RESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive devastating, yet untreatable fibrotic disease of unknown origin. We investigated the contribution of the B-cell activating factor (BAFF), a TNF family member recently implicated in the regulation of pathogenic IL-17-producing cells in autoimmune diseases. The contribution of BAFF was assessed in a murine model of lung fibrosis induced by airway administered bleomycin. We show that murine BAFF levels were strongly increased in the bronchoalveolar space and lungs after bleomycin exposure. We identified Gr1(+) neutrophils as an important source of BAFF upon BLM-induced lung inflammation and fibrosis. Genetic ablation of BAFF or BAFF neutralization by a soluble receptor significantly attenuated pulmonary fibrosis and IL-1ß levels. We further demonstrate that bleomycin-induced BAFF expression and lung fibrosis were IL-1ß and IL-17A dependent. BAFF was required for rIL-17A-induced lung fibrosis and augmented IL-17A production by CD3(+) T cells from murine fibrotic lungs ex vivo. Finally we report elevated levels of BAFF in bronchoalveolar lavages from IPF patients. Our data therefore support a role for BAFF in the establishment of pulmonary fibrosis and a crosstalk between IL-1ß, BAFF and IL-17A.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Fator Ativador de Células B/metabolismo , Bleomicina/efeitos adversos , Fibrose Pulmonar Idiopática/etiologia , Interleucina-17/metabolismo , Animais , Antibióticos Antineoplásicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Fator Ativador de Células B/antagonistas & inibidores , Fator Ativador de Células B/deficiência , Fator Ativador de Células B/genética , Bleomicina/administração & dosagem , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Fibrose , Expressão Gênica , Fibrose Pulmonar Idiopática/patologia , Interleucina-17/genética , Interleucina-1beta/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
We evaluated micro (mi) RNA-mediated regulation of BAFF expression in fibroblasts using two concomitant models: (i) synovial fibroblasts (FLS) isolated from healthy controls (N) or Rheumatoid Arthritis (RA) patients; (ii) human dermal fibroblasts (HDF) isolated from healthy controls (N) or Systemic Sclerosis (SSc) patients. Using RT-qPCR and ELISA, we first showed that SScHDF synthesized and released BAFF in response to Poly(I:C) or IFN-γ treatment, as previously observed in RAFLS, whereas NHDF released BAFF preferentially in response to IFN-γ. Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ. Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model. Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF. Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors. Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc.
Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Fator Ativador de Células B/biossíntese , Fibroblastos/metabolismo , MicroRNAs/genética , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Regiões 3' não Traduzidas/genética , Artrite Reumatoide/imunologia , Fator Ativador de Células B/genética , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Masculino , Pessoa de Meia-Idade , Poli I-C/farmacologia , Escleroderma Sistêmico/imunologiaRESUMO
Follicular helper T cells (Tfh), which play a pivotal role in B cell activation and differentiation in lymphoid structures, secrete IL-21 whose augmented secretion is a hallmark of several autoimmune diseases. To decipher the cellular and molecular interactions occurring in salivary glands of patients suffering from primary Sjögren's syndrome (pSS), we investigated whether salivary gland epithelial cells (SGECs) were capable to induce Tfh differentiation. Co-cultures of naïve CD4(+) T cells and SGECs from both patients with pSS and controls were performed. Here, we report that IL-6 and ICOSL expression by SGECs contributes to naïve CD4(+) T differentiation into Tfh cells, as evidenced by their acquisition of a specific phenotype, characterized by Bcl-6, ICOS and CXCR5 expression and IL-21 secretion, but also but by their main functional feature: the capacity to enhance B lymphocytes survival. We demonstrated an increase of serum IL-21 with systemic activity. Finally, we analyzed the potential occurrence of a genetic association between IL-21 or IL-21R gene polymorphisms and pSS or elevated IL-21 secretion. This study, which demonstrates a direct induction of Tfh differentiation by SGECs, emphasizes a yet unknown pathogenic role of SGECs and suggests that Tfh and IL-21 might be relevant biomarkers and/or therapeutic targets in primary Sjögren's syndrome.
