Jp van Hamburg

Th17 cells, but not Th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin‐17A production

OBJECTIVE Both Th1 cells and Th17 cells have been recognized in rheumatoid arthritis (RA); however, it remains unclear whether Th1 cells and/or Th17 cells are involved in driving disease chronicity and destructiveness. The aim of this study was to identify and characterize the functional role of Th17 cells in early RA. METHODS Flow cytometry analysis was performed on peripheral blood mononuclear cells (PBMCs) from treatment-naive patients with early RA and age-matched healthy volunteers. PBMCs from these patients, naive T cells, and primary CCR6- Th1 cells and CCR6+ Th17 cells were sorted and cultured in the absence or presence of synovial fibroblasts from patients with early RA (RASFs), and cytokine expression and gene transcription were analyzed. In addition, tumor necrosis factor α (TNFα)- and interleukin-17A (IL-17A)-blocking experiments were performed. RESULTS In the PBMCs of treatment-naive patients with early RA, an increased fraction of IL-17A-and TNFα-producing CCR6+ Th17 cells was observed. When cocultured with RASFs, these primary Th17 cells were potent inducers of IL-6 and IL-8 and the tissue-destructive enzymes matrix metalloproteinase 1 (MMP-1) and MMP-3, whereas primary Th1 cells or naive T cells were not. Importantly, specific up-regulation of IL-17A but not TNFα or interferon-γ was observed in RASF/Th17 cell cocultures. In addition to TNFα blocking, IL-17A neutralization was required to further down-regulate Th17 activity in RASF/Th17 cell cocultures. CONCLUSION Th17 cells, but not Th1 cells, cooperated with RASFs in a proinflammatory feedback loop, revealing a potential mechanism by which human Th17 cells drive chronic destructive disease in patients with RA. Furthermore, the neutralization of IL-17A activity is essential in current anti-TNF therapies to suppress Th17 cell activity in patients with early RA and potentially other Th17 cell-mediated disorders.

1,25-Dihydroxyvitamin D3 modulates Th17 polarization and interleukin-22 expression by memory T cells from patients with early rheumatoid arthritis

OBJECTIVE To examine the immunologic mechanism by which 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) may prevent corticosteroid-induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology. METHODS Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO- (non-memory) T cells separated by fluorescence-activated cell sorting (FACS) from treatment-naive patients with early RA were stimulated with anti-CD3/anti-CD28 in the absence or presence of various concentrations of 1,25(OH)(2)D(3), dexamethasone (DEX), and 1,25(OH)(2)D(3) and DEX combined. Levels of T cell cytokines were determined by enzyme-linked immunosorbent assay and flow cytometry. RESULTS The presence of 1,25(OH)(2)D(3) reduced interleukin-17A (IL-17A) and interferon-gamma levels and increased IL-4 levels in stimulated PBMCs from treatment-naive patients with early RA. In addition, 1,25(OH)(2)D(3) had favorable effects on tumor necrosis factor alpha (TNFalpha):IL-4 and IL-17A:IL-4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL-17A- and IL-22-expressing CD4+ T cells and IL-17A-expressing memory T cells were observed in PBMCs from treatment-naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO- cells between these 2 groups. Interestingly, 1,25(OH)(2)D(3), in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL-17A, IL-17F, TNFalpha, and IL-22 production by memory T cells sorted by FACS from patients with early RA. CONCLUSION These data indicate that 1,25(OH)(2)D(3) may contribute its bone-sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL-4.

AB0033 The identification of IL-17A+, IL-17RA+ and IL-17RC+ lymphoid and myeloid cells in blood of treatment naÏve early and in synovial fluid of established psoriatic arthritis patients

