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Basic and translational research
Extended report
Regulatory T cells in rheumatoid arthritis showed increased plasticity toward Th17 but retained suppressive function in peripheral blood
  1. Tian Wang1,2,
  2. Xiaolin Sun1,
  3. Jing Zhao1,
  4. Jing Zhang1,
  5. Huaqun Zhu1,
  6. Chun Li1,
  7. Na Gao2,
  8. Yuan Jia1,
  9. Dakang Xu3,
  10. Fang-Ping Huang4,
  11. Ningli Li5,
  12. Liwei Lu6,
  13. Zhan-Guo Li1
  1. 1Department of Rheumatology & Immunology, Clinical Immunology Center, Peking University People's Hospital, Beijing, China
  2. 2Department of Rheumatology & Immunology, Beijing An Zhen Hospital, Capital Medical University, Beijing, China
  3. 3MIMR-PHI Institute of Medical Research, Monash University, Clayton, Victoria, Australia and Institute of Ageing Research, Hangzhou Normal University School of Medicine, Hangzhou, China
  4. 4Division of Immunology & Inflammation (Rheumatology Section), Department of Medicine, Imperial College Faculty of Medicine, Hammersmith Hospital, London, UK
  5. 5Shanghai Institute of Immunology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China
  6. 6Department of Pathology and Center of Infection and Immunology, The University of Hong Kong, Hong Kong, China
  1. Correspondence to Professor Zhan-Guo Li, Department of Rheumatology & Immunology/Clinical Immunology Center, People's Hospital, Peking University, Beijing 100044, China; li99{at}bjmu.edu.cn or Professor Liwei Lu, Department of Pathology and Center for Infection and Immunology, The University of Hong Kong, China; liweilu@hku.hk

https://doi.org/10.1136/annrheumdis-2013-204228

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    Introduction

    IL-17-producing CD4 T cells (Th17 cells) and Foxp3+ regulatory T cells (Treg cells) have been implicated in the pathogenesis of various chronic autoimmune diseases, including rheumatoid arthritis (RA).1–4 Increased Th17 cells mediate autoreactive inflammation and tissue destruction during RA development,5–8 whereas several types of Treg cells maintain tight regulation of T helper (Th) cells for preventing T cell-mediated autoimmunity and inflammatory damage.9–12 Recent studies have revealed a link between Treg and Th17 cells. Treg and Th17 cells may develop from the same precursors under distinct cytokine conditions,13 and a subset of IL-17-producing CD4 Foxp3+ Treg cells can be generated upon polarisation by cytokines such as IL-6.14–16

    The observed Th17 plasticity of Treg cells has raised the possibility that the stability of Foxp3+ Treg cells may play a potential pathogenic role at sites of inflammation. Treg cells with the plasticity of producing the proinflammatory cytokine IL-17 have been detected in normal human peripheral blood as well as in inflamed intestinal mucosa in patients with Crohn's disease.16 ,17 However, conflicting data exist regarding the functions of the IL-17-producing Treg population. Previous studies have reported that the induction of IL-17 expression in Treg cells under inflammatory conditions was accompanied by impaired or lost suppressive function.18 ,19 By contrast, Hovhannisyan et al17 showed that IL-17-producing Treg cells from patients with Crohn's disease retained suppressive activity despite their increased IL-17 production, although the authors suggested that resident Treg cells under inflammatory conditions may convert to IL-17-producing cells and thus potentially contribute to the pathogenesis of the disease. To date, the exact role of Treg plasticity in immunoregulation is unclear, and the presence and clinical significance of the IL-17-producing Treg subset in RA remains unknown.

    In the present study, we investigated the CD4 Foxp3+ IL-17-producing Treg cell in patients with RA. This Treg population exhibited phenotypic characteristics of Treg and Th17 cells, and retained potent suppressive activity in peripheral blood. However, these Treg cells in RA synovial site lost suppressive activity. The expansion of this Treg population was promoted by the inflammatory cytokines IL-6 and IL-23, which were abnormally elevated in patients with RA. Moreover, the peripheral IL-17-producing Treg subset was associated with decreased RA disease activity, suggesting that these Treg cells are negative regulators in RA peripheral blood, but they might be pathogenic in RA synovial sites.