Assuntos
Diferenciação Celular , Células Epiteliais/metabolismo , Glândulas Salivares/imunologia , Glândulas Salivares/metabolismo , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Estudos de Casos e Controles , Comunicação Celular/imunologia , Sobrevivência Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , Imunofenotipagem , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Interleucina-6/sangue , Interleucina-6/metabolismo , Interleucinas/sangue , Interleucinas/genética , Interleucinas/metabolismo , Ativação Linfocitária/imunologia , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-21/genética , Glândulas Salivares/patologia , Índice de Gravidade de Doença , Síndrome de Sjogren/patologiaRESUMO
INTRODUCTION: B lymphocytes might play a pathogenic role in dermal fibrosis in systemic sclerosis (SSc). B-cell activating factor (BAFF), a key cytokine for B-cell activation, is increased in the serum and the skin of patients with SSc. However, the ability of B cells directly to stimulate dermal fibroblasts and the role of BAFF are not fully understood. We therefore investigated the involvement of B cells and BAFF in the expression of collagen and profibrotic markers by dermal fibroblasts. METHODS: Cocultures of blood B cells from healthy blood donors and normal or SSc dermal fibroblasts stimulated with anti-IgM and BAFF were performed. Alpha-SMA, TIMP1, MMP9, COL1A1, COL1A2, and COL3A1 mRNA expression were determined by quantitative RT-PCR. Soluble collagen, BAFF, IL-6, IL-1ß, TGF-ß1, and CCL2 protein secretion were assessed. RESULTS: Coculture of blood B cells and dermal fibroblasts isolated from SSc patients induced IL-6, TGF-ß1, CCL2, and collagen secretion, as well as Alpha-SMA, TIMP1, and MMP9 expression in dermal fibroblasts. Transwell assays demonstrated that this induction was dependent on cell-cell contact. Addition of anti-IgM and BAFF to the coculture increased IL-6, CCL2, TGF-ß1, and collagen secretion. B cell- and BAFF-induced collagen secretion was highly reduced by anti-TGF-ß1 antibodies. CONCLUSIONS: Our results showed for the first time a direct role of B cells on the production of collagen by dermal fibroblasts, which is further enhanced by BAFF. Thus, these results demonstrate a new pathogenic role of B cells and BAFF in fibrosis and systemic sclerosis.
Assuntos
Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Colágeno/imunologia , Fibroblastos/imunologia , Actinas/genética , Actinas/imunologia , Actinas/metabolismo , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/farmacologia , Fator Ativador de Células B/metabolismo , Fator Ativador de Células B/farmacologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Células Cultivadas , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Colágeno/genética , Colágeno/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/imunologia , Fibrose/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Humanos , Imunoglobulina M/imunologia , Interleucina-6/imunologia , Interleucina-6/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Músculo Liso/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/metabolismo , Pele/imunologia , Pele/metabolismo , Pele/patologia , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/imunologia , Fator de Crescimento Transformador beta1/imunologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
OBJECTIVE: To evaluate whether miR-20a belonging to the cluster miR-17-92 is a negative regulator of inflammation in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) by modulating expression of apoptosis signal-regulating kinase (ASK) 1, a key component of the toll-like receptors 4 pathway, upstream of p38 mitogen-activated protein kinase. METHODS: Evaluation of miR-20a and ASK1 mRNA was performed by RT-qPCR. ASK1 protein expression was assessed by western blotting. Overexpression of miR-20a was performed by transfection of RA FLS and THP-1 cells with miR-20a mimics. Interleukin (IL)-6, CXCL-10, IL-1ß and TNF-α release were measured by ELISA. The role of miR-20a in vivo was assessed by IL-6 release from macrophages obtained from mice injected intraperitoneally with vectorised miR-20a mimics. RESULTS: We showed that stimulation of RA FLS with lipopolysacharide (LPS) and bacterial lipoproteins (BLP) induces a drop in expression of miR-20a and that this decrease is associated with an upregulation of ASK1 expression. Using transfection of Ask1 3'UTR reporters, we demonstrate that Ask1 is a direct target of miR-20a. Overexpression of miR-20a led to a global decrease in ASK1 protein in BLP- and LPS-activated cells indicating that miR-20a regulates the expression of ASK1 at the translational level. Transfection of miR-20a mimics decreases IL-6 and CXCL10 release by RA FLS and IL-1ß and TNF-α by activated THP-1 cells but only in response to LPS. Last, injection of vectorised miR-20a mimics to mice led to a global decrease in ASK1 protein expression and IL-6 secretion in LPS-activated macrophages. CONCLUSIONS: Our data point toward an important role for miR-20a in the regulation of pro-inflammatory cytokines release, by controlling ASK1 expression in RA FLS.
