RESUMO
We profiled gene expression signatures to distinguish rheumatoid arthritis (RA) from non-inflammatory arthralgia (NIA), self-limiting arthritis (SLA), and undifferentiated arthritis (UA) as compared to healthy controls as novel potential biomarkers for therapeutic responsiveness. Global gene expression profiles of PBMCs from 43 drug-naïve patients presenting with joint symptoms were evaluated and differentially expressed genes identified by comparative analysis with 24 healthy volunteers. Patients were assessed at presentation with follow up at 6 and 12 months. Gene ontology and network pathway analysis were performed using DAVID Bioinformatics Resources v6.7. Gene expression profiles were also determined after disease-modifying anti-rheumatic drug (DMARD) treatment in the inflammatory arthritis groups (i.e. RA and UA) and confirmed by qRT-PCR. Receiver operating characteristic (ROC) curves analysis and Area Under the Curve (AUC) estimation were performed to assess the diagnostic value of candidate gene expression signatures. A type I interferon (IFN) gene signature distinguished DMARD-naïve patients who will subsequently develop persistent inflammatory arthritis (i.e. RA and UA) from those with NIA. In patients with RA, the IFN signature is characterised by up-regulation of SIGLEC1 (p = 0.00597) and MS4A4A (p = 0.00000904). We also identified, EPHB2 (p = 0.000542) and PDZK1IP1 (p = 0.0206) with RA-specific gene expression profiles and elevated expression of the ST6GALNAC1 (p = 0.0023) gene in UA. ROC and AUC risk score analysis suggested that MSA4A (AUC: 0.894, 0.644, 0.720), PDZK1IP1 (AUC: 0.785, 0.806, 0.977), and EPHB2 (AUC: 0.794, 0.723, 0.620) at 0, 6, and 12 months follow-up can accurately discriminate patients with RA from healthy controls and may have practical value for RA diagnosis. In patients with early inflammatory arthritis, ST6GALNAC1 is a potential biomarker for UA as compared with healthy controls whereas EPHB2, MS4A4A, and particularly PDZK1IP1 may discriminate RA patients. SIGLEC1 may also be a useful marker of disease activity in UA.
Assuntos
Artralgia/sangue , Artrite/sangue , Interferon Tipo I/sangue , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Artralgia/diagnóstico , Artralgia/genética , Artrite/diagnóstico , Artrite/genética , Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/genética , Biomarcadores , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Ontologia Genética , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Curva ROC , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Toll-like receptors (TLRs) are innate immune receptors that respond to both exogenous and endogenous stimuli and are suggested to contribute to the perpetuation of chronic inflammation associated with rheumatoid arthritis (RA). In particular, the endosomal TLRs 3, 7, 8, and 9 have more recently been postulated to be of importance in RA pathogenesis. In this study, pan inhibition of the endosomal TLRs by a phosphorothioate-modified inhibitory oligodeoxynucleotide (ODN) is demonstrated in primary human B cells, macrophages, and RA fibroblasts. Inhibition of TLR8 was of particular interest as TLR8 has been associated with RA pathogenesis in both human and murine arthritis models. ODN1411 competitively inhibited TLR8 signaling and was observed to directly bind to a purified TLR8 ectodomain, suggesting inhibition was through a direct interaction with the receptor. Addition of ODN1411 to human RA synovial membrane cultures significantly inhibited spontaneous cytokine production from these cultures, suggesting a potential role for one or more of the endosomal TLRs in inflammatory cytokine production in RA and the potential for inhibitory ODNs as novel therapies.
