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1.
Artigo em Inglês | MEDLINE | ID: mdl-32079724

RESUMO

The role of stromal fibroblasts in chronic inflammation is unfolding. In rheumatoid arthritis, leukocyte-derived cytokines TNF and IL-17A work together, activating fibroblasts to become a dominant source of the hallmark cytokine IL-6. However, IL-17A alone has minimal effect on fibroblasts. To identify key mediators of the synergistic response to TNF and IL-17A in human synovial fibroblasts, we performed time series, dose-response, and gene-silencing transcriptomics experiments. Here we show that in combination with TNF, IL-17A selectively induces a specific set of genes mediated by factors including cut-like homeobox 1 (CUX1) and IκBζ (NFKBIZ). In the promoters of CXCL1, CXCL2, and CXCL3, we found a putative CUX1-NF-κB binding motif not found elsewhere in the genome. CUX1 and NF-κB p65 mediate transcription of these genes independent of LIFR, STAT3, STAT4, and ELF3. Transcription of NFKBIZ, encoding the atypical IκB factor IκBζ, is IL-17A dose-dependent, and IκBζ only mediates the transcriptional response to TNF and IL-17A, but not to TNF alone. In fibroblasts, IL-17A response depends on CUX1 and IκBζ to engage the NF-κB complex to produce chemoattractants for neutrophil and monocyte recruitment.

2.
Nat Immunol ; 20(7): 928-942, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31061532

RESUMO

To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.


Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Perfilação da Expressão Gênica , Membrana Sinovial/metabolismo , Transcriptoma , Artrite Reumatoide/patologia , Autoimunidade/genética , Biomarcadores , Biologia Computacional/métodos , Estudos Transversais , Citocinas/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Membrana Sinovial/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fluxo de Trabalho
3.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1516-1524, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30876808

RESUMO

Cadherins are homophilic cell-to-cell adhesion molecules that help cells respond to environmental changes. Newly formed cadherin junctions are associated with increased cell phosphorylation, but the pathways driving this signaling response are largely unknown. Since cadherins have no intrinsic signaling activity, this phosphorylation must occur through interactions with other signaling molecules. We previously reported that cadherin-11 engagement activates joint synovial fibroblasts, promoting inflammatory and degradative pathways important in rheumatoid arthritis (RA) pathogenesis. Our objective in this study was to discover interacting partners that mediate cadherin-11 signaling. Protein array screening showed that cadherin-11 extracellular binding domains linked to an Fc domain (cad11Fc) induced platelet-derived growth factor (PDGFR)-α phosphorylation in synovial fibroblasts and glioblastoma cells. PDGFRs are growth factor receptor tyrosine kinases that promote cell proliferation, survival, and migration in mesodermally derived cells. Increased PDGFR activity is implicated in RA pathology and associates with poor prognosis in several cancers, including sarcoma and glioblastoma. PDGFRα activation by cadherin-11 signaling promoted fibroblast proliferation, a signaling pathway independent from cadherin-11-stimulated IL-6 or matrix metalloproteinase (MMP)-3 release. PDGFRα phosphorylation mediated most of the cad11Fc-induced phosphatidyl-3-kinase (PI3K)/Akt activation, but only part of the mitogen-activated protein kinase (MAPK) response. PDGFRα-dependent signaling did not require cell cadherin-11 expression. Rather, cad11Fc immunoprecipitated PDGFRα, indicating a direct interaction between cadherin-11 and PDGFRα extracellular domains. This study is the first to report an interaction between cadherin-11 and PDGFRα and adds to our growing understanding that cadherin-growth factor receptor interactions help balance the interplay between tissue growth and adhesion.

4.
Nat Commun ; 10(1): 687, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737409

RESUMO

How innate T cells (ITC), including invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells, maintain a poised effector state has been unclear. Here we address this question using low-input and single-cell RNA-seq of human lymphocyte populations. Unbiased transcriptomic analyses uncover a continuous 'innateness gradient', with adaptive T cells at one end, followed by MAIT, iNKT, γδ T and natural killer cells at the other end. Single-cell RNA-seq reveals four broad states of innateness, and heterogeneity within canonical innate and adaptive populations. Transcriptional and functional data show that innateness is characterized by pre-formed mRNA encoding effector functions, but impaired proliferation marked by decreased baseline expression of ribosomal genes. Together, our data shed new light on the poised state of ITC, in which innateness is defined by a transcriptionally-orchestrated trade-off between rapid cell growth and rapid effector function.


