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Basic and translational research
Extended report
TNF blockade requires 1,25(OH)2D3 to control human Th17-mediated synovial inflammation
  1. Jan Piet van Hamburg1,2,
  2. Patrick S Asmawidjaja1,2,
  3. Nadine Davelaar1,2,
  4. Adriana M C Mus1,2,
  5. Ferry Cornelissen1,2,
  6. Johannes P T M van Leeuwen3,
  7. Johanna M W Hazes1,
  8. Radboud J E M Dolhain1,
  9. Pieter A G M Bakx4,
  10. Edgar M Colin1,5,
  11. Erik Lubberts1,2
  1. 1Department of Rheumatology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands
  2. 2Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands
  3. 3Department of Internal Medicine, Erasmus MC, University Medical Center, Rotterdam, The Netherlands
  4. 4Department of Orthopedics, Maasstad Hospital, Rotterdam, The Netherlands
  5. 5Department of Rheumatology, ZGT, Almelo, The Netherlands
  1. Correspondence to Erik Lubberts, Departments of Rheumatology and Immunology, Erasmus MC, University Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands; e.lubberts{at}erasmusmc.nl

Abstract

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.

https://doi.org/10.1136/annrheumdis-2011-200424

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    The cause of rheumatoid arthritis (RA) is largely unknown. However, substantial evidence has emerged supporting the role of T cells and their cytokines in RA initiation and progression.1,–,3 In particular, interleukin (IL)-17A-producing T helper 17 (Th17) cells are attractive targets for RA treatment.4 5 Th17 cells are further characterised by IL-17F and IL-22 production and C-C chemokine receptor6 cell-surface expression.6,–,8 The pathogenic role of IL-17A in murine arthritis has been identified.9 Moreover, mice deficient in factors underlying Th17 differentiation or function are protected against induction and/or progression of arthritis.10 11 In the RA-inflamed joint synovium, IL-17A is expressed and IL-17A-producing T cells have been identified.12,–,16 In addition, we have found increased CCR6+ Th17 cell percentages in peripheral blood of treatment-naive patients with early RA.17 The pathogenic potential of IL-17-producing cells including Th17 cells is further indicated by a decline in disease activity of patients with RA in a clinical anti-IL-17 trial.18

    We recently showed that Th17 cells were potent activators of RA synovial fibroblast (RASF).17 This Th17–RASF interaction revealed a potential Th17 pathogenic activity as shown by: (1) increased production of IL-6 and IL-8 and matrix metalloprotease (MMP)-1 and MMP-3, mediators of cartilage degradation; (2) induction of autocrine IL-17A production, indicating a Th17-induced proinflammatory loop. This loop may be an important pathway in the progression of early inflammatory arthritis to chronic persistent arthritis.17

    A major challenge in medicine is how to control pathogenic Th17 cell activity in human autoimmune-mediated diseases. Interestingly, the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has been shown to affect murine Th17 cytokine expression and function.19 20 The suppressive effects of 1,25(OH)2D3 have been mainly linked to the modulation of functional activities of antigen-presenting cells (APCs).21 22 Although T cell polarisation can indirectly be affected by 1,25(OH)2D3-induced altered APC cytokine expression,23 24 it is becoming clear that T cells, including Th17 cells, are direct 1,25(OH)2D3 targets.25,–,27 In human T cells, 1,25(OH)2D3 suppresses IL-17A and interferon γ (IFNγ) production and stimulates IL-4 and IL-10 production.28,–,30 Moreover, 1,25(OH)2D3 directly reduced the production of the Th17 cytokines, IL-17A, IL-17F and IL-22, by memory T cells of patients with early RA.27 The proinflammatory cytokine, tumour necrosis factor α (TNFα), is a commonly used target for RA treatment.2 However, 1,25(OH)2D3 had no effect on TNFα production by stimulated peripheral blood mononuclear cells, and only limited inhibitory effects on TNFα production by memory T cells.27

    Since 1,25(OH)2D3 has been shown to inhibit IL-17A production by T cells, we hypothesised that VDR pathway activation by 1,25(OH)2D3 may directly regulate the pathogenic activity of Th17 cells from patients with RA. In addition, we hypothesised that 1,25(OH)2D3 has an additional role to TNFα blockade in suppressing synovial inflammation through modulation of Th17 function. These hypotheses were tested using Th17–RASF cocultures from treatment-naïve patients with early RA or RA synovial biopsy cultures from patients with RA undergoing knee joint replacement.

    Here we show direct suppressive effects of 1,25(OH)2D3, but not of TNFα blockade, on IL-17A and IL-22 cytokine expression. Furthermore, the combination of neutralising TNF activity and 1,25(OH)2D3 controls human Th17 activity and additively inhibits synovial inflammation. These data support the development of a clinical trial combining TNF blockade with VDR activation in RA and other Th17-mediated autoimmune diseases.

