF-kB pathway and the NLRP3 inflammasome. This transcriptomic profile was further enhanced in Lecoeur et al. DC Subversion by Leishmania Amazonensis DCs infected with opsonized parasites that displayed a semi-mature phenotype despite absence of inflammasome activation. This paradoxical DC phenotype represents a Leishmania-specific signature, which to our knowledge has not been observed with other opsonized infectious agents. In conclusion, systems-analyses of our transcriptomics data uncovered important and previously unappreciated changes in the DC transcription factor landscape, thus revealing a novel Leishmania immune subversion strategy directly acting on transcriptional control of gene expression. Our data raise important questions on the dynamic and reciprocal interplay between transacting and epigenetic regulators in establishing permissive conditions for intracellular Leishmania infection and polarization of the immune response.

Published
2020

9. Functionally distinct resident macrophage subsets differentially shape responses to infection in the bladder

Author

Matthieu Rousseau, Rachel Legendre, Hugo Varet, Rebecca Gentek, Javier Saenz Coronilla, Livia Lacerda Mariano, Marc Bajénoff, Molly A. Ingersoll, Elisa Gomez Perdiguero, Département d'Immunologie - Department of Immunology, Institut Pasteur [Paris] (IP), Immunité Innée - Innate Immunity, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biomics (plateforme technologique), Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Macrophages et Cellules endothéliales / Macrophages and Endothelial Cells, L.L.M. is part of the Pasteur-Paris University (PPU) International PhD Program, which received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 665807 and from the Labex Milieu Intérieur (ANR-10-LABX-69-01). M.B. was supported by funding from the Agence Nationale de la Recherché (French National Research Agency) ANR-19-CE15-0015. E.G.P. was supported by funding from the Revive Labex (ANR-10-LABX-73), the Fondation Schlumberger (FRM FSER 2017), and the Emergence(s) program from Ville de Paris (2016 DAE 190). M.A.I. was supported by funding from the Agence Nationale de la Recherché (French National Research Agency) ANR-17-CE17-0014 and ANR-19-CE15-0015., ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-17-CE17-0014,PredictUTI,Identification de biomarqueurs sanguins prédictifs des infections uriniaires récidivantes et développement d'immunothérapies préventives personnalisées(2017), ANR-19-CE15-0015,BladderMacs,Fonction, origine, et homéostasie des macrophages résidents de la vessie(2019), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), Institut Pasteur [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Varet, Hugo, Laboratoires d'excellence - GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE - - MILIEU INTERIEUR2010 - ANR-10-LABX-0069 - LABX - VALID, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, Identification de biomarqueurs sanguins prédictifs des infections uriniaires récidivantes et développement d'immunothérapies préventives personnalisées - - PredictUTI2017 - ANR-17-CE17-0014 - AAPG2017 - VALID, Fonction, origine, et homéostasie des macrophages résidents de la vessie - - BladderMacs2019 - ANR-19-CE15-0015 - AAPG2019 - VALID, and Institut Pasteur International Docotal Program - PASTEURDOC - - H20202015-10-01 - 2020-10-01 - 665807 - VALID

Subjects
[SDV.IMM] Life Sciences [q-bio]/Immunology, [SDV]Life Sciences [q-bio], Urinary system, Immunology, Urinary Bladder, urologic and male genital diseases, Protein expression, Transcriptome, Mice, 03 medical and health sciences, 0302 clinical medicine, Immune system, Immunity, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Animals, Macrophage, Research Articles, 030304 developmental biology, 0303 health sciences, Lamina propria, biology, Gene Expression Profiling, Macrophages, SciAdv r-articles, Cell Biology, biology.organism_classification, female genital diseases and pregnancy complications, 3. Good health, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Urinary Tract Infections, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.IMM]Life Sciences [q-bio]/Immunology, Bacteria, Research Article, 030215 immunology
Abstract

Phenotypically and spatially distinct macrophage subsets reside in the bladder with differing roles in response to infection., Resident macrophages are abundant in the bladder, playing key roles in immunity to uropathogens. Yet, whether they are heterogeneous, where they come from, and how they respond to infection remain largely unknown. We identified two macrophage subsets in mouse bladders, MacM in muscle and MacL in the lamina propria, each with distinct protein expression and transcriptomes. Using a urinary tract infection model, we validated our transcriptomic analyses, finding that MacM macrophages phagocytosed more bacteria and polarized to an anti-inflammatory profile, whereas MacL macrophages died rapidly during infection. During resolution, monocyte-derived cells contributed to tissue-resident macrophage pools and both subsets acquired transcriptional profiles distinct from naïve macrophages. Macrophage depletion resulted in the induction of a type 1–biased immune response to a second urinary tract infection, improving bacterial clearance. Our study uncovers the biology of resident macrophages and their responses to an exceedingly common infection in a largely overlooked organ, the bladder.

