Conventional Tregs in treatment-naïve rheumatoid arthritis are deficient in suppressive function with an increase in percentage of CXCR3 and CCR6 expressing Tregs.

Abstract

In rheumatoid arthritis (RA), immune homeostasis is maintained by T regulatory cells (Tregs) that in an inflammatory milieu can change towards T-helper-like phenotypes (Th-like Tregs). Our aim was to examine the phenotypic and functional characteristics of CD4⁺CD25⁺CD127^lo/- Tregs, Th-like Tregs and T effector (Teff) cells in the peripheral blood (PB) and synovial fluid (SF) of treatment-naïve early RA, as compared to osteoarthritis (OA) and healthy control (HC) peripheral blood. Frequencies of Tregs, CXCR3, CCR6 expressing Tregs (Th-like Tregs), and Teff cells were analyzed using flow cytometry in RA (n = 80), OA (n = 20), and HC (n = 40). Cytokine concentrations of the respective T cell subsets in plasma and SF were measured using flow cytometric bead array. Tregs sorted from RA and HC PB using magnetic beads were analyzed for functional capacities by CFSE proliferation assay and FOXP3 gene expression using real-time PCR. We observed that the frequencies of Th17 cells in PB and SF were significantly higher in RA when compared to HC, whereas Tregs were lower in PB and high in SF compared to HC and OA respectively. Th1- and Th17-related pro-inflammatory cytokines IL12p70, INF-γ, TNF-α, and IL-6, and IL-17A were significantly higher in the plasma and SF of RA. Tregs expressing CXCR3 (Th1-like Tregs) and CCR6 (Th17-like Treg) were significantly higher in PB and SF of RA compared to controls and was positively associated with seropositivity and disease activity. Treg cells isolated from peripheral blood of RA showed decreased function and reduced FOXP3 gene expression compared to HC. In our study, we have demonstrated higher frequencies of Th1 and Th17 cells and increased circulatory and SF pro-inflammatory cytokines (IL12P70, INF-γ, IL-6, IL-17A, and TNF-α) in RA. This inflammatory milieu might alter total Tregs frequencies and influence conversion of Tregs into Th-like Tregs.