    Materials and methods

    Flow cytometry

    Isolated PBMCs were stained for CD4 expression using a PE-CF594-labelled antihuman-CD4 antibody, for CD127 expression using a PerCP-Cy5.5-labelled antihuman-CD127 antibody, and for CD25 expression using a PE-Cy7-labelled antihuman-CD25 antibody (all antibodies from BD Pharmingen, San Diego, California, USA). The expression of CTLA-4, IL-10, CD161, and CCR6 was examined using FITC-labelled monoclonal antibodies (BD Pharmingen). To analyse cytokine production, cells were stimulated and incubated for 5 h with 50 ng/mL phorbol myristate acetate, 1 μg/mL ionomycin (both from Sigma–Aldrich, Germany), and 1 μL/mL GolgiStop (BD Biosciences, Franklin Lakes, New Jersey, USA). After stimulation, the cell surface markers CD4, CD25, and CD127 were stained with the fluorescently labelled monoclonal antibodies mentioned above. Next, the cells were fixed and permeabilised for 30 min at room temperature at dark with the Foxp3 Fixation/Permeabilisation Concentrate and Diluent buffer set (eBioscience). Then the cells were stained with PE-labelled antihuman Foxp3 monoclonal antibody (eBioscience) and Alexa Fluor 647-labelled antihuman-IL-17A antibody or PE-labelled antimouse/human RORγt antibody (eBioscience). The cells were examined by flow cytometry (BD Aria II flow cytometer), and the data were analysed using FlowJo V.5.7 software (Tree Star, USA).

    Other detailed information about sample preparation, experimental and statistical methods are available in the online supplementary materials.

    Results

    IL-17-producing Treg cells were present in the peripheral blood of patients with RA

    To verify the plasticity of Treg cells to produce Th17 under autoimmune inflammatory conditions, IL-17 production and Foxp3 expression in CD4 T cells were examined using flow cytometry of PBMCs from patients with RA and healthy subjects. IL-17-producing CD4 Foxp3+ Treg cells were present in patients with RA and healthy controls (HC) (figure 1A), and the frequency of IL-17-producing CD4 Foxp3+ Treg cells was significantly higher in patients with RA than in HCs (figure 1B). Notably, in patients with RA, there is no significant correlation between IL-17-producing Treg cells and CD4 Foxp3+ Treg cells (figure 1C), and this IL-17-producing Treg population showed significantly positive correlation with Th17 cells (figure 1D). We further examined the IL-17-producing Treg cell frequency in matched synovial fluid and peripheral blood from RA patients, and found that the frequency of this Treg subset was significantly elevated in synovial fluid (figure 1E, and see online supplementary figure S1).

    Figure 1
    Figure 1

    IL-17-producing Treg cells in peripheral blood from patients with rheumatoid arthritis (RA) and healthy controls (HC). The expression of Foxp3 and IL-17 in CD4 T cells from patients with RA and HCs was evaluated by intracellular cytokine staining and flow cytometry. (A) Representative flow cytometric profile showing IL-17 expression in CD4 Foxp3+ Treg cells in patients with RA and HCs. (B) Four independent experiments showed that the percentage of IL-17-producing Treg cells in CD4 Foxp3+ Treg cells was significantly higher in patients with RA (n=12) than in healthy control subjects (n=7). *p<0.05. RA: patients with RA; HCs: healthy controls. (C) There is no significant correlation between IL-17-producing Treg cells and CD4 Foxp3+ Treg cells in patients with RA. (D) The IL-17-producing Treg cells were positively correlated with Th17 cells in patients with RA. (E) There are higher frequency of IL-17-producing Tregs in RA synovial fluid than in matched peripheral blood (n=5); SF: synovial fluid; PB: peripheral blood. (F) Distribution of IL-17-producing Treg cells in Treg fractions defined by CD45RA and Foxp3 expression. Numbers on the representative flow cytometry graph indicate IL-17-producing Treg numbers in that Treg fraction, percentages in round brackets indicate the percentages of IL-17-producing Tregs of that fraction in total IL-17-producing Tregs of the representative sample. Fr: Fraction; Fr I: CD45RA+ Foxp3low resting Treg cells; Fr II: CD45RA-Foxp3hi activating Treg cells; Fr III: CD45RA-Foxp3low ‘non-Treg’ cells.