Assuntos
Artrite Reumatoide/fisiopatologia , Citocinas/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Macrófagos/metabolismo , MicroRNAs/fisiologia , RNA Mensageiro/análise , Membrana Sinovial/citologia , Animais , Artrite Reumatoide/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Membrana Sinovial/metabolismo , Membrana Sinovial/fisiopatologiaRESUMO
Resident cells, such as fibroblast-like synoviocytes (FLS), play a crucial role in rheumatoid arthritis (RA). They are implicated in the inflammatory response and play a key role in osteoarticular destruction. Moreover, RA FLS spread RA to unaffected joints. Pathogen-associated molecular patterns and damage-associated molecular patterns have been found to activate RA FLS by interacting with pattern recognition receptors, such as TLR. RA FLS express a large number of TLR, and TLR2 was demonstrated to be involved in RA inflammation. Because microRNA have emerged as important controllers of TLR expression and signaling, the aim of this study was to evaluate their potential involvement in the control of TLR2 expression by RA FLS. We first showed that Tlr2 expression is strongly upregulated in RA FLS in response to TLR2 ligands. Using a microRNA microarray analysis, we identified one miRNA in activated RA FLS, miR-19b, which was downregulated and predicted to target Tlr2 mRNA. Downregulation of miR-19b and miR-19a, which belongs to the same cluster, was confirmed by real-time quantitative PCR. Transfection of RA FLS with miR-19a/b mimics decreased TLR2 protein expression. In parallel, we found that both IL-6 and matrix metalloproteinase 3 secretion was significantly downregulated in activated FLS transfected with either mimic. Moreover, using a luciferase assay, we showed that miR-19a/b directly target Tlr2 mRNA. Taken together, our data point toward an important role for miR-19a/b in the regulation of IL-6 and matrix metalloproteinase 3 release by controlling TLR2 expression, as well as provide evidence that miR-19a/b can act as negative regulators of inflammation in humans.
Assuntos
Artrite Reumatoide/imunologia , Fibroblastos/imunologia , Regulação da Expressão Gênica/imunologia , MicroRNAs/imunologia , Membrana Sinovial/imunologia , Receptor 2 Toll-Like/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Interleucina-6/biossíntese , Interleucina-6/imunologia , Metaloproteinase 3 da Matriz/biossíntese , Metaloproteinase 3 da Matriz/imunologia , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , RNA Mensageiro/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Receptor 2 Toll-Like/biossínteseRESUMO
TNF-α is a major cytokine implicated in rheumatoid arthritis. Its expression is regulated both at the transcriptional and posttranscriptional levels and recent data demonstrated that miRNAs are implicated in TNF-α response in macrophages. LPS-activated FLS isolated from RA patients express TNF-α mRNA but not the mature protein. This prompted us to look for miRNAs which could be implicated in this anti-inflammatory effect. Using a microarray, we found two miRNAs, miR-125b and miR-939 predicted to target the 3'-UTR of TNF-α mRNA, to be up-regulated in RA FLS in response to LPS, but their repression did not restore mature TNF-α expression in FLS. We showed previously that miR-346, which is upregulated in LPS-activated FLS, inhibited Btk expression that stabilized TNF-α mRNA. Blocking miR-346 reestablished TNF-α expression in activated FLS. Interestingly, transfection of miR-346 in LPS-activated THP-1 cells inhibited TNF-α secretion. We also demonstrated that TTP, a RNA binding protein which inhibited TNF-α synthesis, is overexpressed in activated FLS and that inhibition of miR-346 decreases its expression. Conversely, transfection of miR-346 in LPS-activated THP-1 cells increased TTP mRNA expression and inhibited TNF-α release. These results indicate that miR-346 controls TNF-α synthesis by regulating TTP expression.
