ABSTRACT
Regulatory T cells (Treg) are critical players of immune tolerance that develop in the thymus via two distinct developmental pathways involving CD25+Foxp3- and CD25-Foxp3lo precursors. However, the mechanisms regulating the recently identified Foxp3lo precursor pathway remain unclear. Here, we find that the membrane-bound lymphotoxin α1ß2 (LTα1ß2) heterocomplex is upregulated during Treg development upon TCR/CD28 and IL-2 stimulation. We show that Lta expression limits the maturational development of Treg from Foxp3lo precursors by regulating their proliferation, survival, and metabolic profile. Transgenic reporter mice and transcriptomic analyses further reveal that medullary thymic epithelial cells (mTEC) constitute an unexpected source of IL-4. We demonstrate that LTα1ß2-lymphotoxin ß receptor-mediated interactions with mTEC limit Treg development by down-regulating IL-4 expression in mTEC. Collectively, our findings identify the lymphotoxin axis as the first inhibitory checkpoint of thymic Treg development that fine-tunes the Foxp3lo Treg precursor pathway by limiting IL-4 availability.
Subject(s)
Forkhead Transcription Factors , Interleukin-4 , Lymphotoxin beta Receptor , Lymphotoxin-alpha , Signal Transduction , T-Lymphocytes, Regulatory , Animals , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Interleukin-4/metabolism , Mice , Lymphotoxin-alpha/metabolism , Lymphotoxin-alpha/genetics , Lymphotoxin beta Receptor/metabolism , Lymphotoxin beta Receptor/genetics , Thymus Gland/immunology , Thymus Gland/cytology , Thymus Gland/metabolism , Epithelial Cells/metabolism , Mice, Inbred C57BL , Cell Differentiation , Mice, Transgenic , Interleukin-2/metabolism , Cell Proliferation , Lymphotoxin alpha1, beta2 Heterotrimer/metabolism , Lymphotoxin alpha1, beta2 Heterotrimer/geneticsABSTRACT
Intergenic transcription in normal and cancerous tissues is pervasive but incompletely understood. To investigate this, we constructed an atlas of over 180,000 consensus RNA polymerase II (RNAPII)-bound intergenic regions from 900 RNAPII chromatin immunoprecipitation sequencing (ChIP-seq) experiments in normal and cancer samples. Through unsupervised analysis, we identified 51 RNAPII consensus clusters, many of which mapped to specific biotypes and revealed tissue-specific regulatory signatures. We developed a meta-clustering methodology to integrate our RNAPII atlas with active transcription across 28,797 RNA sequencing (RNA-seq) samples from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Encyclopedia of DNA Elements (ENCODE). This analysis revealed strong tissue- and disease-specific interconnections between RNAPII occupancy and transcriptional activity. We demonstrate that intergenic transcription at RNAPII-bound regions is a novel per-cancer and pan-cancer biomarker. This biomarker displays genomic and clinically relevant characteristics, distinguishing cancer subtypes and linking to overall survival. Our results demonstrate the effectiveness of coherent data integration to uncover intergenic transcriptional activity in normal and cancer tissues.
ABSTRACT
Single-cell technologies have revolutionised biological research and applications. As they continue to evolve with multi-omics and spatial resolution, analysing single-cell datasets is becoming increasingly complex. For biologists lacking expert data analysis resources, the problem is even more crucial, even for the simplest single-cell transcriptomics datasets. We propose ShIVA, an interface for the analysis of single-cell RNA-seq and CITE-seq data specifically dedicated to biologists. Intuitive, iterative and documented by video tutorials, ShIVA allows biologists to follow a robust and reproducible analysis process, mostly based on the Seurat v4 R package, to fully explore and quantify their dataset, to produce useful figures and tables and to export their work to allow more complex analyses performed by experts.