    Methods

    Subjects

    In this study, 16 treatment-naive patients with early RA (13 women and three men; mean±SD age 47.8±14.4 years) were studied. All patients fulfilled the American College of Rheumatology 1987 revised criteria for RA. Blood was obtained at the second visit after informed consent had been obtained. Clinical and laboratory data for the patients can be found in online supplementary table S1. Data from patients with established RA are available on request. This study was embedded in the Rotterdam Early Arthritis Cohort Study and approved by the medical ethics committee of the Erasmus MC Rotterdam.

    Flow cytometry antibodies and cell sorting

    Monoclonal antibody preparations, intracellular cytokine detection and flow cytometry have been described previously.11 The following monoclonal antibodies were purchased from BD Biosciences (San Diego, California, USA): CD45RO, CCR6, CD4 and IFNγ. IL-22 and IL-17A monoclonal antibodies were from eBioscience (San Diego, California, USA). Samples were acquired on a FACScantoII flow cytometer (BD Biosciences) and analysed using FlowJo v7.6 research software (Tree Star Inc, Ashland, Oregon, USA). T cell populations were sorted from peripheral blood mononuclear cells using a FACSAria cell sorter (BD Biosciences).

    Cell cultures

    T cells (2.5×104) were cultured for 72 h in Iscove's modified Dulbecco's medium (BioWhittaker, Walkersville, MD, United States), supplemented with 10% fetal calf serum, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine and 50 µM β-mercaptoethanol (Merck, Darmstadt, Germany). Cells were stimulated with soluble αCD3 and αCD28 (0.3 and 0.4 µg/ml, respectively; Sanquin, Amsterdam, The Netherlands) and cultured with or without 100 nM 1,25(OH)2D3 (Leo Pharmaceuticals Products, Ballerup, Denmark).

    RASF isolation and subsequent culture has been described.17 RASFs (1.0×104) were cocultured with 2.5×104 allogeneic Th17 cells for 72 h with soluble αCD3/αCD28 and/or 100 nM 1,25(OH)2D3 and/or 10 µg/ml etanercept (Wyeth Pharmaceuticals Inc, Collegeville, Pennsylvania, USA).

    Synovial biopsy specimens (∼2 mm) were taken randomly from synovial tissue obtained from patients with established RA after knee joint replacement. Three biopsy samples per group were cultured for 72 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 U/ml penicillin/streptomycin, in the presence of αCD3/αCD28 and/or 100 nM 1,25(OH)2D3 and/or 10 µg/ml etanercept.

    Cytokine measurements

    IL-4, IL-6, IL-8, IL-10 and IFNγ production was determined using ELISA (Invitrogen, Carlsbad, California, USA). IL-17A, IL-22, TNFα, MMP-1 and MMP-3 expression was measured using Duoset ELISA (R&D systems, Minneapolis, Minnesota, USA). ELISA was performed according to the manufacturer's instructions.

    Statistical analysis

    Differences between experimental groups were tested with a two-sided paired t test or stated otherwise, using Prism software V.5.04 (GraphPad Software Inc. La Jolla, California, USA). p Values <0.05 were considered significant.

    Results

    1,25(OH)2D3 suppressed Th17-associated cytokine expression by primary memory CCR6+ Th17 cells from patients with early RA

    Recently, we showed that 1,25(OH)2D3 suppressed IL-17A and IL-22 expression by CD4+CD45RO+ (memory) T cells from treatment-naive patients with early RA.27 By using CCR6 expression, it is possible to sort primary IL-17A-producing memory T cells (CCR6+ Th17) from peripheral blood.7 17 When these cells of patients with early RA were stimulated in the presence of 1,25(OH)2D3, a significant reduction was found in the percentage of IL-17A- and IL-22-expressing T cells and in IL-17A and IL-22 protein levels in the supernatant (figure 1A,B). In both Th17 and CCR6 memory T cell cultures, 1,25(OH)2D3 reduced the percentage of IFNγ-producing cells and IFNγ protein production (figure 1A,B).

    Figure 1
    Figure 1

    Effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on T helper 17 (Th17)-, Th1- and Th2-associated cytokine expression by peripheral T cells of patients with early rheumatoid arthritis (RA). Sorted memory CCR6+ Th17, memory CCR6− and naive T cells from peripheral blood were stimulated with αCD3/αCD28 and cultured for 3 days with or without 1,25(OH)2D3. (A) Flow cytometric analysis of intracellular interleukin (IL)-17A, IL-22 and interferon (IFN)γ expression by CCR6+ Th17 cells. Numbers in representative dot plots (upper panel) indicate the proportion of cytokine-expressing cells per quadrant. Mean and SEM (lower panel) are given for CCR6+ Th17 cells obtained from eight patients with early RA, cultured in the absence (white bars) or presence (black bars) of 1,25(OH)2D3. (B) Expression of indicated cytokines in supernatant of memory CCR6+ Th17, memory CCR6− and naive T cells in the absence (white bars) or presence (black bars) of 1,25(OH)2D3. Mean and SEM are given for five treatment-naïve patients with early RA. Results are representative of at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001.