Assuntos
Artrite Reumatoide/imunologia , Citocinas/biossíntese , Oligodesoxirribonucleotídeos/farmacologia , Receptores Toll-Like/antagonistas & inibidores , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Células Cultivadas , Endossomos/imunologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Humanos , Inflamação , Camundongos , Oligodesoxirribonucleotídeos/imunologia , Membrana Sinovial/citologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/imunologia , Receptor 3 Toll-Like/antagonistas & inibidores , Receptor 3 Toll-Like/imunologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/imunologia , Receptor Toll-Like 9/antagonistas & inibidores , Receptor Toll-Like 9/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
OBJECTIVE: We have previously shown, in a cohort of untreated rheumatoid arthritis (RA) patients, that the suppressive function of Treg cells is defective. However, other studies in cohorts of patients with established RA have shown that Treg cell function is normal. We hypothesized that treatment may restore Treg cell function and lead to reduced disease activity. The aim of this study was to investigate whether treatment with methotrexate (MTX) can result in epigenetic changes that lead to restoration of the Treg cell suppressive function in RA. METHODS: Peripheral blood samples from RA patients were assessed using (3) H-thymidine incorporation to measure Treg cell suppression of T cell proliferation, and by enzyme-linked immunosorbent assay to determine Treg cell suppression of interferon-γ production. CTLA-4 and FoxP3 expression was measured by flow cytometry and quantitative polymerase chain reaction (qPCR) in Treg cells from healthy individuals and RA patients. CD4+ T cells isolated from healthy individuals were cultured with interleukin-2 (IL-2), IL-6, and tumor necrosis factor α in the presence or absence of MTX, and FoxP3 expression was determined using qPCR and flow cytometry. Methylation of the FOXP3 upstream enhancer was analyzed by bisulfite sequencing PCR. RESULTS: Defective Treg cell function was observed only in RA patients who had not been treated with MTX, whereas Treg cells from MTX-exposed RA patients had restored suppressive function. This restored suppression was associated with increased expression of FoxP3 and CTLA-4 in Treg cells. Bisulfite sequencing PCR of Treg cells cultured in MTX revealed a significant reduction in methylation of the FOXP3 upstream enhancer. CONCLUSION: This study identifies a novel mechanism of action of MTX, in which treatment of RA patients with MTX restores defective Treg cell function through demethylation of the FOXP3 locus, leading to a subsequent increase in FoxP3 and CTLA-4 expression.
Assuntos
Antirreumáticos/farmacologia , Artrite Reumatoide/imunologia , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Elementos Facilitadores Genéticos/efeitos dos fármacos , Fatores de Transcrição Forkhead/efeitos dos fármacos , Metotrexato/farmacologia , RNA Mensageiro/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Adulto , Idoso , Artrite Reumatoide/tratamento farmacológico , Antígeno CTLA-4/efeitos dos fármacos , Antígeno CTLA-4/metabolismo , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Interferon gama/efeitos dos fármacos , Interferon gama/metabolismo , Leucócitos Mononucleares , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T Reguladores/metabolismoRESUMO
Although strong binding interactions between protein receptor and ligand do not require the participation of a large number of amino acids in either site, short peptide chains are generally poor at recreating the types of protein-protein interactions which take place during cell recognition and signalling process, probably because their flexible backbones prevent the side chains from forming sufficiently rigid and stable epitopes, which can take part in binding with the desired strength and specificity. In a recently-reported study, it was shown that a proto-epitope containing F, R and S amino acids has the ability to down-regulate TNF secretion by macrophages. This paper extends these findings, putting those amino acids into a short cyclic peptide scaffold, and determining the optimal configuration required to overcome the problems of conformational instability, and give rise to molecules which have potential as therapeutic agents in human disease, such as rheumatoid arthritis.
Assuntos
Fatores Imunológicos/farmacologia , Macrófagos/metabolismo , Peptídeos Cíclicos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Desenho de Fármacos , Fatores Imunológicos/síntese química , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Micelas , Peptídeos Cíclicos/síntese químicaRESUMO
Treg-cell function is compromised in rheumatoid arthritis (RA). As the master regulator of Treg cells, FOXP3 controls development and suppressive function. Stable Treg-cell FOXP3 expression is epigenetically regulated; constitutive expression requires a demethylated Treg-specific demethylated region. Here, we hypothesised that methylation of the FOXP3 locus is altered in Treg cells of established RA patients. Methylation analysis of key regulatory regions in the FOXP3 locus was performed on Treg cells from RA patients and healthy controls. The FOXP3 Treg-specific demethylated region and proximal promoter displayed comparable methylation profiles in RA and healthy-donor Treg cells. We identified a novel differentially methylated region (DMR) upstream of the FOXP3 promoter, with enhancer activity sensitive to methylation-induced silencing. In RA Treg cells we observed significantly reduced DMR methylation and lower DNA methyltransferase (DNMT1/3A) expression compared with healthy Treg cells. Furthermore, DMR methylation negatively correlated with FOXP3 mRNA expression, and Treg cells isolated from rheumatoid factor negative RA patients were found to express significantly higher levels of FOXP3 than Treg cells from RhF-positive patients, with an associated decrease in DMR methylation. In conclusion, the novel DMR is involved in the regulation of Treg-cell FOXP3 expression, but this regulation is lost post-transcriptionally in RA Treg cells.
