RESUMO
Population genetics continues to identify genetic variants associated with diseases of the immune system and offers a unique opportunity to discover mechanisms of immune regulation. Multiple genetic variants linked to severe fungal infections and autoimmunity are associated with caspase recruitment domain-containing protein 9 (CARD9). We leverage the CARD9 R101C missense variant to uncover a biochemical mechanism of CARD9 activation essential for antifungal responses. We demonstrate that R101C disrupts a critical signaling switch whereby phosphorylation of S104 releases CARD9 from an autoinhibited state to promote inflammatory responses in myeloid cells. Furthermore, we show that CARD9 R101C exerts dynamic effects on the skin cellular contexture during fungal infection, corrupting inflammatory signaling and cell-cell communication circuits. Card9 R101C mice fail to control dermatophyte infection in the skin, resulting in high fungal burden, yet show minimal signs of inflammation. Together, we demonstrate how translational genetics reveals molecular and cellular mechanisms of innate immune regulation.
Assuntos
Proteínas Adaptadoras de Sinalização CARD , Micoses , Animais , Camundongos , Fosforilação , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Transdução de Sinais , Inflamação , AntifúngicosRESUMO
Multiple studies have established associations between human gut bacteria and host physiology, but determining the molecular mechanisms underlying these associations has been challenging1-3. Akkermansia muciniphila has been robustly associated with positive systemic effects on host metabolism, favourable outcomes to checkpoint blockade in cancer immunotherapy and homeostatic immunity4-7. Here we report the identification of a lipid from A. muciniphila's cell membrane that recapitulates the immunomodulatory activity of A. muciniphila in cell-based assays8. The isolated immunogen, a diacyl phosphatidylethanolamine with two branched chains (a15:0-i15:0 PE), was characterized through both spectroscopic analysis and chemical synthesis. The immunogenic activity of a15:0-i15:0 PE has a highly restricted structure-activity relationship, and its immune signalling requires an unexpected toll-like receptor TLR2-TLR1 heterodimer9,10. Certain features of the phospholipid's activity are worth noting: it is significantly less potent than known natural and synthetic TLR2 agonists; it preferentially induces some inflammatory cytokines but not others; and, at low doses (1% of EC50) it resets activation thresholds and responses for immune signalling. Identifying both the molecule and an equipotent synthetic analogue, its non-canonical TLR2-TLR1 signalling pathway, its immunomodulatory selectivity and its low-dose immunoregulatory effects provide a molecular mechanism for a model of A. muciniphila's ability to set immunological tone and its varied roles in health and disease.
Assuntos
Akkermansia , Homeostase , Imunidade , Fosfatidiletanolaminas , Akkermansia/química , Akkermansia/citologia , Akkermansia/imunologia , Membrana Celular/química , Membrana Celular/imunologia , Citocinas/imunologia , Homeostase/imunologia , Humanos , Mediadores da Inflamação/síntese química , Mediadores da Inflamação/química , Mediadores da Inflamação/imunologia , Fosfatidiletanolaminas/síntese química , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/imunologia , Relação Estrutura-Atividade , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/imunologiaRESUMO
The human immune system detects potentially pathogenic microbes with receptors that respond to microbial metabolites. While the overall immune signaling pathway is known in considerable detail, the initial molecular signals, the microbially produced immunogens, for important diseases like Lyme disease (LD) are often not well-defined. The immunogens for LD are produced by the spirochete Borrelia burgdorferi, and a galactoglycerolipid (1) has been identified as a key trigger for the inflammatory immune response that characterizes LD. This report corrects the original structural assignment of 1 to 3, a change of an α-galactopyranose to an α-galactofuranose headgroup. The seemingly small change has important implications for the diagnosis, prevention, and treatment of LD.