    1,25(OH)2D3 is known to induce IL-4 production in murine naive T cell cultures31 and IL-10 production by T cells from patients with multiple sclerosis.30 In CD4+CD45RO− (naive) T cell, memory CCR6− T cell and memory CCR6+ Th17 cell cultures, 1,25(OH)2D3 was not sufficient to induce IL-4 production. IL-10 production was not significantly increased by 1,25(OH)2D3 in memory CCR6− and CCR6+ Th17 cell cultures (figure 1B). TNFα is expressed by activated T cells, and 1,25(OH)2D3 suppressed TNFα expression in memory CCR6− and CCR6+ Th17 cells, but not in naïve T cells (figure 1B).

    These data show that 1,25(OH)2D3 has direct suppressive effects on the proinflammatory cytokines, IL-17A, IL-22, IFNγ and TNFα, in primary CCR6+ Th17 cell cultures, without affecting IL-4 and IL-10 cytokine production.

    In Th17–RASF cocultures, 1,25(OH)2D3 inhibited autocrine IL-17A production and induced an anti-inflammatory cytokine profile

    We recently reported that, upon coculture with RASFs, Th17 cells induce a proinflammatory feedback loop, resulting in increased IL-17A production.17 Interestingly, in the presence of 1,25(OH)2D3, this proinflammatory loop was inhibited, as both the percentage of IL-17A-producing cells and IL-17A expression levels in Th17–RASF cocultures were significantly lower in the presence of 1,25(OH)2D3 (∼4.1-fold and ∼1.8-fold, respectively, figure 2A,B). Moreover, 1,25(OH)2D3 significantly inhibited the fraction of IL-22- and IFNγ-producing Th17 cells and IL-22 and IFNγ production in Th17–RASF cocultures (figure 2A,B). Of note, 1,25(OH)2D3 significantly induced IL-4 and IL-10 expression in Th17–RASF cocultures, while no effects were observed for TNFα expression (figure 2B).

    Figure 2
    Figure 2

    Effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of T cell-associated cytokines in T helper 17 (Th17)–rheumatoid arthritis (RA) synovial fibroblast (RASF) cocultures. (A) Flow cytometric analysis for intracellular interleukin (IL)-17A, IL-22 and interferon (IFN)γ. Numbers in a representative dot plot (upper panel) represent the percentage of cytokine-producing cells in each quadrant. Mean and SEM (lower panel) are given for Th17–RASF cocultures stimulated with αCD3/αCD28 in the absence (white bars) or presence (black bars) of 1,25(OH)2D3, whereby Th17 cells were obtained from five patients with early RA. (B) Expression of indicated cytokines in Th17–RASF cocultures in the absence (white bars) or presence (black bars) of 1,25(OH)2D3. Mean and SEM are given for five treatment-naïve patients with early RA. Results are representative of at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001.

    In summary, in Th17–RASF cocultures, 1,25(OH)2D3 reduces Th17-associated proinflammatory cytokine production and induces IL-4 and IL-10 production.

    1,25(OH)2D3 suppressed Th17-induced expression of both inflammatory cytokines and mediators of joint destruction by RASFs

    Upon interaction with Th17 cells, RASFs are activated and the expression of IL-6, IL-8, MMP-1 and MMP-3 increased.17 In Th17–RASF cocultures, 1,25(OH)2D3 significantly reduced IL-6, IL-8 and MMP-1 expression (figure 3A). To verify, whether these 1,25(OH)2D3-induced effects were mediated directly via Th17 cells, rather than direct effects on RASFs, we analysed IL-6, IL-8, MMP-1 and MMP-3 expression in RASF cultures with or without 1,25(OH)2D3. In these unstimulated RASF monocultures, no effects of 1,25-(OH)2D3 were found on IL-6, IL-8, MMP-1 and MMP-3 expression (figure 3B). However, in TNFα-stimulated RASF monocultures, inhibitory effects of 1,25-(OH)2D3 were found on IL-6 and IL-8, but not on IL-22, MMP-1 and MMP-3 expression (figure 3B).

    Figure 3
    Figure 3

    1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) inhibits interleukin (IL)-6, IL-8, matrix metalloprotease (MMP)-1 and MMP-3 expression in T helper 17 (Th17)–rheumatoid arthritis synovial fibroblast (RASF) cocultures. Detection of indicated cytokines by ELISA in supernatant of (A) Th17–RASF cocultures or (B) phosphate-buffered saline (PBS) or tumour necrosis factor (TNF)α (1 ng/ml)-stimulated RASF monocultures in the absence (white bars) or presence (black bars) of 1,25(OH)2D3 and stimulated with αCD3/αCD28. (A) Mean and SEM are given for Th17–RASF cocultures, whereby Th17 cells were obtained from five patients with early rheumatoid arthritis. (B) Mean and SEM are given for three RASF cultures. Results are representative of at least three independent experiments. *p<0.05; **p<0.01.

    From this, we concluded that, in Th17–RASF cocultures, 1,25(OH)2D3 inhibits IL-6, IL-8, IL-17A and MMP-1 expression.