Assuntos
Artrite Reumatoide/genética , MicroRNAs/metabolismo , Estabilidade de RNA , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Tirosina Quinase da Agamaglobulinemia , Artrite Reumatoide/patologia , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , Modelos Biológicos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Líquido Sinovial/citologia , Transfecção , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
Fibroblast-like synoviocytes (FLSs) are important actors in rheumatoid arthritis (RA) pathogenesis. The autoimmune nature of RA is attributed to autoantibody production, which confers to B cells a predominant role in RA. Several arguments support an induction of class switch recombination (CSR) in RA synovium, causing--in conjunction with somatic hypermutation--the production of potentially pathogenic IgG. To determine whether RA FLSs can directly promote CSR and to analyze the role of external factors like TLR signals and BAFF (B cell activating factor) family cytokines in this FLS-B cell crosstalk, we performed cocultures of blood B cells (from normal individuals or RA patients) with RA FLSs and analyzed CSR induction by quantification of AICDA (encoding activation-induced cytidine deaminase, AID) and switch circular transcripts expression, and IgG secretion. RA FLSs--and to a lesser extent osteoarthritis or control FLSs--promoted CSR, and TLR3 stimulation potentialized it. In addition, induction of CSR by RA FLSs was totally dependent on cell-cell contact in basal conditions, and partially dependent in the case of TLR3 stimulation. Finally, we showed that the mechanism by which RA FLSs induce CSR is mostly BAFF-dependent. Our results support the hypothesis that CSR can be induced outside the ectopic lymphoid structures in RA.
Assuntos
Artrite Reumatoide/imunologia , Fator Ativador de Células B/metabolismo , Switching de Imunoglobulina , Imunoglobulina G/biossíntese , Membrana Sinovial/citologia , Artrite Reumatoide/patologia , Autoanticorpos/biossíntese , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Técnicas de Cocultura , Citidina Desaminase/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Humanos , Imunoglobulina G/metabolismo , Região de Troca de Imunoglobulinas , Osteoartrite/imunologia , Osteoartrite/patologia , Reação em Cadeia da Polimerase , Hipermutação Somática de Imunoglobulina , Membrana Sinovial/metabolismo , Receptor 3 Toll-Like/metabolismoRESUMO
INTRODUCTION: Interleukin-32 (IL-32) is a recently described cytokine that is a strong inducer of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, IL-1ß, IL-6, and IL-8. The expression of this cytokine is highly increased in the rheumatoid synovium and correlated with the severity of joint inflammation. Little is known regarding the innate immune-related regulation of IL-32 by fibroblast-like synoviocytes (FLSs). We therefore investigated the effect of innate immune stimulation by ligands of Toll-like receptor (TLR)2, TLR3, and TLR4, and cytokines such as TNF-α and interferon (IFN)-γ, on IL-32 expression by FLSs. METHODS: FLSs were isolated from patients with rheumatoid arthritis (RA) according to the ACR criteria. Quantitative RT-PCR, confocal analysis, and ELISA were performed to evaluate IL-32 mRNA induction and IL-32 release by FLSs stimulated with TLR2 (BLP), TLR3 (poly I:C), and TLR4 (lipopolysaccharide) ligands, TNF-α and IFN-γ. RESULTS: TLR2, -3, and -4 ligands as well as IFN-γ and TNF-α induced IL-32 ß, γ and δ mRNA expression by RA FLSs. Mature IL-32 was expressed intracellularly and released by cells stimulated with the various activators. The IL-32α isoform was expressed intracellularly in response to TNF-α and poly I:C and not released in culture supernatants. Stimulation of FLS with TNF-α, BLP, lipopolysaccharide, or poly I:C concomitant with IFN-γ increased IL-32 expression compared with stimulation with IFN-γ alone. CONCLUSIONS: IL-32 synthesis by FLSs is tightly regulated by innate immunity in rheumatoid arthritis. Thus TNF-α, IFN-γ, double-strand RNA, hyaluronic acid, or other damage-associated molecular patterns (DAMPs), highly secreted in synovial tissues of RA patients, might trigger IL-32 secretion by FLSs. IL-32 might therefore represent a relevant therapeutic target in RA.