Assuntos
Artrite Reumatoide/imunologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Artrite Reumatoide/genética , Metilação de DNA/genética , Metilação de DNA/imunologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: Functionally impaired Treg cells expressing abnormally low levels of CTLA-4 have been well documented in rheumatoid arthritis (RA). However, the molecular defect underlying this reduced expression is unknown. The aims of this study were to assess the role of DNA methylation in regulating CTLA-4 expression in Treg cells isolated from RA patients and to elucidate the mechanism of their reduced suppressor function. METHODS: CTLA-4 expression in Treg cells from RA patients and healthy controls was measured by quantitative polymerase chain reaction (PCR) and flow cytometry. Methylation of the CTLA-4 gene promoter was analyzed by bisulfite-specific PCR, followed by sequencing. Methylation-dependent transcriptional activity of the CTLA-4 gene promoter was measured by luciferase assay, and NF-AT binding to the CTLA-4 gene promoter was determined by chromatin immunoprecipitation. The role of CTLA-4 expression in controlling Teff cells was analyzed using an autologous mixed lymphocyte reaction. RESULTS: Down-regulation of CTLA-4 expression in Treg cells from RA patients was caused by methylation of a previously unidentified NF-AT binding site within the CTLA-4 gene promoter. As a consequence, Treg cells were unable to induce expression and activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which in turn resulted in a failure to activate the immunomodulatory kynurenine pathway. CONCLUSION: We show for the first time that epigenetic modifications contribute to defective Treg cell function in RA through an inability to activate the IDO pathway. Therefore, this study sets a precedent for investigating potential therapeutic strategies aimed at reinforcing the IDO pathway in RA patients.
Assuntos
Artrite Reumatoide/imunologia , Antígeno CTLA-4/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/fisiologia , Linfócitos T Reguladores/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Antígeno CTLA-4/genética , Metilação de DNA , Regulação para Baixo , Humanos , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND: Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human T cells is still challenging and methods to achieve efficient gene transfer are often expensive and time-consuming. RESULTS: Here we present a simple optimised protocol for the generation and transduction of lentivirus in primary human CD45RA+ T cells. We show that generation of high-titre lentivirus with improved primary T cell transduction is dependent upon optimised ultracentrifuge speed during viral concentration. Moreover, we demonstrate that transduction efficiency can be increased with simple modifications to the culturing conditions. Overall, a transduction efficiency of up to 89% in primary human CD45RA+ cells is achievable when these modifications are used in conjunction. CONCLUSION: The optimised protocol described here is easy to implement and should facilitate the production of high-titre lentivirus with superior transduction efficiency in primary human T cells without the need for further purification methods.
Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Linfócitos T/citologia , Ácido Butírico/metabolismo , Fosfatos de Cálcio/metabolismo , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Antígenos Comuns de Leucócito/metabolismo , Lipídeos/isolamento & purificação , Plasmídeos/genética , Linfócitos T/metabolismo , Transdução Genética , Transfecção , TransgenesRESUMO
OBJECTIVE: To determine whether selective blockade of tumor necrosis factor receptor I (TNFRI) affects spontaneous proinflammatory cytokine and chemokine production in ex vivo-cultured human rheumatoid arthritis synovial membrane mononuclear cells (MNCs) and to compare this response to that of TNF ligand blockade using etanercept. METHODS: A bispecific, single variable-domain antibody (anti-TNFRI moiety plus an albumin binding moiety [TNFRI-AlbudAb]) was used to selectively block TNFRI. Inhibition of TNFα-mediated responses in cell lines expressing TNFRI/II confirmed TNFRI-AlbudAb potency, human rhabdomyosarcoma cell line KYM-1D4 cytotoxicity, and human umbilical vein endothelial cell (HUVEC) vascular cell adhesion molecule 1 (VCAM-1) upregulation. Eighteen RA synovial membrane MNC suspensions were cultured for 2 days or 5 days, either alone or in the presence of TNFRI-AlbudAb, control-AlbudAb, or etanercept. Proinflammatory cytokines and chemokines in culture supernatants were measured by enzyme-linked immunosorbent assays. A mixed-effects statistical analysis model was used to assess the extent of TNFRI selective blockade, where the results were expressed as the percentage change with 95% confidence intervals (95% CIs). RESULTS: TNFRI-AlbudAb inhibited TNFα-induced KYM-1D4 cell cytotoxicity (50% inhibition concentration [IC50 ] 4 nM) and HUVEC VCAM-1 up-regulation (IC50 12 nM) in a dose-dependent