    Additive effect of 1,25(OH)2D3 treatment combined with TNF blockade in Th17–RASF cocultures

    Recently, we have shown that both IL-17A and TNFα are produced by Th17 cells and that these cytokines are involved in inducing a proinflammatory loop in Th17–RASF cocultures.17 In contrast with 1,25(OH)2D3, TNFα blockade did not influence IL-17A, IL-22 and IFNγ expression in the Th17–RASF cocultures (figure 4). This prompted us to investigate whether combining 1,25(OH)2D3 and TNFα blockade could have an additional effect over TNFα blockade alone in Th17–RASF cocultures. TNFα blockade alone resulted in a significant reduction in IL-6, IL-8, MMP-1 and MMP-3 compared with the control situation. Combining 1,25(OH)2D3 and TNFα blockade had a significant additional effect on IL-6, IL-8, MMP-1 and MMP-3 suppression, when compared with TNFα blockade alone (figure 4).

    Figure 4
    Figure 4

    The combination of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and tumour necrosis factor α (TNFα) blockade has additional effects on interleukin (IL)-6, IL-8, matrix metalloprotease (MMP)-1 and MMP-3 expression compared with TNFα blockade alone in T helper 17 (Th17)– rheumatoid arthritis synovial fibroblast (RASF) cocultures. Expression of the indicated cytokines and MMPs was detected with ELISA in supernatant of Th17–RASF cocultures stimulated with αCD3/αCD28 in the presence or absence 1,25(OH)2D3 and or TNFα blockade. Mean and SEM are given, whereby Th17 cells were obtained from five treatment-naïve patients with early rheumatoid arthritis. Results are representative of at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001.

    These findings show that, in Th17–RASF cocultures, TNF blockade does not suppress Th1 and Th17 cytokines, which can be overcome by 1,25(OH)2D3. The combination of neutralising TNFα and 1,25(OH)2D3 controls human Th17 activity and additively inhibits synovial inflammation.

    Additional effect of 1,25(OH)2D3 over TNF blockade in suppressing IL-6 and IL-8 expression in cultures of RA synovial tissue

    To investigate the functional relevance of 1,25(OH)2D3 effects in the Th17–RASF coculture system, the effects of 1,25(OH)2D3 on synovial tissue of patients with established RA were analysed. Therefore synovial biopsy samples were cultured with αCD3/αCD28 and/or 1,25(OH)2D3 and/or TNFα blockade. As we observed in the Th17–RASF cocultures, 1,25(OH)2D3, but not TNFα blockade, significantly reduced the expression of IL-17A (figure 5). Moreover, both 1,25(OH)2D3 and TNFα blockade resulted in significant suppression of IL-6 and IL-8 expression. Importantly, combining 1,25(OH)2D3 with TNFα blockade had an additive effect compared with TNFα blockade alone in the inhibition of IL-6 and IL-8 expression (figure 5). In contrast, 1,25(OH)2D3 had no effects on MMP-3 expression, and increased MMP-1 expression levels were observed. Consequently, no additional effects of 1,25(OH)2D3 over TNFα blockade were found in the inhibition of MMP-1 and MMP-3 expression. However, combining 1,25(OH)2D3 with TNFα blockade neutralised the 1,25(OH)2D3 effect on MMP-1 induction (figure 5).

    Figure 5
    Figure 5

    Additional effects on the suppression of interleukin (IL)-6 and IL-8 in synovial biopsy cultures in the presence of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and tumour necrosis factor α (TNFα) blockade compared with TNFα blockade alone. Synovial biopsy samples obtained from 14 patients with established rheumatoid arthritis were stimulated with αCD3/αCD28 and cultured with or without 1,25(OH)2D3 and/or TNFα blockade. Expression of the indicated cytokines and matrix metalloproteases (MMPs) was detected in supernatant with ELISA. Mean and SEM are given. *p<0.05; **p<0.01; ***p<0.01, by two-sided Wilcoxon signed-rank test.

    In summary, these findings show that 1,25(OH)2D3 suppresses the expression of IL-6 and IL-8 and autocrine IL-17A expression in RA synovial tissue cultures. Combining 1,25(OH)2D3 and TNFα blockade had additional value over TNFα blockade alone in further reducing IL-6 and IL-8 expression.

    Discussion

    In this study, we found that 1,25(OH)2D3, in contrast with TNFα blockade, directly regulates Th17 cytokine expression. Furthermore, 1,25(OH)2D3 in combination with TNFα blockade is essential to fully neutralise pathological Th17 activity in RA synovial inflammation.

    Evidence is accumulating that 1,25(OH)2D3 has a suppressive role in murine experimental autoimmune models32 33 and in human autoimmune diseases such as RA.34,–,37 However, the mechanism underlying these suppressive effects is not fully elucidated. To analyse the effects of 1,25(OH)2D3 on Th17 cytokine expression and activity, we have used different culturing approaches, including primary Th17 monocultures, Th17–RASF cocultures and synovial biopsy cultures. In all approaches, 1,25(OH)2D3 treatment resulted in suppression of the proinflammatory cytokines, IL-17A, IL-22 and IFNγ. Moreover, the 1,25(OH)2D3 effects in synovial tissue cultures not only show the relevance of the Th17–RASF cocultures as a functional T cell test system in RA, but importantly also show the therapeutic potential of 1,25(OH)2D3/TNF blockade combination as a treatment to control synovial inflammation in RA, in particular in the presence of IL-17/Th17 activity.

