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AIM: We aimed to evaluate the preventive role of the tyrosine kinase inhibitor dasatinib in an animal model of rheumatoid arthritis (RA). METHODS: DBA/1J mice were injected with bovine type II collagen to induce arthritis (collagen-induced arthritis [CIA]). There were four experimental groups of mice, namely negative control (non-CIA), vehicle-treated CIA, dasatinib-pretreated CIA, and dasatinib-treated CIA. After collagen immunization, arthritis progression in the mice was clinically scored twice weekly for 5 weeks. Flow cytometry was used to evaluate in vitro CD4+ T-cell differentiation and ex vivo mast cell/CD4+ T-cell differentiation. Osteoclast formation was evaluated using tartrate-resistant acid phosphatase (TRAP) staining and by estimating the resorption pit area. RESULTS: We found that the clinical arthritis histological scores were lower in the dasatinib pretreatment group than in the vehicle and dasatinib post-treatment groups. Flow cytometry showed that FcεR1+ cells were downregulated and regulatory T cells were upregulated in splenocytes of the dasatinib pretreatment group compared with those in the vehicle group. Additionally, there was a decline in IL-17+ CD4+ T-cell differentiation and an increase in CD4+ CD24high Foxp3+ T-cell differentiation with in vitro dasatinib treatment of human CD4+ T cells. The number of TRAP+ osteoclasts and the area of the resorption were decreased in the bone marrow cells derived from dasatinib-pretreated mice compared with those derived from vehicle group. CONCLUSION: Dasatinib protected against arthritis in an animal model of RA by regulating the differentiation of regulatory T cells and IL-17+ CD4+ T cells and inhibiting osteoclastogenesis, indicating the therapeutic potential of dasatinib in the treatment of early RA.
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Artritis Experimental , Artritis Reumatoide , Humanos , Animales , Bovinos , Ratones , Interleucina-17/uso terapéutico , Dasatinib/farmacología , Dasatinib/uso terapéutico , Ratones Endogámicos DBA , Artritis Reumatoide/inducido químicamente , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/prevención & control , Artritis Experimental/inducido químicamente , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
BACKGROUND: Endothelial cell (EC) dysfunction is a frequent feature in patients with end-stage renal disease (ESRD). The aim of this study was to generate human induced pluripotent stem cells, differentiate ECs (hiPSC-ECs) from patients with ESRD, and appraise the usefulness of hiPSC-ECs as a model to investigate EC dysfunction. METHODS: We generated hiPSCs using peripheral blood mononuclear cells (PBMCs) isolated from three patients with ESRD and three healthy controls (HCs). Next, we differentiated hiPSC-ECs using the generated hiPSCs and assessed the expression of endothelial markers by immunofluorescence. The differentiation efficacy, EC dysfunction, and molecular signatures of EC-related genes based on microarray analysis were compared between the ESRD and HC groups. RESULTS: In both groups, hiPSCs and hiPSC-ECs were successfully obtained based on induced pluripotent stem cell or EC marker expression in immunofluorescence and flow cytometry. However, the efficiency of differentiation of ECs from hiPSCs was lower in the ESRD-hiPSCs than in the HC-hiPSCs. In addition, unlike HC-hiPSC-ECs, ESRD-hiPSC-ECs failed to form interconnecting branching point networks in an in vitro tube formation assay. During microarray analysis, transcripts associated with oxidative stress and inflammation were upregulated and transcripts associated with vascular development and basement membrane extracellular matrix components were downregulated in ESRD-hiPSC-ECs relative to in HC-hiPSC-ECs. CONCLUSION: ESRD-hiPSC-ECs showed a greater level of EC dysfunction than HC-hiPSC-ECs did based on functional assay results and molecular profiles. hiPSC-ECs may be used as a disease model to investigate the pathophysiology of EC dysfunction in ESRD.