manner. In ex vivo-cultured RA synovial membrane MNCs, selective blockade of TNFRI inhibited the production of proinflammatory cytokines and chemokines to levels similar to those obtained with TNF ligand blockade, without inducing cellular toxicity. Changes in cytokine levels were as follows: -23.5% (95% CI -12.4, -33.2 [P = 0.004]) for granulocyte-macrophage colony-stimulating factor, -33.4% (95% CI -20.6, -44.2 [P ≤ 0.0001]) for interleukin-10 (IL-10), -17.6% (95% CI 3.2, -34.2 [P = 0.0880]) for IL-1ß, and -19.0% (95% CI -3.4, -32.1 [P = 0.0207]) for IL-6. Changes in chemokine levels were as follows: -34.2% (-14.4, -49.4 [P = 0.0030]) for IL-8, -56.6% (-30.7, -72.9 [P = 0.0011]) for RANTES, and -24.9% (2, -44.8 [P = 0.0656]) for monocyte chemotactic protein 1. CONCLUSION: In ex vivo-cultured RA synovial membrane MNCs, although a limited role of TNFRII cannot be ruled out, TNFRI signaling was found to be the dominant pathway leading to proinflammatory cytokine and chemokine production. Thus, selective blockade of TNFRI could potentially be therapeutically beneficial over TNF ligand blockade by retaining the beneficial TNFRII signaling.
Assuntos
Artrite Reumatoide/metabolismo , Quimiocinas/biossíntese , Citocinas/biossíntese , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Membrana Sinovial/efeitos dos fármacos , Adulto , Idoso , Antirreumáticos/farmacologia , Artrite Reumatoide/patologia , Linhagem Celular Tumoral , Células Cultivadas , Etanercepte , Feminino , Humanos , Imunoglobulina G/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Receptores do Fator de Necrose Tumoral/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
Previously, we demonstrated that spontaneous TNF-α production by macrophages in rheumatoid arthritis (RA) synovial tissue is largely driven by contact-dependent activation with T cells in that tissue. Whereas abundant IL-10 is present in these RA synovial cultures, it does not adequately control the production of TNF-α. In this study, we have compared the mechanisms involved in IL-10-mediated TNF-α regulation in LPS-stimulated macrophages with macrophages stimulated with activated T cells. We confirm that in LPS-stimulated macrophages the 3' enhancer region of tnf is essential for tnf transcription, and its regulation by IL-10 is dominated by a STAT3-dependent pathway. However, in contrast, we have found that tnf transcription in macrophages stimulated by activated T cells or by RA synovial T cells does not require the 3' enhancer region of tnf, and that its regulation by IL-10 is subsequently altered and clearly is not mediated by a dominant STAT3 pathway. These observations have very important implications for our understanding as to how IL-10 regulates TNF-α production at sites of chronic inflammation, such as the synovial tissue of patients with RA. Furthermore, these distinct IL-10 mechanisms will have bearing upon the identification of potential therapeutic targets in RA synovial macrophages where the activation stimulus is clearly not LPS.
Assuntos
Interleucina-10/imunologia , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Artrite Reumatoide/imunologia , Humanos , Inflamação , Lipopolissacarídeos , Fator de Necrose Tumoral alfa/imunologiaRESUMO
OBJECTIVE: To examine the migratory properties of cytokine-activated T (Tck) cells. METHODS: Tck cells were generated by culture of peripheral blood T cells in the presence of interleukin-6 (IL-6), tumor necrosis factor α, and IL-2. Changes in cell surface phenotype were analyzed by flow cytometry. Chemotactic responsiveness was measured using in vitro chemotaxis assays and transendothelial migration through human umbilical vein endothelial cell monolayers. Levels of vascular cell adhesion molecule 1 (VCAM-1) were measured by sandwich enzyme-linked immunosorbent assay. RESULTS: Cytokine stimulation up-regulated the expression of chemokine receptors and integrins on Tck cells, including CXCR4, very late activation antigen 4 (VLA-4), and lymphocyte function-associated antigen 1. Increased expression of CXCR4 and VLA-4 integrin resulted in concentration-dependent chemotaxis to their ligands, stromal cell-derived factor 1 (SDF-1) and VCAM-1, which could be selectively blocked using a specific CXCR4 inhibitor and antibodies against VLA-4. Increased expression of VLA-4 also resulted in increased transendothelial migration of Tck cells, which could be abrogated using blocking antibodies against VLA-4. Tck cells also showed an increased chemotactic response to rheumatoid arthritis (RA) fibroblast-like synoviocytes cultured in vitro, which could be blocked using inhibitors against VLA-4 and CXCR4. CONCLUSION: The activated phenotype of Tck cells results in increased migratory responsiveness to SDF-1 and soluble VCAM-1, which are among the chemokines and proteins found elevated in the RA synovial joint environment. Cytokine-dependent activation may contribute to RA pathogenicity by promoting T cell recruitment to and retention in the joint, perpetuating the inflammatory cascade in RA.