Background Interleukin (IL)-17A is a pro-inflammatory cytokine and is involved in the pathogenesis of psoriatic arthritis (PsA) (1,2). Various cells can produce IL-17A. However, it is not clear which cell types in PsA patients are responsible for the production of IL-17A. In addition, the expression of IL-17RA and IL-17RC on different cell types is not well defined. Objectives To identify IL-17A, IL-17RA and IL-17RC positive cells in blood of first diagnosed PsA patients with arthritis and in synovial fluid of established PsA patients with active disease. Methods Fresh blood was taken from first diagnosed DMARD and steroid naïve PsA patients (n=10), having arthritis in 1 or more joints (PsA blood). The diagnosis was made by a rheumatologist according to the CASPAR-criteria. In addition, fresh synovial fluid was obtained from established PsA patients (PsA SF) with active disease (n=10) and treated with either methotrexate (n=3) or adalumimab (n=3) or NSAIDs (n=4). Multicolor flow cytometric analysis was performed on PsA blood and PsA SF. For the detection of IL-17A, IL-17RA or IL-17RC the following antibodies were used: IL-17A-PE (eBioscience), IL-17RA or isotype control IgG1k (both Biolegend), IL-17RC or isotype control IgG2b (both R&D systems). The following markers were used to discriminate between different cell populations: T cell subsets (CD3, CD4, CD8, CD45RO, CCR6, TCRγδ), B cells (CD19), NK cells (CD15-CD16+), neutrophils (CD15+CD16+), monocytes (CD33+CD14+CD16+/-), mast cells (CD117+FcER1a+) and eosinophils (CD15+FcER1a+). Results Different lymphoid and myeloid cell types were IL-17A positive in PsA blood of first diagnosed PsA patients such as CD3+, TCRγδ+, CD4+, CD8+ lymphoid cells, CD14+ monocytes and eosinophils. In PsA SF of established PsA patients TCRγδ+ T cells, neutrophils, NK cells and eosinophils were IL-17A positive. In both groups, no difference in expression of IL-17RA and IL-17RC was found on CD4+, CD8+, CD4+CD45RO+CCR6+/-, TCRγδ+ and CD19+ lymphoid cells compared to their isotype control. In contrast, the expression of IL-17RA and IL-17RC was increased compared to their isotype control on neutrophils and monocytes in PsA blood and on neutrophils, monocytes, mast cells and eosinophils in PsA SF. Conclusions These preliminary data show that not only lymphoid cells but also specific myeloid cell types may be sources of IL-17A in PsA. Furthermore, not lymphoid cells but IL-17RA/IL-17RC positive myeloid cells such as monocytes, neutrophils, mast cells and eosinophils may be potential target cells for IL-17A. Together, these data suggest a more broad, but specific IL-17A-IL-17RA/RC signaling network between different cell types important in the IL-17A-driven pathogenesis of PsA. References Lubberts E. Nat Rev Rheumatol 2015, 11: 415–29. McInnes IB, et al. Lancet 2015, 386: 1137–46. Disclosure of Interest None declared

A2.19 IL-17A-low CCR6+ TH cell populations of patients with rheumatoid arthritis are pathogenic, multidrug resistant and associated with dmard and glucocorticoid treatment response

Background and objectives CCR6+ T-helper (Th) cells and their pro-inflammatory cytokines, including IL-17A, are implicated in the pathogenesis of rheumatoid arthritis (RA). However, within CCR6+ Th cells various subpopulations are present and their clinical relevance in RA is unclear. Therefore, we characterised CCR6+ Th subpopulations with regard to pathogenic potential and disease-modifying antirheumatic drugs (DMARDs) and glucocorticoid (GC) therapy outcome in RA. Material and methods Within total CCR6+ Th cells from patients with RA, CCR4+CXCR3- (Th17), CCR4-CXCR3+ (Th17.1), CCR4/CXCR3 double-positive (DP) and double-negative (DN) cells were distinguished and/or sorted by flow cytometry. These subpopulations were: analysed for Th17/Th1-associated factors; co-cultured with RA-derived synovial fibroblasts (RASF); related to disease-modifying antirheumatic drugs (DMARDs) and glucocorticoid (GC) therapy response; analysed regarding the expression of multidrug transporters MDR1 and MRP1 and drug efflux potential. Results All these populations expressed the transcription factor RORC and were present in RA peripheral blood and synovial fluid. Despite differential IL-17A, IL-17F, IFNg and TBX21 expression, all subpopulations, including the IL-17A low-producing Th17.1, DP and DN cells, showed pathogenic activity in the induction of IL-1β, IL-6, IL-8, COX-2 and MMP-3 expression by synovial fibroblasts. MDR1 (ABCB1) and MRP1 (ABCC1) are cellular efflux transporters of glucocorticoids and the DMARD methotrexate. In particular Th17.1 and DN cells expressed relatively high levels of MDR1, whereas MRP1 was expressed at similar levels among the subpopulations. Interestingly, increased drug efflux potential by CCR6+ Th cells, as measured by rhodamine123 and calcein transport, was associated with the lack of DMARD/GC therapy response. Conclusions Therefore, we conclude that in addition to IL-17-high Th17 cells, IL-17-low CCR6+ Th cells display pathogenic activity in the context of RA. Moreover, we identified efflux transporter expression and efflux activity by RA CCR6+ Th cells. These findings suggest that pathogenic and multidrug resistant CCR6+ Th cells are associated with the lack of DMARD/GC therapy response in patients with RA.