    A previous study has defined three cell fractions with different functional characteristics in CD4CD25+ Treg cells with CD45RA and Foxp3 expression,20 (figure 1F upper panel). Our study showed that more than 60% of IL-17-producing Treg cells were enriched in CD45RA− Foxp3low ‘non-Treg’ fraction, and around 30% IL-17-producing Treg cells distributed in CD45RA+ Foxp3low ‘resting Treg’ fraction. Few Tregs in CD45RA− Foxp3hi ‘activating Treg’ fraction expressed IL-17 (figure 1F lower panel).

    IL-17-producing Treg cells from the peripheral blood of patients with RA exhibited phenotypic characteristics of conventional Th17 and Treg cells

    To examine the phenotypic characteristics of the IL-17-producing CD4 Foxp3+ Treg cells, we used flow cytometry to assess the expression of Foxp3 and surface markers or cytokines known to be associated with either Th17 or Treg cells. The expression levels of the Treg-associated regulatory molecules CTLA-4 and IL-10 were significantly higher in IL-17-producing CD4 Foxp3+ Treg cells (mean fluorescence intensity (MFI): 6.11±0.92 and 4.46±0.39, respectively) than in either conventional CD4 Foxp3+ Treg cells (MFI: 2.77±0.10 and 2.69±0.21, respectively; p<0.05 for each) or Th17 cells (MFI: 3.67±0.48 and 3.25±0.07, respectively; p<0.05 for each), whereas Foxp3 expression was slightly but significantly lower in IL-17-producing CD4 Foxp3+ Treg cells than in conventional Treg cells (MFI: 68.74±13.80 vs 77.88±16.00, p<0.05) (figure 2). CCR6 and CD161 are human Th17 cell markers,21 whereas conventional Treg cells express characteristically low levels of CD127.22 Similar to Th17 cells, IL-17-producing CD4 Foxp3+ Treg cells expressed higher levels of CCR6, CD161 and CD127 compared with conventional Treg cells (CCR6 MFI: 3.40±0.16 vs 1.86±0.07, p<0.05; CD161 MFI: 13.99±2.98 vs 1.86±0.07, p<0.05; CD127 MFI: 32.29±5.68 vs 13.48±2.36, p<0.05; figure 2A,B). These results show that IL-17-producing CD4 Foxp3+ Treg cells exhibit phenotypes of conventional Th17 and Treg cells.

    Figure 2
    Figure 2

    IL-17-producing Treg cells display phenotypic similarities to Th17 and Treg cells. Surface expression of CTLA-4, CD161, CCR6, and CD127, and intracellular expression of Foxp3 and IL-10 were measured in IL-17-producing CD4 Foxp3+ Treg cells. The CD4 Foxp3+ Treg cells and CD4IL17+ Th17 cells were examined by flow cytometry 5 h after stimulation with phorbol myristate acetate (PMA)/ionomycin. The expression levels of the surface markers or cytokines were quantified as mean fluorescence intensity (MFI). Data are representative of at least five independent experiments (n=5; *p<0.05). (A) Representative overlaid flow cytometric histograms showing expression of CTLA-4, CD161, CCR6, CD127, Foxp3 and IL-10 in CD4 Foxp3+ Treg cells, IL-17-producing Treg cells and CD4IL17+ Th17 cells; red histogram, CD4 Foxp3+ Treg cells; green histogram, IL-17-producing Treg cells; blue histogram, Th17 cells. (B) The expression levels of the molecular measured above were quantified as MFI.

    Inflammatory cytokines in RA promoted the induction of IL-17-producing Treg cells

    To characterise the inflammatory milieu contributing to the plasticity of Treg cells in the peripheral blood from patients with RA, we assessed the concentrations of different proinflammatory cytokines in serum samples. Serum levels of IL-6 and IL-23 were significantly elevated in patients with RA compared with HCs (IL-23: 32.67±34.50 vs 1.82±1.51 pg/mL, p<0.001; IL-6: 157.83±27.07 vs 21.97±3.52 pg/mL, p<0.001; figure 3A).