Assuntos
Quimiotaxia de Leucócito/imunologia , Receptores CXCR4/metabolismo , Linfócitos T/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina alfa4beta1/metabolismo , Interleucina-2/farmacologia , Interleucina-6/farmacologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/imunologia , Linfócitos T/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Increasing evidence suggests that regulatory T cell (Treg) function is impaired in chronic inflammatory diseases such as rheumatoid arthritis (RA). Here we demonstrate that Tregs are unable to modulate the spontaneous production of TNF-α from RA synovial cells cultured from the diseased synovium site. Cytokine (IL-2, IL-6, TNF-α) activated T cells (Tck), cells we previously demonstrated to mimic the effector function of pathogenic RA synovial T cells, contained Tregs that survived and divided in this cytokine environment; however, the up-regulation of key molecules associated with Treg function (CTLA-4 and LFA-1) was impaired. Furthermore, Tregs were unable to suppress the function of Tcks, including contact-dependent induction of TNF-α from macrophages, supporting the concept that impaired Treg function/responsiveness contributes to chronicity of RA. However, ectopic foxp3 expression in both Tcks and pathogenic RA synovial T cells attenuated their cytokine production and function, including contact-dependent activation of macrophages. This diminished response to cytokine activation after ectopic foxp3 expression involved inhibited NF-κB activity and differed mechanistically from that displayed endogenously in conventional Tregs. These results suggest that diseases such as RA may perpetuate owing to the inability of Tregs to control cytokine-activated T-cell function. Understanding the mechanism whereby foxp3 attenuates the pathogenic function of synovial T cells may provide insight into the mechanisms of chronicity in inflammatory disease and potentially reveal new therapeutic candidates.
Assuntos
Artrite Reumatoide/imunologia , Fatores de Transcrição Forkhead/imunologia , Cápsula Articular/imunologia , Linfócitos T Reguladores/imunologia , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Cápsula Articular/citologia , Cápsula Articular/metabolismo , Lentivirus , Luciferases , NF-kappa B/imunologia , NF-kappa B/metabolismo , Transdução Genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To assess the role of the activating receptor NKG2D in arthritis. METHODS: Levels of NKG2D and its ligands were determined by fluorescence-activated cell sorting, real-time polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti-NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days. RESULTS: We demonstrated expression of NKG2D and its ligands on human RA synovial cells and extended this finding to the paws of arthritic mice. Expression of messenger RNA for the NKG2D ligand Rae-1 was up-regulated, and NKG2D was present predominantly on natural killer (NK) and CD4+ T cells, in arthritic paw cell isolates. NKG2D was down-modulated during the progression of collagen-induced arthritis (CIA). NKG2D expression in arthritic paws was demonstrated by immunohistochemistry. Blockade of NKG2D ameliorated established CIA, with significant reductions in clinical scores and paw swelling. Histologic analysis of arthritic joints from anti-NKG2D-treated mice demonstrated significant joint protection, compared with control mice. Moreover, anti-NKG2D treatment significantly reduced both interleukin-17 production from CD4+ T cells in arthritic paws and splenic NK cell cytotoxic effector functions in vivo and in vitro. CONCLUSION: Our findings indicate that blockade of NKG2D in a murine model and in human explants has beneficial therapeutic potential that merits further investigation in RA.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Artrite Experimental/tratamento farmacológico , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Membrana Sinovial/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Células Cultivadas , Técnicas de Cocultura , Articulações/imunologia , Articulações/patologia , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Membrana Sinovial/patologiaRESUMO
Natural Killer (NK) cells are lymphocytes of the innate immune system, originally described by their capacity to control tumour cells and eliminate virus-infected cells. However accumulating evidence suggests that NK cells can interact with various components of the immune system and play a critical role in autoimmune diseases by limiting or exacerbating immune responses. Rheumatoid arthritis is a chronic inflammatory disease characterised by joint inflammation and cartilage and bone destruction. NK cells are enriched within the joints of patients with rheumatoid arthritis but how they contribute to disease pathology is currently not fully elucidated. This review will outline the current understanding of NK cell biology and how these cells may modulate disease pathogenesis in rheumatoid arthritis through interactions with other immune cells.