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BACKGROUND: Patients with rheumatoid arthritis (RA) have increased levels of interleukin-18 (IL-18) and decreased levels of IL-18 binding protein (IL-18BP) in the serum and synovial fluid (SF) compared to those in patients with osteoarthritis (OA) or in healthy controls. In this study, we evaluated the effects of IL-18BP on osteoclastogenesis and T cell differentiation in RA in vitro. METHODS: Serum and SF of patients with RA and OA were collected to compare IL-18 and IL-18BP levels by the enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells (PBMCs) and SF mononuclear cells (SFMCs) of RA patients were cultured under type 17 helper T cell (Th17) polarisation conditions with or without IL-18BP. In addition, PBMCs were cultured in the presence of receptor activator of nuclear factor kappa-Β ligand (RANKL) or IL-17A with or without IL-18BP, and tartrate-resistant acid phosphatase (TRAP) staining and real-time quantitative polymerase chain reaction for expression levels of osteoclast-related genes were performed. RESULTS: IL-18 levels were higher in the serum and SF of patients with RA, whereas IL-18BP was lower in the SF of patients with RA than in the control group. Treatment of patients' PBMCs with IL-18BP decreased the differentiation of CD4+ IL-17A+ and CD4+ RANKL+ T cells, whereas the differentiation of CD4+CD25highFOXP3+ T cell population increased in a dose-dependent manner. These changes in CD4+ T cell differentiation were also observed in the SFMCs of patients with RA. The levels IL-17A and soluble RANKL in the culture medium were significantly decreased by IL-18BP. IL-18BP administration decreased TRAP+ cell counts in a dose-dependent manner on the background of stimulation with RANKL-and IL-17A. In addition, expression levels of TRAP, NFATC1, CTSK, and TNFRSF11A (RANK) genes were lower in the IL-18BP treated cells. CONCLUSION: We showed that IL-18BP can rectify the Th17/Treg imbalance and decrease IL-17-induced osteoclastogenesis in PBMCs from patients with RA. Therefore, IL-18BP may have therapeutic potential for RA treatment.
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Artritis Reumatoide , Interleucina-17 , Células Cultivadas , Humanos , Péptidos y Proteínas de Señalización Intercelular , Leucocitos Mononucleares , Osteoclastos , Osteogénesis , Ligando RANK , Linfocitos T Reguladores , Células Th17RESUMEN
BACKGROUND: In the pathogenesis of rheumatoid arthritis (RA), the role of mast cells has not been revealed clearly. We aimed to define the inflammatory and tissue-destructive roles of mast cells in rheumatoid arthritis (RA). METHODS: Serum and synovial fluid (SF) concentration levels of tryptase, chymase, and histamine were quantified using ELISA. After activating mast cells using IL-33, the production of TNF-α, IL-1ß, IL-6, IL-17, RANKL, and MMPs was determined using real-time PCR and ELISA. Osteoclastogenesis was assessed in CD14+ monocytes from peripheral blood and SF, which were cultured with IL-33-activated mast cells, by counting TRAP-positive multinucleated cells. RESULTS: The concentration levels of serum tryptase, chymase, and histamine and SF histamine were higher in patients with RA than in controls. FcεR1 and c-kit-positive mast cells were higher in RA synovium than in osteoarthritic (OA) synovium. Stimulation of mast cells by IL-33 increased the number of trypatse+chymase- and tryptase+chymase+ mast cells. IL-33 stimulation also increased the gene expression levels of TNF-α, IL-1ß, IL-6, IL-17, RANKL, and MMP-9 in mast cells. Furthermore, IL-33 stimulated human CD14+ monocytes to differentiate into TRAP+ multinucleated osteoclasts. When CD14+ monocytes were co-cultured with mast cells, osteoclast differentiation was increased. Additionally, IL-33-activated mast cells stimulated osteoclast differentiation. The inhibition of intercellular contact between mast cells and monocytes using inserts reduced osteoclast differentiation. CONCLUSIONS: IL-33 increased inflammatory and tissue-destructive cytokines by activation of mast cells. Mast cells stimulated osteoclast differentiation in monocytes. Mast cells could stimulate osteoclastogenesis indirectly through production of tissue-destructive cytokines and directly through stimulation of osteoclast precursors.
