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03.10 Regulation of the th17 cell differentiation in rheumatoid arthritis

Background Th17 cells have a central role in the inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)−17A, −21,–22, and tumour necrosis factor-α. We studied in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA). Materials and methods Mononuclear cells (PBMC) were isolated from peripheral blood of healthy volunteers and rheumatoid arthritis (RA) patients. CD45RO- (naive) and CD45RO+ (memory) T cells were isolated from PBMC with a two-step negative magnetic separation method. They were activated with anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml) and with crosslinking (1 µg/ml) antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with anti-IL-4 (10 µg/ml) blocking antibody. After 5 and 10 days the IL-17 and IL-22 production were measured by ELISA and the RORc and Tbet expression were measured by qPCR. The expression of CCR6, CCR4 and CXCR3 of the cells were determined by flow cytometric analysis. Cell viability was monitored by impendance-based cell analyzer (CASY-TT). Results In healthy donors the memory T cells were characterised by higher RORc (p<0.05) and Tbet (p<0.05) expression levels compared to the naive T cells. This difference between the naive and memory T cells was not detected in RA patients (p=0.5625). The naive T cells from RA patients expressed more RORc (p<0.05), but not Tbet than the healthy donor derived naive cells. The CCR6 and CCR4 expression were higher in memory T cells of healthy donors than those of the naive T-cells (p<0.05) however this difference between T cell subpopulations of RA patients was not significant (p=0.06). The IL17 and IL22 production was increased by IL1+IL23+IL6+anti-IL4 treatment in healthy donors and RA patients (p<0.05) as well. Interestingly in RA the IL1+IL-23+anti-IL4 treatment also promoted the IL17 and IL22 production (p<0.05), suggesting that in vitro IL6 treatment is not required for the IL-17 and IL-22 production. Conclusions Our data suggest that the increased baseline RORc expression of the CD45RO- cells may contribute to the accelerated Th17 differentiation in RA. The IL-17 and IL-22 production are differently regulated in RA compared to the healthy donors.