Assuntos
Proliferação de Células/fisiologia , Linfócitos/metabolismo , Feminino , Ontologia Genética , Humanos , Imunidade Inata/fisiologia , Imunofenotipagem , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/fisiologia , Masculino , Células T Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/metabolismo
5.
Proc Natl Acad Sci U S A ; 114(31): 8348-8353, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28716901

RESUMO

Glycolipid antigens recognized by αß T-cell receptors (TCRs) drive the activation of invariant natural killer T (iNKT) cells, a specialized subset of innate T lymphocytes. Glycolipids with α-linked anomeric carbohydrates have been identified as potent microbial lipid antigens for iNKT cells, and their unusual α-anomeric linkage has been thought to define a "foreign" lipid antigen motif. However, mammals use endogenous lipids to select iNKT cells, and there is compelling evidence for iNKT cell responses in various types of sterile inflammation. The nature of endogenous or environmental lipid antigens encountered by iNKT cells is not well defined. Here, we sought to identify lipid antigens in cow's milk, a prominent part of the human diet. We developed a method to directly capture lipid antigens within CD1d-lipid-TCR complexes, while excluding CD1d bound to nonantigenic lipids, followed by direct biochemical analysis of the lipid antigens trapped at the TCR-CD1d interface. The specific antigens captured by this "TCR trap" method were identified as α-linked monohexosylceramides by mass spectrometry fragmentation patterns that distinguished α- from ß-anomeric monohexosylceramides. These data provide direct biochemical evidence for α-linked lipid antigens from a common dietary source.


Assuntos
Antígenos CD1d/imunologia , Galactosilceramidas/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Bioensaio/métodos , Dieta , Humanos , Camundongos , Leite/química , Espectrometria de Massas por Ionização por Electrospray
6.
Immunity ; 46(2): 220-232, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28228280

RESUMO

Fibroblasts are major contributors to and regulators of inflammation and dominant producers of interleukin-6 (IL-6) in inflammatory diseases like rheumatoid arthritis. Yet, compared to leukocytes, the regulation of inflammatory pathways in fibroblasts is largely unknown. Here, we report that analyses of genes coordinately upregulated with IL-6 pointed to STAT4 and leukemia inhibitory factor (LIF) as potentially linked. Gene silencing revealed that STAT4 was required for IL-6 transcription. STAT4 was recruited to the IL-6 promoter after fibroblast activation, and LIF receptor (LIFR) and STAT4 formed a molecular complex that, together with JAK1 and TYK2 kinases, controlled STAT4 activation. Importantly, a positive feedback loop involving autocrine LIF, LIFR, and STAT4 drove sustained IL-6 transcription. Besides IL-6, this autorine loop also drove the production of other key inflammatory factors including IL-8, granulocyte-colony stimulating factor (G-CSF), IL-33, IL-11, IL-1α, and IL-1ß. These findings define the transcriptional regulation of fibroblast-mediated inflammation as distinct from leukocytes.


Assuntos
Comunicação Autócrina/imunologia , Fibroblastos/imunologia , Regulação da Expressão Gênica/imunologia , Fator Inibidor de Leucemia/imunologia , Receptores de OSM-LIF/imunologia , Artrite Reumatoide/imunologia , Células Cultivadas , Citocinas/biossíntese , Perfilação da Expressão Gênica , Humanos , Inflamação/imunologia , Interleucina-6/imunologia , Fator de Transcrição STAT4/imunologia , Membrana Sinovial/imunologia , Transcriptoma
7.
Proc Natl Acad Sci U S A ; 112(48): 14948-53, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26578807

