Nadine Davelaar

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.

TNF blockade requires 1,25(OH)2D3 to control human Th17-mediated synovial inflammation

Objectives T helper 17 (Th17) cells from patients with early rheumatoid arthritis (RA) induce a proinflammatory feedback loop upon RA synovial fibroblast (RASF) interaction, including autocrine interleukin (IL)-17A production. A major challenge in medicine is how to control the pathogenic Th17 cell activity in human inflammatory autoimmune diseases. The objective of this study was to examine whether tumour necrosis factor (TNF) blockade and/or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) controls Th17-mediated synovial inflammation. Methods Peripheral CD4+CD45RO+CCR6+ Th17 cells of patients with early RA, Th17–RASF cocultures and synovial biopsy specimens were cultured with or without 1,25(OH)2D3 and/or TNFα blockade. Intracellular cytokine expression was detected by flow cytometry. Cytokine and matrix metalloprotease (MMP) production was determined by ELISA. Results The authors show that the 1,25(OH)2D3, but not TNFα blockade, significantly suppressed autocrine IL-17A production in Th17–RASF and synovial biopsy cultures. Combining 1,25(OH)2D3 and TNFα blockade had a significant additive effect compared with single treatment in controlling synovial inflammation, indicated by a further reduction in IL-6, IL-8, MMP-1 and MMP-3 in Th17–RASF cocultures and IL-6 and IL-8 expression in cultures of RA synovial tissue. Conclusions These data show that TNF blockade does not suppress IL-17A and IL-22, which can be overcome by 1,25(OH)2D3. The combination of neutralising TNF activity and 1,25(OH)2D3 controls human Th17 activity and additively inhibits synovial inflammation. This indicates more valuable therapeutic potential of activation of Vitamin D receptorsignalling over current TNF neutralisation strategies in patients with RA and potentially other Th17-mediated inflammatory diseases.

T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?

IntroductionA hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6+ memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature.MethodsIn total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN+) (n = 16) and negative (IFN-) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4+CD45RO+CCR6+ T cells (CCR6+ cells) was measured with flow cytometry and compared between IFN+, IFN- patients and HCs.ResultsIncreased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6+ cells were observed in IFN+ patients compared with IFN- patients and HCs. IL-17A and IL-17F expression within CCR6+ cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)–a factor strongly correlating with IFN type I - and IL-21 producing CCR6+ cells.ConclusionsWe show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6+ memory T-helper cells in IFN+ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.

CCR6(+) Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis.

IntroductionPatients with rheumatoid arthritis (RA) can be separated into two major subpopulations based on the absence or presence of serum anti-citrullinated protein antibodies (ACPAs). The more severe disease course in ACPA+ RA and differences in treatment outcome between these subpopulations suggest that ACPA+ and ACPA− RA are different disease subsets. The identification of T-helper (Th) cells specifically recognizing citrullinated peptides, combined with the strong association between HLA-DRB1 and ACPA positivity, point toward a pathogenic role of Th cells in ACPA+ RA. In this context we recently identified a potential pathogenic role for CCR6+ Th cells in RA. Therefore, we examined whether Th cell population distributions differ by ACPA status.MethodsWe performed a nested matched case–control study including 27 ACPA+ and 27 ACPA− treatment-naive early RA patients matched for disease activity score in 44 joints, presence of rheumatoid factor, sex, age, duration of complaints and presence of erosions. CD4+CD45RO+ (memory) Th cell distribution profiles from these patients were generated based on differential chemokine receptor expression and related with disease duration.ResultsACPA status was not related to differences in total CD4+ T cell or memory Th cell proportions. However, ACPA+ patients had significantly higher proportions of Th cells expressing the chemokine receptors CCR6 and CXCR3. Similar proportions of CCR4+ and CCR10+ Th cells were found. Within the CCR6+ cell population, four Th subpopulations were distinguished based on differential chemokine receptor expression: Th17 (CCR4+CCR10−), Th17.1 (CXCR3+), Th22 (CCR4+CCR10+) and CCR4/CXCR3 double-positive (DP) cells. In particular, higher proportions of Th22 (p = 0.02), Th17.1 (p = 0.03) and CCR4/CXCR3 DP (p = 0.01) cells were present in ACPA+ patients. In contrast, ACPA status was not associated with differences in Th1 (CCR6−CXCR3+; p = 0.90), Th2 (CCR6−CCR4+; p = 0.27) and T-regulatory (CD25hiFOXP3+; p = 0.06) cell proportions. Interestingly, CCR6+ Th cells were inversely correlated with disease duration in ACPA− patients (R2 = −0.35; p < 0.01) but not in ACPA+ (R2 < 0.01; p = 0.94) patients.ConclusionsThese findings demonstrate that increased peripheral blood CCR6+ Th cells proportions distinguish ACPA+ RA from ACPA− RA. This suggests that CCR6+ Th cells are involved in the differences in disease severity and treatment outcome between ACPA+ and ACPA− RA.