A2.25 Vitamin D induces a TR1-like phenotype in human CCR6+T cells and promotes their migration to an inflammatory environment

Background and objectives CCR6+ T helper (Th) cells, including Th17 and Th17.1 cells, are thought to play an important role in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). They produce cytokines like IL-17A and TNFα and activate synovial fibroblasts to induce a pro-inflammatory feedback loop. This interaction may underlie the chronic joint inflammation in RA and therefore CCR6+ Th cells are an interesting therapeutic target in this disease. Vitamin D is known to have suppressive effects on autoimmune diseases and its active metabolite directly inhibits the pathogenicity of CCR6+ Th cells. Here we further investigated the effect of 1,25(OH)2D3 on CCR6+ Th cells to understand how 1,25(OH)2D3 suppresses the inflammatory responses in autoimmune diseases. Materials and methods We cultured CCR6+ Th cells from treatment-naïve early RA patients with or without 1,25(OH)2D3 and generated gene-expression profiles. These profiles were validated using RT-PCR, ELISA and flow cytometry. Functional effects were evaluated via co-culture with RA synovial fibroblasts (RASF) and Boyden chamber-based migration assays. Results The gene-expression profiles confirmed that 1,25(OH)2D3 downregulated pro-inflammatory cytokines like IL-17A, IL-17F and IL-22, but also genes important for the pathogenic Th17 phenotype like RORC and IL23R. In contrast, 1,25(OH)2D3 induced the anti-inflammatory cytokine IL-10, but not the classical Treg transcription factor FoxP3. Instead, upregulation of genes like LAG3, c-MAF, CTLA4, and IRF8 suggests a Tr1-like phenotype. Because of the decreased pathogenicity of these cells, we next investigated whether they migrate towards the site of inflammation. Indeed, the gene-expression profiles indicate a shift in the chemokine receptor profile upon treatment with 1,25(OH)2D3. This altered profile was accompanied by increased migration towards an inflammatory environment as modelled by the CCR6+ Th – RASF co-culture. Interestingly, CCR6+ Th cells that were pre-treated with 1,25(OH)2D3 were less capable of inducing the pro-inflammatory loop upon interaction with RASF. Conclusions 1,25(OH)2D3 inhibits the pathogenic Th17 phenotype in CCR6+ Th cells, while inducing a regulatory Tr1-like phenotype. These cells will then migrate towards the site of inflammation, where they are less potent activators of RASF. This effect of 1,25(OH)2D3 on CCR6+ Th cells may underlie the suppression of RA by vitamin D.

A6.32 1,25(OH)2D3 suppresses the pro-inflammatory TH17-RASF feedback loop via IL-4-dependent and –independent mechanisms

Background and objectives Vitamin D has suppressive effects on autoimmune diseases, such as rheumatoid arthritis (RA). Within these diseases, Th17 cells are considered to play a crucial role in the processes underlying chronic inflammation. We have shown that Th17 cells from RA patients induce a pro-inflammatory feedback loop upon interaction with RA synovial fibroblasts (RASF). Interestingly, 1,25(OH)2D3 is able to inhibit this feedback loop by modulating Th17 cell activity. However the molecular mechanisms underlying this inhibition by vitamin D are currently unclear. Materials and methods CD4+CD45RO+ (memory) and CCR6+ memory T-helper cells were sorted from peripheral blood of patients with early RA and healthy volunteers. They were cultured for three days in the presence or absence of 1,25(OH)2D3 together with synovial fibroblasts from RA patients. The expression of cytokines and transcription factors of interest was analysed using microarray based gene expression profiling, flow cytometry, ELISA and/or RT-PCR. Results In the presence of 1,25(OH)2D3 the pro-inflammatory cytokines IL-17A, IL-17F and IL-22 were inhibited. Also the expression of Th17 signature genes like RORgt and IL-23R was reduced. On the other hand we find an increase in IL-4 and IL-10 expression. Interestingly neutralisation of IL-4 partly reversed the effect of 1,25(OH)2D3 on the inhibition of IL-17A, IL22 and RORγt expression. In addition, the inhibition of IL-17F by 1,25(OH)2D3 was almost completely absent when IL-4 was blocked. In contrast to IL-4, IL-10 neutralisation had limited effects in these cultures. Because the effect of 1,25(OH)2D3 is only partially dependent on IL-4, we examined factors that could play a role independent of IL-4. Gene expression profiling revealed that two transcription factors that are known to play a role in Th17 differentiation, EOMES and IRF8, are upregulated by 1,25(OH)2D3. EOMES and IRF8 are direct regulators of RORgt expression. Blocking IL-4 does not affect this up regulation, indicating that EOMES and IRF8 might be important in the IL-4 independent regulating of Th17 polarisation by 1,25(OH)2D3. Conclusions From these findings, we conclude that 1,25(OH)2D3 inhibits the pro-inflammatory feedback loop between Th17 cells and synovial fibroblast. This modulation is partly dependent on up regulation of IL-4. IL-4 independent mechanisms may include the down-regulation of RORgt expression via up regulation of IRF8 and EOMES.