RESUMO

Interleukin (IL)-6 blockade is an effective treatment for rheumatoid arthritis (RA), and synovial fibroblasts are a major IL-6 producer in the inflamed joint. We found that human RA and osteoarthritis (OA) synovial fibroblasts derived from independent donors reproducibly segregated into low, medium, and high IL-6 producers, independent of stimulus, cell passage, or disease state. IL-6 expression pattern correlated strongly with total mRNA expression, not mRNA stability, suggesting transcriptional rather than posttranscriptional regulation. High-fibroblast IL-6 expression was significantly associated with the IL-6 proximal promoter single nucleotide polymorphism (SNP) rs1800795 minor allele (CC) genotype. In contrast, no association between this SNP and IL-6 production was detected in CD14(+) monocytes, another major producer of synovial IL-6. Luciferase expression assays confirmed that this SNP was associated with differential IL-6 expression in fibroblasts. To date, several association studies examining rs1800795 allele frequency and disease risk have reported seemingly conflicting results ranging from no association to association with either the major or minor allele across a spectrum of conditions, including cancer and autoimmune, cardiovascular, infectious, and metabolic diseases. This study points to a prominent contribution from promoter genetic variation in fibroblast IL-6 regulation, but not in other IL-6-producing cell types. We propose that some of the heterogeneity in these clinical studies likely reflects the cellular source of IL-6 in specific diseases, much of which may be produced by nonhematopoietic cells. These results highlight that functional analysis of disease-associated SNPs on gene expression and pathologic processes must consider variation in diverse cell types.


Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Interleucina-6 , Polimorfismo de Nucleotídeo Único , Artrite Reumatoide/patologia , Células Cultivadas , Feminino , Fibroblastos/patologia , Humanos , Interleucina-6/biossíntese , Interleucina-6/genética , Masculino , Estabilidade de RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
8.
Arthritis Res Ther ; 17: 126, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25975695

RESUMO

INTRODUCTION: Engagement of the homotypic cell-to-cell adhesion molecule cadherin-11 on rheumatoid arthritis (RA) synovial fibroblasts with a chimeric molecule containing the cadherin-11 extracellular binding domain stimulated cytokine, chemokine, and matrix metalloproteinases (MMP) release, implicating cadherin-11 signaling in RA pathogenesis. The objective of this study was to determine if cadherin-11 extracellular domain fragments are found inside the joint and if a physiologic synovial fibroblast cleavage pathway releases those fragments. METHODS: Cadherin-11 cleavage fragments were detected by western blot in cell media or lysates. Cleavage was interrupted using chemical inhibitors or short-interfering RNA (siRNA) gene silencing. The amount of cadherin-11 fragments in synovial fluid was measured by western blot and ELISA. RESULTS: Soluble cadherin-11 extracellular fragments were detected in human synovial fluid at significantly higher levels in RA samples compared to osteoarthritis (OA) samples. A cadherin-11 N-terminal extracellular binding domain fragment was shed from synovial fibroblasts after ionomycin stimulation, followed by presenilin 1 (PSN1)-dependent regulated intramembrane proteolysis of the retained membrane-bound C-terminal fragments. In addition to ionomycin-induced calcium flux, tumor necrosis factor (TNF)-α also stimulated cleavage in both two- and three-dimensional fibroblast cultures. Although cadherin-11 extracellular domains were shed by a disintegrin and metalloproteinase (ADAM) 10 in several cell types, a novel ADAM- and metalloproteinase-independent activity mediated shedding in primary human fibroblasts. CONCLUSIONS: Cadherin-11 undergoes ectodomain shedding followed by regulated intramembrane proteolysis in synovial fibroblasts, triggered by a novel sheddase that generates extracelluar cadherin-11 fragments. Cadherin-11 fragments were enriched in RA synovial fluid, suggesting they may be a marker of synovial burden and may function to modify cadherin-11 interactions between synovial fibroblasts.


Assuntos
Artrite Reumatoide/metabolismo , Caderinas/metabolismo , Fibroblastos/metabolismo , Membrana Sinovial/metabolismo , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoprecipitação , Osteoartrite/metabolismo , Fragmentos de Peptídeos/metabolismo , RNA Interferente Pequeno , Líquido Sinovial/química , Líquido Sinovial/metabolismo , Transfecção
9.
Proc Natl Acad Sci U S A ; 111(37): 13433-8, 2014 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-25197085

RESUMO

Invariant natural killer T (iNKT) cells are a specialized T-cell subset that recognizes lipids as antigens, contributing to immune responses in diverse disease processes. Experimental data suggests that iNKT cells can recognize both microbial and endogenous lipid antigens. Several candidate endogenous lipid antigens have been proposed, although the contextual role of specific antigens during immune responses remains largely unknown. We have previously reported that mammalian glucosylceramides (GlcCers) activate iNKT cells. GlcCers are found in most mammalian tissues, and exist in variable molecular forms that differ mainly in N-acyl fatty acid chain use. In this report, we purified, characterized, and tested the GlcCer fractions from multiple animal species. Although activity was broadly identified in these GlcCer fractions from mammalian sources, we also found activity properties that could not be reconciled by differences in fatty acid chain use. Enzymatic digestion of ß-GlcCer and a chromatographic separation method demonstrated that the activity in the GlcCer fraction was limited to a rare component of this fraction, and was not contained within the bulk of ß-GlcCer molecular species. Our data suggest that a minor lipid species that copurifies with ß-GlcCer in mammals functions as a lipid self antigen for iNKT cells.