Vitamin D suppresses the pathogenic behaviour of primary Th17 cells from patients with early rheumatoid arthritis

Background and objectives Recently, the authors showed that CCR6+ Th17 cells from early rheumatoid arthritis (RA) patients are potent inducers of a pro-inflammatory feed-back loop upon RA synovial fibroblast (RASF) interaction, including autocrine interleukin (IL)-17A production. In this study, the effect of vitamin D on this pathogenic behaviour of Th17 cells was investigated. Materials and methods Peripheral CCR6+ Th17 cells of patients with early RA, CCR6+ Th17/RASF co-cultures and synovial biopsies of patients with established RA, were cultured in the absence or presence of 1,25(OH)2D3, the active vitamin D metabolite and/or Etanercept. Intracellular cytokine expression was detected by flow cytometry. Cytokine and matrix metalloprotease (MMP) expression was determined by ELISA. Transcription of factors involved in Th differentiation and function was analysed by quantitative PCR analysis. Results In Th17 cultures and Th17/RASF co-cultures vitamin D suppressed the expression of IL-17A, IL-22 and IFN-γ. Furthermore, in Th17/RASF co-cultures, vitamin D induced IL-4 and IL-10 expression, which was accompanied with reduced Rorγt and induced Gata3 transcription. Moreover, vitamin D suppressed IL-6, IL-8, MMP-1 and MMP-3 expression in the Th17/RASF co-cultures and synovial biopsies. Interestingly, the enhanced autocrine production of IL-17A in Th17/RASF co-cultures was inhibited by vitamin D, but not with tumour necrosis factor (TNF)-α neutralisation. Vitamin D in combination with TNF-α neutralisation has an additive effect on the suppression of Th17 activity as indicated by a significant further reduction in IL-6, IL-8, MMP-1 and MMP-3 expression in the co-cultures and IL-6 and IL-8 in the synovial biopsy cultures. Conclusions These data show that vitamin D modulates the pathogenic behaviour of Th17 cells and may have additional therapeutic potential when used in combination with TNF treatment in RA and potentially other Th17-mediated inflammatory diseases.

The COX/prostaglandin-E2 pathway is critical for autocrine IL-17A production by TH17 cells upon synovial fibroblast interaction

