RESUMO
OBJECTIVE: This study aimed to understand the role of mammalian target of rapamycin (mTOR) in CD8+ cells in the pathogenicity of RA and the changes after treatment with biologic drugs. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 17 healthy controls and 86 patients with RA. Phosphorylation of mTOR (p-mTOR) and its clinical relevance were evaluated. The role of mTOR in CD8+ cells was also examined in vitro. RESULTS: Patients with RA who had a moderate or high disease activity, were biologic-naïve, and were refractory to MTX were enrolled in this study. The p-mTOR levels in CD8+ cells were higher in patients with RA than in healthy controls, and they positively correlated with the disease activity in such patients. However, after one year of treatment with TNF inhibitors, the p-mTOR levels in CD8+ cells were suppressed and showed a positive correlation with the treatment response, which was not observed in the abatacept-treatment group. In vitro stimulation of CD8+ cells with anti-CD3 and anti-CD28 antibodies induced mTOR phosphorylation and increased the production of granzyme B, granulysin, TNF-α and IFN-γ but decreased the production of granzyme K. However, on treatment with TNF inhibitors, p-mTOR levels in CD8+ cells and granzyme B production decreased, while granzyme K production increased. The production of granulysin and IFN-γ was not affected by the TNF inhibitors. CONCLUSION: These results suggested that mTOR activation in CD8+ cells may be a novel evaluation marker for RA disease activity and a predictive marker of therapeutic response to TNF inhibitors.
Assuntos
Artrite Reumatoide , Inibidores do Fator de Necrose Tumoral , Artrite Reumatoide/tratamento farmacológico , Linfócitos T CD8-Positivos , Granzimas , Humanos , Leucócitos Mononucleares , Serina-Treonina Quinases TORRESUMO
OBJECTIVES: B cells play an important pathological role in RA. In this study, we investigated the role of metabolic regulator mTOR in B cells and its relevance to the pathology of RA. METHODS: Peripheral blood mononuclear cells were isolated from 31 normal subjects and 86 RA patients and the gated B cells were assessed for mTOR phosphorylation and chemokine receptor expression. In vitro studies on peripheral blood B cells isolated from the control and RA patients investigated the molecular mechanisms. RESULTS: Higher concentrations of CXCL10 (CXCR3 ligands) and lower percentages of CXCR3+ memory B cells were present in the peripheral blood of RA patients relative to the control. RA patients with high CXCL10 concentrations had smaller percentage of CXCR3+ memory B cells and high disease activity. One-year treatment with TNF inhibitors increased the percentage of CXCR3+ memory B cells and reduced serum CXCL10 concentrations. mTOR phosphorylation in B cells was further enhanced in RA patients, compared with the control, and was selectively enhanced in CXCR3+ memory B cells. mTOR phosphorylation in CXCR3+ memory B cells correlated with disease activity. In vitro, mTOR phosphorylation in B cells enhanced IL-6 production and increased RANKL expression. CONCLUSION: mTOR activation in CXCR3+ memory B cells of RA patients is associated with disease activity, mediated through IL-6 production and RANKL expression. The obtained results also suggest that TNF inhibitors mediate an impact on the association between CXCL10 and mTOR activated CXCR3+ memory B cells.