Assuntos
Glucosilceramidas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Animais , Bovinos , Cromatografia em Camada Delgada , Glucosilceramidase/metabolismo , Glucosilceramidas/química , Humanos , Ativação Linfocitária/imunologia , Espectrometria de Massas , Camundongos , Leite/química , Células T Matadoras Naturais/efeitos dos fármacos
10.
Proc Natl Acad Sci U S A ; 110(5): 1827-32, 2013 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-23307809

RESUMO

CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18(+) invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and ß-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens. Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection.


Assuntos
Antígenos de Bactérias/imunologia , Células T Matadoras Naturais/imunologia , Fosfolipídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Bactérias/metabolismo , Antígenos CD1d/genética , Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Cardiolipinas/imunologia , Cardiolipinas/metabolismo , Linhagem Celular , Células Cultivadas , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/imunologia , Corynebacterium glutamicum/metabolismo , Galactosilceramidas/imunologia , Galactosilceramidas/metabolismo , Hibridomas/imunologia , Hibridomas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Células T Matadoras Naturais/metabolismo , Fosfatidilgliceróis/imunologia , Fosfatidilgliceróis/metabolismo , Fosfolipídeos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/imunologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
11.
Proc Natl Acad Sci U S A ; 109(52): 21354-9, 2012 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-23223633

RESUMO

Intracellular recycling pathways play critical roles in internalizing membrane and fluid phase cargo and in balancing the inflow and outflow of membrane and cell surface molecules. To identify proteins involved in the regulation of endocytic recycling, we used an shRNA trafficking library and screened for changes in the surface expression of CD1a antigen-presenting molecules that follow an endocytic recycling route. We found that silencing of the ADP-ribosylation factor (Arf)-like small GTPase Arl13b led to a decrease in CD1a surface expression, diminished CD1a function, and delayed CD1a recycling, suggesting that Arl13b is involved in the regulation of endocytic recycling traffic. Arl13b appears to be required for the major route of endocytic trafficking, causing clustering of early endosomes and leading to the accumulation of endocytic cargo. Moreover, Arl13b colocalized with markers of the endocytic recycling pathway followed by CD1a, namely Arf6 and Rab22a. We also detected an interaction between Arl13b and the actin cytoskeleton. Arl13b was previously implicated in cilia formation and function. Our present results indicate a previously unidentified role for Arl13b in endocytic recycling traffic and suggest a link between Arl13b function and the actin cytoskeleton.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Endocitose , Fatores de Ribosilação do ADP/química , Citoesqueleto de Actina/metabolismo , Antígenos CD1/metabolismo , Membrana Celular/metabolismo , Análise por Conglomerados , Endossomos/metabolismo , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
12.
Arthritis Rheum ; 63(12): 3768-78, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22127696

RESUMO

OBJECTIVE: Cadherin 11 is a homophilic cell-to-cell adhesion molecule expressed on joint synovial fibroblasts. Absence of cadherin 11 in a mouse rheumatoid arthritis (RA) model led to striking reductions in cartilage erosion. Matrix metalloproteinases (MMPs) are enzymes expressed by synovial fibroblasts important for cartilage erosion. The objective of this study was to determine if synovial fibroblast MMP production is regulated by cadherin 11. METHODS: To mimic cadherin 11 engagement, human RA synovial fibroblasts were stimulated with a chimeric construct consisting of the cadherin 11 extracellular domain linked to the human IgG1 Fc domain (Cad-11-Fc). Effects on MMP production were measured by enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction analysis, and immunoblotting. RESULTS: Human Cad-11-Fc up-regulated MMP-1 and MMP-3 protein production by RA synovial fibroblasts, both alone and in synergy with tumor necrosis factor α. This up-regulation required cell cadherin 11 engagement, since a mutant Cad-11-Fc with reduced binding affinity stimulated significantly less MMP production. Also, short hairpin RNA (shRNA) cadherin 11 silencing almost completely inhibited Cad-11-Fc-induced MMP expression. Cad-11-Fc stimulation increased RA synovial fibroblast MMP messenger RNA levels. It also increased the phosphorylation of the MAPKs JNK, ERK, and p38 kinase, the phosphorylation of NF-κB p65, and the nuclear translocation of activator protein 1 transcription factor. MAPK and NF-κB inhibitors partially blocked RA synovial fibroblast MMP expression. CONCLUSION: Cadherin 11 engagement stimulates increased synthesis of several MMPs by RA synovial fibroblasts in a MAPK- and NF-κB-dependent manner. These results underscore the existence of a pathway by which cadherin 11 regulates MMP production and has important implications for joint destruction in RA.


