The Role Of Adapter Protein SLAT In Th17 Differentiation In Rheumatoid Arthritis

Background Rheumatoid arthritis(RA) is a common autoimmune disease that is associated with progressive disability, systemic complications, early death, and socioeconomic costs. The cause and pathogenesis of rheumatoid arthritis is not completely understood, however, CD4+ T cells play an important role in the pathogenesis of RA. T helper 17 cells(Th17 cells) are a subset of T helper cells, distinct from Th1 and Th2 cells, producing interlukin 17(IL-17). Th17 cells create inflammation and tissue injury and play central role in rheumatoid arthritis. Similar to Th1 and Th2 cells, Th17 cells require specific cytokines and transcription factors for their differentiation. However, little is known regarding the T cell receptor(TCR) activation pathway. Adaptor proteins contain a variety of protein-binding modules that link protein-binding partners together and facilitate the creation of larger signaling complexes in a signal transduction pathway. SLAT(SWAP-70-like adaptor protein of T cells) is an adaptor protein expressed in cells of the hematopoietic system. SLAT is a guanine nucleotide exchange factor for Rho GTPases that regulates the development of Th1 and Th2 cell inflammatory responses by controlling the Ca2+-NFAT signaling pathway. Recent studies show SLAT cause bone destruction via activation of effector cells. Therefore, SLAT plays a pivotal role in T cell immune response, implications for rheumatoid arthritis.Objective The aim of this study is to explore the role of SLAT in Th17 cells differentiation in rheumatoid arthritis.Methods: We collected synovial fluid and peripheral blood from 20 patients with active rheumatoid arthritis. Peripheral blood from 15 normal healthy donors were collected as control. Clinical and immunological features were assessed as well. Peripheral blood mononuclear cells(PBMC) and synovial fluid mononuclear cells(SFMC) were isolated and pretreated with PMA, lonomycin and monensin for 4 hours. Flow cytometry was used to measure Th17 cells and SLAT expression by using BV510 anti-human CD4, PE-Cy7 anti-human IL-17, primary anti-SLAT antibody and secondary antibody Donkey Anti-Rabbit 488. We analyze the correlation between SLAT expression and Th17 cells or disease activity score 28(DAS28).We isolated PBMC from normal healthy donors and created Th17 differentiation system in vitro. Cells were treated with T-Activator CD3/CD28 as Group A; treated with T-Activator CD3/CD28 plus TGF-β, IL-23 and IL-6 as Group B; treated with CD4 Aptamer-SLAT sh RNA chimera as Group C. Flow cytometry was used to measure IL-17+, IFN-γ+, TNF-α+, RORγt+, IRF4+ or SLAT+ CD4+ T cells in all 3 groups. In the Th17 differentiation system, CD4+ T cells were isolated by magnetic-activated cell sorting(MACS) and cultured for 72 hours. ELISA was used to measure IL-17, TNF-α and IFN-γ concentration in culture supernatants. Flow cytometry and Real time-PCR were used to measure SLAT protein and m RNA expression, respectively. Moreover, immunofluorescence microscopy was used to observe silencing SLAT expression in CD4+ T cells through CD4 Aptamer-SLAT sh RNA chimera. SPSS 20.0 was used for statistical analysis.Results: 1. The proportion of SLAT+ CD4+ T cells in the peripheral blood(52±3.6%) and synovial fluid(47±3.8%) of patients with active RA was higher than that in peripheral blood(21±2.4%) of normal control( P<0.01).2. The proportion of Th17+ CD4+ T cells in the synovial fluid(2.01±0.42%) and peripheral blood(1.46±0.21%) of patients with active RA was higher than that in peripheral blood(0.43±0.14%) of normal control( P<0.01). The proportion of Th17+ CD4+ T cells in the synovial fluid was even higher than in peripheral blood in RA(P<0.05).3. The proportion of SLAT+ CD4+ T cells was correlated with Th17 cells(r=0.582,P=0.05) in the peripheral blood and DAS28(r=0.732 P=0.006) in patients with RA.4. Percentage of IL-17+, IFN-γ+, TNF-α+, RORγt+, IRF4+ or SLAT+ CD4+ T cells in group A and B cells were higher than in group C(P<0.01, P<0.05).5. IL-17, IFN-γ and TNF-α secretion in group A and B were higher than in group C(P<0.05, P<0.01). IL-17, IFN-γ and TNF-α expression in group B were even higher than in group A(P<0.05).6. IL-17, IFN-γ, TNF-α, RORγt, IRF4 and SLAT m RNA relative expression in group A and group B were higher than in group C(P<0.05, P<0.01).7. The mean fluorescence intensity(MFI) of SLAT+ CD4+ T cells in group A(236±38) and group B(541±24) were higher than in group C(105±23)(P<0.05, P<0.01). The MFI of SLAT+ CD4+ T cells in group B was higher than in group A(P<0.01)8. Immunofluorescence microscopy showed a significant decrease in SLAT expression in CD4+ T cells after silencing SLAT gene.Conclusions 1. The proportion of SLAT+ CD4+ T cells in peripheral blood and paired synovial fluid from patients with active RA was higher than that in normal control. The proportion of SLAT+ CD4+ T cells was positively correlated with Th17 cells in peripheral blood and DAS28 in RA as well. These results indicate that SLAT could be a marker to evaluate RA activity.2. Th17 related cells, cytokines and m RNA expression significantly increased after activation of CD4+ T cells, while declined significantly after SLAT silenecing. These indicate that SLAT plays a pivotal role in the differentiation of Th17 cells via IL-17, IFN-γ and TNF-α secretion.3. SLAT expression in synovial fluid was higher than in peripheral blood from patients with active RA. This may imply that different mechanisms involved in SLAT induced Th17 cells differentiation.