Assuntos
Artrite Reumatoide/imunologia , Linfócitos B/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Linfócitos B/efeitos dos fármacos , Estudos de Casos e Controles , Quimiocina CXCL10/sangue , Humanos , Interleucina-6/metabolismo , Ligante RANK/metabolismo , Receptores CXCR3/metabolismo , Índice de Gravidade de Doença , Inibidores do Fator de Necrose Tumoral/farmacologia , Inibidores do Fator de Necrose Tumoral/uso terapêuticoRESUMO
Mesenchymal stem cells (MSC) can differentiate into chondrocytes. Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed during chondrogenic differentiation and can be produced by MSC. EBI3 is also a subunit of interleukin (IL)-27 and IL-35, and it accumulates in the endoplasmic reticulum (ER) when its partners, such as IL-27 p28 and IL-35 p35, are insufficient. ER stress induced by protein accumulation is responsible for chondrogenic differentiation. However, the role of EBI3 and its relevance to the ER stress in chondrogenic differentiation of MSC have never been addressed. Here, we demonstrate that EBI3 protein is expressed in the early stage of chondrogenic differentiation of MSC. Additionally, knockdown, overexpression, or induction of EBI3 through IL-1ß inhibits chondrogenesis. We show that EBI3 localizes and accumulates in the ER of MSC after overexpression or induction by IL-1ß and TNF-α, whereas ER stress inhibitor 4-phenylbutyric acid decreases its accumulation in MSC. Moreover, EBI3 modulates ER stress sensor inositol-requiring enzyme 1 α (IRE1α) after induced by IL-1ß, and MSC-like cells coexpress EBI3 and IRE1α in rheumatoid arthritis (RA) synovial tissue. Altogether, these data demonstrate that intracellular EBI3 commits to chondrogenic differentiation by regulating ER stress sensor IRE1α.
Assuntos
Diferenciação Celular , Condrócitos , Condrogênese , Estresse do Retículo Endoplasmático , Interleucinas , Células-Tronco Mesenquimais , Antígenos de Histocompatibilidade Menor , Humanos , Condrócitos/citologia , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Interleucinas/genética , Interleucinas/fisiologia , Células-Tronco Mesenquimais/citologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/fisiologia , Diferenciação Celular/genética , Condrogênese/genéticaRESUMO
BACKGROUND: Highly regulated gene expression program underlies osteogenesis of mesenchymal stem cells (MSCs), but the regulators in the program are not entirely identified. As enhancer RNAs (eRNAs) have recently emerged as a key regulator in gene expression, we assume a commitment of an eRNA in osteogenesis. METHODS: We performed in silico analysis to identify potential osteogenic microRNA (miRNA) gene predicted to be regulated by super-enhancers (SEs). SE inhibitor treatment and eRNA knocking-down were used to confirm the regulational mechanism of eRNA. miRNA function in osteogenesis was elucidated by miR mimic and inhibitor transfection experiments. RESULTS: miR-3129 was found to be located adjacent in a SE (osteoblast-specific SE_46171) specifically activated in osteoblasts by in silico analysis. A RT-quantitative PCR analysis of human bone marrow-derived MSC (hBMSC) cells showed that eRNA_2S was transcribed from the SE with the expression of miR-3129. Knockdown of eRNA_2S by locked nucleic acid as well as treatment of SE inhibitors JQ1 or THZ1 resulted in low miR-3129 levels. Overexpression of miR-3129 promoted hBMSC osteogenesis, while knockdown of miR-3129 inhibited hBMSC osteogenesis. Solute carrier family 7 member 11 (SLC7A11), encoding a bone formation suppressor, was upregulated following miR-3129-5p inhibition and identified as a target gene for miR-3129 during differentiation of hBMSCs into osteoblasts. CONCLUSIONS: miR-3129 expression is regulated by SEs via eRNA_2S and this miRNA promotes hBMSC differentiation into osteoblasts through downregulating the target gene SLC7A11. Thus, the present study uncovers a commitment of an eRNA via a miR-3129/SLC7A11 regulatory pathway during osteogenesis of hBMSCs.
