Wendy Dankers

The role and modulation of CCR6+ Th17 cell populations in rheumatoid arthritis

The IL-17A producing T-helper-17 (Th17) cell population plays a major role in rheumatoid arthritis (RA) pathogenesis and has gained wide interest as treatment target. IL-17A expressing Th cells are characterized by the expression of the chemokine receptor CCR6 and the transcription factor RORC. In RA, CCR6+ Th cells were identified in peripheral blood, synovial fluid and inflamed synovial tissue. CCR6+ Th cells might drive the progression of an early inflammation towards a persistent arthritis. The CCR6+ Th cell population is heterogeneous and several subpopulations can be distinguished, including Th17, Th22, Th17.1 (also called non-classic Th1 cells), and unclassified or intermediate populations. Interestingly, some of these populations produce low levels of IL-17A but are still very pathogenic. Furthermore, the CCR6+ Th cells phenotype is unstable and plasticity exists between CCR6+ Th cells and T-regulatory (Treg) cells and within the CCR6+ Th cell subpopulations. In this review, characteristics of the different CCR6+ Th cell populations, their plasticity, and their potential impact on rheumatoid arthritis are discussed. Moreover, current approaches to target CCR6+ Th cells and future directions of research to find specific CCR6+ Th cell targets in the treatment of patients with RA and other CCR6+ Th cell mediated autoimmune diseases are highlighted.

Vitamin D in Autoimmunity: Molecular Mechanisms and Therapeutic Potential

Over the last three decades, it has become clear that the role of vitamin D goes beyond the regulation of calcium homeostasis and bone health. An important extraskeletal effect of vitamin D is the modulation of the immune system. In the context of autoimmune diseases, this is illustrated by correlations of vitamin D status and genetic polymorphisms in the vitamin D receptor with the incidence and severity of the disease. These correlations warrant investigation into the potential use of vitamin D in the treatment of patients with autoimmune diseases. In recent years, several clinical trials have been performed to investigate the therapeutic value of vitamin D in multiple sclerosis, rheumatoid arthritis, Crohn’s disease, type I diabetes, and systemic lupus erythematosus. Additionally, a second angle of investigation has focused on unraveling the molecular pathways used by vitamin D in order to find new potential therapeutic targets. This review will not only provide an overview of the clinical trials that have been performed but also discuss the current knowledge about the molecular mechanisms underlying the immunomodulatory effects of vitamin D and how these advances can be used in the treatment of autoimmune diseases.

1,25(OH)2D3 modulates gene expression involved in cytokine production, proliferation, survival and migration of TH17 cells from patients with rheumatoid arthritis

Background 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, the authors 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, the authors aim to identify molecular targets of 1,25(OH)2D3 signaling underlying this suppressive action of 1,25(OH)2D3 in Th17 cells. Materials and 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, Rorγt, 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, the authors concluded that 1,25(OH)2D3 modulates the expression of genes involved in cytokine production, proliferation, survival and migration of Th17 cells. These data indicate that 1,25(OH)2D3 not only suppresses Th17 cell activity but also regulates migration of these cells to sites of tissue inflammation in RA.

T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention

Interleukin-17-expressing CD4+ T helper 17 (Th17) cells are considered as critical regulators of multiple sclerosis disease activity. However, depending on the species and pro-inflammatory milieu, Th17 cells are functionally heterogeneous, consisting of subpopulations that differentially produce interleukin-17, interferon-gamma and granulocyte macrophage colony-stimulating factor. In the current study, we studied distinct effector phenotypes of human Th17 cells and their correlation with disease activity in multiple sclerosis patients. T helper memory populations single- and double-positive for C-C chemokine receptor 6 (CCR6) and CXC chemokine receptor 3 (CXCR3) were functionally assessed in blood and/or cerebrospinal fluid from a total of 59 patients with clinically isolated syndrome, 35 untreated patients and 24 natalizumab-treated patients with relapsing-remitting multiple sclerosis, and nine patients with end-stage multiple sclerosis. Within the clinically isolated syndrome group, 23 patients had a second attack within 1 year and 26 patients did not experience subsequent attacks during a follow-up of >5 years. Low frequencies of T helper 1 (Th1)-like Th17 (CCR6+CXCR3+), and not Th17 (CCR6+CXCR3-) effector memory populations in blood strongly associated with a rapid diagnosis of clinically definite multiple sclerosis. In cerebrospinal fluid of clinically isolated syndrome and relapsing-remitting multiple sclerosis patients, Th1-like Th17 effector memory cells were abundant and showed increased production of interferon-gamma and granulocyte macrophage colony-stimulating factor compared to paired CCR6+ and CCR6-CD8+ T cell populations and their blood equivalents after short-term culturing. Their local enrichment was confirmed ex vivo using cerebrospinal fluid and brain single-cell suspensions. Across all pro-inflammatory T helper cells analysed in relapsing-remitting multiple sclerosis blood, Th1-like Th17 subpopulation T helper 17.1 (Th17.1; CCR6+CXCR3+CCR4-) expressed the highest very late antigen-4 levels and selectively accumulated in natalizumab-treated patients who remained free of clinical relapses. This was not found in patients who experienced relapses during natalizumab treatment. The enhanced potential of Th17.1 cells to infiltrate the central nervous system was supported by their predominance in cerebrospinal fluid of early multiple sclerosis patients and their preferential transmigration across human brain endothelial layers. These findings reveal a dominant contribution of Th1-like Th17 subpopulations, in particular Th17.1 cells, to clinical disease activity and provide a strong rationale for more specific and earlier use of T cell-targeted therapy in multiple sclerosis.