Backgroundand objectives Recently, the authors have shown that Th17, but not Th1 cells, from patients with early rheumatoid arthritis (RA) are potent activators of RA synovial fibroblasts (RASF) resulting in autocrine IL-17A production. This autocrine IL-17A production by Th17 cells is critical for the perseverance of the pro-inflammatory loop, but the mechanism underlying the autocrine IL-17A induction is unclear. Objective To investigate the mechanism responsible for the autocrine IL-17A induction upon Th17-RASF interaction. Materials and 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 neutralising antibodies directed against soluble IL-6R (anti-sIL-6R) and/or IL-1β, and celecoxib. Gene expression profiles were generated and supernatant was collected for cytokine analyses by ELISA. Results Gene expression analyses revealed that the genes encoding for IL-6 and IL-1β were up-regulated in Th17-RASF cultures. These data were confirmed by ELISA and Q-PCR, respectively. Since IL-1β and IL-6 may be involved in IL-17/Th17 polarisation the authors examined the contribution of these cytokines on the autocrine IL-17A loop. Blockade of IL-1β and IL-6 activity, but not TNFα significantly suppressed IL-17A production in the co-culture, but the inhibitory effects were limited. Of note, the combination of anti-IL-1β and anti-sIL-6R markedly suppressed IL-17F. No effects were found on IFNγ. Interestingly, the genes encoding for cyclo-oxygenase-2 (COX-2) and prostaglandin-E-synthase (PTGES), which are involved in prostaglandin-E2 (PGE2) synthesis, were also up-regulated in Th17-RASF cultures. This was associated with a dramatic increase of PGE2 production in Th17-RASF cultures compared to Th1-RASF cultures. Treatment of Th17-RASF cultures with celecoxib, an inhibitor of cyclo-oxygenase-2 (COX-2) activity, resulted in a significant decrease of IL-17A, but not of IFNγ and TNFα production. Furthermore, the decrease in IL-17A production caused by celecoxib treatment was much more pronounced compared to neutralising IL-1 and IL-6 activity. Flow cytometry revealed a decrease in Th17 cells in celecoxib treated co-cultures, while no effects on Th1 or Th2 cells were found. Moreover, celecoxib treatment significantly inhibited the pro-inflammatory loop as less induction of IL-6, IL-8 and matrix metalloproteinase-3 production was observed in the Th17-RASF co-cultures. Conclusions These findings show the critical role of the COX/PGE2 pathway in the autocrine IL-17A production upon Th17 and synovial fibroblast interaction. Inhibition of this pathway down-regulates the pro-inflammatory feedback loop induced by Th17-RASF interaction leading to less production of pro-inflammatory cytokines and destructive mediators.

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.

A1.76 Higher proportions of pathogenic CCR6+ T cell subpopulations in ACPA+ compared to ACPA- patients with early rheumatoid arthritis

Background and Objectives Presence of serum anti-citrullinated protein antibodies (ACPAs) in patients with rheumatoid arthritis (RA) predicts worse disease course and a more erosive disease. In the pathogenesis of RA, inflammatory T cells play a central role. In this context, we recently found increased proportions of pathogenic peripheral CCR6+ memory T helper cells in patients with early RA compared to healthy individuals. However, it is unclear how T cell proportions are distributed between ACPA+ and ACPA- patients. Therefore alterations in peripheral T cell populations and their pathogenic potential were examined in ACPA+ and ACPA- patient groups. Materials and Methods A nested matched case control study was performed including 27 ACPA+ and 27 ACPA- patients with early RA. T cell profiles (Treg, Th1, Th2, Th17, Th22 and various less well classified populations) from these ACPA+ and ACPA- patients were generated based on chemokine receptor, cytokine and transcription factor expression. Differentially present T cell populations were isolated from peripheral blood and analysed for their pathologic potential in a co-culture system with RA derived synovial fibroblasts (RASF). Results In comparison to ACPA- patients, ACPA+ patients have higher proportions of regulatory T cells (Treg) and CD4+ memory CCR6+ T cells. Since the CCR6+ T cell population is still a heterogeneous population, four CCR6+ T cell subpopulations were distinguished by differential expression of CXCR3 and CCR4. All four CCR6+ subpopulations shared Th17 cell characteristics such as Rorγt and CCL20 expression, but IL-17A, IL-17F, IL-22 and IFN-γ expression differed greatly between these subpopulations. However, even the population with lowest expression of these cytokines showed high pathological potential as shown by stimulating IL-1β, IL-6, IL-8, COX-2 and MMP-3 expression upon co-culture with RASF. Indeed, despite dissimilar Th17 and Th1 characteristics between the CCR6+ subpopulations, all four showed highly increased pathological potential in co-culture compared to naive and Th1 cells. Conclusions ACPA+ and ACPA- patients can be distinguished by the distribution of Treg and CCR6+ T cell subpopulations. These CCR6+ subpopulations exhibit dissimilar Th17 and Th1 characteristics, but all possess high pathological potential, including the population that has low IL-17A/F and IL-22 expression. These findings indicate a prominent role of CCR6+ T cells in the pathogenesis of ACPA+ patients with early RA and may contribute to the worse disease outcome in ACPA+ patients.