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Artritis Reumatoide , Osteogénesis , Diferenciación Celular , Células Cultivadas , Citocinas , Humanos , Mastocitos , Osteoclastos , Membrana SinovialRESUMEN
Clinical trials of biologic agents for chronic active antibody-mediated rejection (CAMR) in kidney transplant recipients (KTRs) have been disappointing. We performed a clinical trial of mesenchymal stem cell (MSC) treatment in KTRs with CAMR unresponsive to rituximab and intravenous immunoglobulin. This study was a phase 1 clinical trial to confirm patient safety. Two patients with CAMR unresponsive to rituximab and intravenous immunoglobulin were included. Each patient received allogeneic MSCs for 4 cycles (1 × 106 cells/kg every other week) via the peripheral vein in the distal arm. We observed adverse events and renal function for 6 months after the final MSC infusion and analyzed changes in immunomodulatory parameters in the peripheral blood between the start of treatment and 3 months after the final MSC infusion. There were no serious adverse events during the study period. Renal function was stable during MSC treatment but gradually decreased between the final MSC infusion and the study endpoint (patient 1: creatinine levels ranged from 3.01 mg/dL to 7.81 mg/dL, patient 2: 2.87 mg/dL to 3.91 mg/dL). In peripheral blood sample analysis between the start of treatment and 3 months after the final MSC infusion, there were similar trends for immunomodulatory markers. Our study showed that there were no serious adverse events for six months after allogeneic MSC treatment in KTRs with CAMR refractory to rituximab and intravenous immunoglobulin, but further studies need to define the efficacy of MSC treatment in CAMR.
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BACKGROUND/AIMS: The present study aimed to investigate whether tocotrienol regulates interleukin 17 (IL-17)-induced osteoclastogenesis in rheumatoid arthritis (RA). METHODS: We evaluated the effect of tocotrienol on IL-17-induced receptor activator of nuclear factor kappa B ligand (RANKL) production using RA fibroblast-like synoviocyte (FLS), together with real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Osteoclast differentiation was confirmed after culturing IL-17-treated RA FLS and Th17 cells with tocotrienol and monocytes. We analyzed the suppressive effect of tocotrienol on Th17 cells percentage or Th17-cytokine levels among peripheral blood mononuclear cells using flow cytometry. RESULTS: We found that IL-17 stimulated FLS to produce RANKL and tocotrienol decreased this IL-17-induced RANKL production. Tocotrienol decreased the IL-17-induced activation of mammalian target of rapamycin, extracellular signal-regulated kinase, and inhibitor of kappa B-alpha. When monocytes were incubated with IL-17, RANKL, IL-17-treated FLS or Th17 cells, osteoclasts were differentiated and tocotrienol decreased this osteoclast differentiation. Tocotrienol reduced Th17 cell differentiation and the production of IL-17 and sRANKL; however, tocotrienol did not affect Treg cell differentiation. CONCLUSION: Tocotrienol inhibited IL-17- activated RANKL production in RA FLS and IL-17-activated osteoclast formation. In addition, tocotrienol reduced Th17 differentiation. Therefore, tocotrienol could be a new therapeutic choice to treat bone destructive processes in RA.
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Artritis Reumatoide , Tocotrienoles , Artritis Reumatoide/tratamiento farmacológico , Diferenciación Celular , Humanos , Leucocitos Mononucleares , Osteoclastos , Osteogénesis , Tocotrienoles/farmacologíaRESUMEN
BACKGROUND: The present study aimed to evaluate the suppressive role of interleukin (IL)-25 in IL-22-induced osteoclastogenesis and receptor activator of nuclear factor κB ligand (RANKL) expression in rheumatoid arthritis (RA). METHODS: Serum from patients with RA and osteoarthritis (OA), and healthy controls, and synovial fluid from patients with RA and OA were collected, and the levels of IL-22 and IL-25 were measured. RA and OA synovial tissues were stained against IL-25. Fibroblast-like synoviocytes (FLSs) of patients with RA were cultured with IL-22, in the presence or absence of IL-25, and RANKL expression was measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA). Human peripheral blood monocytes were cultured under IL-22/RANKL + M-CSF, with or without IL-25, and tartrate-resistant acid phosphatase (TRAP)-positive cells and osteoclast-related markers were investigated to determine osteoclastogenesis. RESULTS: Serum and synovial IL-25 levels in RA were upregulated compared to those in OA and healthy control, and elevated expression of IL-25 in RA synovial tissue was re-confirmed. IL-25 and IL-22 levels showed significant correlation in serum and synovial fluid. Pre-treatment of FLS with IL-25 reduced IL-22-induced RANKL expression at the RNA level. The suppressive effects of IL-25 were confirmed to occur through the STAT3 and p38 MAPK/IκBα pathways. IL-25 reduced osteoclast differentiation and suppressed the expression of osteoclast-related markers. CONCLUSION: In the current study, we demonstrated the regulatory effect of IL-25 on IL-22-induced osteoclastogenesis. Therapeutic approach involving augmentation of IL-25 regulatory response may serve as a novel treatment option for RA, especially by suppressing osteoclastogenesis.