Subject(s)
Data Analysis , Single-Cell Gene Expression Analysis , Humans , Gene Expression Profiling , Health Personnel , MultiomicsABSTRACT
Chagas disease is a parasitical disease caused by Trypanosoma cruzi which affects â¼7 million people worldwide. Per year, â¼10 000 people die from this pathology. Indeed, â¼30% of humans develop severe chronic forms, including cardiac, digestive or neurological disorders, for which there is still no treatment. In order to facilitate research on Chagas disease, a manual curation of all papers corresponding to 'Chagas disease' referenced on PubMed has been performed. All deregulated molecules in hosts (all mammals, humans, mice or others) following T. cruzi infection were retrieved and included in a database, named ChagasDB. A website has been developed to make this database accessible to all. In this article, we detail the construction of this database, its contents and how to use it. Database URL https://chagasdb.tagc.univ-amu.fr.
Subject(s)
Chagas Disease , Humans , Animals , Mice , Databases, Factual , PubMed , MammalsABSTRACT
Peyer's patches (PPs) are secondary lymphoid organs in contact with the external environment via the intestinal lumen, thus combining antigen sampling and immune response initiation sites. Therefore, they provide a unique opportunity to study the entire process of phagocyte differentiation and activation in vivo. Here, we deciphered the transcriptional and spatial landscape of PP phagocyte populations from their emergence in the tissue to their final maturation state at homeostasis and under stimulation. Activation of monocyte-derived Lysozyme-expressing dendritic cells (LysoDCs) differs from that of macrophages by their upregulation of conventional DC (cDC) signature genes such as Ccr7 and downregulation of typical monocyte-derived cell genes such as Cx3cr1. We identified gene sets that distinguish PP cDCs from the villus ones and from LysoDCs. We also identified key immature, early, intermediate, and late maturation markers of PP phagocytes. Finally, exploiting the ability of the PP interfollicular region to host both villous and subepithelial dome emigrated cDCs, we showed that the type of stimulus, the subset, but also the initial location of cDCs shape their activation profile and thus direct the immune response. Our study highlights the importance of targeting the right phagocyte subset at the right place and time to manipulate the immune response.
Subject(s)
Dendritic Cells , Peyer's Patches , Phagocytes , Macrophages , Mononuclear Phagocyte SystemABSTRACT
The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II- MM were abundant neonatally, whereas MHC-II+ MM appeared over time. These barrier macrophages differentially responded to in vivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II+ MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation.
Subject(s)
COVID-19 , Lymphocytic Choriomeningitis , Animals , Mice , Lipopolysaccharides , Mice, Inbred C57BL , SARS-CoV-2 , Lymphocytic choriomeningitis virus/physiology , Macrophages , MeningesABSTRACT
Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. We investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP). Genome-wide associations studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that we identified were used to map potential regulatory non-coding regions, which in turn were employed to predict transcription factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart-specific knockdown. Additionally, we showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, we showed that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, we documented correlations between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 and EGR2 in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans.
Subject(s)
Drosophila melanogaster , Heart , Quantitative Trait Loci , Animals , Humans , Drosophila melanogaster/physiology , Gene Regulatory Networks , Genetic Variation , Genome-Wide Association Study , Phenotype , Heart/physiologyABSTRACT
Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology.
Subject(s)
Chagas Cardiomyopathy , Chagas Disease , Adenosine Triphosphate/metabolism , Biomarkers/metabolism , Chagas Cardiomyopathy/diagnosis , Chagas Cardiomyopathy/genetics , Chagas Disease/genetics , DNA Methylation , HumansABSTRACT
Understanding the relationship between genetic variations and variations in complex and quantitative phenotypes remains an ongoing challenge. While Genome-wide association studies (GWAS) have become a vital tool for identifying single-locus associations, we lack methods for identifying epistatic interactions. In this article, we propose a novel method for higher-order epistasis detection using mixed effect conditional inference forest (epiMEIF). The proposed method is fitted on a group of single nucleotide polymorphisms (SNPs) potentially associated with the phenotype and the tree structure in the forest facilitates the identification of n-way interactions between the SNPs. Additional testing strategies further improve the robustness of the method. We demonstrate its ability to detect true n-way interactions via extensive simulations in both cross-sectional and longitudinal synthetic datasets. This is further illustrated in an application to reveal epistatic interactions from natural variations of cardiac traits in flies (Drosophila). Overall, the method provides a generalized way to identify higher-order interactions from any GWAS data, thereby greatly improving the detection of the genetic architecture underlying complex phenotypes.