Assuntos
Antígenos de Bactérias/química , Borrelia burgdorferi/química , Galactolipídeos/química , Animais , Antígenos de Bactérias/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Galactolipídeos/síntese química , Galactolipídeos/farmacologia , Inflamação/induzido quimicamente , Doença de Lyme/imunologia , Camundongos , Receptor 2 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Genome-wide association studies have identified common genetic variants impacting human diseases; however, there are indications that the functional consequences of genetic polymorphisms can be distinct depending on cell type-specific contexts, which produce divergent phenotypic outcomes. Thus, the functional impact of genetic variation and the underlying mechanisms of disease risk are modified by cell type-specific effects of genotype on pathological phenotypes. In this study, we extend these concepts to interrogate the interdependence of cell type- and stimulation-specific programs influenced by the core autophagy gene Atg16L1 and its T300A coding polymorphism identified by genome-wide association studies as linked with increased risk of Crohn's disease. We applied a stimulation-based perturbational profiling approach to define Atg16L1 T300A phenotypes in dendritic cells and T lymphocytes. Accordingly, we identified stimulus-specific transcriptional signatures revealing T300A-dependent functional phenotypes that mechanistically link inflammatory cytokines, IFN response genes, steroid biosynthesis, and lipid metabolism in dendritic cells and iron homeostasis and lysosomal biogenesis in T lymphocytes. Collectively, these studies highlight the combined effects of Atg16L1 genetic variation and stimulatory context on immune function.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Doença de Crohn/metabolismo , Células Dendríticas/fisiologia , Genótipo , Linfócitos T/fisiologia , Animais , Proteínas Relacionadas à Autofagia/genética , Células Cultivadas , Doença de Crohn/genética , Predisposição Genética para Doença , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Fenótipo , Polimorfismo Genético , Risco , Ativação TranscricionalRESUMO
The formation of red blood cells begins with the differentiation of multipotent haematopoietic progenitors. Reconstructing the steps of this differentiation represents a general challenge in stem-cell biology. Here we used single-cell transcriptomics, fate assays and a theory that allows the prediction of cell fates from population snapshots to demonstrate that mouse haematopoietic progenitors differentiate through a continuous, hierarchical structure into seven blood lineages. We uncovered coupling between the erythroid and the basophil or mast cell fates, a global haematopoietic response to erythroid stress and novel growth factor receptors that regulate erythropoiesis. We defined a flow cytometry sorting strategy to purify early stages of erythroid differentiation, completely isolating classically defined burst-forming and colony-forming progenitors. We also found that the cell cycle is progressively remodelled during erythroid development and during a sharp transcriptional switch that ends the colony-forming progenitor stage and activates terminal differentiation. Our work showcases the utility of linking transcriptomic data to predictive fate models, and provides insights into lineage development in vivo.
Assuntos
Eritrócitos/citologia , Células Precursoras Eritroides/citologia , Eritropoese , Animais , Basófilos/citologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Eritropoese/efeitos dos fármacos , Feminino , Citometria de Fluxo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Mastócitos/citologia , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Citoplasmático Pequeno/análise , RNA Citoplasmático Pequeno/genética , Análise de Célula Única , TranscriptomaRESUMO
SETD1A is a member of trithorax-related histone methyltransferases that methylate lysine 4 at histone H3 (H3K4). We showed previously that Setd1a is required for mesoderm specification and hematopoietic lineage differentiation in vitro. However, it remains unknown whether or not Setd1a controls specific hematopoietic lineage commitment and differentiation during animal development. Here, we reported that homozygous Setd1a knockout (KO) mice are embryonic lethal. Loss of the Setd1a gene in the hematopoietic compartment resulted in a blockage of the progenitor B-cell-to-precursor B-cell development in bone marrow (BM) and B-cell maturation in spleen. The Setd1a-cKO (conditional knockout) mice exhibited an enlarged spleen with disrupted spleen architecture and leukocytopenia. Mechanistically, Setd1a deficiency in BM reduced the levels of H3K4me3 at critical B-cell gene loci, including Pax5 and Rag1/2, which are critical for the IgH (Ig heavy-chain) locus contractions and rearrangement. Subsequently, the differential long-range looped interactions of the enhancer Eµ with proximal 5' DH region and 3' regulatory regions as well as with Pax5-activated intergenic repeat elements and 5' distal VH genes were compromised by the Setd1a-cKO. Together, our findings revealed a critical role of Setd1a and its mediated epigenetic modifications in regulating the IgH rearrangement and B-cell development.