Assuntos
Artrite Reumatoide/metabolismo , Caderinas/farmacologia , Fibroblastos/efeitos dos fármacos , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Membrana Sinovial/metabolismo , Artrite Reumatoide/patologia , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Fragmentos Fc das Imunoglobulinas/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/fisiologia , Membrana Sinovial/patologia
13.
Nat Immunol ; 13(1): 44-50, 2011 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-22120118

RESUMO

Mouse invariant natural killer T cells (iNKT cells) provide cognate and noncognate help for lipid and protein-specific B cells, respectively. However, the long-term outcome for B cells after cognate help is provided by iNKT cells is unknown at present. Here we found that cognate iNKT cell help resulted in a B cell differentiation program characterized by extrafollicular plasmablasts, germinal-center formation, affinity maturation and a robust primary immunoglobulin G (IgG) antibody response that was uniquely dependent on iNKT cell-derived interleukin 21 (IL-21). However, cognate help from iNKT cells did not generate an enhanced humoral memory response. Thus, cognate iNKT cell help for lipid-specific B cells induces a unique signature that is a hybrid of classic T cell-dependent and T cell-independent type 2 B cell responses.


Assuntos
Antígenos/imunologia , Linfócitos B/imunologia , Interleucinas/fisiologia , Lipídeos/imunologia , Células T Matadoras Naturais/imunologia , Animais , Centro Germinativo/imunologia , Imunidade Humoral , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Baço/imunologia
14.
Cell Host Microbe ; 10(5): 437-50, 2011 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-22100160

RESUMO

iNKT cells are innate T lymphocytes recognizing endogenous and foreign lipid antigens presented in the MHC-like molecule CD1d. The semi-invariant iNKT cell TCR can detect certain bacterial and parasitic lipids and drive iNKT cell responses. How iNKT cells respond to fungi, however, is unknown. We found that CD1d-deficient mice, which lack iNKT cells, poorly control infection with the fungal pathogen Aspergillus fumigatus. Furthermore, A. fumigatus rapidly activates iNKT cells in vivo and in vitro in the presence of APCs. Surprisingly, despite a requirement for CD1d recognition, the antifungal iNKT cell response does not require fungal lipids. Instead, Dectin-1- and MyD88-mediated responses to ß-1,3 glucans, major fungal cell-wall polysaccharides, trigger IL-12 production by APCs that drives self-reactive iNKT cells to secrete IFN-γ. Innate recognition of ß-1,3 glucans also drives iNKT cell responses against Candida, Histoplasma, and Alternaria, suggesting that this mechanism may broadly define the basis for antifungal iNKT cell responses.


Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Parede Celular/imunologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/microbiologia , beta-Glucanas/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD1d/imunologia , Aspergilose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
15.
J Exp Med ; 208(6): 1163-77, 2011 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-21555485

RESUMO

Invariant natural killer T cells (iNKT cells) are critical for host defense against a variety of microbial pathogens. However, the central question of how iNKT cells are activated by microbes has not been fully explained. The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection. However, when we compared the role of microbial antigens versus innate cytokine-driven mechanisms, we found that iNKT cell interferon-γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor-driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12-induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections.


