The heterogeneous human memory CCR6+ T helper-17 populations differ in T-bet and cytokine expression but all activate synovial fibroblasts in an IFNγ-independent manner

Background: Chronic synovial inflammation is an important hallmark of inflammatory arthritis, but the cells and mechanisms involved are incompletely understood. Previously, we have shown that CCR6+ memory T-helper (memTh) cells and synovial fibroblasts (SF) activate each other in a pro-inflammatory feedforward loop, which potentially drives persistent synovial inflammation in inflammatory arthritis. However, the CCR6+ memTh cells are a heterogeneous population, containing Th17/Th22 and Th17.1 cells. Currently, it is unclear which of these subpopulations drive SF activation and how they should be targeted. In this study, we examined the individual contribution of these CCR6+ memTh subpopulations to SF activation and examined ways to regulate their function. Methods: Th17/Th22 (CXCR3−CCR4+), Th17.1 (CXCR3+CCR4−), DP (CXCR3+CCR4+), and DN (CXCR3−CCR4−) CCR6+ memTh, cells sorted from PBMC of healthy donors or treatment-naïve early rheumatoid arthritis (RA) patients, were cocultured with SF from RA patients with or without anti-IL17A, anti-IFNγ, or 1,25(OH)2D3. Cultures were analyzed by RT-PCR, ELISA, or flow cytometry. Results: Th17/Th22, Th17.1, DP, and DN cells equally express RORC but differ in production of TBX21 and cytokines like IL-17A and IFNγ. Despite these differences, all the individual CCR6+ memTh subpopulations, both from healthy individuals and RA patients, were more potent in activating SF than the classical Th1 cells. SF activation was partially inhibited by blocking IL-17A, but not by inhibiting IFNγ or TBX21. However, active vitamin D inhibited the pathogenicity of all subpopulations leading to suppression of SF activation. Conclusions: Human CCR6+ memTh cells contain several subpopulations that equally express RORC but differ in TBX21, IFNγ, and IL-17A expression. All individual Th17 subpopulations are more potent in activating SF than classical Th1 cells in an IFNγ-independent manner. Furthermore, our data suggest that IL-17A is not dominant in this T cell-SF activation loop but that a multiple T cell cytokine inhibitor, such as 1,25(OH)2D3, is able to suppress CCR6+ memTh subpopulation-driven SF activation

Synovial fibroblasts directly induce TH17 pathogenicity via the cyclooxygenase/prostaglandin E2 pathway, independent of IL-23

Th17 cells are critically involved in autoimmune disease induction and severity. Recently, we showed that Th17 cells from patients with rheumatoid arthritis (RA) directly induced a proinflammatory loop upon interaction with RA synovial fibroblasts (RASF), including increased autocrine IL-17A production. To unravel the mechanism driving this IL-17A production, we obtained primary CD4+CD45RO+CCR6+ (Th17) cells and CD4+CD45RO+CCR62 (CCR62) T cells from RA patients or healthy individuals and cocultured these with RASF. IL-1b, IL-6, IL-23p19, and cyclooxygenase (COX)-2 expression and PGE2 production in Th17-RASF cultures were higher than in CCR62 T cell-RASF cultures. Cytokine neutralization showed that IL-1b and IL-6, but not IL-23, contributed to autocrine IL-17A induction. Importantly, treatment with celecoxib, a COX-2 inhibitor, resulted in significantly lower PGE2 and IL-17A, but not IFN-g, production. Combined celecoxib and TNF-A blockade more effectively suppressed the proinflammatory loop than did single treatment, as shown by lower IL-6, IL-8, matrix metalloproteinase-1 and matrix metalloproteinase-3 production. These findings show a critical role for the COX-2/PGE2 pathway in driving Th17-mediated synovial inflammation in an IL-23- and monocyte-independent manner. Therefore, it would be important to control PGE2 in chronic inflammation in RA and potentially other Th17-mediated autoimmune disorders

Enhanced Bruton's Tyrosine Kinase Activity in Peripheral Blood B Lymphocytes From Patients With Autoimmune Disease

ObjectiveBruton's tyrosine kinase (BTK) transmits crucial survival signals from the B cell receptor (BCR) in B cells. Pharmacologic BTK inhibition effectively diminishes disease symptoms in mouse models of autoimmunity; conversely, transgenic BTK overexpression induces systemic autoimmunity in mice. We undertook this study to investigate BTK expression and activity in human B cells in the context of autoimmune disease. MethodsUsing intracellular flow cytometry, we quantified BTK expression and phosphorylation in subsets of peripheral blood B cells from 30 patients with rheumatoid arthritis (RA), 26 patients with primary Sjogren's syndrome (SS), and matched healthy controls. ResultsIn circulating B cells, BTK protein expression levels correlated with BTK phosphorylation. BTK expression was up-regulated upon BCR stimulation in vitro and was significantly higher in CD27+ memory B cells than in CD27-IgD+ naive B cells. Importantly, BTK protein and phospho-BTK were significantly increased in B cells from anti-citrullinated protein antibody (ACPA)-positive RA patients but not in B cells from ACPA-negative RA patients. BTK was increased both in naive B cells and in memory B cells and correlated with frequencies of circulating CCR6+ Th17 cells. Likewise, BTK protein was increased in B cells from a major fraction of patients with primary SS and correlated with serum rheumatoid factor levels and parotid gland T cell infiltration. Interestingly, targeting T cell activation in patients with primary SS using the CTLA-4Ig fusion protein abatacept restored BTK protein expression in B cells to normal levels. ConclusionThese data indicate that autoimmune disease in humans is characterized by enhanced BTK activity, which is linked not only to autoantibody formation but also to T cell activity