RESUMEN
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.
Asunto(s)
Artritis Reumatoide/inmunología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Anciano , Artritis Reumatoide/genética , Metilación de ADN/genética , Metilación de ADN/inmunología , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
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.
Asunto(s)
Artritis Reumatoide/inmunología , Factores de Transcripción Forkhead/inmunología , Cápsula Articular/inmunología , Linfocitos T Reguladores/inmunología , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Humanos , Cápsula Articular/citología , Cápsula Articular/metabolismo , Lentivirus , Luciferasas , FN-kappa B/inmunología , FN-kappa B/metabolismo , Transducción Genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Subfamilia K de Receptores Similares a Lectina de Células NK/antagonistas & inhibidores , Membrana Sinovial/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Artritis Experimental/inmunología , Artritis Experimental/patología , Células Cultivadas , Técnicas de Cocultivo , Articulaciones/inmunología , Articulaciones/patología , Células Asesinas Naturales/inmunología , Masculino , Ratones , Ratones Endogámicos DBA , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Membrana Sinovial/patologíaRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/enzimología , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Citocinas/biosíntesis , Activación Enzimática , Humanos , Antígenos Comunes de Leucocito/inmunología , Transducción de Señal/inmunologíaRESUMEN
Adaptive immune responses are initiated by resident myeloid tissue DC. A major fraction of tissue DC express CD1c+ and is thought to be derived from blood CD1c DC, an idea supported here by the observation that they express tissue-homing molecules and rapidly differentiate into cells with a tissue DC phenotype. Responses are thought to be augmented/modulated further by inflammatory moDC. Although much accepted human myeloid DC cell biology is based on moDC studies, we find these 2 DC populations to be functionally distinct. Stimulated moDC produce high levels of IL-10 and the Th1-promoting cytokine IL-12. Under identical conditions, CD1c DC synthesized no IL-10 and no or low levels of IL-12. Despite this, CD1c DC stimulated a strong Th1 response, demonstrated by IL-12 neutralization to be IL-12 independent, whereas the response induced by moDC was IL-12 dependent. This finding was supported by studies on a patient with a highly reduced ability to synthesize IL-12, whose CD1c DC induced a good Th1 response contrasting with the failure of his moDC, which were impaired in IL-12 production, to induce IFN-γ-secreting T cells. The IL-10 and IL-12 data were confirmed by microarray analysis, which also showed that stimulated moDC produced inflammatory-associated chemokines and cytokines, whereas stimulated CD1c DC showed minimal up-regulation of these genes. Thus, moDC, widely used as a human myeloid DC model, do not faithfully reflect the properties of CD1c tissue DC, making the initial response to a pathogen or vaccine.
RESUMEN
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.
Asunto(s)
Antirreumáticos/farmacología , Artritis Reumatoide/inmunología , Proliferación Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Elementos de Facilitación Genéticos/efectos de los fármacos , Factores de Transcripción Forkhead/efectos de los fármacos , Metotrexato/farmacología , ARN Mensajero/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Adulto , Anciano , Artritis Reumatoide/tratamiento farmacológico , Antígeno CTLA-4/efectos de los fármacos , Antígeno CTLA-4/metabolismo , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Factores de Transcripción Forkhead/genética , Humanos , Interferón gamma/efectos de los fármacos , Interferón gamma/metabolismo , Leucocitos Mononucleares , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Linfocitos T Reguladores/metabolismoRESUMEN
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.
Asunto(s)
Artritis Reumatoide/inmunología , Antígeno CTLA-4/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal/fisiología , Linfocitos T Reguladores/metabolismo , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Antígeno CTLA-4/genética , Metilación de ADN , Regulación hacia Abajo , Humanos , Linfocitos T Reguladores/inmunologíaRESUMEN
IL-10 is a potent anti-inflammatory molecule, which regulates TNF-alpha at multiple levels. We investigated whether IL-10 also modulated the activity of the TNF-alpha-converting enzyme (TACE). Using an ex vivo fluorogenic assay we observed that LPS rapidly induced TACE activity in monocytes coinciding with release of soluble TNF-alpha. In the presence of IL-10, TNF-alpha production and activation of surface TACE was significantly inhibited. Paradoxically, both LPS with or without IL-10 led to accumulation of surface TACE (albeit catalytically inactive) over a 24 h period. We investigated whether this was mediated through induction of endogenous tissue inhibitor metalloproteinase-3 (TIMP-3). We found that the inhibition of TACE activity at 2 h by IL-10 was not TIMP-3 dependent but that the late accumulation of surface TACE was prevented with TIMP-3 antibodies. Furthermore, induction of endogenous TIMP-3 was observed by western blotting in both LPS- and in LPS with IL-10-treated monocytes from 6 to 8 h of culture. These results indicate that IL-10 further regulates TNF-alpha by modulating TACE activation at early time points and by contributing to the induction of TIMP-3, the natural inhibitor of active TACE, at later time points. These observations add to our understanding of inflammation and the importance of homeostatic regulators of these events.
Asunto(s)
Proteínas ADAM/metabolismo , Interleucina-10/farmacología , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Proteína ADAM17 , Anticuerpos/inmunología , Catálisis , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Células Cultivadas , Humanos , Lipopolisacáridos/farmacología , Microscopía Confocal , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Solubilidad , Inhibidor Tisular de Metaloproteinasa-3/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia ArribaRESUMEN
BACKGROUND: Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue. METHODS: Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4+CD45RO+, CCR7-, CD49dhigh population, and that these cells are derived from the effector memory CD4+ T cells in resting blood. RESULTS: After stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-alpha production from monocytes stimulated with CD4+CD45RO+ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-alpha production in RA synovial mononuclear cell cultures. CONCLUSION: Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.