    Figure 3
    Figure 3

    Induction of peripheral CD4 regulatory T cells into IL-17-producing CD4 Treg cells under rheumatoid arthritis (RA) inflammatory conditions. IL-6 and IL-23 levels in sera from patients with RA and healthy controls (HC) were quantified by ELISA. CD4 T cells were stimulated with either IL-6 or IL-23, or both in vitro in the presence of anti-CD3 and anti-CD28 mAb-coated beads, IL-2 and IL-1β. The expression of Foxp3 and IL-17 was determined by intracellular cytokine staining and flow cytometry. (A) IL-6 and IL-23 concentrations in sera of 39 patients with RA (RA) and 27 HCs were assessed by ELISA (*p<0.01). (B) IL-17 and Foxp3 expression levels in CD4CD25+ Treg cells from RA patients and HCs were examined in the presence or absence of IL-6 or IL-23 in vitro with anti-CD3 and anti-CD28 mAb-coated beads, IL-2 and IL-1β. Data are representative of three independent experiments. (C) The frequencies of IL-17-producing Treg cells from RA patients and HCs were measured in the presence or absence of IL-6 or IL-23 in vitro. Data are from three independent experiments (*p<0.05). (D) Comparison of RORγt expression in CD4CD25+ Treg cells in the presence or absence of IL-6 or IL-23 in vitro. Data are representative of two independent experiments.

    Because IL-6 and IL-23 together promote the development and expansion of Th17 cells,9 ,10 ,11 elevated levels of these two cytokines in the sera of patients with RA may also provide the polarising environment for the expansion of IL-17-producing Treg cells. To test this hypothesis, CD4CD25+ Treg cells from the peripheral blood of patients with RA, or HCs, were purified by cell sorting and cultured in vitro in the presence of IL-2, IL-1β and anti-CD3 and anti-CD28 antibody-coated beads and supplemented with IL-6, IL-23, or both for 5 days. As expected, relatively low frequency of IL-17-producing Foxp3+ Treg cells was detected in cultures without the addition of polarising cytokines. In the presence of IL-23, there was a slight but not significant increase in the IL-17-producing Treg frequency. However, under the polarising culture condition supplemented with IL-6 or IL-6 and IL-23, a significantly increased IL-17-producing Treg cell frequency was observed (figure 3B,C), implicating the polarising function of IL-6 and IL-23, and that IL-6 should be a major player in the induction of IL-17-producing Treg cells. However, we did not observe any significant correlation between the serum levels of these polarising cytokines and the IL-17-producing Treg subset (see online supplementary figure S2). Compared with Tregs from RA patients, Tregs from HCs showed similar plasticity toward Th17 under the same polarising culture conditions (figure 3B,C), though the IL-17-producing Treg frequencies were modestly lower than those from RA patients without statistical significance. We also examined the expression of Th17-lineage specific transcription factor RORγt in Treg cells cultured in polarising conditions. The RORgt levels were obviously increased when IL-6 or IL-6 together with IL-23 was added to culture (figure 3D).

    CD161 expression defined IL-17-producing Treg subset

    We found that CD161, a surface marker of Th17 precursor cells,21 was dramatically increased on IL-17-producing Treg cells (figure 2), and it could also be used as the surface marker to characterise IL-17-producing Treg cells (figure 4A). With CD161 expression, we sorted CD4CD25+ Treg cells from RA peripheral blood into two subsets: CD4CD25+CD161+ Treg and CD4CD25+CD161− Treg (figure 4A upper panel). Most IL-17-producing Treg cells were enriched in the CD161 Treg population and few CD161− Treg cells expressed IL-17 (figure 4A middle panel). Besides IL-17 production, the CD4CD25+CD161+ Treg cells also expressed significantly higher RORγt than the CD161− Treg population (figure 4A lower panel), which further confirmed that CD4CD25+CD161+ Treg population was the IL-17-producing Treg subset and processed increased plasticity toward Th17. Similar results were also obtained in Treg cells from synovial fluid of RA patients and peripheral blood of HCs (data not shown).