Assuntos
Artrite Reumatoide/imunologia , Células Matadoras Naturais/imunologia , Animais , Doenças Autoimunes/imunologia , Homeostase/imunologia , Humanos , Imunomodulação , Imunofenotipagem , Ativação Linfocitária/imunologiaRESUMO
Peripheral blood CD4(+) CD45RO(+) T cells activated in vitro are able to induce expression of tumour necrosis factor-α (TNF-α) in monocytes via a contact-dependent mechanism. Activation is achieved either with interleukin-2 (IL-2)/IL-6/TNF-α over an 8-day period or cross-linking CD3 using anti-CD3 antibody for 48 hr. In this paper, we show that the p38 mitogen-activated protein kinase (MAPK) signalling pathway played different roles in the generation of effector function in these two types of activated T cells. In anti-CD3 activated T cells, p38 MAPK is a negative regulator for anti-CD3 induced cell proliferation and has no significant effect on the acquisition of either the effector function (induction of monocyte-derived TNF-α) or production of T-cell cytokines. In contrast, the p38 MAPK signalling pathway is required for the acquisition of cytokine-induced effector function and promotes cell proliferation and cytokine production.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/enzimologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Ativação Enzimática , Humanos , Antígenos Comuns de Leucócito/imunologia , Transdução de Sinais/imunologiaRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase activity and hence PG production. However, the ability of NSAIDs to ameliorate pain and tenderness does not prevent disease progression in rheumatoid arthritis, a disease whose pathogenesis is linked to the presence of proinflammatory cytokines, such as TNF-alpha. To understand this observation, we have examined the effect of NSAIDs on the production of clinically validated proinflammatory cytokines. We show that a variety of NSAIDs superinduce production of TNF from human peripheral blood monocytes and rheumatoid synovial membrane cultures. A randomized, double-blinded, crossover, placebo-controlled trial in healthy human volunteers also revealed that the NSAID drug celecoxib increased LPS-induced TNF production in whole blood. NSAID-mediated increases in TNF are reversed by either the addition of exogenous PGE(2) or by a PGE(2) EP2 receptor agonist, revealing that PGE(2) signaling via its EP2 receptor provides a valuable mechanism for controlling excess TNF production. Thus, by reducing the level of PGE(2), NSAIDs can increase TNF production and may exacerbate the proinflammatory environment both within the rheumatoid arthritis joint and the systemic environment.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antirreumáticos/farmacologia , Artrite Reumatoide/imunologia , Membrana Sinovial/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/sangue , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Adulto , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Celecoxib , Células Cultivadas , Estudos Cross-Over , Citocinas/biossíntese , Citocinas/sangue , Método Duplo-Cego , Humanos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
INTRODUCTION: Type 4 phosphodiesterases (PDE4) play an important role in immune cells through the hydrolysis of the second messenger, cAMP. Inhibition of PDE4 has previously been shown to suppress immune and inflammatory responses, demonstrating PDE4 to be a valid therapeutic target for immune-mediated pathologies. We assessed the anti-inflammatory effects of a novel PDE4 inhibitor, apremilast, in human synovial cells from rheumatoid arthritis (RA) patients, as well as two murine models of arthritis. METHODS: Cells liberated from tissue excised from arthritic joints of RA patients were cultured in the presence of increasing concentrations of apremilast for 48 hours and spontaneous tumour necrosis factor-alpha (TNFalpha) production was analysed in culture supernatants by ELISA. In addition, arthritis was induced in BALB/c and DBA/1 mice by passive transfer of anti-type II collagen mAb and immunisation with type II collagen, respectively. Mice with established arthritis received 5 or 25 mg/kg apremilast and disease severity was monitored relative to mice receiving vehicle alone. At the end of the study, paws were removed and processed for histopathological assessment. Behavioural effects of apremilast, relative to rolipram, were assessed in naïve DBA/1 mice using an automated activity monitor (LABORAS). RESULTS: Apremilast dose dependently inhibited spontaneous release of TNFalpha from human rheumatoid synovial membrane cultures. Furthermore, apremilast significantly reduced clinical score in both murine models of arthritis over a ten day treatment period and maintained a healthy joint architecture in a dose-dependent manner. Importantly, unlike rolipram, apremilast demonstrated no adverse behavioural effects in naïve mice. CONCLUSIONS: Apremilast is an orally available PDE4 inhibitor that reduces TNFalpha production from human synovial cells and significantly suppresses experimental arthritis. Apremilast appears to be a potential new agent for the treatment of rheumatoid arthritis.