Assuntos
Artrite Reumatoide/imunologia , Fibroblastos/imunologia , Imunidade Inata/imunologia , Interleucinas/imunologia , Membrana Sinovial/imunologia , Artrite Reumatoide/genética , Células Cultivadas , Sinergismo Farmacológico , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Humanos , Indutores de Interferon/imunologia , Indutores de Interferon/farmacologia , Interferon gama/imunologia , Interferon gama/farmacologia , Interleucinas/genética , Lipopolissacarídeos/farmacologia , Poli I-C/imunologia , Poli I-C/farmacologia , RNA Mensageiro/metabolismo , Membrana Sinovial/citologia , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/imunologia , Receptor 3 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
INTRODUCTION: In the present study, we investigated the ability of microparticles isolated from synovial fluids from patients with rheumatoid arthritis or osteoarthritis to induce the synthesis and release of key cytokines of B-lymphocyte modulation such as B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor by rheumatoid fibroblast-like synoviocytes. METHODS: Microparticles were analyzed in synovial fluids from patients with rheumatoid arthritis, osteoarthritis, microcristalline arthritis, and reactive arthritis. In addition, microparticle release after activation from various cell lines (CEM lymphocyte and THP-1 cells) was assessed. Microparticles were isolated by differential centrifugation, and quantitative determinations were carried out by prothrombinase assay after capture on immobilized annexin V. B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor release was evaluated by enzyme-linked immunosorbent assay. RESULTS: Microparticles isolated from synovial fluids obtained from rheumatoid arthritis and osteoarthritis patients or microparticles derived from activated THP-1 cells were able to induce B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor release by rheumatoid arthritis fibroblast-like synoviocytes. Conversely, CEM-lymphocytes-derived microparticles generated by treatment with a combination of PHA, PMA and Adt-D did not promote the release of B cell-activating factor but favored the secretion of thymic stroma lymphopoietin and secretory leukocyte protease inhibitor by rheumatoid arthritis fibrobast-like synoviocytes. However, microparticles isolated from actinomycin D-treated CEM lymphocytes were not able to induce B cell-activating factor, thymic stroma lymphopoietin, or secretory leukocyte protease inhibitor release, indicating that microparticles derived from apoptotic T cells do not function as effectors in B-cell activation. CONCLUSIONS: These results demonstrate that microparticles are signalling structures that may act as specific conveyors in the triggered induction and amplification of autoimmunity. This study also indicates that microparticles have differential effects in the crosstalk between B lymphocytes and target cells of autoimmunity regarding the parental cells from which they derive.