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.

AB0118 The lymphatic system: a gatekeeper for migration of pathogenic t-cells towards synovial joints and entheses in psoriasis

Background The factors underlying the transition of psoriasis (PsO) to psoriatic arthritis (PsA) are poorly understood. The lymphatic system may control the homing of disease-associated T-cells to skin and extra-cutaneous sites like synovial joints and entheses. Objectives To study the capacity of lymphatic endothelial cells (LEC) to regulate T-cell homing capabilities in PsA. Methods Human dermal LEC (0.5x104), and fibroblast-like synoviocytes of a patient with PsA (PsA-FLS; 1.0x104) were pre-incubated for 3 days with PsA synovial fluid (PsA-SF; 0/10/20% v/v) and co-cultured with 2.5x104 allogeneic CD4+CD45RO+CD25- T-cells that were sorted from peripheral blood mononuclear cells of 3 donors (with or without stimulation with αCD3/αCD28). After 72 h, T-cells were analyzed by flow cytometry. The CCR6+ T-helper (Th) subsets Th17.1 (CCR4-/CXCR3+), Th17/Th22 (CCR4+/CXCR3-), Th17 (CCR4+/CXCR3-/CCR10-) and Th22 (CCR4+/CXCR3-/CCR10+), and CCR6- subsets Th1 (CCR4-/CXCR3+), and Th2 (CCR4+/CXCR3-) were identified. We also looked at cutaneous lymphocyte- associated antigen (CLA). IL-17A, IL-22, and TNF protein levels were determined by ELISA. Statistical analysis included unpaired t-test (two-sided) for two-group comparison or one-way ANOVA with the Tukey-Kramer post hoc test for multi-group comparisons. Results Stimulation of CD4+CD45RO+ T-cells in co-culture with PsA-FLS skewed towards the CCR6+ subset Th17/Th22, which were predominantly Th17 cells. Th17 differentiation upon stimulation was suppressed in co-culture with LEC, even when LEC were pre-incubated with PsA-SF. T-cell stimulation in co-culture with LEC, as compared to PsA-FLS, promoted the generation of the Th22 subset. Upon co-culture with untreated LEC, stimulated T-cells showed higher expression of the skin homing receptor CLA than those that were co-cultured with PsA-FLS. The proportional reduction in CLA expression on T-cells in the co-cultures with LEC pre-incubated with PsA-SF 20% was comparable to PsA-FLS, however LEC conserved CLA expression on CD4+CD45RO T-cells at a higher level than PsA-FLS; this phenomenon particularly affected the CCR6+ T-cells. In line, a trend towards lower IL-17A and higher IL-22 levels were observed in the co-cultures with LEC that were pretreated with PsA-SF 20%, as compared to the co-culture with PsA-FLS. No differences were seen for TNF protein levels. Conclusions LECs are directly involved in T-cell differentiation and homing capabilities, as shown by suppression of Th17 differentiation upon stimulation, as compared to PsA-FLS. Also, LEC promoted Th22 generation, and conserved CLA expression in CCR6+ T-cells, even when LEC were preincubated with PsA-SF. Studies are underway to confirm that LECs from relevant biological tissues (e.g. synovium and lymph nodes) are critical for tissue-restricted T-cell migration to skin and synovial membranes in PsA. Disclosure of Interest None declared