A1.2 Prominent role of pathogenic memory CCR6+ TH17 cell populations in the pathogenesis of ACPA+ patients with rheumatoid arthritis

Background and objectives Patients with rheumatoid arthritis (RA) positive for serum anti-citrullinated protein antibodies (ACPAs) have a worse disease course than ACPA- patients. Association of ACPA and HLA-DRB1 suggests the involvement of T cells in ACPA+ RA. Therefore we examined possible differences in memory CD4+ T (Th) cell distribution between ACPA+ and ACPA- patients and their functional relevance. Materials and methods Th cell distribution profiles from ACPA+ and ACPA- patients with early RA were generated based on chemokine receptor, cytokine and transcription factor expression. On the basis of CXCR3 and CCR4 expression, four CCR6+ subpopulations were distinguished: Th17, Th17.1, CCR4/CXCR3 double positive (DP) and double negative (DN) cells. These Th cell populations were analysed for their pathological potential. Results ACPA+ patients had higher proportions of peripheral CCR6+ Th cells, notably Th22, Th17.1 and DP cells. All CCR6+ subpopulations were also present in RA synovial fluid. These subpopulations shared Th17 characteristics including RORC and CD161 expression. However, expression of IL-17A, IL-17F, IFNg and the Th1-associated transcription factor TBX21 differed markedly between CCR6+ subpopulations. Nevertheless, all CCR6+ Th cell subpopulations showed higher pathological activity than naive and Th1 cells, as they were more effective in stimulating IL-1β, IL-6, IL-8, COX-2 and MMP-3 expression by synovial fibroblasts. Interestingly, CCR6+ Th populations are inverse correlated with disease duration in ACPA- patients but not in ACPA+ patients. Conclusions ACPA+ and ACPA- patients can be distinguished by differences in pathogenic memory CCR6+ Th cell proportions, suggesting that these cells are involved in the worse disease course observed in ACPA+ RA.

A4.1 1.25(OH)2D3 Modulates Gene Expression Involved in Phenotype Stability and Migration of Th17 Cells from Patients with Rheumatoid Arthritis

Background and Objectives Vitamin D has suppressive effects on autoimmune diseases, such as rheumatoid arthritis (RA). Within these diseases, T-helper-17 (Th17) cells have been implicated to play a crucial role in the development and progression of chronic inflammation. Recently, we have found that the active vitamin D compound, 1.25(OH)2D3, has direct suppressive effects on both human and mouse Th17 cytokine expression and activity. Using gene-expression profiling, we aim to identify molecular targets of 1.25(OH)2D3 signalling underlying this suppressive action of 1.25(OH)2D3 in Th17 cells. Methods Primary Th17 cells were sorted from peripheral blood of treatment naïve patients with early RA and cultured with or without 1.25(OH)2D3. From these cultures gene-expression profiles were generated. Expression of genes of interest was confirmed by Q-PCR and/or specific ELISA. Results In the presence of 1.25(OH)2D3, protein expression of Th17 associated cytokines IL-17A and IL-22 was inhibited, while in contrast the anti-inflammatory cytokine IL-10 was induced. These findings were supported by the gene-expression profiles from these cultures. Furthermore, 1.25(OH)2D3 inhibited transcription of the cytokine receptors IL-23R and IL-7R, which are involved in Th17 survival and proliferation. Chemokines CCL20 and CXCL10 were down-regulated and chemokine receptors CCR2, CXCR6, CXCR3 and CCR10 were up-regulated. Importantly, RORγ t, which is critically involved in Th17 differentiation and function and the cell-size regulator and oncogene c-Myc were down-regulated by 1.25(OH)2D3. Conclusions From these findings, we concluded that 1.25(OH)2D3 modulates the expression of genes involved in cytokine production, proliferation, and migration of Th17 cells. These data indicate that 1.25(OH)2D3 not only suppresses Th17 cell activity but also regulates Th17 phenotype stability and migration of these cells to sites of tissue inflammation in RA.