Subject(s)
Epistasis, Genetic , Genome-Wide Association Study , Genome-Wide Association Study/methods , Multifactorial Inheritance/genetics , Cross-Sectional Studies , Polymorphism, Single Nucleotide , ForestsABSTRACT
Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic parasitic disease of Latin America, affecting 7 million people. Although most patients are asymptomatic, 30% develop complications, including the often-fatal Chronic Chagasic Cardiomyopathy (CCC). Although previous studies have demonstrated some genetic deregulations associated with CCCs, the causes of their deregulations remain poorly described. Based on bulk RNA-seq and whole genome DNA methylation data, we investigated the genetic and epigenetic deregulations present in the moderate and severe stages of CCC. Analysis of heart tissue gene expression profile allowed us to identify 1407 differentially expressed transcripts (DEGs) specific from CCC patients. A tissue DNA methylation analysis done on the same tissue has permitted the identification of 92 regulatory Differentially Methylated Regions (DMR) localized in the promoter of DEGs. An in-depth study of the transcription factors binding sites (TFBS) in the DMRs corroborated the importance of TFBS's DNA methylation for gene expression in CCC myocardium. TBX21, RUNX3 and EBF1 are the transcription factors whose binding motif appears to be affected by DNA methylation in the largest number of genes. By combining both transcriptomic and methylomic analysis on heart tissue, and methylomic analysis on blood, 4 biological processes affected by severe CCC have been identified, including immune response, ion transport, cardiac muscle processes and nervous system. An additional study on blood methylation of moderate CCC samples put forward the importance of ion transport and nervous system in the development of the disease.
Subject(s)
Chagas Cardiomyopathy , Chagas Disease , Trypanosoma cruzi , Chagas Disease/genetics , Epigenesis, Genetic , Humans , Transcription Factors/geneticsABSTRACT
Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology.
Subject(s)
Chagas Cardiomyopathy , Chagas Disease , Methylation , Parasitic Diseases , Therapeutics , BiomarkersABSTRACT
Abstract: Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic parasitic disease of Latin America, affecting 7 million people. Although most patients are asymptomatic, 30% develop complications, including the often-fatal Chronic Chagasic Cardiomyopathy (CCC). Although previous studies have demonstrated some genetic deregulations associated with CCCs, the causes of their deregulations remain poorly described. Based on bulk RNA-seq and whole genome DNA methylation data, we investigated the genetic and epigenetic deregulations present in the moderate and severe stages of CCC. Analysis of heart tissue gene expression profile allowed us to identify 1407 differentially expressed transcripts (DEGs) specific from CCC patients. A tissue DNA methylation analysis done on the same tissue has permitted the identification of 92 regulatory Differentially Methylated Regions (DMR) localized in the promoter of DEGs. An in-depth study of the transcription factors binding sites (TFBS) in the DMRs corroborated the importance of TFBS's DNA methylation for gene expression in CCC myocardium. TBX21, RUNX3 and EBF1 are the transcription factors whose binding motif appears to be affected by DNA methylation in the largest number of genes. By combining both transcriptomic and methylomic analysis on heart tissue, and methylomic analysis on blood, 4 biological processes affected by severe CCC have been identified, including immune response, ion transport, cardiac muscle processes and nervous system. An additional study on blood methylation of moderate CCC samples put forward the importance of ion transport and nervous system in the development of the disease.
