RESUMO
TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or host-derived ligands found in arthritic joints. Here, we show that TLR2 and TLR4, but not TLR9, are involved in the pathogenesis of autoimmune arthritis and play distinct roles in the regulation of T cells and cytokines. We investigated the involvement of TLR2, TLR4, and TLR9 in the progression of arthritis using IL-1 receptor antagonist-knockout (IL1rn-/-) mice, which spontaneously develop an autoimmune T cell-mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. Clinical and histopathological evaluation of IL1rn-/-Tlr2-/- mice revealed more severe arthritis, characterized by reduced suppressive function of Tregs and substantially increased IFN-gamma production by T cells. IL1rn-/-Tlr4-/- mice were, in contrast, protected against severe arthritis and had markedly lower numbers of Th17 cells and a reduced capacity to produce IL-17. A lack of Tlr9 did not affect the progression of arthritis. While any therapeutic intervention targeting TLR2 still seems complicated, the strict position of TLR4 upstream of a number of pathogenic cytokines including IL-17 provides an interesting potential therapeutic target for rheumatoid arthritis.
Assuntos
Artrite Reumatoide/imunologia , Vida Livre de Germes/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/microbiologia , Modelos Animais de Doenças , Vida Livre de Germes/genética , Humanos , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Interleucina-17/imunologia , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologiaRESUMO
Rheumatoid arthritis (RA) is a common autoimmune disease leading to profound disability and premature death. Although a role for FcgammaRs and TLRs is accepted, their precise involvement remains to be elucidated. FcgammaRIIb is an inhibitory FcR important in the maintenance of tolerance. We hypothesized that the inhibitory FcgammaRIIb inhibits TLR responses on monocyte-derived dendritic cells (DC) and serves as a counterregulatory mechanism to dampen inflammation, and we surmised that this mechanism might be defective in RA. The expression of the inhibitory FcgammaRIIb was found to be significantly higher on DCs from RA patients having low RA disease activity in the absence of treatment with antirheumatic drugs. The expression of activating FcgammaRs was similarly distributed among all RA patients and healthy controls. Intriguingly, only DCs with a high expression of FcgammaRIIb were able to inhibit TLR4-mediated secretion of proinflammatory cytokines when stimulated with immune complexes. In addition, when these DCs were coincubated with the combination of a TLR4 agonist and immune complexes, a markedly inhibited T cell proliferation was apparent, regulatory T cell development was promoted, and T cells were primed to produce high levels of IL-13 compared with stimulation of the DCs with the TLR4 agonist alone. Blocking FcgammaRIIb with specific Abs fully abrogated these effects demonstrating the full dependence on the inhibitory FcgammaRIIb in the induction of these phenomena. This TLR4-FcgammaRIIb interaction was shown to dependent on the PI3K and Akt pathway.
Assuntos
Artrite Reumatoide/imunologia , Células Dendríticas/imunologia , Regulação para Baixo/imunologia , Inibidores do Crescimento/fisiologia , Receptores de IgG/fisiologia , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/fisiologia , Regulação para Cima/imunologia , Idoso , Complexo Antígeno-Anticorpo/fisiologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Células Cultivadas , Estudos de Coortes , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Regulação para Baixo/genética , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/genética , Humanos , Mediadores da Inflamação/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Estudos Prospectivos , Receptores de IgG/biossíntese , Receptores de IgG/genética , Regulação para Cima/genéticaRESUMO
BACKGROUND: The T cell attracting chemokine CCL18 is produced by antigen presenting cells and a role for CCL18 has been suggested in the pathogenesis of a variety of diseases. Rheumatoid arthritis (RA) is one of these conditions, in which abundant CCL18 production is present. Although Th2 cytokines and IL-10 are known to have an effect on CCL18 production, there are several gaps in our knowledge regarding the exact regulation of CCL18 secretion, both in general and in RA. In this study we provide new insights in the regulation of CCL18 secretion by monocytes and dendritic cells. RESULTS: In contrast to a large panel of pro-inflammatory stimuli (IL-1beta, TNF-alpha, IL-10, IL-13, IL-15, IL-17, IL-18, IFN-gamma), T cell mimicking molecules (RANKL, CD40L) or TLR driven maturation, the anti-inflammatory IL-10 strongly stimulated DC to secrete CCL18. On freshly isolated monocytes, CCL18 secretion was induced by IL-4 and IL-13, in strong synergy with IL-10. This synergistic effect could already be observed after only 24 hours, indicating that not only macrophages and dendritic cells, but also monocytes secrete CCL18 under these stimulatory conditions. A high CCL18 expression was detected in RA synovial tissue and incubation of monocytes with synovial fluid from RA patients clearly enhanced the effects of IL-4, IL-13 and IL-10. Surprisingly, the effect of synovial fluid was not driven by IL-10 of IL-13, suggesting the presence of another CCL18 inducing factor in synovial fluid. CONCLUSION: In summary, IL-10 synergistically induces CCL18 secretion in combination with IL-4 of IL-13 on monocytes and monocyte derived cells. The effects of IL-14, IL-13 and IL-10 are strongly enhanced by synovial fluid. This synergy may contribute to the high CCL18 expression in RA.
