The Roles And Mechanisms Of Methotrexate Or Simvastatin On Th17/IL-17 In Peripheral Blood Mononuclear Cells From Rheumatoid Arthritis In Vitro Study

PartⅠ:The effect of various concentrations of methrotrexate on the Th17/IL-17 levels in peripheral blood mononuclear cells from RA patients and healthy individualsBackground Rheumatoid arthritis (RA) is a systemic autoimmune and inflammatory disease. It is characterized by recurrenting synovitis, which makes bone destruction and joint disability. Methotrexate (MTX), a first-line DMARDs in the treatment of RA, has been proved to be effective and safe. But the mechanism of action of MTX for attenuating the disease process in RA remains elusive. Th17 response dysfunction leads to overwhelming IL-17 production and induce RA inflammation. It may be one of mechanism in MTX therapy on RA.Objective To investigate whether the inhibition of Thl7/interleukin (IL)-17 contributes to the beneficial effects of MTX in the treatment of RA.Methods Peripheral blood mononuclear cells (PBMCs) from healthy donors and RA patients were collected. The cells were stimulated with monoclonal antibodies to CD3 and CD28 in the absence or presence of MTX. After co-incubation, IL-17 production was detected at both the mRNA and protein levels, and the percentage of cells positive for both CD4 and IL-17 in PBMCs was analyzed by flow cytometry.Results PBMCs of healthy donors and RA patients were stimulated with CD3 and CD28 monoclonal antibodies to produce high levels of IL-17 mRNA (RA:0.78±0.14 vs.0.54±0.15,p=0.02; HC:0.71±0.17 vs.0.34±0.14,p<0.01) and high levels of IL-17 protein (RA:436.16±205.08 vs.15.36±6.57, p<0.01; HC:271.83±145.11 vs. 11.48±7.12,p<0.01). The augmentation of IL-17 at the mRNA and protein levels was significantly inhibited when PBMC cultures were pre-incubated with MTX, especially at 5.0 and 25.0μg/ml MTX groups(p<0.05 respectively). Compared with PBMCs of healthy donors, PBMCs of RA patients produced higher levels of IL-17, and this increase in IL-17 levels was more inhibited by MTX pretreatment. lgMTX was negatively correlated with IL-17mRNA(RA:R2=0.96,p=0.02; HC:R2=0.94,p=0.03), but not at the protein level, in both PBMCs of healthy donors and RA patients. MTX did not affect the percentage of CD4-and IL-17-positive cells in PBMCs.Conclusions MTX suppressed the production of IL-17 at the mRNA levels and protein levels by PBMCs from healthy donors and RA patients. Suppression of IL-17 by MTX may contribute to its potent anti-inflammatory role in RA therapy. PartⅡ:Effects of Simvastatin on IL-17 production and expression of Th-17 transcription factor BATF in PBMCs from RA patients and healthy individualsBackground RA, an inflammatory disease mainly destroys peripheral joints and also increases the risk of cardiovascular diseases. Being effective cholesterol-lowering agents, statins have been extensively used for primary or secondary prevention of cardiovascular disease. More recently, many studies revealed that statins exert several immunomodulatory effects by kinds of singal passways and performed anti-inflammatory effect in RA therapy. IL-17 is a crucial inflammatory cytokine involving in pathophysiological processes of RA. BATF possessing is a member of the AP-1 transcription factor family. It regulates Th17 cells differentiation and IL-17 production. Statins can inhibit some cytokines production via AP-1 ways and whether statins also can inhibit IL-17 production by BATF to attenuate inflammatory response in RA.Objective To investigate the effects of simvastatin on the production of IL-17 and BATF in peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients and healthy individuals.Methods PBMCs were isolated from heparinized blood of healthy donors or RA patients using Ficoll-Hypaque density gradient centrifugation. The cells were stimulated by PMA and ionomycin in absence or presence of simvastatin or MVA at 37℃5%CO2. The mRAN level of IL-17, BATF and GAPDH was detected by RT-PCR; the protein level of IL-17 in supernatants was assayed by ELISA kit;the protein level of BATF was detected by Western Blot.Results After treatment with simvastatin, the expression of IL-17mRNA in RA PBMCs was all markedly decreased from 0.60±0.18 to 0.31±0.17, with p=0.04. Meanwhile, the secretion of IL-17 was also reduced significantly (79.64±12.67 vs. 44.61±3.90,p=0.01). The mRNA level of BATF in RA PBMCS was higher than BATF mRNA in PBMCs of healthy controls (0.39±0.05 vs.0.25±0.07,p=0.04), but no significant changes of BATF after treating with simvastatin. The inhibition of simvastatin on production of IL-17 was reversed by mevalonic acid (MVA).Conclusions Simvastatin inhibited production of IL-17 in RA PBMCs at gene and protein level, which was not targeted at suppressing expression of IL-17 transcription factor BATF, though BATF was highly expressed on RA PBMCs.