Assuntos
Rearranjo Gênico de Cadeia Pesada de Linfócito B , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Canais Iônicos/metabolismo , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/metabolismo , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Proteínas de Homeodomínio/metabolismo , Canais Iônicos/deficiência , Canais Iônicos/genética , Leucopoese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição PAX5/metabolismo , Células Precursoras de Linfócitos B/citologiaRESUMO
JMJD3 (KDM6B) antagonizes Polycomb silencing by demethylating lysine 27 on histone H3. The interplay of methyltransferases and demethylases at this residue is thought to underlie critical cell fate transitions, and the dynamics of H3K27me3 during neurogenesis posited for JMJD3 a critical role in the acquisition of neural fate. Despite evidence of its involvement in early neural commitment, however, its role in the emergence and maturation of the mammalian CNS remains unknown. Here, we inactivated Jmjd3 in the mouse and found that its loss causes perinatal lethality with the complete and selective disruption of the pre-Bötzinger complex (PBC), the pacemaker of the respiratory rhythm generator. Through genetic and electrophysiological approaches, we show that the enzymatic activity of JMJD3 is selectively required for the maintenance of the PBC and controls critical regulators of PBC activity, uncovering an unanticipated role of this enzyme in the late structuring and function of neuronal networks.
Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Neurônios/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/deficiência , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mortalidade Perinatal , Explosão Respiratória/fisiologia , Insuficiência Respiratória/patologia , Somatostatina/metabolismoRESUMO
Oncogene-induced senescence (OIS) is a barrier for tumor development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and, accordingly, ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes, however, inhibit ARF/p53 and only infrequently select for ARF or p53 mutations, suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK, the initiating oncogene of anaplastic large cell lymphomas (ALCLs), induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly, human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a, which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence, and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Linfoma/genética , Proteínas Tirosina Quinases/fisiologia , Proteína do Retinoblastoma/fisiologia , Animais , Células Cultivadas , Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma/metabolismo , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/prevenção & controle , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/fisiologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Mammalian genomes are pervasively transcribed outside mapped protein-coding genes. One class of extragenic transcription products is represented by long non-coding RNAs (lncRNAs), some of which result from Pol_II transcription of bona-fide RNA genes. Whether all lncRNAs described insofar are products of RNA genes, however, is still unclear. Here we have characterized transcription sites located outside protein-coding genes in a highly regulated response, macrophage activation by endotoxin. Using chromatin signatures, we could unambiguously classify extragenic Pol_II binding sites as belonging to either canonical RNA genes or transcribed enhancers. Unexpectedly, 70% of extragenic Pol_II peaks were associated with genomic regions with a canonical chromatin signature of enhancers. Enhancer-associated extragenic transcription was frequently adjacent to inducible inflammatory genes, was regulated in response to endotoxin stimulation, and generated very low abundance transcripts. Moreover, transcribed enhancers were under purifying selection and contained binding sites for inflammatory transcription factors, thus suggesting their functionality. These data demonstrate that a large fraction of extragenic Pol_II transcription sites can be ascribed to cis-regulatory genomic regions. Discrimination between lncRNAs generated by canonical RNA genes and products of transcribed enhancers will provide a framework for experimental approaches to lncRNAs and help complete the annotation of mammalian genomes.
Assuntos
Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , RNA não Traduzido/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Animais , Sítios de Ligação , Feminino , Regulação da Expressão Gênica , Humanos , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Camundongos , RNA Polimerase II/metabolismoRESUMO
Jmjd3, a JmjC family histone demethylase, is induced by the transcription factor NF-kB in response to microbial stimuli. Jmjd3 erases H3K27me3, a histone mark associated with transcriptional repression and involved in lineage determination. However, the specific contribution of Jmjd3 induction and H3K27me3 demethylation to inflammatory gene expression remains unknown. Using chromatin immunoprecipitation-sequencing we found that Jmjd3 is preferentially recruited to transcription start sites characterized by high levels of H3K4me3, a marker of gene activity, and RNA polymerase II (Pol_II). Moreover, 70% of lipopolysaccharide (LPS)-inducible genes were found to be Jmjd3 targets. Although most Jmjd3 target genes were unaffected by its deletion, a few hundred genes, including inducible inflammatory genes, showed moderately impaired Pol_II recruitment and transcription. Importantly, most Jmjd3 target genes were not associated with detectable levels of H3K27me3, and transcriptional effects of Jmjd3 absence in the window of time analysed were uncoupled from measurable effects on this histone mark. These data show that Jmjd3 fine-tunes the transcriptional output of LPS-activated macrophages in an H3K27 demethylation-independent manner.
