RESUMO
Arginase 1 (Arg1), the enzyme catalyzing the conversion of arginine to ornithine, is a hallmark of IL-10-producing immunoregulatory M2 macrophages. However, its expression in T cells is disputed. Here, we demonstrate that induction of Arg1 expression is a key feature of lung CD4+ T cells during mouse in vivo influenza infection. Conditional ablation of Arg1 in CD4+ T cells accelerated both virus-specific T helper 1 (Th1) effector responses and its resolution, resulting in efficient viral clearance and reduced lung pathology. Using unbiased transcriptomics and metabolomics, we found that Arg1-deficiency was distinct from Arg2-deficiency and caused altered glutamine metabolism. Rebalancing this perturbed glutamine flux normalized the cellular Th1 response. CD4+ T cells from rare ARG1-deficient patients or CRISPR-Cas9-mediated ARG1-deletion in healthy donor cells phenocopied the murine cellular phenotype. Collectively, CD4+ T cell-intrinsic Arg1 functions as an unexpected rheostat regulating the kinetics of the mammalian Th1 lifecycle with implications for Th1-associated tissue pathologies.
Assuntos
Arginase , Influenza Humana , Animais , Humanos , Camundongos , Arginase/genética , Arginase/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Glutamina , Cinética , Pulmão/metabolismo , MamíferosRESUMO
OBJECTIVES: Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE. METHODS: Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively. RESULTS: Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM. CONCLUSIONS: Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy.
Assuntos
Granulócitos/patologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas de Membrana/análise , Neutrófilos/patologia , Proteoma/análise , Estudos de Casos e Controles , Heterogeneidade Genética , Granulócitos/fisiologia , Humanos , Interferon Tipo I/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Microvasos/metabolismo , Neutrófilos/fisiologia , Fosforilação , ProteômicaRESUMO
Pathway analysis is a key analytical stage in the interpretation of omics data, providing a powerful method for detecting alterations in cellular processes. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package single-cell pathway analysis (SCPA). We demonstrate the effectiveness of SCPA over commonly used methods, generate a scRNA-seq T cell dataset, and characterize pathway activity over early cellular activation. This reveals regulatory pathways in T cells, including an intrinsic type I interferon system regulating T cell survival and a reliance on arachidonic acid metabolism throughout T cell activation. A systems-level characterization of pathway activity in T cells across multiple tissues also identifies alpha-defensin expression as a hallmark of bone-marrow-derived T cells. Overall, this work provides a widely applicable tool for single-cell pathway analysis and highlights regulatory mechanisms of T cells.
Assuntos
Análise de Célula Única , Software , Análise de Célula Única/métodos , Ativação Linfocitária , Sequenciamento do Exoma/métodos , Linfócitos TRESUMO
Regulatory B cells restrict immune and inflammatory responses across a number of contexts. This capacity is mediated primarily through the production of IL-10. Here we demonstrate that the induction of a regulatory program in human B cells is dependent on a metabolic priming event driven by cholesterol metabolism. Synthesis of the metabolic intermediate geranylgeranyl pyrophosphate (GGPP) is required to specifically drive IL-10 production, and to attenuate Th1 responses. Furthermore, GGPP-dependent protein modifications control signaling through PI3Kδ-AKT-GSK3, which in turn promote BLIMP1-dependent IL-10 production. Inherited gene mutations in cholesterol metabolism result in a severe autoinflammatory syndrome termed mevalonate kinase deficiency (MKD). Consistent with our findings, B cells from MKD patients induce poor IL-10 responses and are functionally impaired. Moreover, metabolic supplementation with GGPP is able to reverse this defect. Collectively, our data define cholesterol metabolism as an integral metabolic pathway for the optimal functioning of human IL-10 producing regulatory B cells.