Published
2020
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10. Targeting Macrophage Histone H3 Modification as a Leishmania Strategy to Dampen the NF-κB/NLRP3-Mediated Inflammatory Response

Author

Paya N’Diaye, Guangxun Meng, Nathalie Aulner, Eric Prina, Hugo Varet, Kossiwa Kokou, Robert Weil, Yue Xing, Thibault Rosazza, Gerald F. Späth, Geneviève Milon, Giovanni Bussotti, Hervé Lecoeur, Parasitologie moléculaire et Signalisation / Molecular Parasitology and Signaling, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Key Laboratory of Molecular Virology and Immunology [Shanghai, China], Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Institut Pasteur [Paris], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie et de Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), This project was supported by a fund of the Institut Pasteur International Direction to the International Mixed Unit 'Inflammation and Leishmania Infection' and by the International Partnership Program (153831KYSB20190008) of the Chinese Academy of Sciences. The UtechS PBI/C2RT is part of the FranceBioImaging infrastructure, supported by the French National Research Agency (ANR-10-INSB-04-01, Investments for the Future) and is supported by Conseil de la Région Ile-de-France (Domaine d’Intérêt Majeur DIM1HEALTH) and Fondation Française pour la Recherche Médicale (Programme Grands Equipements)., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Parasitologie moléculaire et Signalisation, Key Laboratory of Molecular Virology & Immunology (LMVI), Pasteur International Mixed Unit 'Inflammation and Leishmania infection' (IMU-InflaLeish), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Innate Immunity [China], Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Leishmania Lab [Corée du sud], Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), Technologie et Service BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], This project was supported by a fund of the Institut Pasteur International Direction to the International Mixed Unit 'Inflammation and Leishmania Infection' and by the International Partnership Program (153831KYSB20190008) of the Chinese Academy of Sciences. We would like to thank Jean-Marc Cavaillon for support and helpful discussions and Drs. Emmanuel Laplantine and Anastassia V. Komarova for providing antibodies. The UtechS PBI/C2RT is part of the FranceBioImaging infrastructure, supported by the French National Research Agency (ANR-10-INSB-04-01, Investments for the Future) and is supported by Conseil de la Région Ile-de-France (Domaine d’Intérêt Majeur DIM1HEALTH) and Fondation Française pour la Recherche Médicale (Programme Grands Equipements)., ANR-10-INSB-04-01,INSB,Investissements d'Avenir, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie et des Maladies Infectieuses (CIMI), Varet, Hugo, and Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID

Subjects
0301 basic medicine, [SDV.IMM] Life Sciences [q-bio]/Immunology, Inflammasomes, [SDV]Life Sciences [q-bio], H3 methylation, amastigotes, histone, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Biochemistry, Genetics and Molecular Biology, NF-κB, Histones, 03 medical and health sciences, Histone H3, chemistry.chemical_compound, 0302 clinical medicine, Immune system, Animals, Macrophage, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Epigenetics, lesional macrophage, lcsh:QH301-705.5, Leishmania, biology, epigenetics, Macrophages, NF-kappa B, H3 acetylation, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, NLRP3 inflammasome, 3. Good health, Human morbidity, Cell biology, [SDV] Life Sciences [q-bio], 030104 developmental biology, Histone, chemistry, lcsh:Biology (General), biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, 030217 neurology & neurosurgery, Leishmania amazonensis
Abstract

Summary: Aberrant macrophage activation during intracellular infection generates immunopathologies that can cause severe human morbidity. A better understanding of immune subversion strategies and macrophage phenotypic and functional responses is necessary to design host-directed intervention strategies. Here, we uncover a fine-tuned transcriptional response that is induced in primary and lesional macrophages infected by the parasite Leishmania amazonensis and dampens NF-κB and NLRP3 inflammasome activation. Subversion is amastigote-specific and characterized by a decreased expression of activating and increased expression of de-activating components of these pro-inflammatory pathways, thus revealing a regulatory dichotomy that abrogates the anti-microbial response. Changes in transcript abundance correlate with histone H3K9/14 hypoacetylation and H3K4 hypo-trimethylation in infected primary and lesional macrophages at promoters of NF-κB-related, pro-inflammatory genes. Our results reveal a Leishmania immune subversion strategy targeting host cell epigenetic regulation to establish conditions beneficial for parasite survival and open avenues for host-directed, anti-microbial drug discovery. : Lecoeur et al. demonstrate that the parasite Leishmania amazonensis modifies expression of NF-kB-related genes and prevents NLRP3 inflammasome activation of host macrophages in vitro and in vivo by changing histone H3 modifications at the promotor of pro-inflammatory genes. This mechanism of immune subversion opens avenues for host-directed, anti-leishmanial therapies. Keywords: Leishmania amazonensis, amastigotes, lesional macrophage, NF-κB, NLRP3 inflammasome, H3 acetylation, H3 methylation, epigenetics, histone