Subject(s)
Humans , Chagas Cardiomyopathy , Chagas Disease/genetics , Transcription Factors/genetics , Trypanosoma cruzi , Epigenesis, Genetic , MethylationABSTRACT
Lung-resident memory B cells (MBCs) provide localized protection against reinfection in respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. We found that two main transcriptionally distinct subsets of MBCs colonized the lung peribronchial niche after infection. These subsets arose from different progenitors and were both class switched, somatically mutated, and intrinsically biased in their differentiation fate toward plasma cells. Combined analysis of antigen specificity and B cell receptor repertoire segregated these subsets into "bona fide" virus-specific MBCs and "bystander" MBCs with no apparent specificity for eliciting viruses generated through an alternative permissive process. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.
Subject(s)
COVID-19 , Immunologic Memory , Animals , B-Lymphocytes , Lung , Memory B Cells , Mice , Reinfection , SARS-CoV-2ABSTRACT
Natural killer (NK) cells are known to be able to kill established tumor cell lines, but important caveats remain regarding their roles in the detection and elimination of developing primary tumors. Using a genetic model of selective ILC1 and NK cell deficiency, we showed that these cells were dispensable for tumor immunosurveillance and immunoediting in the MCA-induced carcinogenesis model. However, we were able to generate primary cell lines derived from MCA-induced tumors with graded sensitivity to NK1.1+ cells (including NK cells and ILC1). This differential sensitivity was associated neither with a modulation of intratumoral NK cell frequency, nor the capacity of tumor cells to activate NK cells. Instead, ILC1 infiltration into the tumor was found to be a critical determinant of NK1.1+ cell-dependent tumor growth. Finally, bulk tumor RNAseq analysis identified a gene expression signature associated with tumor sensitivity to NK1.1+ cells. ILC1 therefore appear to play an active role in inhibiting the antitumoral immune response, prompting to evaluate the differential tumor infiltration of ILC1 and NK cells in patients to optimize the harnessing of immunity in cancer therapies.
Subject(s)
Cytotoxicity, Immunologic/immunology , Lymphocytes/immunology , Sarcoma, Experimental/immunology , Animals , Cell Line, Tumor , Gene Expression Profiling , Humans , Immunity, Innate , Killer Cells, Natural/immunology , Lymphocyte Activation , Lymphocytes, Tumor-Infiltrating/immunology , Mice , Mice, Inbred C57BLABSTRACT
The development of high-throughput technologies revealed the existence of non-canonical short open reading frames (sORFs) on most eukaryotic ribonucleic acids. They are ubiquitous genetic elements conserved across species and suspected to be involved in numerous cellular processes. MetamORF (https://metamorf.hb.univ-amu.fr/) aims to provide a repository of unique sORFs identified in the human and mouse genomes with both experimental and computational approaches. By gathering publicly available sORF data, normalizing them and summarizing redundant information, we were able to identify a total of 1 162 675 unique sORFs. Despite the usual characterization of ORFs as short, upstream or downstream, there is currently no clear consensus regarding the definition of these categories. Thus, the data have been reprocessed using a normalized nomenclature. MetamORF enables new analyses at locus, gene, transcript and ORF levels, which should offer the possibility to address new questions regarding sORF functions in the future. The repository is available through an user-friendly web interface, allowing easy browsing, visualization, filtering over multiple criteria and export possibilities. sORFs can be searched starting from a gene, a transcript and an ORF ID, looking in a genome area or browsing the whole repository for a species. The database content has also been made available through track hubs at UCSC Genome Browser. Finally, we demonstrated an enrichment of genes harboring upstream ORFs among genes expressed in response to reticular stress. Database URL https://metamorf.hb.univ-amu.fr/.
Subject(s)
Genome , Software , Animals , Databases, Factual , Mice , Open Reading Frames/geneticsABSTRACT
[This corrects the article DOI: 10.3389/fimmu.2020.00216.].