1,25(OH) 2 D 3 and dexamethasone additively suppress synovial fibroblast activation by CCR6 + T helper memory cells and enhance the effect of tumor necrosis factor alpha blockade

BackgroundDespite recent improvements in the treatment of rheumatoid arthritis (RA), an insufficient treatment response and the development of treatment resistance in many patients illustrates the need for new therapeutic strategies. Chronic synovial inflammation could be suppressed by targeting RA synovial fibroblast (RASF) activation by, for example, interleukin (IL)-17A-producing CCR6+ T helper memory (memTh) cells. Here, we modulated this interaction by combining the active vitamin D metabolite 1,25(OH)2D3 with dexamethasone (DEX) and explored the potential therapeutic applications.MethodsCCR6+ memTh cells from peripheral blood mononuclear cells (PBMCs) of healthy donors or treatment-naive early RA patients were cultured alone or with RASF from established RA patients for 3 days and treated with or without 1,25(OH)2D3, DEX, or etanercept. Treatment effects were assessed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry.Results1,25(OH)2D3, and to lesser extent DEX, reduced production of the pro-inflammatory cytokines IL-17A, IL-22, and interferon (IFN)γ in CCR6+ memTh cells. Tumor necrosis factor (TNF)α was only inhibited by the combination of 1,25(OH)2D3 and DEX. In contrast, DEX was the strongest inhibitor of IL-6, IL-8, and tissue-destructive enzymes in RASF. As a result, 1,25(OH)2D3 and DEX additively inhibited inflammatory mediators in CCR6+ memTh-RASF cocultures. Interestingly, low doses of mainly DEX, but also 1,25(OH)2D3, combined with etanercept better suppressed synovial inflammation in this coculture model compared with etanercept alone.ConclusionThis study suggests that 1,25(OH)2D3 and DEX additively inhibit synovial inflammation through targeting predominantly CCR6+ memTh cells and RASF, respectively. Furthermore, low doses of DEX and 1,25(OH)2D3 enhance the effect of TNFα blockade in inhibiting RASF activation, thus providing a basis to improve RA treatment.

OP0115 Improved response to etanercept is associated with serum vitamin d levels in rheumatoid arthritis

Background Although treatment of rheumatoid arthritis (RA) has significantly improved during the past decades, many patients do not adequately respond or become resistant to current treatments. It is currently unknown why some patients respond well and others do not, and how the response rate could be improved. Vitamin D has strong immunomodulatory properties and it has been shown RA patients have a lower serum 25(OH)D level than healthy individuals. Moreover, vitamin D levels are correlated with disease severity.1 2 Interestingly, in vitro studies have shown that vitamin D augments the suppressive effects of etanercept in a simplified model for synovial inflammation.3 This suggests that vitamin D could improve the therapeutic response to etanercept in RA patients. Objectives To investigate if etanercept response is related to serum vitamin D (25(OH)D) levels in RA patients. Methods For this study, data were used from the tREACH trial, a multicenter stratified single blinded randomised clinical trial. RA patients, according to the 2010 classification criteria, who started with etanercept within the first 12 months of the study were included in the analysis. Serum vitamin D (25(OH)D) levels were determined at the start of treatment (Tstart) and 3 months later using the LIAISON 25 OH Vitamin D TOTAL assay. Correlation coefficients between vitamin D levels and the disease activity score (DAS) were calculated. Treatment response was determined with the EULAR response criteria, and difference in response rates was assessed using Chi-Square tests. Results 91 patients started etanercept in the first 12 months of the study, of which 24 did not have serum for 25(OH)D measurements at both start of treatment and three months later. Therefore, a total of 67 patients was included in this study, of which 82% was female. At baseline, 45 (67%) and 48 (73%) were positive for rheumatoid factor and anti-citrullinated protein antibodies, respectively. DAS after etanercept treatment was weakly inversely correlated with serum 25(OH)D after treatment (r=-0.29, p=0.02) and the change in 25(OH)D during treatment (r=-0.25, p=0.04). After correcting for DAS and serum 25(OH)D at the start of treatment the aforementioned correlations were still found. Importantly, EULAR response rate was significantly lower in patients who were vitamin d-deficient at the start of treatment (34.6% vs 59.4%) and in patients with decreasing 25(OH)D levels during treatment (39.2% vs 57.7%) (figure 1). Conclusions RA patients with a serum 25(OH)D level below 50 nmol/L at the start of etanercept treatment or a decreasing level during treatment have a lower EULAR response rate. Therefore, increasing serum 25(OH)D level in vitamin D deficient patients may be important to achieve optimal effects of TNFα blocking therapy. References [1] Lee YH, Bae SC. Clin Exp Rheumatol2016;34(5):827–33. [2] Lin J, Liu J, Davies ML, et al. PLoS One2016;11(1):e0146351. [3] van Hamburg JP, Asmawidjaja PS, Davelaar N, et al. Ann Rheum Dis2012;71(4):606–12. Disclosure of Interest None declared