Assuntos
Linfócitos B Reguladores/metabolismo , Colesterol/metabolismo , Interleucina-10/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Animais , Antígenos CD19/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Técnicas de Cocultura , Doenças Hereditárias Autoinflamatórias/metabolismo , Humanos , Macrófagos/metabolismo , Síndrome Metabólica/metabolismo , Deficiência de Mevalonato Quinase/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Análise de Componente Principal , Transdução de Sinais , Células Th1/metabolismo , Receptor Toll-Like 9/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Dendritic cells (DCs) are specialized antigen presenting cells that instruct T cell responses through sensing environmental and inflammatory danger signals. Maintaining the homeostasis of the multiple functionally distinct conventional dendritic cells (cDC) subsets that exist in vivo is crucial for regulating immune responses, with changes in numbers sufficient to break immune tolerance. Using Ptpn22-/- mice we demonstrate that the phosphatase PTPN22 is a highly selective, negative regulator of cDC2 homeostasis, preventing excessive population expansion from as early as 3 weeks of age. Mechanistically, PTPN22 mediates cDC2 homeostasis in a cell intrinsic manner by restricting cDC2 proliferation. A single nucleotide polymorphism, PTPN22R620W, is one of the strongest genetic risk factors for multiple autoantibody associated human autoimmune diseases. We demonstrate that cDC2 are also expanded in mice carrying the orthologous PTPN22619W mutation. As a consequence, cDC2 dependent CD4+ T cell proliferation and T follicular helper cell responses are increased. Collectively, our data demonstrate that PTPN22 controls cDC2 homeostasis, which in turn ensures appropriate cDC2-dependent T cell responses under antigenic challenge. Our findings provide a link between perturbations in DC development and susceptibility to a broad spectrum of PTPN22R620W associated human autoimmune diseases.
Assuntos
Autoimunidade/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Homeostase/imunologia , Tolerância Imunológica/imunologia , Camundongos , Camundongos Transgênicos , Polimorfismo de Nucleotídeo Único , Proteína Tirosina Fosfatase não Receptora Tipo 22/genéticaRESUMO
The mechanisms controlling CD4+ T cell switching from an effector to an anti-inflammatory (IL-10+) phenotype play an important role in the persistence of chronic inflammatory diseases. Here, we identify the cholesterol biosynthesis pathway as a key regulator of this process. Pathway analysis of cultured cytokine-producing human T cells reveals a significant association between IL-10 and cholesterol metabolism gene expression. Inhibition of the cholesterol biosynthesis pathway with atorvastatin or 25-hydroxycholesterol during switching from IFNγ+ to IL-10+ shows a specific block in immune resolution, defined as a significant decrease in IL-10 expression. Mechanistically, the master transcriptional regulator of IL10 in T cells, c-Maf, is significantly decreased by physiological levels of 25-hydroxycholesterol. Strikingly, progression to rheumatoid arthritis is associated with altered expression of cholesterol biosynthesis genes in synovial biopsies of predisposed individuals. Our data reveal a link between sterol metabolism and the regulation of the anti-inflammatory response in human CD4+ T cells.
Assuntos
Colesterol/biossíntese , Interferon gama/metabolismo , Interleucina-10/metabolismo , Células Th1/metabolismo , Atorvastatina/farmacologia , Humanos , Hidroxicolesteróis/farmacologia , Células Th1/efeitos dos fármacosRESUMO
The PTPN22R620W single nucleotide polymorphism increases the risk of developing multiple autoimmune diseases including type 1 diabetes, rheumatoid arthritis and lupus. PTPN22 is highly expressed in antigen presenting cells (APCs) where the expression of the murine disease associated variant orthologue (Ptpn22R619W) is reported to dysregulate pattern recognition receptor signalling in dendritic cells (DCs) and promote T-cell proliferation. Because T-cell activation is dependent on DC antigen uptake, degradation and presentation, we analysed the efficiency of these functions in splenic and GM-CSF bone marrow derived DC from wild type (WT), Ptpn22-/- or Ptpn22R619W mutant mice. Results indicated no differential ability of DCs to uptake antigen via macropinocytosis or receptor-mediated endocytosis. Antigen degradation and presentation was also equal as was WT T-cell conjugate formation and subsequent T-cell proliferation. Despite the likely presence of multiple phosphatase-regulated pathways in the antigen uptake, processing and presentation pathways that we investigated, we observed that Ptpn22 and the R619W autoimmune associated variant were dispensable. These important findings indicate that under non-inflammatory conditions there is no requirement for Ptpn22 in DC dependent antigen uptake and T-cell activation. Our findings reveal that perturbations in antigen uptake and processing, a fundamental pathway determining adaptive immune responses, are unlikely to provide a mechanism for the risk associated with the Ptpn22 autoimmune associated polymorphism.