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Nat Immunol ; 20(7): 902-914, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209404


Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

Rim/imunologia , Nefrite Lúpica/imunologia , Biomarcadores , Biópsia , Análise por Conglomerados , Biologia Computacional/métodos , Células Epiteliais/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Interferons/metabolismo , Rim/metabolismo , Rim/patologia , Leucócitos/imunologia , Leucócitos/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Anotação de Sequência Molecular , Células Mieloides/imunologia , Células Mieloides/metabolismo , Análise de Célula Única , Transcriptoma
Arthritis Res Ther ; 20(1): 139, 2018 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-29996944


BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 µg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

Artrite Reumatoide/patologia , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Membrana Sinovial/patologia , Criopreservação , Humanos
Immunity ; 41(6): 1001-12, 2014 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-25526311


Decreased HIV-specific CD8(+) T cell proliferation is a hallmark of chronic infection, but the mechanisms of decline are unclear. We analyzed gene expression profiles from antigen-stimulated HIV-specific CD8(+) T cells from patients with controlled and uncontrolled infection and identified caspase-8 as a correlate of dysfunctional CD8(+) T cell proliferation. Caspase-8 activity was upregulated in HIV-specific CD8(+) T cells from progressors and correlated positively with disease progression and programmed cell death-1 (PD-1) expression, but negatively with proliferation. In addition, progressor cells displayed a decreased ability to upregulate membrane-associated caspase-8 activity and increased necrotic cell death following antigenic stimulation, implicating the programmed cell death pathway necroptosis. In vitro necroptosis blockade rescued HIV-specific CD8(+) T cell proliferation in progressors, as did silencing of necroptosis mediator RIPK3. Thus, chronic stimulation leading to upregulated caspase-8 activity contributes to dysfunctional HIV-specific CD8(+) T cell proliferation through activation of necroptosis and increased cell death.

Linfócitos T CD8-Positivos/imunologia , Caspase 8/metabolismo , Infecções por HIV/imunologia , HIV/fisiologia , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T CD8-Positivos/virologia , Proliferação de Células/genética , Células Cultivadas , Progressão da Doença , Ativação Enzimática , Regulação da Expressão Gênica , Proteína do Núcleo p24 do HIV/imunologia , Humanos , Necrose , Fragmentos de Peptídeos/imunologia , Receptor de Morte Celular Programada 1/genética , RNA Interferente Pequeno/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transcriptoma , Carga Viral
Nature ; 487(7407): 325-9, 2012 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-22763456


During progression of atherosclerosis, myeloid cells destabilize lipid-rich plaques in the arterial wall and cause their rupture, thus triggering myocardial infarction and stroke. Survivors of acute coronary syndromes have a high risk of recurrent events for unknown reasons. Here we show that the systemic response to ischaemic injury aggravates chronic atherosclerosis. After myocardial infarction or stroke, Apoe-/- mice developed larger atherosclerotic lesions with a more advanced morphology. This disease acceleration persisted over many weeks and was associated with markedly increased monocyte recruitment. Seeking the source of surplus monocytes in plaques, we found that myocardial infarction liberated haematopoietic stem and progenitor cells from bone marrow niches via sympathetic nervous system signalling. The progenitors then seeded the spleen, yielding a sustained boost in monocyte production. These observations provide new mechanistic insight into atherogenesis and provide a novel therapeutic opportunity to mitigate disease progression.

Aterosclerose/etiologia , Aterosclerose/patologia , Infarto do Miocárdio/complicações , Infarto do Miocárdio/patologia , Animais , Apolipoproteínas E/genética , Células-Tronco Hematopoéticas/citologia , Inflamação/complicações , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Baço/citologia , Células-Tronco/citologia
Science ; 335(6068): 597-601, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22245738


Recognition and clearance of a bacterial infection are a fundamental properties of innate immunity. Here, we describe an effector B cell population that protects against microbial sepsis. Innate response activator (IRA) B cells are phenotypically and functionally distinct, develop and diverge from B1a B cells, depend on pattern-recognition receptors, and produce granulocyte-macrophage colony-stimulating factor. Specific deletion of IRA B cell activity impairs bacterial clearance, elicits a cytokine storm, and precipitates septic shock. These observations enrich our understanding of innate immunity, position IRA B cells as gatekeepers of bacterial infection, and identify new treatment avenues for infectious diseases.

Subpopulações de Linfócitos B/imunologia , Infecções por Escherichia coli/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunidade Inata , Peritonite/imunologia , Sepse/imunologia , Animais , Subpopulações de Linfócitos B/metabolismo , Linhagem da Célula , Separação Celular , Feminino , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Imunoglobulina M/metabolismo , Imunofenotipagem , Integrina alfa4beta1/imunologia , Integrina alfa4beta1/metabolismo , Lipopolissacarídeos , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Parabiose , Choque Séptico/imunologia , Baço/imunologia , Receptor 4 Toll-Like/imunologia
J Immunol ; 186(2): 969-76, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21148801


The expression of CD127, the IL-7-binding subunit of the IL-7 R, is tightly regulated during the development and activation of T cells and is reduced during chronic viral infection. However, the molecular mechanism regulating the dynamic expression of CD127 is still poorly understood. In this study, we report that the transcription factor Ets-1 is required for maintaining the expression of CD127 in murine peripheral T cells. Ets-1 binds to and activates the CD127 promoter, and its absence leads to reduced CD127 expression, attenuated IL-7 signaling, and impaired IL-7-dependent homeostatic proliferation of T cells. The expression of CD127 and Ets-1 is strongly correlated in human T cells. Both CD127 and Ets-1 expression are decreased in CD8(+) T cells during HIV infection. In addition, HIV-associated loss of CD127 is only observed in Ets-1(low) effector memory and central memory but not in Ets-1(high) naive CD8(+) T cells. Taken together, our data identify Ets-1 as a critical regulator of CD127 expression in T cells.

Interleucina-7/biossíntese , Proteína Proto-Oncogênica c-ets-1/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Células Cultivadas , Feminino , HIV-1/imunologia , Humanos , Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/imunologia , Ligação Proteica/imunologia , Proteína Proto-Oncogênica c-ets-1/deficiência , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Subpopulações de Linfócitos T/virologia