Assuntos
Artrite Reumatoide/imunologia , Micropartículas Derivadas de Células/imunologia , Líquido Sinovial/imunologia , Idoso , Idoso de 80 Anos ou mais , Fator Ativador de Células B/biossíntese , Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Fibroblastos/imunologia , Humanos , Interleucina-6/biossíntese , Interleucina-6/imunologia , Interleucina-8/biossíntese , Interleucina-8/imunologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Receptor Cross-Talk/imunologia , Inibidor Secretado de Peptidases Leucocitárias/biossíntese , Inibidor Secretado de Peptidases Leucocitárias/imunologia , Líquido Sinovial/citologia , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Linfopoietina do Estroma do TimoRESUMO
OBJECTIVE: It was recently demonstrated that synoviocytes (FLS) from rheumatoid arthritis (RA) patients express BAFF transcripts that are up-regulated by tumor necrosis factor alpha (TNFalpha) and interferon-gamma (IFNgamma). Thus, BAFF increases in RA target cells might be related to activation of the receptors of innate immunity. The purpose of this study was to determine whether ligands of Toll-like receptor 2 (TLR-2), TLR-4, TLR-9, and alpha5beta1 integrin are able to induce BAFF synthesis by RA FLS. METHODS: Quantitative reverse transcription-polymerase chain reaction analyses and enzyme-linked immunosorbent assays were performed to evaluate BAFF messenger RNA induction and BAFF release from FLS after stimulation by ligands for TLR-2, TLR-4, TLR-9, alpha5beta1 integrin (bacterial lipopeptide [BLP] palmitoyl-3-cysteine-serine-lysine-4, lipopolysaccharide [LPS], CpG, and protein I/II, respectively), TNFalpha, and IFNgamma. RESULTS: In contrast to IFNgamma, neither TNFalpha, LPS, BLP, nor CpG induced the de novo synthesis and release of BAFF by FLS. Priming of cells with IFNgamma did not have a synergistic effect on BAFF synthesis by FLS stimulated with bacterial products known as pathogen-associated molecular patterns. Moreover, we found that IFNgamma-induced BAFF synthesis is inhibited by simultaneous stimulation with either TLR ligands or TNFalpha. We also showed that interplay between TLRs, TNF receptors, and IFNgamma signaling induces the expression of suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 and reduces IFNgamma-dependent STAT-1 phosphorylation, which might explain this inhibition. In contrast, we demonstrated that stimulation of alpha5beta1 integrin can induce BAFF synthesis and release per se and that stimulation of this pathway has no inhibitory effect on IFNgamma-induced BAFF synthesis. CONCLUSION: Our findings indicate that BAFF secretion by resident cells in target organs of autoimmunity is tightly regulated by innate immunity, with positive and negative controls, depending on the receptors and the pathways triggered.
Assuntos
Artrite Reumatoide/imunologia , Fator Ativador de Células B/biossíntese , Integrina alfa5beta1/imunologia , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Técnicas de Cultura de Células , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Interferon gama/metabolismo , Ligantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismoRESUMO
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in signaling downstream of integrins, linking bacterial detection, cell entry, and initiation of proinflammatory response through MAPKs and NF-kappaB activation. In this study, using protein I/II from Streptococcus mutans as a model activator of FAK, we investigated the potential link between FAK and TLR pathways. Using macrophages from TLR- or MyD88-deficient mice, we report that MyD88 plays a major role in FAK-dependent protein I/II-induced cytokine release. However, response to protein I/II stimulation was independent of TLR4, TLR2, and TLR6. The data suggest that there is a cross talk between FAK and MyD88 signaling pathways. Moreover, MyD88-dependent, LPS-induced IL-6 secretion by human and murine fibroblasts required the presence of FAK, confirming that MyD88 and FAK pathways are interlinked.