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Artritis Reumatoide , Osteogénesis , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Interleucina-17 , Interleucinas/metabolismo , Inhibidor NF-kappaB alfa , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Factor de Transcripción STAT3 , Membrana Sinovial/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos , Interleucina-22RESUMEN
We investigated the phenotype and molecular signatures of CD8+ T cell subsets in kidney-transplant recipients (KTRs) with biopsy-proven T cell-mediated rejection (TCMR). We included 121 KTRs and divided them into three groups according to the pathologic or clinical diagnosis: Normal biopsy control (NC)(n = 32), TCMR (n = 50), and long-term graft survival (LTGS)(n = 39). We used flowcytometry and microarray to analyze the phenotype and molecular signatures of CD8+ T cell subsets using peripheral blood from those patients and analyzed significant gene expressions according to CD8+ T cell subsets. We investigated whether the analysis of CD8+ T cell subsets is useful for predicting the development of TCMR. CCR7+CD8+ T cells significantly decreased, but CD28nullCD57+CD8+ T cells and CCR7-CD45RA+CD8+ T cells showed an increase in the TCMR group compared to other groups (p<0.05 for each); hence CCR7+CD8+ T cells showed significant negative correlations to both effector CD8+ T cells. We identified genes significantly associated with the change of CCR7+CD8+ T, CCR7-CD45RA+CD8+ T, and CD28nullCD57+CD8+ T cells in an ex vivo study and found that most of them were included in the significant genes on in vitro CCR7+CD8+ T cells. Finally, the decrease of CCR7+CD8+ T cells relative to CD28nullCD57+ T or CCR7-CD45RA+CD8+ T cells can predict TCMR significantly in the whole clinical cohort. In conclusion, phenotype and molecular signature of CD8+ T subsets showed a significant relationship to the development of TCMR; hence monitoring of CD8+ T cell subsets may be a useful for predicting TCMR in KTRs.
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Linfocitos T CD8-positivos/inmunología , Rechazo de Injerto/inmunología , Trasplante de Riñón/efectos adversos , Subgrupos de Linfocitos T/inmunología , Adulto , Antígenos CD28/genética , Antígenos CD28/inmunología , Antígenos CD57/genética , Antígenos CD57/inmunología , Linfocitos T CD8-positivos/clasificación , Estudios Transversales , Femenino , Perfilación de la Expresión Génica , Rechazo de Injerto/etiología , Rechazo de Injerto/genética , Voluntarios Sanos , Humanos , Inmunofenotipificación , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Receptores CCR7/genética , Receptores CCR7/inmunología , Subgrupos de Linfocitos T/clasificaciónRESUMEN
The human-induced pluripotent stem cell (KIN-hiPSCs) line (CMCi001-A), derived from peripheral blood mononuclear cells (PBMCs) of a 42-year-old woman with karyomegalic interstitial nephritis (KIN) caused by the mutation of FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene, was generated using Sendai virus. KIN-hiPSCs showed a typical human embryonic stem cell like morphology and expressed all pluripotency-associated markers, and directly differentiated into all three germ layers. Karyotyping of PBMCs of the patient and KIN-hiPSCs showed 47, XXX. In summary, we generated a novel patient-specific hiPSC line containing the mutation of FAN1 gene and it can be used to provide additional insights for KIN pathophysiology.
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Células Madre Pluripotentes Inducidas , Nefritis Intersticial , Adulto , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Femenino , Humanos , Leucocitos Mononucleares , Nefritis Intersticial/genética , Eliminación de SecuenciaRESUMEN
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory arthritis, and the complex interaction and activation of innate and adaptive immune cells are involved in RA pathogenesis. Mast cells (MCs) are one of the tissue-resident innate immune cells, and they contribute to RA pathogenesis. In the present review, the evidence of the pathologic role of MC in RA is discussed based on human and animal data. In addition, the potential role of MC in RA pathogenesis and the research area that should be focused on in the future are suggested.