    In contrast, IL-4 and IL-10 production was not affected by 1,25(OH)2D3 in Th17 cultures, but was enhanced in Th17–RASF cocultures after 1,25(OH)2D3 treatment. These findings implicate that, in the coculture system, 1,25(OH)2D3 supported Th2 function and/or inhibited factors that can negatively regulate IL-4 expression, such as IFNγ and T-Bet. On the other hand, in this study no effects were found of 1,25(OH)2D3 treatment on TNFα production by Th17–RASF cocultures, whereas, in Th17 monocultures, TNFα production was significantly lower after 1,25(OH)2D3 treatment. This suggests that, besides direct effects of 1,25(OH)2D3 on Th17 cytokines, other factors that are induced in Th17–RASF cocultures and in the inflamed RA synovium are involved in the regulation of IL-4 and TNFα. We found no upregulation of Foxp3expression in our Th17–RASF cocultures (data not shown), which indicates that the observed suppression is independent of T regulatory cell activity. Moreover, it has to be taken into account that the CCR6+ Th17 population is a heterogeneous population in which cells are present that are negative and positive for the production of either IL-17A or IFNγ or both. The molecular mechanism responsible for the induction of IL-4 by 1,25(OH)2D3 in CCR6+ Th17 cocultures is at present under investigation.

    1,25(OH)2D3 suppressed Th17 activity as observed by lower IL-6, IL-8 and MMP-1 levels in Th17–RASF cocultures. In an earlier study, we showed that both IL-17A and TNFα blockade are required to neutralise Th17 activity in Th17–RASF cocultures.17 These cytokines were shown to have synergistic effects on fibroblast activation.38 Since TNFα blockade had limited effects on the Th17 cytokines, IL-17A and IL-22, and 1,25(OH)2D3 had limited effects on TNFα expression, the combination of TNFα blockade and 1,25(OH)2D3 additionally downregulated Th17 activity. This indicates more valuable therapeutic potential of VDR signalling activation over the current TNF neutralisation strategies in patients with RA.

    In contrast with Th17–RASF cocultures, 1,25(OH)2D3 treatment resulted in an increase in MMP-1 expression in synovial biopsy cultures, which was overcome by the combination 1,25(OH)2D3 and TNFα blockade. These different effects on MMP-1 expression in the synovial biopsy cultures compared with the Th17–RASF cocultures is still not well understood, but may be affected by the composition of the synovial biopsy samples. Besides T cells and synovial fibroblasts, these consist of other cells, such as inflammatory CD68+ macrophages. Of note, when TNF-stimulated RASFs were used, no stimulatory effect on MMP-1 expression by 1,25(OH)2D3 was found. In line with our study, articular chondrocyte cultures, but not RASF cultures, have been shown to increase MMP-1 expression after 1,25(OH)2D3 treatment.39 Further experiments are needed to explain the phenomenon of increased MMP-1 expression by 1,25(OH)2D3 in synovial tissue, focusing on the cellular composition and cellular interaction between cells present in the inflamed synovium.

    Progression of joint damage and disease activity has been shown to correlate with the levels of IL-17A and Th17 cells in patients with RA.16 40 The present study shows that the presence of IL-17A in the synovial biopsy samples has a marked influence on the outcome and demonstrates the additional value of the combination of TNFα blocking and 1,25(OH)2D3 treatment compared with TNFα blocking alone. The more IL-17A produced by the synovial biopsy samples after T cell receptorand costimulatory activation, the more valuable the therapeutic potential effect on synovial inflammation attained by adding 1,25(OH)2D3 to TNFα blocking. This suggests less effective anti-TNF therapy in patients with RA under increased IL-17A levels in these patients. In addition, we recently showed raised levels of memory Th17 cells in treatment-naïve patients with early RA compared with healthy controls.17 Therefore together these data support the development of a clinical trial combining TNF blockade with VDR activation in patients with RA, in particular at the early stage of the disease.

    TNFα blockade is a commonly used treatment in RA.2 However, a large fraction (∼20–40%) of patients do not respond to this therapy.41 The present study shows that TNFα blockade has no effect on expression of Th17 cytokines such as IL-17A. This may explain why TNFα blockade alone is not effective in all patients with RA and is in line with the suggestion based on preliminary data that TNFα blockade may be less effective in patients with RA who have especially raised levels of IL-17A.5 Therefore it would be of great interest to investigate whether the group of anti-TNF non-responders has raised levels of Th17 cytokines and/or Th17 cell activity. This study further shows that, for full neutralisation of Th17 activity in RA synovial inflammation, TNFα blockade needs to be combined with 1,25(OH)2D3 signalling. This underlines the importance of finding combinations of therapeutic approaches to improve the efficacy of current treatment strategies for RA, such as TNFα blockade.