Assuntos
Antígenos de Diferenciação/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptor Cross-Talk/imunologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Diferenciação/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Células Cultivadas , Fibroblastos/enzimologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Integrina alfa5beta1/metabolismo , Interleucina-6/metabolismo , Ligantes , Lipopolissacarídeos/farmacologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide , Óxido Nítrico/biossíntese , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Streptococcus mutans/imunologia , Membrana Sinovial/enzimologia , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptor 6 Toll-Like , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Fibroblast-like synoviocytes (FLSs) play a major role in the pathogenesis of rheumatoid arthritis (RA) by secreting effector molecules that promote inflammation and joint destruction. How these cells become and remain activated is still elusive. Both genetic and environmental factors probably play a role in transforming FLSs into inflammatory matrix-degrading cells. As bacterial products have been detected in the joint and shown to trigger joint inflammation, this study was undertaken to investigate whether a bacterial ligand of integrin alpha5beta1, protein I/II, could contribute to the aggressive behavior of RA FLSs. Protein I/II is a pathogen-associated molecular pattern (PAMP) isolated from oral streptococci that have been identified in the joints of RA patients. The response of RA and osteoarthritis FLSs to protein I/II was analyzed using human cancer cDNA expression arrays. RT-PCR and pro-MMP-3 (pro-matrix metalloproteinase) assays were then performed to confirm the up-regulation of gene expression. Protein I/II modulated about 6% of all profiled genes. Three of these, those encoding IL-6, leukemia inhibitory factor, and MMP-3, showed a high expression level in all RA FLSs tested, whereas the expression of genes encoding other members of the cytokine or MMP-family was not affected. Furthermore, the up-regulation of MMP-3 gene expression was followed by an increase of pro-MMP-3 release. The expression of interferon regulatory factor 1 and fibroblast growth factor-5 was also up-regulated, although the expression levels were lower. Only one gene, that for insulin-like growth factor binding protein-4, was down-regulated in all RA FLSs. In contrast, in osteoarthritis FLSs only one gene, that for IL-6, was modulated. These results suggest that a bacterial ligand of integrin alpha5beta1 may contribute to the aggressive behavior of RA FLSs by inducing the release of pro-inflammatory cytokines and a cartilage-degrading enzyme, such as IL-6 and MMP-3, respectively.
Assuntos
Artrite Reumatoide/enzimologia , Proteínas de Bactérias/farmacologia , Fibroblastos/enzimologia , Integrina alfa5beta1/efeitos dos fármacos , Metaloproteinase 3 da Matriz/biossíntese , Streptococcus mutans/química , Membrana Sinovial/patologia , Artrite Reumatoide/patologia , Proteínas de Bactérias/genética , Parede Celular/química , Indução Enzimática/efeitos dos fármacos , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/genética , Fator 5 de Crescimento de Fibroblastos/biossíntese , Fator 5 de Crescimento de Fibroblastos/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfa5beta1/fisiologia , Fator Regulador 1 de Interferon/biossíntese , Fator Regulador 1 de Interferon/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Fator Inibidor de Leucemia , Ligantes , Metaloproteinase 3 da Matriz/metabolismo , Osteoartrite/enzimologia , Osteoartrite/patologia , Proteínas Recombinantes de Fusão/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismoRESUMO
Rheumatoid arthritis is a common and severe inflammatory rheumatic disease, for which the immune mechanisms are being decoded little by little. The pathogenic ncludes significant cellular actors of innate immunity (fibroblastic synoviocytes, macrophages, mastocytes...) and adaptive immunity (T and B lymphocytes). These actors interact through the production of and response to specific (cytokines, chemokines and auto-antibodies) and non-specific (prostaglandins, nitrous oxide [NO], complement, proteases) mediators. The chronology of this rheumatoid synovitis is becoming progressively clearer. Its initiation could be the consequence of a precocious activation of the innate immunity, induced by bacterial agents or debris (PAMP). The activation of the synoviocytes and the macrophages via specific receptors (PPR) unleashes an intense inflammatory reaction that triggers a cascade of events. The ongoing nature of this synovitis leads to the intra-articular recruitment of different cells of immunity. This