Assuntos
Antígenos de Bactérias/metabolismo , Citocinas/metabolismo , Células T Matadoras Naturais/microbiologia , Animais , Feminino , Galactosilceramidas/química , Interferon gama/metabolismo , Interleucina-12/metabolismo , Ligantes , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células T Matadoras Naturais/metabolismo , Fosforilação , Fator de Transcrição STAT4/metabolismo , Transdução de Sinais , Espectrometria de Massas por Ionização por Electrospray/métodos , Sphingomonas/metabolismo , Streptococcus pneumoniae/metabolismo
16.
Arthritis Rheum ; 62(3): 742-52, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20131230

RESUMO

OBJECTIVE: To define the intrinsic capacity of fibroblast-like synoviocytes (FLS) to establish a 3-dimensional (3-D) complex synovial lining architecture characterized by the multicellular organization of the compacted synovial lining and the elaboration of synovial fluid constituents. METHODS: FLS were cultured in spherical extracellular matrix (ECM) micromasses for 3 weeks. The FLS micromass architecture was assessed histologically and compared with that of dermal fibroblast controls. Lubricin synthesis was measured via immunodetection. Basement membrane matrix and reticular fiber stains were performed to examine ECM organization. Primary human and mouse monocytes were prepared and cocultured with FLS in micromass to investigate cocompaction in the lining architecture. Cytokine stimuli were applied to determine the capacity for inflammatory architecture rearrangement. RESULTS: FLS, but not dermal fibroblasts, spontaneously formed a compacted lining architecture over 3 weeks in the 3-D ECM micromass organ cultures. These lining cells produced lubricin. FLS rearranged their surrounding ECM into a complex architecture resembling the synovial lining and supported the survival and cocompaction of monocyte/macrophages in the neo-lining structure. Furthermore, when stimulated by cytokines, FLS lining structures displayed features of the hyperplastic rheumatoid arthritis synovial lining. CONCLUSION: This 3-D micromass organ culture method demonstrates that many of the phenotypic characteristics of the normal and the hyperplastic synovial lining in vivo are intrinsic functions of FLS. Moreover, FLS promote survival and cocompaction of primary monocytes in a manner remarkably similar to that of synovial lining macrophages. These findings provide new insight into inherent functions of the FLS lineage and establish a powerful in vitro method for further investigation of this lineage.


Assuntos
Fibroblastos/fisiologia , Líquido Sinovial/química , Membrana Sinovial/citologia , Animais , Matriz Extracelular/ultraestrutura , Glicoproteínas/biossíntese , Humanos , Inflamação/fisiopatologia , Macrófagos/citologia , Camundongos , Técnicas de Cultura de Órgãos , Membrana Sinovial/anatomia & histologia
17.
Proc Natl Acad Sci U S A ; 107(7): 3052-7, 2010 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-20133624

RESUMO

Myeloid antigen-presenting cells (APC) express CD1d molecules that present exogenous and endogenous lipid antigens that activate CD1d-restricted T cells, natural killer T (NKT) cells. NKT cell activation has been shown to mediate the potent downstream activation of other immune cells through cell-cell interactions and rapid, prolific cytokine production. Foreign antigens are not required for NKT cell activation. The endogenous lipids bound to CD1d are sufficient for activation of NKT cells in the setting of Toll-like receptor-induced cytokines. The most potent NKT cell antigens identified are glycosphingolipids (GSL). The GSL repertoire of endogenous ligands bound to CD1d molecules that are expressed in myeloid APC at steady state and in the setting of activation has not been delineated. This report identifies the range of GSL bound to soluble murine CD1d (mCD1d) molecules that sample the endoplasmic reticulum/secretory routes and cell surface-cleaved mCD1d that also samples the endocytic system. Specific GSL species are preferentially bound by mCD1d and do not solely reflect cellular GSL. GM1a and GD1a are prominent CD1d ligands for molecules following both the ER/secretory and lysosomal trafficking routes, whereas GM2 was eluted from soluble CD1d but not lysosomal trafficking CD1d. Further, after LPS activation, the quantities of soluble CD1d-bound GM3 and GM1a markedly increased. A unique alpha-galactose-terminating GSL was also found to be preferentially bound to mCD1d at steady state, and it increased with APC activation. Together, these studies identify the range of GSL presented by CD1d and how presentation varies based on CD1d intracellular trafficking and microbial activation.