03.22 Vitamin d utilises different pathways to inhibit th17 differentiation dependent on the maturation stage of the cd4+t cell

Background In many human autoimmune diseases, including rheumatoid arthritis (RA), vitamin D is associated with disease suppression. Furthermore, experimental collagen-induced arthritis (CIA) is prevented by 1,25(OH)2D3 supplementation, the active form of vitamin D. Since high doses of 1,25(OH)2D3 in humans can cause serious side effects, understanding the mechanism behind the suppression may lead to new therapeutic targets. Although currently this mechanism is not known, it is hypothesised that Th17 cells play an important role in the immunomodulatory effect of vitamin D. Here we study the effect of 1,25(OH)2D3 on the differentiation of Th17 cells and the underlying mechanism in the context of the autoimmune-mediated CIA model. Materials and methods DBA/1 mice were immunised with type II collagen (CII) in complete Freund’s adjuvant. 10 days after immunisation the mice were sacrificed and spleens were harvested. Total CD4+, naïve CD4+ or effector CD4+ T cells were isolated and cultured for 3–7 days under Th17 polarising conditions with or without 1,25(OH)2D3. Results 1,25(OH)2D3 inhibits Th17 differentiation in total CD4+ T cells in CII-immunised mice. Interestingly, 1,25(OH)2D3 induces IL-4 and IL-10 in these cells. Therefore we investigated whether the effect of 1,25(OH)2D3 on Th17 differentiation was dependent on IL-4 or IL-10. In both naïve and effector CD4+ T cells from CII-immunised mice, 1,25(OH)2D3 inhibited Th17 differentiation as demonstrated by decreased IL-17A, IL-17F and IL-22. Blocking IL-10 did not prevent this effect in either subsets. However, in the effector cells, but not the naïve cells, blocking IL-4 partially reduced the effect of 1,25(OH)2D3 on Th17 differentiation. Conclusions 1,25(OH)2D3 similarly inhibits Th17 differentiation of naïve and effector CD4+ T cells from CII-immunised mice. However, only the inhibition in effector cells, not in naïve cells, is partially dependent on IL-4. These data show that 1,25(OH)2D3utilises different pathways to inhibit Th17 differentiation, depending on the maturation stage of the CD4+ T cell.

A2.31 Immunisation with type II collagen (CII) alters the IL-23 receptor expression profile compared to naïve conditions

Background and objectives Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterised by synovial infiltration of pro-inflammatory cells. In RA, the interleukin-23/17A (IL-23/IL-17A) pathway plays a central role. While in human RA increased IL-23 levels correlate with higher disease activity, IL-23deficient mice are protected against collagen induced arthritis. Under naïve conditions, it was shown that IL-23 receptor (IL-23R) positivecells were mainly located in the lymph nodes (LNs), but not in the spleens of IL-23R GFP-reporter mice. While these cells were mainly composed of T cells, macrophages and dendritic cells (DCs), no IL-23R expression was found on CD8+ T cells, B cells or NK cells. However, since IL-23R expression can be altered in an inflammatory condition, we aimed to analyse expression of IL-23R on immune cells in lymphoid tissues of type II collagen (CII) immunised mice. Materials and methods IL-23R GFP-reporter mice and littermate wild type controls were immunised with 100 µg chicken type II collagen in complete Freund’s adjuvant. After 10 days, LNs and spleens of mice were harvested and IL-23R expressing cell types were analysed using flow cytometry. In addition, splenocytes of IL-23R GFP-reporter mice were cultured overnight with IL-23, in order to assess their responsiveness. Results In contrast to naïve mice, IL-23R expression was detected on B cells and CD8+ T cells in LNs of CII immunised mice. While T cells were the most prominent IL-23R-expressing cell type in the LNs, DCs formed the main IL-23R+ population in the spleens. Furthermore, IL-23R expression was found on splenic B cells. Within the T cell population, γδ T cells form the largest IL-23R+ cell type in both the LNs and spleens. In vitro, splenocytes of IL-23R GFP-reporter mice respond to IL-23 by upregulating IL-23R gene expression. Conclusions In contrast to naïve mice, IL-23R+ cells such as DCs, B cells and γδ T cells were detected in the spleens of CII immunised mice. Furthermore, γδ T cells, B cells and CD8+ T cells express IL-23R in the LNs. These data suggest that immunisation with CII alters the IL-23R expression profile compared to naïve conditions.

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.