cellular afflux amplifies the macrophagic and synoviocytic activation and proliferation. All of these interactive phenomena end in the production of large quantities of pro-inflammatory cytokines (TNFa, IL1, IL6, IL15, IL17, IL18) but also other pathogenic mediators (auto-antibodies, complement, prostaglandins, nitrous oxide...). This synovitis persists, as it is no longer regulated by a sufficient production of physiological regulators (soluble receptors and inhibitors of cytokines). The consequence of this intense inflammation and synovial proliferation leads to osteo-articular destruction by the production of proteases and the activation of osteoclasts by the RANK/RANK-ligand pathway under the effect of cytokines (TNFa, IL5, IL1, IL6, IL17) and other mediators (prostaglandins) liberated by synoviocytes, macrophages and lymphocytes. The decryption of this puzzle has already created new therapeutic orientations. The identification of new targets is one of the major consequences of this progress in immuno-rheumatology.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/etiologia , Artrite Reumatoide/patologia , Artrite Reumatoide/terapia , Autoanticorpos/imunologia , Citocinas/imunologia , Humanos , Imunidade Celular , Imunoterapia , Ativação de Macrófagos , Macrófagos/imunologia , Neutrófilos/imunologia , Membrana Sinovial/citologia , Sinovite/complicações , Sinovite/imunologiaRESUMO
Proinflammatory cytokines such as tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 beta and IL-18 are key mediators of joint inflammation during rheumatoid arthritis (RA). This chronic inflammation may result from a non-specific innate immune response that could be triggered by a wide variety of microorganisms, because numerous bacterial fragments have been identified in the joints of RA patients. As we have demonstrated previously that protein I/II, a pathogen-associated molecular pattern (PAMP) from oral streptococci, triggers IL-6 and IL-8 gene expression and release from either THP-1 cells or fibroblast-like synoviocytes (FLSs), we next explored the capacity of protein I/II to induce the synthesis and release of IL-18 in THP-1 cells and in FLSs isolated from either RA or osteoarthritis (OA) patients. We demonstrate that protein I/II induced IL-18 mRNA in both THP-1 cells and FLSs but, in contrast to THP-1 cells, gene expression was not associated with the synthesis of the corresponding protein in FLSs. Furthermore, our studies revealed that FLSs did not express the biologically inactive precursor, pro-IL-18, in response to protein I/II. Using actinomycin D, we also showed that IL-18 mRNA is unstable in FLSs. Taken together, these data indicate that lack of IL-18 release from activated FLSs results from a defect in translation of IL-18 mRNA into pro-IL-18 because of rapid degradation of IL-18 mRNA.
Assuntos
Proteínas de Bactérias/fisiologia , Fibroblastos/imunologia , Interleucina-18/metabolismo , Streptococcus mutans/patogenicidade , Membrana Sinovial/imunologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Linhagem Celular , Células Cultivadas , Dactinomicina/farmacologia , Regulação da Expressão Gênica , Humanos , Interleucina-18/biossíntese , Interleucina-18/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Osteoartrite/imunologia , Osteoartrite/patologia , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/análise , RNA Mensageiro/isolamento & purificação , Membrana Sinovial/citologia , Transcrição GênicaRESUMO
Protein I/II, a pathogen-associated molecular pattern from oral streptococci, is a potent inducer of interleukin-6 (IL-6) and IL-8 synthesis and release from fibroblast-like synoviocytes (FLSs), cells that are critically involved in joint inflammation. This synthesis implicates ERK 1/2 and JNKs as well as AP-1-binding activity and nuclear translocation of NF-kappaB. The mechanisms by which protein I/II activates MAPKs remain, however, elusive. Because focal adhesion kinase (FAK) was proposed to play a role in signaling to MAPKs, we examined its ability to contribute to the MAPKs-dependent synthesis of IL-6 and IL-8 in response to protein I/II. We used FAK-/- fibroblasts as well as FAK+/+ fibroblasts and FLSs transfected with FRNK, a dominant negative form of FAK. The results demonstrate that IL-6 and IL-8 release in response to protein I/II was strongly inhibited in both protein I/II-stimulated FAK-/- and FRNK-transfected cells. Cytochalasin D, which inhibits protein I/II-induced phosphorylation of FAK (Tyr-397), had no effect either on activation of ERK 1/2 and JNKs or on IL-6 and IL-8 release. Taken together, these results indicate that IL-6 and IL-8 release by protein I/II-activated FLSs is regulated by FAK independently of Tyr-397 phosphorylation.