Assuntos
Apresentação do Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos CD1d/imunologia , Glicoesfingolipídeos/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Animais , Transporte Biológico/imunologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Fluorescência , Glicoesfingolipídeos/metabolismo , Humanos , Camundongos , Microscopia Confocal , Células T Matadoras Naturais/metabolismo
18.
Science ; 327(5965): 580-3, 2010 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-20110505

RESUMO

In addition to their pivotal role in thrombosis and wound repair, platelets participate in inflammatory responses. We investigated the role of platelets in the autoimmune disease rheumatoid arthritis. We identified platelet microparticles--submicrometer vesicles elaborated by activated platelets--in joint fluid from patients with rheumatoid arthritis and other forms of inflammatory arthritis, but not in joint fluid from patients with osteoarthritis. Platelet microparticles were proinflammatory, eliciting cytokine responses from synovial fibroblasts via interleukin-1. Consistent with these findings, depletion of platelets attenuated murine inflammatory arthritis. Using both pharmacologic and genetic approaches, we identified the collagen receptor glycoprotein VI as a key trigger for platelet microparticle generation in arthritis pathophysiology. Thus, these findings demonstrate a previously unappreciated role for platelets and their activation-induced microparticles in inflammatory joint diseases.


Assuntos
Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Plaquetas/fisiologia , Micropartículas Derivadas de Células/fisiologia , Colágeno/metabolismo , Citocinas/metabolismo , Líquido Sinovial/imunologia , Animais , Artrite/sangue , Artrite/imunologia , Artrite Reumatoide/fisiopatologia , Plaquetas/citologia , Plaquetas/ultraestrutura , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Interleucina-1/metabolismo , Camundongos , Camundongos Transgênicos , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Colágeno/metabolismo , Líquido Sinovial/citologia , Membrana Sinovial/citologia , Membrana Sinovial/imunologia
19.
J Immunol ; 180(7): 4885-91, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18354212

RESUMO

Although the innate immune function of mast cells in the acute phase of parasitic and bacterial infections is well established, their participation in chronic immune responses to indolent infection remains incompletely understood. In parasitic infection with Trichinella spiralis, the immune response incorporates both lymphocyte and mast cell-dependent effector functions for pathogen eradication. Among the mechanistic insights still unresolved in the reaction to T. spiralis are the means by which mast cells respond to parasites and the mast cell effector functions that contribute to the immunologic response to this pathogen. We hypothesized that mast cell elaboration of tryptase may comprise an important effector component in this response. Indeed, we find that mice deficient in the tryptase mouse mast cell protease-6 (mMCP-6) display a significant difference in their response to T. spiralis larvae in chronically infected skeletal muscle tissue. Mechanistically, this is associated with a profound inability to recruit eosinophils to larvae in mMCP-6-deficient mice. Analysis of IgE-deficient mice demonstrates an identical defect in eosinophil recruitment. These findings establish that mast cell secretion of the tryptase mMCP-6, a function directed by the activity of the adaptive immune system, contributes to eosinophil recruitment to the site of larval infection, thereby comprising an integral link in the chronic immune response to parasitic infection.


Assuntos
Adaptação Biológica/imunologia , Imunidade Inata/imunologia , Mastócitos/enzimologia , Mastócitos/imunologia , Trichinella spiralis/imunologia , Triquinelose/imunologia , Triptases/metabolismo , Animais , Doença Crônica , Eosinofilia/genética , Eosinofilia/imunologia , Eosinofilia/metabolismo , Eosinofilia/patologia , Eosinófilos/imunologia , Feminino , Imunoglobulina E/deficiência , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Intestinos/imunologia , Intestinos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Triquinelose/metabolismo , Triquinelose/parasitologia , Triquinelose/patologia , Triptases/deficiência , Triptases/genética , Triptases/imunologia
20.
Science ; 315(5814): 1006-10, 2007 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-17255475

RESUMO

The normal synovium forms a membrane at the edges of joints and provides lubrication and nutrients for the cartilage. In rheumatoid arthritis, the synovium is the site of inflammation, and it participates in an organized tissue response that damages cartilage and bone. We identified cadherin-11 as essential for the development of the synovium. Cadherin-11-deficient mice have a hypoplastic synovial lining, display a disorganized synovial reaction to inflammation, and are resistant to inflammatory arthritis. Cadherin-11 therapeutics prevent and reduce arthritis in mouse models. Thus, synovial cadherin-11 determines the behavior of synovial cells in their proinflammatory and destructive tissue response in inflammatory arthritis.


Assuntos
Artrite Reumatoide/patologia , Caderinas/antagonistas & inibidores , Caderinas/fisiologia , Membrana Sinovial/citologia , Membrana Sinovial/patologia , Animais , Anticorpos Monoclonais/uso terapêutico , Artrite Experimental , Artrite Reumatoide/metabolismo , Artrite Reumatoide/terapia , Caderinas/biossíntese , Caderinas/deficiência , Adesão Celular/fisiologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos
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