    Figure 4
    Figure 4

    IL-17-producing Treg cells were enriched in the CD4CD25+CD161+ Treg subset. CD4 CD25− effector T (Tresp) cells stained with carboxyfluorescein diacetatesuccinimidyl ester (CFSE) were cultured alone or with IL-17-producing CD4CD25+CD161+ Treg cells or CD4CD25+CD161− Treg cells with rare IL-17 production. Tresp cells were stimulated with anti-CD3 and anti-CD28 mAb coated beads, and their proliferation in 5 days was determined by flow cytometry. (A) CD4CD25+ Treg cells could be isolated into CD4CD25+CD161+ subset and CD4CD25+CD161− subset. IL-17-producing Treg cells were enriched in the CD4CD25+CD161+ Treg subset. RORgt expression was significantly elevated in CD4CD25+CD161+ Treg cells (*p<0.05). (B) The CD4CD25+CD161+ and CD4CD25+CD161− Treg subsets from peripheral blood of rheumatoid arthritis (RA) patients and HCs showed comparable suppression activity on CD4CD25− Tresp cell proliferation, while the suppressive activity of CD4CD25+CD161+ and CD4CD25+CD161− Treg subsets from RA synovial fluid was dramatically impaired. Data are representative of 3–5 independent experiments (*p<0.05). RA: patients with RA; HCs: healthy controls; Tresp: responder T cell.

    Peripheral IL-17-producing Treg cells from patients with RA retained suppressive function in vitro

    We next investigated whether the IL-17-producing Treg cells from patients with RA possessed suppressive function. Although IL-17-producing Treg cells expressed IL-17, we also observed significantly elevated immunosuppressive molecular IL-10 and CTLA-4 expression in this Treg subset than in conventional Treg cells (figure 2B), which implicated the possibility that IL-17-producing Treg cells might play a role in immunosuppression.

    By CD161 staining, it was feasible to sort and enrich fresh IL-17-producing Treg cells from peripheral blood or synovial fluid. We set up Treg suppression assay with CD4CD25+CD161+ IL-17-producing Treg cells and CD4CD25+CD161− Treg cells from peripheral blood of RA patients and HCs as well as these two Treg subsets from RA synovial fluid. The CD4CD25+CD161+ Treg cells and CD4CD25+CD161− Treg cells from RA and healthy peripheral blood showed comparable suppression activity on CD4CD25− responder T-cell (Tresp) proliferation (figure 4B). By contrast with Tregs from peripheral blood, CD161 IL-17-producing Tregs and CD161− Tregs from RA synovial fluid did not inhibit Tresp proliferation (figure 4B). This result shows that IL-17-producing Treg cells circulating in peripheral blood retained a potent suppressive function to inhibit T cell proliferation, but the immunosuppressive activity of Treg cells in RA synovial fluid was seriously impaired.

    Associations between circulating IL-17-producing Treg cells and RA clinical features

    To determine the clinical relevance of IL-17-producing Treg cells in RA, the percentages of this Treg subset among CD4 Foxp3+ Treg cells in the peripheral blood were measured in a cohort of 42 patients with RA. The percentages of IL-17-producing Treg cells among CD4 Foxp3+ Treg cells were negatively correlated with ESR (r=−0.50, p<0.01), CRP (r=−0.32, p=0.036), and total IgG (r=−0.371, p=0.022) (figure 5A). There were also weak but significant positive correlations between this subset and circulating complement levels (C3, r=0.33, p=0.048; C4, r=0.32, p=0.045; figure 5A), suggesting that C3 and C4 levels would decrease with fewer IL-17-producing Treg cells. No significant correlation was found with patient age, disease duration, anti-cyclic citrullinated peptide (anti-CCP) antibody, RF-IgM, RF-IgG, total IgA, total IgM, tender joint count, swollen joint count, or DAS28. These correlations indicate that the increased ratio of IL-17-producing Treg cells is associated with milder inflammation in patients with RA, which suggests that the circulating IL-17-producing Treg subset plays a negative regulatory role in the autoimmunity of RA. Additionally, the ratio of IL-17-producing Treg cells was significantly lower in CD4 Treg cells from patients with high RA disease activity (DAS28>5.1) than in cells from patients with intermediate or low disease activity (DAS28<5.1; figure 5B). These results were consistent with our data from in vitro suppressive assays, suggesting that circulating IL-17-producing Treg cells in patients with RA retain their regulatory function in the inflammatory conditions of RA.

    Figure 5
    Figure 5

    Negative correlation of IL-17-producing Treg cells with rheumatoid arthritis (RA) disease activity. The correlations between the percentage of IL-17-producing CD4 Foxp3+ Treg cells in circulating CD4 Foxp3+ Treg cells and clinical features of patients with RA (n=42) were analysed by Spearman's rank correlation test. RA disease activity was evaluated by the 28-joint count disease activity score (DAS28), and the IL-17-producing CD4 Foxp3+ Treg cell frequencies were compared between patients with high RA disease activity and patients with intermediate and low disease activity. (A) The ratio of IL-17-producing CD4 Foxp3+ Treg cells in circulating CD4 Foxp3+ Treg cells in patients with RA was negatively correlated with the inflammation markers ESR, CRP, and total IgG, and was positively correlated with C3 and C4 levels. (B) Patients with higher circulating IL-17-producing Treg cell ratios had lower RA disease activity. *p<0.05.