    Currently, there is considerable interest in targeting IL-17A or Th17 cells in the treatment of RA and other Th17-mediated disorders.4 8 In line with this, activation of VDR signalling might be a contending approach. In relation to IL-17A blockade alone, activation of VDR signalling has the advantage that, in addition to IL-17A, other Th17 cytokines, such as IL-17F and IL-22, are downregulated, whereas cytokines such as IL-4 and IL-10 are induced (present study).27 However, activation of VDR signalling with 1,25(OH)2D3 can have severe side effects such as hypercalcaemia.42 For this reason, future research should focus on the identification of 1,25(OH)2D3 targets in T cells, and Th17 cells in particular, which may have therapeutic potential for the treatment of RA. Moreover, it should be taken into account that cells of the immune system such as macrophages express the vitamin D-converting enzyme, 1α-hydroxylase, enabling the conversion of 25(OH)D3 into 1,25(OH)2D3.24 43 44 This may imply increased local levels of 1,25(OH)2D3 after administration of 25(OH)D3.

    In conclusion, this study shows direct suppressive effects of 1,25(OH)2D3, in contrast with TNFα blockade, on Th17 cytokine expression and activity by Th17 cells from patients with RA. This implies that adding activation of VDR signalling to current TNF blockade therapy may have an additional role in fully neutralising pathogenic Th17 activity in RA and other Th17-mediated autoimmune diseases. Moreover, our data suggest less effective anti-TNF therapy in patients with RA with increased IL-17A levels and provide a rationale for a therapeutic trial combining TNF blockade with VDR activation in patients with RA and potentially also in other Th17-mediated autoimmune disorders.

    Acknowledgments

    We thank E de Haas, B Bartol, K X Wen, and A Kleinjan for excellent technical assistance during various stages of the project. This work was supported by a research grant from the Dutch Arthritis Association and a research grant from the Dutch Zilveren Kruis Foundation Achmea.