Published
2020

11. Cellular and molecular profiling of T-cell subsets at the onset of human acute GVHD

Author

Gérard Socié, Régis Peffault de Latour, Elisabetta Bianchi, Eleonora Latis, Lars Rogge, Hugo Varet, David Michonneau, C. Leloup, Varet, Hugo, Programme de Recherche Translationnelle en Santé - Biomarqueurs de la maladie du greffon contre l'hôte et de la tolérance immunitaire chez l'homme - - BioGvHD2013 - ANR-13-PRTS-0002 - PRTS - VALID, Immunorégulation - Immunoregulation, Institut Pasteur [Paris] (IP), Université Paris Diderot, Sorbonne Paris Cité, Paris, France, Université Paris Diderot - Paris 7 (UPD7), Inserm UMR-S 976, Institut National de la Santé et de la Recherche Médicale (INSERM), Service d'Hématologie Greffe, Hospital Saint Louis, Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants from the Agence National de la Recherche (ANR-PRTS 13002), the Institut National du Cancer (INCa 2014-1-PL BIO-07-IP-1), and the Direction Générale de l’Offre de Soins, as well as by institutional funds from Institut Pasteur. E.L. was supported by a grant from the Université Paris Diderot and the Ministère de la Recherche. G.S. received a research grant from ALEXION Pharmaceuticals. The CRYOSTEM project is supported by a grant from the Institut National du Cancer and the Agence Nationale de la Recherche and is under the auspices of the Société Francophone de Greffe de Moelle et de Thérapie Cellulaire., The authors thank all the members of the CRYOSTEM Consortium and the Francophone Society of Marrow Transplantations and Cellular Therapy for providing patient samples used in this study, as well as the Center for Translational Science/Cytometry Biomarkers Unit of Technology and Service (Institut Pasteur) for support in conducting this study., ANR-13-PRTS-0002,BioGvHD,Biomarqueurs de la maladie du greffon contre l'hôte et de la tolérance immunitaire chez l'homme(2013), Institut Pasteur [Paris], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, phenotype, T cell, molecular profiling, [SDV]Life Sciences [q-bio], Graft vs Host Disease, Human leukocyte antigen, Biology, CD8-Positive T-Lymphocytes, Flow cytometry, memory, 03 medical and health sciences, 0302 clinical medicine, Immunophenotyping, hemic and lymphatic diseases, medicine, graft-versus-host disease, Humans, genes, donors, t-lymphocytes, medicine.diagnostic_test, Effector, flow cytometry, Hematopoietic Stem Cell Transplantation, acute, Hematology, Phenotype, Tissue Donors, Transplantation, [SDV] Life Sciences [q-bio], 030104 developmental biology, medicine.anatomical_structure, surgical procedures, operative, t-lymphocyte subsets, 030220 oncology & carcinogenesis, Immunology, CD8, transplantation
Abstract

The cellular and molecular processes involved in acute graft-versus-host disease (aGVHD) development early after allogeneic hematopoietic cell transplantation (HCT) in humans remain largely unknown. We have performed multiparameter immunophenotyping and molecular profiling of CD4+ and CD8+ T cells in 2 independent cohorts of patients undergoing HCT, as well as in their HLA-identical sibling donors. Cellular profiling using spectral flow cytometry showed an incomplete reconstitution of the T-cell compartment in recipients without aGVHD early after transplantation, as well as a shift toward an effector memory phenotype, paralleled by depletion of the naive T-cell pool. Molecular profiling of T-cell populations in donors vs recipients without aGVHD revealed increased pathway activity of >40 gene modules in recipients. These pathways were associated in particular with T-cell activation, adhesion, migration, and effector functions. Cellular profiles from recipients developing aGVHD displayed an enrichment of cells with a T memory stem cell–like phenotype compared with recipients without aGVHD. Comparison of gene profiles from these recipients revealed that transforming growth factor-β (TGF-β) signaling was most significantly downregulated, whereas the pathway activity of NF-κB–associated transcription factors and signaling pathways were increased, at aGVHD onset. This study suggests that the integration of cellular and molecular profiles provides new insights into the development of aGVHD in humans.