Assuntos
Regulação da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lipopolissacarídeos/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Animais , Células Cultivadas , Feminino , Técnicas de Inativação de Genes , Histona Desmetilases com o Domínio Jumonji/análise , Histona Desmetilases com o Domínio Jumonji/genética , Macrófagos/metabolismo , CamundongosRESUMO
PURPOSE: To investigate the role of MYOC and CYP1B1 in Iranian juvenile open angle glaucoma (JOAG) patients. METHODS: Twenty-three JOAG probands, their available affected and unaffected family members, and 100 ethnically matched control individuals without history of ocular disease were recruited. Clinical examinations of the probands included slit lamp biomicroscopy, intraocular pressure (IOP) measurement, gonioscopic evaluation, fundus examination, and perimetry measurement. Familial cases were classified according to the mode of inheritance. Exons of MYOC and CYP1B1 were sequenced, and novel variations assessed in the control individuals. Potential disease-associated variations were tested for segregation with disease status in available family members. RESULTS: The mode of inheritance of the disease in the families of four probands (17.4%) appeared to be autosomal dominant and in at least eight (34.8%) to be autosomal recessive. Four patients carried MYOC mutations, and an equal number carried CYP1B1 mutations. The MYOC mutations were heterozygous; two of them (p.C8X and p.L334P) are novel, and one codes for the shortest truncated protein so far reported. Autosomal recessive inheritance was consistent with inheritance observed in families of patients carrying CYP1B1 mutations. All these patients carried homozygous mutations. CONCLUSIONS: MYOC and CYP1B1 contributed equally to the disease status of the Iranian JOAG patients studied. The contribution of the two genes appeared to be independent in that no patient carried mutations in both genes. The fraction of Iranian patients carrying MYOC mutations was comparable to previously reported populations.
Assuntos
Árabes/genética , Sistema Enzimático do Citocromo P-450/genética , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/epidemiologia , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , Adolescente , Adulto , Idade de Início , Hidrocarboneto de Aril Hidroxilases , Criança , Citocromo P-450 CYP1B1 , Genes Dominantes , Genes Recessivos , Heterozigoto , Homozigoto , Humanos , Recém-Nascido , Irã (Geográfico)RESUMO
The mutation spectrum of CYP1B1 among 104 primary congenital glaucoma patients of the genetically heterogeneous Iranian population was investigated by sequencing. We also determined intragenic single nucleotide polymorphism (SNP) haplotypes associated with the mutations and compared these with haplotypes of other populations. Finally, the frequency distribution of the haplotypes was compared among primary congenital glaucoma patients with and without CYP1B1 mutations and normal controls. Genotype classification of six high-frequency SNPs was performed using the PHASE 2.0 software. CYP1B1 mutations in the Iranian patients were very heterogeneous. Nineteen nonconservative mutations associated with disease, and 10 variations not associated with disease were identified. Ten mutations and three variations not associated with disease were novel. The 13 novel variations make a notable contribution to the approximately 70 known variations in the gene. CYP1B1 mutations were identified in 70% of the patients. The four most common mutations were G61E, R368H, R390H, and R469W, which together constituted 76.2% of the CYP1B1 mutated alleles found. Six unique core SNP haplotypes were identified, four of which were common to the patients with and without CYP1B1 mutations and controls studied. Three SNP blocks determined the haplotypes. Comparison of haplotypes with those of other populations suggests a common origin for many of the mutations.