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Artritis Reumatoide , Mastocitos , Animales , Artritis Reumatoide/diagnóstico , HumanosRESUMEN
BACKGROUND: The inflammatory cascade in the rheumatoid arthritis (RA) synovium is modulated by a variety of cytokine and chemokine networks; however, the roles of IL-26, in RA pathogenesis, are poorly defined. Here, we investigated the functional role of interleukin-26 (IL)-26 in osteoclastogenesis in RA. METHODS: We analyzed levels of IL-20 receptor subunit A (IL-20RA), CD55, and receptor activator of nuclear factor kappaB (NF-κB) ligand (RANKL) in RA fibroblast-like synoviocytes (FLSs) using confocal microscopy. Recombinant human IL-26-induced RANKL expression in RA-FLSs was examined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Human peripheral blood monocytes were cultured with macrophage colony-stimulating factor (M-CSF) and IL-26, after which osteoclastogenesis was evaluated by counting the number of tartrate-resistant acid phosphatase-positive multinucleated cells. Additionally, osteoclastogenesis was evaluated by monocytes co-cultured with IL-26-prestimulated FLSs. RESULTS: The expression of IL-20RA in RA-FLSs was higher than that in osteoarthritis-FLSs. Additionally, in IL-26-pretreated RA-FLSs, the expression of IL-20RA (but not IL-10 receptor subunit B) and RANKL increased in a dose-dependent manner, with IL-26-induced RANKL expression reduced by IL-20RA knockdown. Moreover, IL-26-induced RANKL expression was significantly downregulated by inhibition of signal transducer and activator of transcription 1, mitogen-activated protein kinase, and NF-κB signaling. Furthermore, IL-26 promoted osteoclast differentiation from peripheral blood monocytes in the presence of low dose of RANKL, with IL-26 exerting an additive effect. Furthermore, co-culture of IL-26-pretreated RA-FLSs with peripheral blood monocytes also increased osteoclast differentiation in the absence of addition of RANKL. CONCLUSIONS: IL-26 regulated osteoclastogenesis in RA through increased RANKL expression in FLSs and direct stimulation of osteoclast differentiation. These results suggest the IL-26/IL-20RA/RANKL axis as a potential therapeutic target for addressing RA-related joint damage.
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Interleucinas/farmacología , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Proteínas Recombinantes/farmacología , Sinoviocitos/efectos de los fármacos , Adulto , Anciano , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Interleucinas/genética , Masculino , Microscopía Confocal , Persona de Mediana Edad , Osteoartritis/genética , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoclastos/metabolismo , Osteogénesis/genética , Ligando RANK/genética , Ligando RANK/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Sinoviocitos/metabolismoRESUMEN
The purpose of this study was to determine the regulatory role of intravenous Ig (IVIg) in Th17 cytokine-induced RANK ligand (RANKL) expression and osteoclast (OC) differentiation from OC precursors (pre-OC). Human CD14+ monocytes were isolated and stimulated by Th17 cytokines (IL-17, IL-21, and IL-22) and RANKL expression was investigated using a real-time PCR. CD14+ monocytes were incubated with RANKL, Th17 cytokines, and M-CSF, with/without IVIg, and OC differentiation was determined by counting tartrate-resistant acid phosphatase-positive multinucleated cells. OC differentiation was investigated after monocytes were cocultured with Th17 cells in the presence of IVIg. Th17 cell differentiation was determined using enzyme-linked immunosorbent assay and flow cytometry after CD4+ T cells were cultured with IVIg under Th17 condition. Th17 cytokines stimulated monocytes to express RANKL and IVIg suppressed the Th17 cytokine-induced RANKL expression. OCs were differentiated when pre-OC were cocultured with RANKL or Th17 cytokines and IVIg reduced the osteoclastogenesis. IVIg also decreased osteoclastogenesis when pre-OC were cocultured with Th17 cells. IVIg decreased both Th17 and Th1 cell differentiation while it did not affect Treg cell differentiation. In summary, IVIg inhibited Th17 cytokine-induced RANKL expression and OC differentiation. IVIg reduced osteoclastogenesis when monocytes were cocultured with Th17 cells. IVIg also reduced Th17 polarization. IVIg could be a new therapeutic option for Th17 cell-mediated osteoclastogenesis.