Published
2020

12. Immune parameters and outcomes during Ebola virus disease

Author

Hervé Raoul, Emilie Gloaguen, Cédric Laouénan, Justine Schaeffer, Alexandra Journeaux, Sylvain Baize, Stéphanie Reynard, Natalia Pietrosemoli, Jimmy Mullaert, Mathieu Mateo, Hugo Varet, Nicolas Baillet, Biologie des Infections Virales Émergentes - Biology of Emerging Viral Infections (UBIVE), Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Infection, Anti-microbiens, Modélisation, Evolution (IAME (UMR_S_1137 / U1137)), Université Paris 13 (UP13)-Université Paris Diderot - Paris 7 (UPD7)-Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Département d'épidémiologie, biostatistique et recherche clinique, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-AP-HP - Hôpital Bichat - Claude Bernard [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Laboratoire P4 - Jean Mérieux, Centre Européen de Virologie/Immunologie-Institut National de la Santé et de la Recherche Médicale (INSERM), French Ministry of Foreign Affairs, the Agence Française de Développement, and the Institut Pasteur., École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Varet, Hugo, Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)

Subjects
0301 basic medicine, [SDV.IMM] Life Sciences [q-bio]/Immunology, [SDV]Life Sciences [q-bio], viruses, Disease, medicine.disease_cause, Virus, 03 medical and health sciences, 0302 clinical medicine, Immune system, Virology, Medicine, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Innate immunity, Infectious disease, Innate immune system, Ebola virus, business.industry, Septic shock, Outbreak, General Medicine, medicine.disease, 3. Good health, [SDV] Life Sciences [q-bio], 030104 developmental biology, Infectious disease (medical specialty), 030220 oncology & carcinogenesis, Immunology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Cytokines, [SDV.IMM]Life Sciences [q-bio]/Immunology, Chemokines, Clinical Medicine, business
Abstract

Background The West African Ebola virus epidemic from 2014-2016 highlighted the lack of knowledge about the pathogenicity of the virus and the factors responsible for outcome. A performant and rapid diagnosis is of crucial importance, as is overcoming the difficulty of providing high-quality patient management during such an extensive outbreak. Here, we propose to study the role of the immune mediators during Ebola virus disease and to define some molecules of importance in the outcome. Methods Plasma from Guinean patients sampled during the outbreak were analyzed using RT-qPCR, magnetic bead assay, ELISA, and high-quality statistical analyses. We also performed a transcriptomic analysis in leukocytes samples. Therefore, we deeply characterized the immune responses involved in Ebola virus disease. Results We evaluated the immune patterns depending on the outcome of the disease. Survivors presented an efficient and well-balanced immune response, whereas fatalities were characterized by an intense inflammatory response, overexpression of multiple cytokines, and a "chemokine storm." The plasma concentration of most of the parameters tested increased until death. Statistical analyses also allowed us to define a panel of markers highly predictive of outcome. Conclusion The immune response observed in fatalities was highly similar to that characterizing septic shock syndrome. Our results suggest that immune responses can play a major pathogenic role during severe Ebola virus infection and argue in favor of therapeutic approaches that act on both viral replication and the induction of shock syndrome. Funding French Ministry of Foreign Affairs, the Agence Francaise de Developpement, and the Institut Pasteur.