Assuntos
Artrite Reumatoide/imunologia , Quimiocinas CC/biossíntese , Células Dendríticas/imunologia , Interleucinas/farmacologia , Monócitos/imunologia , Líquido Sinovial/imunologia , Células Cultivadas , Citocinas/farmacologia , Células Dendríticas/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Interleucina-10/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Monócitos/efeitos dos fármacos , Receptores Toll-Like/metabolismoRESUMO
OBJECTIVE: To investigate functional consequences of the Toll-like receptor 4 (TLR4) variant (Asp299Gly) in rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells from 28 patients with RA carrying or not carrying the TLR4 variant were incubated with lipopolysaccharide (LPS) and heat shock protein B8 (HSPB8). Concentrations of interleukin 6 (IL-6), tumor necrosis factor-alpha(TNF-alpha), and IL-10 were determined along with TLR4 and CD14 expression. RESULTS: TLR4 expression was similar in patients carrying or not carrying the variant. In contrast, both LPS and HSPB8 resulted in significantly lower secretion of IL-6, TNF-alpha, and IL-10 in those who carried the variant, whereas the frequency of CD14+ cells was higher in these individuals. CONCLUSION: TLR4 variant clearly reduces its potency to mediate signaling. Correction for CD14+ cells is necessary in comparable experiments.
Assuntos
Artrite Reumatoide/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Mutação de Sentido Incorreto , Receptor 4 Toll-Like/genética , Idoso , Artrite Reumatoide/sangue , Feminino , Proteínas de Choque Térmico/farmacologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares , Proteínas Serina-Treonina Quinases/farmacologia , Transdução de Sinais , Receptor 4 Toll-Like/metabolismoRESUMO
OBJECTIVE: Degeneration of extracellular matrix of cartilage leads to the production of molecules capable of activating the immune system via Toll-like receptor 4 (TLR-4). The objective of this study was to investigate the involvement of TLR-4 activation in the development and progression of autoimmune destructive arthritis. METHODS: A naturally occurring TLR-4 antagonist, highly purified lipopolysaccharide (LPS) from Bartonella quintana, was first characterized using mouse macrophages and human dendritic cells (DCs). Mice with collagen-induced arthritis (CIA) and mice with spontaneous arthritis caused by interleukin-1 receptor antagonist (IL-1Ra) gene deficiency were treated with TLR-4 antagonist. The clinical score for joint inflammation, histologic characteristics of arthritis, and local expression of IL-1 in joints were evaluated after treatment. RESULTS: The TLR-4 antagonist inhibited DC maturation induced by Escherichia coli LPS and cytokine production induced by both exogenous and endogenous TLR-4 ligands, while having no effect on these parameters by itself. Treatment of CIA using TLR-4 antagonist substantially suppressed both clinical and histologic characteristics of arthritis without influencing the adaptive anti-type II collagen immunity crucial for this model. Treatment with TLR-4 antagonist strongly reduced IL-1beta expression in articular chondrocytes and synovial tissue. Furthermore, such treatment inhibited IL-1-mediated autoimmune arthritis in IL-1Ra(-/-) mice and protected the mice against cartilage and bone pathology. CONCLUSION: In the present study, we demonstrate for the first time that inhibition of TLR-4 suppresses the severity of experimental arthritis and results in lower IL-1 expression in arthritic joints. Our data suggest that TLR-4 might be a novel target in the treatment of rheumatoid arthritis.
Assuntos
Artrite/tratamento farmacológico , Artrite/imunologia , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Escherichia coli , Lipopolissacarídeos/uso terapêutico , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Artrite/sangue , Doenças Autoimunes/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBARESUMO
Macrophage migration inhibitory factor (MIF) is clearly associated with rheumatoid arthritis (RA) disease severity. However, the regulation of MIF during the course of RA has not been subjected to similar scientific scrutiny. The aim of our study was to investigate the role of various Toll-like receptors (TLRs) and inflammatory mediators on MIF production by dendritic cells (DCs) in healthy controls and RA patients. DCs were cultured from 12 healthy donors and 12 RA patients. Triggering via TLR mediated pathways was achieved using various TLR specific ligands alone or in combination: Pam3Cys for TLR2, LPS and recombinant extra domain A containing fibronectin for TLR4 and Poly(I:C) and R848 for TLR3 and TLR7, respectively. In addition, iDCs from healthy controls were incubated with various cytokines, RANKL and CD40L for 48 h. MIF levels were measured using an ELISA assay. Stimulation of DCs by TLR4 ligands resulted in higher MIF production compared to immature DCs from healthy controls (p<0.002) and RA patients (p<0.002). DCs from RA patients produced higher MIF levels than healthy controls both at the immature stage (p<0.04) as well after full maturation via TLR2 (p<0.04) and TLR4 (p<0.001) triggering. Incubation with TLR3 and TLR7 ligands resulted in a significantly decreased secretion of MIF in RA patients and controls. Simultaneous incubation of TLR4 with either TLR3 or TLR7 ligands resulted in a decrease of MIF secretion when compared to TLR4 stimulation alone. The secretion of MIF increased when DCs were stimulated with TNF-alpha, RANKL and CD40L. The secretion of MIF by dendritic cells is differentially regulated by TLRs. In addition, TNF-alpha, RANKL, and CD40L augment MIF production by DCs and thus play a potential role in the amplification of the inflammatory loop in RA.