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/metabolismo , Inibidores da Fosfodiesterase 4 , Membrana Sinovial/metabolismo , Talidomida/análogos & derivados , Fator de Necrose Tumoral alfa/metabolismo , Animais , Artrite Reumatoide/patologia , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Inibidores de Fosfodiesterase/efeitos adversos , Inibidores de Fosfodiesterase/uso terapêutico , Rolipram/efeitos adversos , Rolipram/uso terapêutico , Índice de Gravidade de Doença , Membrana Sinovial/efeitos dos fármacos , Talidomida/farmacologia , Talidomida/uso terapêuticoRESUMO
OBJECTIVE: Single-immunoglobulin interleukin-1 receptor-related (SIGIRR), which is also known as Toll/interleukin-1 receptor 8 (TIR-8), is a member of the TIR domain-containing family of receptors and was first characterized as an inhibitor of interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) signaling. In the Dextran sulfate sodium-induced colitis model, SIGIRR(-/-) mice were shown to have increased inflammation and to be more susceptible to endotoxin challenge. Increasing evidence implicates TLR and IL-1R signaling in the pathology of rheumatoid arthritis (RA). Therefore, the purpose of this study was to investigate the involvement of SIGIRR in regulating inflammation in disease-relevant models. METHODS: Primary human monocyte-derived macrophages and dendritic cells (DCs) were used to overexpress SIGIRR as well as to knock down endogenously expressed SIGIRR using small interfering RNAs. SIGIRR was also overexpressed in synovial cells derived from RA patients. To investigate the role of SIGIRR in vivo, zymosan-induced arthritis (ZIA) and collagen antibody-induced arthritis (CAIA) were induced in SIGIRR-knockout mice. RESULTS: SIGIRR overexpression inhibited TLR-induced cytokine production in macrophages and DCs, while SIGIRR knockdown resulted in increased cytokine production following TLR stimulation. Moreover, SIGIRR overexpression inhibited the spontaneous release of cytokines by human RA synovial cells. The role of SIGIRR as an inhibitor of inflammation was confirmed in vivo, since SIGIRR(-/-) mice developed a more severe disease in both the ZIA and CAIA models. CONCLUSION: Our study is the first to show the expression pattern and function of SIGIRR in primary human cells. Furthermore, this investigation defines the role of SIGIRR in disease-relevant cell types and demonstrates that SIGIRR is a potential therapeutic target for RA.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Inflamação/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores Toll-Like/metabolismo , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Western Blotting , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Técnicas de Transferência de Genes , Humanos , Inflamação/genética , Inflamação/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Interferência de RNA , RNA Interferente Pequeno , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
OBJECTIVE: Selective serotonin reuptake inhibitors (SSRIs), in addition to their antidepressant effects, have been reported to have antiinflammatory effects. The aim of this study was to assess the antiarthritic potential of 2 SSRIs, fluoxetine and citalopram, in murine collagen-induced arthritis (CIA) and in a human ex vivo disease model of rheumatoid arthritis (RA). METHODS: Following therapeutic administration of SSRIs, paw swelling was assessed and clinical scores were determined daily in DBA/1 mice with CIA. Joint architecture was examined histologically at the end of the treatment period. Cultures of human RA synovial membranes were treated with SSRIs, and cytokine production was measured. Toll-like receptor (TLR) function was examined in murine and human macrophages, human B cells, and human fibroblast-like synovial cells treated with SSRIs. RESULTS: Both SSRIs significantly inhibited disease progression in mice with CIA, with fluoxetine showing the greatest degree of efficacy at the clinical and histologic levels. In addition, both drugs significantly inhibited the spontaneous production of tumor necrosis factor, interleukin-6, and interferon-gamma-inducible protein 10 in human RA synovial membrane cultures. Fluoxetine and citalopram treatment also inhibited the signaling of TLRs 3, 7, 8, and 9, providing a potential mechanism for their antiinflammatory action. CONCLUSION: Fluoxetine and citalopram treatment selectively inhibit endosomal TLR signaling, ameliorate disease in CIA, and suppress inflammatory cytokine production in human RA tissue. These data highlight the antiarthritic potential of the SSRI drug family and provide further evidence of the involvement of TLRs in the pathogenesis of RA. The SSRIs may provide a template for potential antiarthritic drug development.