Asunto(s)
Artritis Reumatoide/inmunología , Linfocitos T CD4-Positivos/citología , Linaje de la Célula/inmunología , Membrana Sinovial/inmunología , Subgrupos de Linfocitos T/citología , Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Citometría de Flujo , Humanos , Memoria Inmunológica , Activación de Linfocitos/inmunología , Monocitos/inmunología , Monocitos/metabolismo , Fenotipo , Subgrupos de Linfocitos T/inmunologíaRESUMEN
The widespread distribution of Toll-like receptors (TLRs) and their ligands raises the question whether they contribute to the production of inflammatory and tissue destructive molecules in rheumatoid arthritis (RA). We examined the expression and function of TLR2 and TLR4 and their downstream signaling adaptors MyD88 and Mal/TIRAP in synovial membrane cultures from RA tissue. Both TLR2 and TLR4 were detected by flow cytometry, and stimulation with TLR2 and TLR4 ligands augmented the spontaneous production of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8, indicating that TLR2 and TLR4 are functional in these cultures. In addition, overexpression of dominant-negative forms of MyD88 and Mal/TIRAP significantly down-regulated the spontaneous production of cytokines tumor necrosis factor-alpha, IL-6, and vascular endothelial growth factor, and enzymes MMP-1, MMP-2, MMP-3, and MMP-13 in RA synovial membrane cell cultures. Because TLR2 and TLR4 require both MyD88 and Mal/TIRAP for signaling, this study suggests that TLR function may regulate the expression of these factors in the RA synovium. Conditioned media from synovial membrane cell cultures stimulated human macrophages in a MyD88- and Mal-dependent manner, suggesting the release of a TLR ligand(s) from these cells. Thus, TLRs not only protect against infection but may also promote the inflammatory and destructive process in RA.
Asunto(s)
Artritis Reumatoide/metabolismo , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores de Interleucina-1/metabolismo , Membrana Sinovial/metabolismo , Artritis Reumatoide/patología , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Macrófagos/patología , Metaloproteinasas de la Matriz/metabolismo , Transducción de Señal , Membrana Sinovial/patología , Técnicas de Cultivo de Tejidos , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
The T-cell receptor zeta (TCRzeta) chain is a master sensor and regulator of lymphocyte responses. Loss of TCRzeta expression has been documented in infectious, inflammatory, and malignant diseases, suggesting that it may serve to limit T-cell reactivity and effector responses at sites of tissue damage. These observations prompted us to explore the relationship between TCRzeta expression and effector function in T cells. We report here that TCRzeta(dim) lymphocytes are enriched for antigen-experienced cells refractory to TCR-induced proliferation. Compared to their TCRzeta(bright) counterparts, TCRzeta(dim) cells share characteristics of differentiated effector T cells but use accessory pathways for transducing signals for inflammatory cytokine gene expression and cell contact-dependent pathways to activate monocytes. TCRzeta(dim) T cells accumulate in inflamed tissues in vivo and have intrinsic migratory activity in vitro. Whilst blocking leukocyte trafficking with anti-TNF therapy in vivo is associated with the accumulation of TCRzeta(dim) T cells in peripheral blood, this T-cell subset retains the capacity to migrate in vitro. Taken together, the functional properties of TCRzeta(dim) T cells make them promising cellular targets for the treatment of chronic inflammatory disease.
Asunto(s)
Movimiento Celular/inmunología , Inflamación/inmunología , Subgrupos Linfocitarios/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Linfocitos T/fisiología , Adulto , Animales , Artritis Psoriásica/sangre , Artritis Psoriásica/inmunología , Artritis Reactiva/sangre , Artritis Reactiva/inmunología , Artritis Reumatoide/sangre , Artritis Reumatoide/inmunología , Células CHO , Cricetinae , Cricetulus , Citometría de Flujo , Fluorescencia , Humanos , Persona de Mediana Edad , TransfecciónRESUMEN
Current immunological opinion holds that myeloid dendritic cell (mDC) precursors migrate from the blood to the tissues, where they differentiate into immature dermal- and Langerhans-type dendritic cells (DC). Tissue DC require appropriate signals from pathogens or inflammatory cytokines to mature and migrate to secondary lymphoid tissue. We show that purified blood mDC cultured in vitro with GM-CSF and IL-4, but in the absence of added exogenous maturation stimuli, rapidly differentiate into two maturational and phenotypically distinct populations. The major population resembles immature dermal DC, being positive for CD11b, CD1a, and DC-specific ICAM-3-grabbing nonintegrin. They express moderate levels of MHC class II and low levels of costimulatory molecules. The second population is CD11b(-/low) and lacks CD1a and DC-specific ICAM-3-grabbing nonintegrin but expresses high levels of MHC class II and costimulatory molecules. Expression of CCR7 on the CD11b(-/low) population and absence on the CD11b(+) cells further supports the view that these cells are mature and immature, respectively. Differentiation into mature and immature populations was not blocked by polymyxin B, an inhibitor of LPS. Neither population labeled for Langerin, E-cadherin, or CCR6 molecules expressed by Langerhans cells. Stimulation of 48-h cultured DC with LPS, CD40L, or poly(I:C) caused little increase in MHC or costimulatory molecule expression in the CD11b(-/low) DC but caused up-regulated expression in the CD11b(+) cells. In HIV-infected individuals, there was a marked decrease in the viability of cultured blood mDC, a failure to differentiate into the two populations described for normal donors, and an impaired ability to stimulate T cell proliferation.