ABSTRACT
During embryogenesis, lymphoid tissue inducer (LTi) cells are essential for lymph node organogenesis. These cells are part of the innate lymphoid cell (ILC) family. Although their earliest embryonic hematopoietic origin is unclear, other innate immune cells have been shown to be derived from early hemogenic endothelium in the yolk sac as well as the aorta-gonad-mesonephros. A proper model to discriminate between these locations was unavailable. In this study, using a Cxcr4-CreERT2 lineage tracing model, we identify a major contribution from embryonic hemogenic endothelium, but not the yolk sac, toward LTi progenitors. Conversely, embryonic LTi cells are replaced by hematopoietic stem cell-derived cells in adults. We further show that, in the fetal liver, common lymphoid progenitors differentiate into highly dynamic alpha-lymphoid precursor cells that, at this embryonic stage, preferentially mature into LTi precursors and establish their functional LTi cell identity only after reaching the periphery.
Subject(s)
Hemangioblasts/metabolism , Hematopoiesis/physiology , Lymphoid Tissue/embryology , Receptors, CXCR4/metabolism , Animals , Embryonic Development/physiology , Hemangioblasts/cytology , Hematopoietic Stem Cells/metabolism , Humans , Immunity, Innate , Liver/embryology , Lymphocytes/metabolism , T-Lymphocytes, Helper-Inducer/metabolism , Yolk Sac/embryologyABSTRACT
[This corrects the article DOI: 10.3389/fimmu.2020.00216.].
ABSTRACT
Located within red pulp cords, splenic red pulp macrophages (RPMs) are constantly exposed to the blood flow, clearing senescent red blood cells (RBCs) and recycling iron from hemoglobin. Here, we studied the mechanisms underlying RPM homeostasis, focusing on the involvement of stromal cells as these cells perform anchoring and nurturing macrophage niche functions in lymph nodes and liver. Microscopy revealed that RPMs are embedded in a reticular meshwork of red pulp fibroblasts characterized by the expression of the transcription factor Wilms' Tumor 1 (WT1) and colony stimulating factor 1 (CSF1). Conditional deletion of Csf1 in WT1+ red pulp fibroblasts, but not white pulp fibroblasts, drastically altered the RPM network without altering circulating CSF1 levels. Upon RPM depletion, red pulp fibroblasts transiently produced the monocyte chemoattractants CCL2 and CCL7, thereby contributing to the replenishment of the RPM network. Thus, red pulp fibroblasts anchor and nurture RPM, a function likely conserved in humans.
Subject(s)
Fibroblasts/metabolism , Macrophage Colony-Stimulating Factor/metabolism , Macrophages/immunology , Spleen/cytology , WT1 Proteins/metabolism , Animals , Chemokine CCL2/metabolism , Chemokine CCL7/metabolism , Gene Expression Regulation , Humans , Immunity, Innate/immunology , Iron/metabolism , Macrophage Colony-Stimulating Factor/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Monocytes/immunology , Rats , Signal Transduction/immunology , Spleen/metabolismABSTRACT
The monocyte-derived phagocytes termed LysoDCs are hallmarks of Peyer's patches, where their main function is to sample intestinal microorganisms. Here, we study their differentiation pathways in relation with their sampling, migratory, and T cell-priming abilities. Among four identified LysoDC differentiation stages displaying similar phagocytic activity, one is located in follicles, and the others reside in subepithelial domes (SED), where they proliferate and mature as they get closer to the epithelium. Mature LysoDCs but not macrophages express a gene set in common with conventional dendritic cells and prime naive helper T cells in vitro. At steady state, they do not migrate into naive T cell-enriched interfollicular regions (IFRs), but upon stimulation, they express the chemokine receptor CCR7 and migrate from SED to the IFR periphery, where they strongly interact with proliferative immune cells. Finally, we show that LysoDCs populate human Peyer's patches, strengthening their interest as targets for modulating intestinal immunity.