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This study aimed to investigate the regulatory effect of SKI305X, a mixed extract of three herbs, in T helper (Th)17 cytokine-induced inflammation and joint destruction in rheumatoid arthritis (RA). Synovial fibroblasts were isolated from RA patients and cultured with Th17 cytokines including interleukin (IL)-17, IL-21, and IL-22 and SKI306X, and tumor necrosis factor (TNF)-, IL-1, and receptor activator of nuclear factor kappa-Β ligand (RANKL) expression and production were investigated using real-time PCR and ELISA of culture media. After peripheral blood (PB) cluster of differentiation (CD)14+ monocytes were cultured in media supplemented with Th17 cytokines and SKI306X, tartrate-resistant acid phosphatase positive (TRAP+) multinucleated giant cells (mature osteoclasts) were enumerated and gene expression associated with osteoclast maturation was assessed via real-time PCR analysis. After PB monocytes were co-cultured with IL-17-stimulated RA synovial fibroblasts in the presence of SKI306, osteoclast differentiation was assessed. When RA synovial fibroblasts were cultured with IL-17, IL-21, and IL-22, TNF-, IL-1, and RANKL expression and production were increased; however, SKI306X reduced cytokine expression and production. When PB monocytes were cultured in media supplemented with Th17 cytokines, osteoclast differentiation was stimulated; however, SKI306X decreased osteoclast differentiation and osteoclast maker expression. When PB monocytes were co-cultured with IL-17-stimulated RA synovial fibroblasts, osteoclast differentiation was increased; however, SKI306X decreased osteoclast differentiation and osteoclast maker expression. SKI306X reduced Th17 cytokine-induced TNF-, IL-1, and RANKL expression and osteoclast differentiation, providing novel insights into adjuvant therapy for regulating inflammation and joint destruction in RA.
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This study aimed to determine the regulatory role of toll-like receptor 7 (TLR7) in receptor activator of nuclear factor kappa-B ligand (RANKL) production and osteoclast differentiation in rheumatoid arthritis (RA). In confocal microscopy, the co-expression of TLR7, CD55 and RANKL was determined in RA synovial fibroblasts. After RA synovial fibroblasts were treated with imiquimod, the RANKL gene expression and protein production were determined by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Osteoclastogenesis from peripheral blood CD14+ monocytes which were cultured with imiquimod was assessed by determining the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. The signal pathways mediating the TLR7-induced RANKL expression and osteoclastogenesis were analysed after inhibition of intracellular signal molecules and their phosphorylation. Imiquimod stimulated the expression of TLR7 and RANKL and production of RANKL in RA synovial fibroblasts, increasing the phosphorylation of TRAF6, IRF7, mitogen-activated protein kinases (MAPK), c-Jun and NFATc1. When CD14+ monocytes were cultured with imiquimod or co-cultured with imiquimod-pre-treated RA synovial fibroblasts, they were differentiated into TRAP+ multinucleated osteoclasts in the absence of RANKL. TLR7 activation-induced osteoclastogenesis in RA through direct induction of osteoclast differentiation from its precursors and up-regulation of RANKL production in RA synovial fibroblasts. Thus, the blockage of TLR7 pathway could be a promising therapeutic strategy for preventing bone destruction in RA.
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Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Osteogénesis , Receptor Toll-Like 7/metabolismo , Anciano , Artritis Reumatoide/patología , Células Cultivadas , Femenino , Fibroblastos/patología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: We previously reported that tacrolimus (Tac) does not decrease T helper 17 cells (Th17) response in kidney transplantation. In this study, we evaluated whether Resveratrol (Resv) has immunosuppressive effects by decreasing Th17 responses in Tac-based immunosuppression. METHODS: We investigated the effects of Resv under Tac-treatment conditions, on CD4+ T cell differentiation to Th17 cells in peripheral blood mononuclear cells (PBMCs), and proliferation of CD4+ T cells co-cultured with human renal proximal tubular epithelial cells (HRPTEpiCs). The effects of Resv on Th17 cells were tested in the murine skin transplant model. RESULTS: In PBMCs, Tac did not but combination of Tac and Resv further suppressed Th17 immune response. In the co-culture study, combination of Resv to Tac significantly decreased HRPTEpiC-induced T cell proliferation compared to Tac alone. Resv treatment in the Jurkat cell induced the expression of AMP-activated protein kinase and suppressed the expression of mammalian target of rapamycin (mTOR), suggesting blocking Th17 pathway by Resv. In the murine skin transplant model, combination of Resv to Tac significantly prolonged skin graft survival accompanied by the suppression of Th17 cells, compared to either the Tac-alone or control groups. CONCLUSION: The results of our study suggest that Resv provides additional immunosuppressive effects to Tac by suppressing effector CD4+ T cells, especially Th17 cells, in the transplantation setting.