THU0048 1,25(OH)2D3 Inhibits Th17 Polarization and RORC Expression Through GATA3-Dependent and -Independent Mechanisms

Background and Objectives Vitamin D has suppressive effects on autoimmune diseases, such as rheumatoid arthritis (RA). Regulation of Th17 cell activity is an important mechanism by which vitamin D exerts these effects. Aside from inhibiting Th17 cytokines and the Th17 transcription factor RoRγ t, vitamin D induces IL-4 and GATA3. Since GATA3 over-expression inhibits experimental Th17-mediated autoimmunity, we studied the contribution of GATA3 in vitamin D-mediated suppression of Th17 polarisation. Methods Therefore CD4+ T cells were sorted from patients with early RA, naïve DBA-1 mice, DBA-1 mice immunised with collagen type II (CII) or naïve CD2-GATA3 transgenic mice and cultured under T helper cell polarising conditions with or without 1.25(OH)2D3, the active form of vitamin D. Results 1.25(OH)2D3 inhibits Th17 polarisation in CD4+ cells from both non-immunised and CII-immunised mice, while up-regulating IL-4 and GATA3 expression. In these cultures, IL-4 inhibition partly reversed the vitamin D-mediated inhibition of Th17 polarisation. Moreover, GATA3 over-expression reduces Th17 differentiation to a lower level than 1.25(OH)2D3. Interestingly, combining GATA3 over-expression and 1.25(OH)2D3 treatment reduced IL-17A and RoRγ t expression even further. Furthermore, gene-expression analysis showed that NFAT-C2, which is involved in IL-17A production, was down-regulated by 1.25(OH)2D3. In addition, in T cells from patients with RA, 1.25(OH)2D3 inhibited Th17 cytokine and RORγ t expression and induced IL-4 and GATA3 expression. Conclusions These data show that vitamin D-mediated regulation of Th17 polarisation occurs through GATA3-dependent mechanisms, including direct effects on RORγ t expression and IL-4-mediated inhibition of Th17 polarisation. Moreover, GATA3-independent mechanisms are involved that may include modulation of NFAT-C2 expression.

THU0036 Synovial Fibroblasts Directly Induce Th17 Pathogenicity VIA the Cyclooxygenase/Prostaglandin-E2 Pathway, Independent of IL-23

Background Th17 cells are critically involved in autoimmune disease induction and severity. Recently, we showed that Th17 cells, but not Th1 cells, from patients with rheumatoid arthritis (RA) upon interactionwith RA-synovial-fibroblasts (RASF) directly induced autocrine IL-17A production. This autocrine IL-17A production in turn creates a pro-inflammatory loop characterized by up-regulation of the pro-inflammatory cytokines IL-6 and IL-8, and the cartilage degrading enzymes MMP-1 and MMP-3. This loop may be an important pathway in the progression of an early inflammatory arthritis, towards a chronic destructive arthritis. However, the mechanism underlying the autocrine IL-17A production in the pro-inflammatory loop upon Th17-RASF interaction is currently unknown. Objectives To investigate the mechanism responsible for the autocrine IL-17A induction upon Th17-RASF interaction. Methods CD4+CD45RO+CCR6+ (Th17) and CD4+CD45RO+CCR6- (Th1) cells were isolated by FACS sorting from healthy controls and early RA patients. These cells were co-cultured with RASF, in the presence of neutralizing antibodies directed against soluble IL-6R (anti-sIL-6R), and/or IL-1β, and/or IL-23, etanercept, and celecoxib. Gene expression profiles were generated and supernatant was collected for cytokine analyses by ELISA. Results IL-6, IL-1β and cyclooxygenase-2 (COX-2) expressions and prostaglandin-E2 (PGE2) production in Th17-RASF cultures were higher than in Th1-RASF cultures. Cytokine neutralization showed that IL-1β, IL-6 and IL-23 contributed marginally to the IL-17A induction. In contrast, treatment with celecoxib, a COX-2 inhibiter, resulted in significantly lower PGE2 and IL-17A production. This effect was IL-17A specific as no inhibitory effects were found on IFN-γ and TNF-α production. Combined celecoxib and TNF-α blockade more effectively suppressed the pro-inflammatory loop than did single treatment, as shown by lower IL-6, IL-8, matrix metalloproteinase-1 (MMP-1) and MMP-3 production. Conclusions 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 other Th17-mediated autoimmune disorders. Disclosure of Interest None Declared