Published
2018

13. In Situ Fixation Redefines Quiescence and Early Activation of Skeletal Muscle Stem Cells

Author

Léo Machado, Joana Esteves de Lima, Odile Fabre, Caroline Proux, Rachel Legendre, Anikó Szegedi, Hugo Varet, Lars Roed Ingerslev, Romain Barrès, Frédéric Relaix, Philippos Mourikis, Biologie du système neuromusculaire - Biology of the neuromuscular system [Maisons-Alfort] (BNMS - Team 10), École nationale vétérinaire - Alfort (ENVA)-Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Novo Nordisk Foundation Center for Basic Metabolic Research (CBMR), Faculty of Health and Medical Sciences, University of Copenhagen = Københavns Universitet (UCPH)-University of Copenhagen = Københavns Universitet (UCPH), Transcriptome et Epigénome (PF2), Institut Pasteur [Paris] (IP), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by funding to F.R. from the Association Française contre les Myopathies (AFM) via TRANSLAMUSCLE (PROJECT 19507), Labex REVIVE (ANR-10-LABX-73), Fondation pour la Recherche Médicale (FRM, grants FDT20130928236 and DEQ20130326526), Agence Nationale pour la Recherche (ANR) grant Epimuscle (ANR 11 BSV2 017 02), Bone-muscle-repair (ANR-13-BSV1-0011-02), BMP-myomass (ANR-12-BSV1-0038-04), Satnet (ANR-15-CE13-0011-01), Crestnetmetabo (ANR-15-CE13-0012-02), and RHU CARMMA (ANR-15-RHUS-0003). O.F. was a recipient of a research grant from the Danish Diabetes Academy (1077471001) supported by the Novo Nordisk Foundation., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-11-BSV2-0017,epimuscle,Contrôle épigenetique du développement et de la physiologie musculaire par les histone demethylases.(2011), ANR-13-BSV1-0011,Bone-Muscle-Repair,Interactions os-muscle au cours de la régénération musculosquelettique(2013), ANR-12-BSV1-0038,BMP-MyoMass,Contôle de la mase musculaire par les BMPs (Bone Morphogenetic Proteins)(2012), ANR-15-CE13-0011,SatNet,Hétérogénéité et quiescence des cellules souches du muscle(2015), ANR-15-CE13-0012,CRESTNETMETABO,Nouveaux Défis du Réseau Régulateur du Développement Précoce de la Crete Neurale(2015), ANR-15-RHUS-0003,CARMMA,Role de la sénescence du tissu adipeux dans les co-morbidités des pathologies métaboliques(2015), Varet, Hugo, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, BLANC - Contrôle épigenetique du développement et de la physiologie musculaire par les histone demethylases. - - epimuscle2011 - ANR-11-BSV2-0017 - BLANC - VALID, Blanc 2013 - Interactions os-muscle au cours de la régénération musculosquelettique - - Bone-Muscle-Repair2013 - ANR-13-BSV1-0011 - Blanc 2013 - VALID, BLANC - Contôle de la mase musculaire par les BMPs (Bone Morphogenetic Proteins) - - BMP-MyoMass2012 - ANR-12-BSV1-0038 - BLANC - VALID, Hétérogénéité et quiescence des cellules souches du muscle - - SatNet2015 - ANR-15-CE13-0011 - AAPG2015 - VALID, Nouveaux Défis du Réseau Régulateur du Développement Précoce de la Crete Neurale - - CRESTNETMETABO2015 - ANR-15-CE13-0012 - AAPG2015 - VALID, Role de la sénescence du tissu adipeux dans les co-morbidités des pathologies métaboliques - - CARMMA2015 - ANR-15-RHUS-0003 - RHUS - VALID, École nationale vétérinaire d'Alfort (ENVA)-Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-IFR10-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-IFR10, University of Copenhagen = Københavns Universitet (KU)-University of Copenhagen = Københavns Universitet (KU), Institut Pasteur [Paris], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Tissue Fixation, early response genes, Myoblasts, Skeletal, [SDV]Life Sciences [q-bio], Primary Cell Culture, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Primary Cell Culture, Mice, MESH: DNA Methylation, Journal Article, Animals, quiescence, MESH: Animals, lcsh:QH301-705.5, MESH: Mice, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Cells, Cultured, satellite cells, MESH: Myoblasts, Skeletal, MESH: Transcriptome, DNA Methylation, Histone Code, ChIP-seq, [SDV] Life Sciences [q-bio], muscle stem cells, lcsh:Biology (General), MESH: Histone Code, Female, methylation, RNA-seq, MESH: Tissue Fixation, Transcriptome, MESH: Female, MESH: Cells, Cultured
Abstract

Summary: State of the art techniques have been developed to isolate and analyze cells from various tissues, aiming to capture their in vivo state. However, the majority of cell isolation protocols involve lengthy mechanical and enzymatic dissociation steps followed by flow cytometry, exposing cells to stress and disrupting their physiological niche. Focusing on adult skeletal muscle stem cells, we have developed a protocol that circumvents the impact of isolation procedures and captures cells in their native quiescent state. We show that current isolation protocols induce major transcriptional changes accompanied by specific histone modifications while having negligible effects on DNA methylation. In addition to proposing a protocol to avoid isolation-induced artifacts, our study reveals previously undetected quiescence and early activation genes of potential biological interest. : Machado et al. demonstrate that muscle stem cells undergo changes in transcripts and histone modifications during isolation. The authors develop an in situ fixation-based methodology, which allows capture of cells in their native state. In light of these findings, some high-throughput analyses of tissue extracted cells may need to be revisited. Keywords: muscle stem cells, quiescence, early response genes, RNA-seq, ChIP-seq, methylation, satellite cells

Published
2017

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