RESUMO
Recent reports have shown the importance of IFN-γ and T-bet+ B cells in the pathology of SLE, suggesting the involvement of IFN-γ-producing T-bet+ CD4+ cells, i.e., Th1 cells. This study determined the changes in Th1 subsets with metabolic shift and their potential as therapeutic targets in SLE. Compared with healthy donors, patients with SLE had higher numbers of T-bethiCXCR3lo effector cells and T-bet+Foxp3lo non-suppressive cells, which excessively produce IFN-γ, and lower number of non-IFN-γ-producing T-bet+Foxp3hi activated-Treg cells. These changes were considered to be involved in treatment resistance. The differentiation mechanism of Th1 subsets was investigated in vitro using memory CD4+ cells obtained from healthy donors and patients with SLE. In memory CD4+ cells of healthy donors, both rapamycin and 2-deoxy-D-glucose (2DG) suppressed T-bet+Foxp3- cells, and induced T-bet+Foxp3+(lo/hi) cells. Rapamycin induced IFN-γ-producing T-bet+Foxp3lo cells accompanied with enhanced lipid metabolism, whereas 2DG induced IFN-γ-non-producing T-bet+Foxp3hi cells. In memory CD4+ cells of SLE patients, inhibition of fatty acid synthesis, but not ß-oxidation, suppressed IFN-γ production, and up-regulated of Foxp3 expression in T-bet+Foxp3+ cells. Metabolic regulators such as fatty acid synthesis inhibitors may improve the pathological status by correcting Th1 subset imbalance and overproduction of IFN-γ in SLE.
Assuntos
Ácidos Graxos/biossíntese , Interferon gama/biossíntese , Contagem de Linfócitos , Subpopulações de Linfócitos T/metabolismo , Células Th1/metabolismo , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Memória Imunológica , Imunofenotipagem , Leucócitos Mononucleares , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Receptores CXCR3/metabolismo , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologiaRESUMO
BACKGROUND: 14-3-3η is an intracellular protein also detected in the serum and synovial fluid of patients with rheumatoid arthritis (RA). It is closely related to disease activity and anti-cyclic citrullinated peptide antibody levels. However, the main source of 14-3-3η and the mechanism of its release into the extracellular space remain unclear. Addressing these two points was the main goal of the current study. METHODS: The source of 14-3-3η was investigated by immunostaining RA synovial tissue. Fibroblast-like synoviocytes, CD4+ cells, and macrophages were selected as candidates among the various cell types in the synovial tissue. Phosphorylation of mixed-lineage kinase domain-like pseudokinase (MLKL) and cell death of macrophages were studied by phalloidin staining and electron microscopy after stimulation with an oxidative stress inducer (diamide) or tumour necrosis factor (TNF)-α. Extracellular 14-3-3η protein levels were examined by western blotting. RESULTS: Macrophages from the synovial tissue from RA, but not osteoarthritis, showed dense and widespread cytoplasmic staining for the 14-3-3η protein, co-localized with peptidylarginine deiminase 4. Swelling and membrane rupture of macrophages were induced by treatment with TNF-α, but not interleukin (IL) 6/soluble IL-6 receptor (sIL-6R). Increased MLKL phosphorylation followed by necroptosis was also induced in TNF-α-stimulated macrophages. Necrostatin-1, a necroptosis inhibitor, antagonized MLKL phosphorylation. High levels of 14-3-3η were detected in the culture supernatants of macrophages stimulated with diamide and TNF-α, but not IL-6/sIL-6R. CONCLUSIONS: Macrophages that highly express 14-3-3η undergo TNF-α-induced necroptosis with damage to the cellular structure, resulting in the secretion of 14-3-3η into the extracellular space. The current study provides a novel mechanism for 14-3-3η level increase in the RA synovial fluid.