    References

    1. ↵
      1. Annunziato F,
      2. Cosmi L,
      3. Liotta F,
      4. et al
      . Type 17 T helper cells-origins, features and possible roles in rheumatic disease. Nat Rev Rheumatol 2009;5:325–31.
      OpenUrlCrossRefPubMedWeb of Science
    2. ↵
      1. McInnes IB,
      2. Schett G
      . Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol 2007;7:429–42.
      OpenUrlCrossRefPubMed
    3. ↵
      1. Isaacs JD
      . The changing face of rheumatoid arthritis: sustained remission for all? Nat Rev Immunol 2010;10:605–11.
      OpenUrlCrossRefPubMed
    4. ↵
      1. Lubberts E
      . IL-17/Th17 targeting: on the road to prevent chronic destructive arthritis? Cytokine 2008;41:84–91.
      OpenUrlCrossRefPubMedWeb of Science
    5. ↵
      1. van den Berg WB,
      2. Miossec P
      . IL-17 as a future therapeutic target for rheumatoid arthritis. Nat Rev Rheumatol 2009;5:549–53.
      OpenUrlCrossRefPubMedWeb of Science
    6. ↵
      1. Zhu J,
      2. Yamane H,
      3. Paul WE
      . Differentiation of effector CD4 T cell populations (*). Annu Rev Immunol 2010;28:445–89.
      OpenUrlCrossRefPubMedWeb of Science
    7. ↵
      1. Acosta-Rodriguez EV,
      2. Rivino L,
      3. Geginat J,
      4. et al
      . Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol 2007;8:639–46.
      OpenUrlCrossRefPubMedWeb of Science
    8. ↵
      1. Miossec P,
      2. Korn T,
      3. Kuchroo VK
      . Interleukin-17 and type 17 helper T cells. N Engl J Med 2009;361:888–98.
      OpenUrlCrossRefPubMedWeb of Science
    9. ↵
      1. Lubberts E
      . Th17 cytokines and arthritis. Semin Immunopathol 2010;32:43–53.
      OpenUrlCrossRefPubMed
    10. ↵
      1. Murphy CA,
      2. Langrish CL,
      3. Chen Y,
      4. et al
      . Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med 2003;198:1951–7.
      OpenUrlAbstract/FREE Full Text
    11. ↵
      1. van Hamburg JP,
      2. Mus AM,
      3. de Bruijn MJ,
      4. et al
      . GATA-3 protects against severe joint inflammation and bone erosion and reduces differentiation of Th17 cells during experimental arthritis. Arthritis Rheum 2009;60:750–9.
      OpenUrlCrossRefPubMedWeb of Science
    12. ↵
      1. Nistala K,
      2. Moncrieffe H,
      3. Newton KR,
      4. et al
      . Interleukin-17-producing T cells are enriched in the joints of children with arthritis, but have a reciprocal relationship to regulatory T cell numbers. Arthritis Rheum 2008;58:875–87.
      OpenUrlCrossRefPubMedWeb of Science
    13. ↵
      1. Chabaud M,
      2. Durand JM,
      3. Buchs N,
      4. et al
      . Human interleukin-17: A T cell-derived proinflammatory cytokine produced by the rheumatoid synovium. Arthritis Rheum 1999;42:963–70.
      OpenUrlCrossRefPubMedWeb of Science
    14. ↵
      1. Kotake S,
      2. Udagawa N,
      3. Takahashi N,
      4. et al
      . IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J Clin Invest 1999;103:1345–52.
      OpenUrlCrossRefPubMedWeb of Science
    15. ↵
      1. Raza K,
      2. Falciani F,
      3. Curnow SJ,
      4. et al
      . Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin. Arthritis Res Ther 2005;7:R784–95.
      OpenUrlCrossRefPubMedWeb of Science
    16. ↵
      1. Leipe J,
      2. Grunke M,
      3. Dechant C,
      4. et al
      . Role of Th17 cells in human autoimmune arthritis. Arthritis Rheum 2010;62:2876–85.
      OpenUrlCrossRefPubMedWeb of Science
    17. ↵
      1. van Hamburg JP,
      2. Asmawidjaja PS,
      3. Davelaar N,
      4. et al
      . 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. Arthritis Rheum 2011;63:73–83.
      OpenUrlCrossRefPubMedWeb of Science
    18. ↵
      1. Genovese MC,
      2. Van den Bosch F,
      3. Roberson SA,
      4. et al
      . LY2439821, a humanized anti-interleukin-17 monoclonal antibody, in the treatment of patients with rheumatoid arthritis: A phase I randomized, double-blind, placebo-controlled, proof-of-concept study. Arthritis Rheum 2010;62:929–39.
      OpenUrlCrossRefPubMedWeb of Science
    19. ↵
      1. Tang J,
      2. Zhou R,
      3. Luger D,
      4. et al
      . Calcitriol suppresses antiretinal autoimmunity through inhibitory effects on the Th17 effector response. J Immunol 2009;182:4624–32.
      OpenUrlAbstract/FREE Full Text
    20. ↵
      1. Penna G,
      2. Amuchastegui S,
      3. Cossetti C,
      4. et al
      . Treatment of experimental autoimmune prostatitis in nonobese diabetic mice by the vitamin D receptor agonist elocalcitol. J Immunol 2006;177:8504–11.
      OpenUrlAbstract/FREE Full Text
    21. ↵
      1. Gauzzi MC,
      2. Purificato C,
      3. Donato K,
      4. et al
      . Suppressive effect of 1alpha,25-dihydroxyvitamin D3 on type I IFN-mediated monocyte differentiation into dendritic cells: impairment of functional activities and chemotaxis. J Immunol 2005;174:270–6.
      OpenUrlAbstract/FREE Full Text
    22. ↵
      1. Mathieu C,
      2. van Etten E,
      3. Decallonne B,
      4. et al
      . Vitamin D and 1,25-dihydroxyvitamin D3 as modulators in the immune system. J Steroid Biochem Mol Biol 2004;89-90:449–52.
      OpenUrlCrossRefPubMed
    23. ↵
      1. D'Ambrosio D,
      2. Cippitelli M,
      3. Cocciolo MG,
      4. et al
      . Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-kappaB downregulation in transcriptional repression of the p40 gene. J Clin Invest 1998;101:252–62.
      OpenUrlCrossRefPubMedWeb of Science
    24. ↵
      1. Mora JR,
      2. Iwata M,
      3. von Andrian UH