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Inmunosupresores/farmacología , Resveratrol/farmacología , Tacrolimus/farmacología , Células Th17/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Adulto , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Supervivencia de Injerto/efectos de los fármacos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Trasplante de Piel , Serina-Treonina Quinasas TOR/metabolismo , Células Th17/inmunología , Células Th17/metabolismoRESUMEN
We investigated the immune-regulatory function of quercetin, in interleukin (IL)-17-produced osteoclastogenesis in rheumatoid arthritis (RA). RA fibroblasts-like synoviocytes (RA-FLS) were stimulated with IL-17, and the mRNA expression and secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. CD14+ monocytes (osteoclast precursors) were stimulated with IL-17, RANKL, with/without quercetin, and tartrate-resistant acid phosphatase activity was evaluated to assess osteoclast differentiation. Osteoclast differentiation was investigated after coculturing IL-17-stimulated RA-FLS and Th17 cells with monocytes. CD4+ T cells were cocultured with quercetin under Th17-inducing conditions, and their differentiation to Th17 cells and Treg cells was determined by flow cytometry analysis. We found that IL-17 stimulated RA-FLS to produce RANKL and quercetin decreased the IL-17-induced RANKL protein levels. Quercetin decreased the IL-17-produced activation of mammalian target of rapamycin, extracellular signal-regulated kinase and inhibitor of kappa B-alpha. When monocytes were stimulated with IL-17, macrophage colony-stimulating factor or RANKL, mature osteoclasts were formed, and quercetin decreased this osteoclastogenesis. When monocytes were cultured with IL-17-prestimulated RA-FLS or Th17 cells, osteoclasts were produced, and quercetin decreased this osteoclast differentiation. In Th17-differentiation conditions, quercetin suppressed Th17 cell and the production of IL-17, but quercetin did not affect Treg cells. Quercetin inhibits IL-17-stimulated RANKL production in RA-FLS and IL-17-stimulated osteoclast formation. Quercetin reduces Th17 differentiation. Quercetin could be an additional therapeutic option for bone destructive processes in RA.
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Artritis Reumatoide , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Fitoterapia , Extractos Vegetales/farmacología , Quercetina/farmacología , Fosfatasa Ácida/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Diferenciación Celular , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Interleucina-17/efectos adversos , Interleucina-17/metabolismo , Monocitos , Extractos Vegetales/uso terapéutico , Quercetina/uso terapéutico , Ligando RANK/metabolismo , Sinoviocitos/efectos de los fármacos , Sinoviocitos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Fosfatasa Ácida Tartratorresistente/metabolismo , Células Th17RESUMEN
BACKGROUND/AIMS: This study aimed to determine the regulatory role of N-acetyl-l-cysteine (NAC), an antioxidant, in interleukin 17 (IL-17)-induced osteoclast differentiation in rheumatoid arthritis (RA). METHODS: After RA synovial fibroblasts were stimulated by IL-17, the expression and production of receptor activator of nuclear factor κ-B ligand (RANKL) was determined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). Osteoclastogenesis was also determined after co-cultures of IL-17-stimulated RA synovial fibroblasts, Th17 cells and various concentrations of NAC with monocytes. After human peripheral CD4+ T cells were cultured with NAC under Th17 condition, IL-17, interferon γ, IL-4, Foxp3, RANKL, and IL-2 expression and production was determined by flow cytometry or ELISA. RESULTS: When RA synovial fibroblasts were stimulated by IL-17, IL-17 stimulated the production of RANKL, and NAC reduced the IL-17-induced RANKL production in a dose-dependent manner. NAC decreased IL-17-activated phosphorylation of mammalian target of rapamycin, c-Jun N-terminal kinase, and inhibitor of κB. When human peripheral blood CD14+ monocytes were cultured with macrophage colony-stimulating factor and IL-17 or RANKL, osteoclasts were differentiated, and NAC reduced the osteoclastogenesis. After human peripheral CD4+ T cells were co-cultured with IL-17-pretreated RA synovial fibroblasts or Th17 cells, NAC reduced their osteoclastogenesis. Under Th17 polarizing condition, NAC decreased Th17 cell differentiation and IL-17 and RANKL production. CONCLUSION: NAC inhibits the IL-17-induced RANKL production in RA synovial fibroblasts and IL-17-induced osteoclast differentiation. NAC also reduced Th17 polarization. NAC could be a supplementary therapeutic option for inflammatory and bony destructive processes in RA.