Assuntos
Artrite Reumatoide/metabolismo , Células Dendríticas/metabolismo , Fatores Inibidores da Migração de Macrófagos/biossíntese , Receptores Toll-Like/metabolismo , Feminino , Saúde , Humanos , Fatores Inibidores da Migração de Macrófagos/metabolismo , Masculino , Transdução de SinaisRESUMO
Dendritic cells (DCs) are specialized APCs that can be activated upon pathogen recognition as well as recognition of endogenous ligands, which are released during inflammation and cell stress. The recognition of exogenous and endogenous ligands depends on TLRs, which are abundantly expressed in synovial tissue from rheumatoid arthritis (RA) patients. Furthermore TLR ligands are found to be present in RA serum and synovial fluid and are significantly increased, compared with serum and synovial fluid from healthy volunteers and patients with systemic sclerosis and systemic lupus erythematosus. Identification of novel endogenous TLR ligands might contribute to the elucidation of the role of TLRs in RA and other autoimmune diseases. In this study, we investigated whether five members of the small heat shock protein (HSP) family were involved in TLR4-mediated DC activation and whether these small HSPs were present in RA synovial tissue. In vitro, monocyte-derived DCs were stimulated with recombinant alphaA crystallin, alphaB crystallin, HSP20, HSPB8, and HSP27. Using flow cytometry and multiplex cytokine assays, we showed that both alphaA crystallin and HSPB8 were able to activate DCs and that this activation was TLR4 dependent. Furthermore, Western blot and immunohistochemistry showed that HSPB8 was abundantly expressed in synovial tissue from patients with RA. With these experiments, we identified sHSP alphaA crystallin and HSPB8 as two new endogenous TLR4 ligands from which HSPB8 is abundantly expressed in RA synovial tissue. These findings suggest a role for HSPB8 during the inflammatory process in autoimmune diseases such as RA.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Proteínas de Choque Térmico/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor 4 Toll-Like/metabolismo , Animais , Artrite Reumatoide/patologia , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/metabolismo , Humanos , Ligantes , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Chaperonas Moleculares , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Membrana Sinovial/metabolismo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Regulação para Cima/imunologia , Cadeia A de alfa-Cristalina/fisiologiaRESUMO
OBJECTIVE: To study the expression of Toll-like receptor 2 (TLR-2) and TLR-4 and its association with proinflammatory cytokines in synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals. METHODS: Synovial tissue specimens from 29 RA patients were stained for TLR-2, TLR-4, and proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-12, IL-17, IL-18, and tumor necrosis factor alpha [TNFalpha]). The expression of TLR-2, TLR-4, and cytokines as well as the degree of inflammation in synovial tissue were compared between patients with RA, patients with OA (n = 5), and healthy individuals (n = 3). Peripheral blood mononuclear cells (PBMCs) were incubated with IL-12 and IL-18, and TLR expression was assessed using fluorescence-activated cell sorter analysis. Production of TNFalpha and IL-6 was measured using Luminex bead array technology. RESULTS: In RA synovial tissue, the expression of TLR-2 was slightly higher than that of TLR-4. Interestingly, both TLR-2 and TLR-4 were expressed at higher levels in moderately inflamed synovium, as compared with synovial tissue with no or severe inflammation. TLR expression in both the lining and the sublining was associated with the presence of IL-12 and IL-18, but no other cytokines, in the lining. The expression of both TLRs was low in synovial tissue from OA patients and healthy donors. Stimulation of PBMCs with IL-12 and IL-18 resulted in increased expression of both TLR-2 and TLR-4; this could be blocked with anti-interferon-gamma (anti-IFNgamma) antibodies, suggesting a role for IFNgamma. Lipopolysaccharide- or lipoteichoic acid-mediated triggering of PBMCs incubated with IL-12/IL-18 or IFNgamma led to an increased production of both TNFalpha and IL-6, indicating the functionality of TLR-2 and TLR-4. CONCLUSION: TLR-2 and TLR-4 are expressed in synovial tissue of patients with clinically active disease and are associated with the levels of both IL-12 and IL-18. The synergistic effect of IL-12 and IL-18 on T cell IFNgamma production seems to regulate expression of TLR-2 and TLR-4 in the synovial tissue of RA patients.