Assuntos
Proteínas 14-3-3/metabolismo , Artrite Reumatoide , Macrófagos/metabolismo , Necroptose/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas 14-3-3/imunologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
T helper (Th) cells can differentiate into functionally distinct subsets and play a pivotal role in inflammatory and autoimmune diseases such as rheumatoid arthritis (RA). Th22 cells have been identified as a new subset secreting interleukin (IL)-22. Although elevated levels of IL-22 in the synovial fluids of RA patients were reported, its pathological roles remain unclear. Here, we demonstrated that IL-22 was characteristically produced from CD3+CD4+CC-chemokine receptor (CCR)4+CCR6+CCR10+ cells and their ability of the production of IL-22 markedly exceeded that of other Th subsets and the subset, thereby, designated Th22 cells. Th22 cells were efficiently induced by the stimulation with tumor necrosis factor-α, IL-6, and IL-1ß. Th22 cells were markedly infiltrated in synovial tissue in patients with active RA, but not in patients with osteoarthritis (OA). CCL17, CCL20, and CCL28, which are chemokine ligands of CCR4, CCR6, and CCR10, respectively, were abundantly expressed in RA synovial tissue compared to OA. By in vitro Trans-well migration assay, Th22 cells efficiently migrated toward CCL28. Co-culture of Th22 cells, which were sorted from peripheral blood, with monocytes in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor (NF)-κB ligand induced osteoclasts formation more efficiently than that of either Th1 cells or Th17 cells. Furthermore, IL-22 markedly augmented osteoclast differentiation by promoting nuclear factor of activated T cells c1 expression in CD14+ monocytes. Contrarily, the addition of IFN-γ to the culture significantly decreased osteoclasts number, whereas IL-17 had marginal effects. IL-22 neutralizing antibody inhibited osteoclast formation in the co-culture of Th22 cells with CD14+ monocytes. Collectively, the results indicated that Th22 cells, which co-express chemokine receptors CCR4, CCR6, and CCR10, possess strong potency of tissue migration and accumulate into inflamed synovial tissues where the ligands such as CCL28 are highly expressed. Thus, Th22 cells have the capacity to promote osteoclast differentiation through production of IL-22 and thus play a pivotal role in bone destruction in patients with RA.
Assuntos
Artrite Reumatoide/imunologia , Interleucinas/metabolismo , Osteoclastos/fisiologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/complicações , Artrite Reumatoide/patologia , Artrite Reumatoide/cirurgia , Artroplastia do Joelho , Diferenciação Celular/imunologia , Células Cultivadas , Quimiocinas CC/imunologia , Quimiocinas CC/metabolismo , Técnicas de Cocultura , Humanos , Interleucinas/imunologia , Masculino , Pessoa de Meia-Idade , Osteoartrite/imunologia , Osteoartrite/patologia , Osteoartrite/cirurgia , Cultura Primária de Células , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Interleucina 22RESUMO
BACKGROUND: ß2-glycoprotein I (ß2GPI)-dependent antiphospholipid antibodies (aPLs) are considered to play a pivotal pathogenic role in antiphospholipid syndrome (APS) by inducing the expression of tissue factor, inflammatory cytokines, and chemokines, most of which are dependent upon the NF-κB pathway. Therefore, the NF-κB is regarded as a promising target for the development of a novel therapeutic strategy. However, progress has been limited owing to the fact that there are no widely-used in vivo models, or highly specific inhibitors. OBJECTIVE: This study aimed to test the effects of an NF-κB-specific inhibitor, DHMEQ, in preventing thrombus formation using an original mouse model of APS. MATERIALS AND METHODS: Specificity of a monoclonal aPL WB-6 was examined by ELISA. WB-6 was injected into normal BALB/c mice with or without DHMEQ treatment. A pulse laser was radiated to a cutaneous vein in the window of a dorsal skinfold chamber attached to the mouse and thrombus formation was observed and recorded under a microscope. RESULTS: WB-6 bound preferentially to the caldiolipin (CL)-ß2GPI complex rather than to CL alone, or ß2GPI alone. WB-6, but not isotype-matched control antibody, induced a prothrombotic state in the mice by inducing tissue factor expression upon circulating monocytes, resulting in thrombus formation at the site of laser-induced endothelial injury. This diathesis was almost completely ameliorated by DHMEQ treatment. CONCLUSIONS: Inhibition of the NF-κB pathway is a promising strategy for the development of a novel treatment for APS.