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Acetilcisteína/farmacología , Artritis Reumatoide/tratamiento farmacológico , Osteogénesis/efectos de los fármacos , Ligando RANK/metabolismo , Células Th17/efectos de los fármacos , Adulto , Anciano , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/patología , Humanos , Técnicas In Vitro , Interleucina-17/metabolismo , Persona de Mediana Edad , Osteogénesis/inmunología , Ligando RANK/genética , Transducción de Señal/efectos de los fármacos , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Células Th17/inmunología , Células Th17/patologíaRESUMEN
OBJECTIVES: Macrophage migration inhibitory factor (MIF) is a proinflammatory, chemotactic, and tissue destructive cytokine. This study determined monosodium urate crystal-induced MIF production and its interaction with interleukin (IL)-8 in gout. METHODS: Peripheral blood (PB), synovial fluid (SF), and clinical data were obtained from 98 patients with gout. SF and serum concentrations of MIF and IL-8 were measured using ELISA. SF monocytes and neutrophils were cultured with monosodium urate (MSU) crystals and the cytokine production was determined. The signalling pathways involved were determined using signal inhibitors. The interaction between MIF and IL-8 was investigated. RESULTS: SF MIF was higher in acute gout and that in serum was higher in patients with intercritical gout compared with controls. SF MIF was positively correlated with SF leukocyte and neutrophil counts and IL-8. The expression of MIF was similar in SF neutrophils and monocytes, while IL-8 was higher in monocytes. MSU crystals induced MIF production in monocytes and IL-8 production in neutrophils. This effect was decreased by inhibiting Fc-gamma receptor 1 and toll-like receptor 4. IL-8 increased MIF production in monocytes while MIF increased interleukin-8 production in neutrophils. CONCLUSIONS: MIF and IL-8 are highly produced in acute gout. MSU crystals induced MIF production in monocytes and IL-8 production in neutrophils with a reciprocal interaction between the two cytokines.
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Gota , Interleucina-8/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Gota/metabolismo , Humanos , Neutrófilos , Ácido ÚricoRESUMEN
OBJECTIVES: The aim of this study was to investigate the diagnostic accuracy of rheumatoid factor (RF) isotype for the detection of primary Sjogren's syndrome (pSS) and evaluate the clinical and serological associations of immunoglobulin (Ig) A RF in patients with pSS. MATERIALS AND METHODS: RF levels were measured in 77 and 37 patients with pSS and idiopathic sicca symptoms, respectively, using ELISA and analysed with respect to clinical and laboratory disease characteristics. Receiver operating characteristic curves were used to determine and compare the diagnostic accuracy of IgA RF with other diagnostic tests. RESULTS: Serum levels of IgA RF were significantly higher in patients with pSS than in those with idiopathic sicca symptoms. IgA RF showed sensitivity, specificity, positive, and negative predictive values of 83.1, 78.4, 88.9, and 69.0%, respectively, for pSS diagnosis. IgA RF was associated with xerostomia, severe sialoscintigraphic grade, low unstimulated salivary flow rate (USFR), antinuclear antibody, high IgG and IgM/G RF levels, and low C3 levels in patients with pSS. IgA RF titres had positive correlations with sialoscintigraphic grade and IgG and IgG/M RF levels and had negative correlations with USFR and C3 levels. CONCLUSION: Our findings confirmed the potential of IgA RF to distinguish pSS from idiopathic sicca symptoms. The presence of IgA RF in patients with pSS was associated with significantly worse exocrine function and active serologic profile. No association between IgA RF and extra-glandular manifestations was noted. CLINICAL RELEVANCE: IgA RF should be the predictive and diagnostic marker in patients with pSS.