      . Vitamin effects on the immune system: vitamins A and D take centre stage. Nat Rev Immunol 2008;8:685–98.
      OpenUrlCrossRefPubMedWeb of Science
    25. ↵
      1. Chang SH,
      2. Chung Y,
      3. Dong C
      . Vitamin D suppresses Th17 cytokine production by inducing C/EBP homologous protein (CHOP) expression. J Biol Chem 2010;285:38751–5.
      OpenUrlAbstract/FREE Full Text
    26. ↵
      1. von Essen MR,
      2. Kongsbak M,
      3. Schjerling P,
      4. et al
      . Vitamin D controls T cell antigen receptor signaling and activation of human T cells. Nat Immunol 2010;11:344–9.
      OpenUrlCrossRefPubMedWeb of Science
    27. ↵
      1. Colin EM,
      2. Asmawidjaja PS,
      3. van Hamburg JP,
      4. et al
      . 1,25-dihydroxyvitamin D3 modulates Th17 polarization and interleukin-22 expression by memory T cells from patients with early rheumatoid arthritis. Arthritis Rheum 2010;62:132–42.
      OpenUrlCrossRefPubMedWeb of Science
    28. ↵
      1. Borgogni E,
      2. Sarchielli E,
      3. Sottili M,
      4. et al
      . Elocalcitol inhibits inflammatory responses in human thyroid cells and T cells. Endocrinology 2008;149:3626–34.
      OpenUrlCrossRefPubMedWeb of Science
    29. ↵
      1. Rausch-Fan X,
      2. Leutmezer F,
      3. Willheim M,
      4. et al
      . Regulation of cytokine production in human peripheral blood mononuclear cells and allergen-specific th cell clones by 1alpha,25-dihydroxyvitamin D3. Int Arch Allergy Immunol 2002;128:33–41.
      OpenUrlCrossRefPubMed
    30. ↵
      1. Correale J,
      2. Ysrraelit MC,
      3. Gaitán MI
      . Immunomodulatory effects of vitamin D in multiple sclerosis. Brain 2009;132(Pt 5):1146–60.
      OpenUrlAbstract/FREE Full Text
    31. ↵
      1. Boonstra A,
      2. Barrat FJ,
      3. Crain C,
      4. et al
      . 1alpha,25-Dihydroxyvitamin d3 has a direct effect on naive CD4(+) T cells to enhance the development of Th2 cells. J Immunol 2001;167:4974–80.
      OpenUrlAbstract/FREE Full Text
    32. ↵
      1. Spach KM,
      2. Nashold FE,
      3. Dittel BN,
      4. et al
      . IL-10 signaling is essential for 1,25-dihydroxyvitamin D3-mediated inhibition of experimental autoimmune encephalomyelitis. J Immunol 2006;177:6030–7.
      OpenUrlAbstract/FREE Full Text
    33. ↵
      1. Cantorna MT,
      2. Hayes CE,
      3. DeLuca HF
      . 1,25-Dihydroxycholecalciferol inhibits the progression of arthritis in murine models of human arthritis. J Nutr 1998;128:68–72.
      OpenUrlAbstract/FREE Full Text
    34. ↵
      1. Rossini M,
      2. Maddali Bongi S,
      3. La Montagna G,
      4. et al
      . Vitamin D deficiency in rheumatoid arthritis: prevalence, determinants and associations with disease activity and disability. Arthritis Res Ther 2010;12:R216.
      OpenUrlCrossRefPubMed
    35. ↵
      1. Lee YH,
      2. Bae SC,
      3. Choi SJ,
      4. et al
      . Associations between vitamin D receptor polymorphisms and susceptibility to rheumatoid arthritis and systemic lupus erythematosus: a meta-analysis. Mol Biol Rep 2011;38:3643–51.
      OpenUrlCrossRefPubMed
    36. ↵
      1. Cutolo M
      . Vitamin D and autoimmune rheumatic diseases. Rheumatology (Oxford) 2009;48:210–12.
      OpenUrlFREE Full Text
    37. ↵
      1. Andjelkovic Z,
      2. Vojinovic J,
      3. Pejnovic N,
      4. et al
      . Disease modifying and immunomodulatory effects of high dose 1 alpha (OH) D3 in rheumatoid arthritis patients. Clin Exp Rheumatol 1999;17:453–6.
      OpenUrlPubMedWeb of Science
    38. ↵
      1. Miossec P
      . Interleukin-17 in rheumatoid arthritis: if T cells were to contribute to inflammation and destruction through synergy. Arthritis Rheum 2003;48:594–601.
      OpenUrlCrossRefPubMedWeb of Science
    39. ↵
      1. Tetlow LC,
      2. Woolley DE
      . The effects of 1 alpha,25-dihydroxyvitamin D(3) on matrix metalloproteinase and prostaglandin E(2) production by cells of the rheumatoid lesion. Arthritis Res 1999;1:63–70.
      OpenUrlCrossRefPubMed
    40. ↵
      1. Kirkham BW,
      2. Lassere MN,
      3. Edmonds JP,
      4. et al
      . Synovial membrane cytokine expression is predictive of joint damage progression in rheumatoid arthritis: a two-year prospective study (the DAMAGE study cohort). Arthritis Rheum 2006;54:1122–31.
      OpenUrlCrossRefPubMedWeb of Science
    41. ↵
      1. Klareskog L,
      2. van der Heijde D,
      3. de Jager JP,
      4. et al
      . Therapeutic effect of the combination of etanercept and methotrexate compared with each treatment alone in patients with rheumatoid arthritis: double-blind randomised controlled trial. Lancet 2004;363:675–81.
      OpenUrlCrossRefPubMedWeb of Science
    42. ↵
      1. Vieth R
      . Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety. Am J Clin Nutr 1999;69:842–56.
      OpenUrlAbstract/FREE Full Text
    43. ↵
      1. Cutolo M,
      2. Plebani M,
      3. Shoenfeld Y,
      4. et al
      . Vitamin D endocrine system and the immune response in rheumatic diseases. Vitam Horm 2011;86:327–51.
      OpenUrlCrossRefPubMed
    44. ↵
      1. Nelson CD,
      2. Reinhardt TA,
      3. Beitz DC,
      4. et al
      . In vivo activation of the intracrine vitamin D pathway in innate immune cells and mammary tissue during a bacterial infection. PLoS ONE 2010;5:e15469.
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    Footnotes

    • Competing interests None.

    • Patient consent Obtained.

    • Ethics approval Medical ethics committee of the Erasmus MC, Rotterdam, The Netherlands.

    • Provenance and peer review Not commissioned; externally peer reviewed.