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/metabolismo , Artrite Reumatoide/fisiopatologia , Citalopram/farmacologia , Fluoxetina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Receptores Toll-Like/antagonistas & inibidores , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Artrite Reumatoide/tratamento farmacológico , Células Cultivadas , Citalopram/uso terapêutico , Citocinas/biossíntese , Modelos Animais de Doenças , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Fluoxetina/uso terapêutico , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Membrana Sinovial/metabolismo , Receptores Toll-Like/fisiologiaRESUMO
OBJECTIVE: IL-23 is a pro-inflammatory cytokine proposed to be central to the development of autoimmune disease. We investigated whether IL-23, together with the downstream mediator IL-17A, was present and functional in RA in humans. METHODS: RA synovial cells were cultured in the presence or absence of antibodies directed against IL-23p19 or -23R and -17. IL-23, -12, -17, and their receptors, and IL-6, -1beta and TNF-alpha were measured by ELISA and/or PCR. RESULTS: Small amounts of cell-associated IL-23 (median 110 pg/ml) were detected in RA synovial cultures, and found to be functional as IL-23R blockade resulting in a significant inhibition of TNF-alpha (57%), IL-1beta (51%) and IL-6 (30%). However, there was a considerable variability between individual patient samples, and anti-IL-23p19 was found to be considerably less effective. IL-17A protein was detected in approximately 40% of the supernatants and IL-17A blockade, in IL-17A-producing cultures, resulted in a small but significant inhibition of TNF-alpha (38%), IL-1beta (23%) and IL-6 (22%). Addition of recombinant IL-23 to cultures had a variable effect on the spontaneous production of endogenous IL-17A with enhancement observed in some but not all cultures, suggesting that either the low levels of endogenous IL-23 are sufficient to support cytokine production and/or that the relevant Th17 cells were not present. CONCLUSIONS: These results suggest that although IL-23 may have pathogenic activity in a proportion of patients with late-stage RA, it is not abundantly produced in this inflammatory tissue, nor does it have a dominant role in all patient tissues analysed.
Assuntos
Artrite Reumatoide/imunologia , Interleucina-17/imunologia , Interleucina-23/imunologia , Adulto , Idoso , Bioensaio/métodos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-17/biossíntese , Interleucina-17/genética , Interleucina-23/biossíntese , Interleucina-23/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Receptores de Interleucina/biossíntese , Receptores de Interleucina/genética , Proteínas Recombinantes/imunologia , Membrana Sinovial/imunologiaRESUMO
OBJECTIVES: To investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) in the synovium of human RA patients as well as the level of soluble TREM-1 in the plasma of RA patients. METHODS: Twenty-four RA synovial samples were analysed by gene expression oligonucleotide microarrays. Expression levels of TREM-1 mRNA in murine CIA paws were determined by quantitative PCR (qPCR). TREM-1 protein expression was detected by immunohistochemistry in five RA synovial samples and two OA synovial samples. TREM-1-positive cells from five RA synovial tissues were analysed by FACS staining to determine the cell type. Activation of TREM-1 was tested in five RA synovial samples. Soluble TREM-1 was measured in serum from 32 RA patients. RESULTS: The expression of TREM-1 mRNA was found to increase 6.5-fold in RA synovial samples, whereas it was increased 132-fold in CIA paws. Increased numbers of TREM-1-positive cells were seen in RA synovium sections and these cells co-expressed CD14. Using a TREM-1-activating cross-linking antibody in RA synovial cultures, multiple pro-inflammatory cytokines were induced. The average amount of soluble TREM-1 in plasma from RA patients was found to be higher than that in plasma from healthy volunteers. CONCLUSIONS: These findings suggest that the presence of high levels of functionally active TREM-1 in RA synovium may contribute to the development or maintenance of RA, or both. Inhibiting TREM-1 activity may, therefore, have a therapeutic effect on RA. High levels of soluble TREM-1 in the plasma of RA patients compared with healthy volunteers may indicate disease activity.