    Discussion

    Recent studies have reported plasticity in the differentiation of Treg and Th17 cells. Foxp3+ Treg cells can be converted into inflammatory cytokine-producing cells in the specific inflammatory microenvironment, and may potentially contribute to disease pathogenesis.14–17 ,22–24 However, the contributions of Treg plasticity to the pathogenesis of autoimmune diseases require further study. In this study, we report for the first time the identification of IL-17-producing CD4 Foxp3+ Treg cells in patients with RA. The frequency of this IL-17-producing Treg population is significantly increased in peripheral blood and synovial fluid of RA patients compared with that in HCs, which suggests that this Treg cell subset is associated with RA pathology.

    We evaluated the distribution of IL-17-producing Treg in CD4CD25+ Treg subpopulations defined with different CD45RA and Foxp3 expression by Sakaguchi group.20 The majority of IL-17-producing Treg cells (over 60%) were from the CD45RA− Foxp3low ‘non-Treg’ fraction, which was considered transiently and unstably expressing Foxp3 and was shown to lack suppressive activity and had Th17 cell potential,20 while relatively fewer IL-17-producing Treg cells were also distributed in the CD45RA+ Foxp3low ‘resting Treg’ fraction (around 30%) and CD45RA- Foxp3hi ‘activating Treg’ fraction (less than 10%), both of which stably expressed Foxp3 and were suppressive.20 This result clearly revealed that IL-17-producing Treg cells derived from different Treg lineages, and Treg plasticity toward Th17 existed in all these subpopulations.

    We observed that circulating IL-17-producing Foxp3+ Treg cells in peripheral blood exhibited phenotypes of Th17 cells, including IL-17 secretion and elevated expression of RORγt, CD161 and CCR6. CD161 has been identified to be an important Th17 molecular marker. Nearly all Th17 cells were contained in CD161 population and originated from CD161 naive T cells.21 In this study, we also identified CD161 as the key surface marker to characterise IL-17-producing Treg cells. The CD4CD25+CD161+ Treg subset enriched nearly all the IL-17-producing Treg cells, and the RORγt expression was significantly increased in this subset, which indicated that the CD161 specifically defined the Treg subset producing IL-17 and possessing high plasticity toward Th17.

    Besides the Th17 lineage markers, the IL-17-producing Treg cells from peripheral blood of RA patients continued to express the anti-inflammatory factors IL-10 and CTLA-4 and to retain functional suppressive activity in vitro. Additionally, we found a significant negative correlation between this Treg subset in peripheral blood and RA inflammation markers (ESR, CRP, total IgG and C3/C4 decreasement), which indicated that inflammation and autoimmune responses might be milder with increased peripheral IL-17-producing Treg cells in patients with RA. This clinical relevance is consistent with the potent suppressive function of RA peripheral IL-17-producing Treg cells. Our data are in agreement with the recent finding that IL-17-positive Treg cell clones from the inflamed intestinal mucosa of patients with Crohn's disease remained suppressive in vitro,17 and our results support those of an in vitro investigation by Walter et al,25 which showed that the IL-17-producing Treg cells induced by RA-related cytokine-activated monocytes in vitro exhibited enhanced inflammatory cytokine expression and suppressive function. Together with findings from different experimental systems, our study suggests that an increased proinflammatory cytokine IL-17-expressing Treg population in peripheral blood acts as a negative regulator in RA development by maintaining or enhancing suppressive capacity.

    The IL-17-producing Treg subset exhibits phenotypic characteristics of Treg and Th17 cells. As a result of the overproduction of proinflammatory cytokines in RA, it is possible that Treg cells may be skewed toward IL-17-producing Foxp3+ Treg cells in the presence of inflammatory mediators, thereby favouring Th17 differentiation in patients with RA. Although these cells in peripheral blood could play a regulatory role in RA, we cannot exclude the possibility that some cells in some circumstances may functionally shift from regulatory T cells to inflammatory effectors expressing IL-17 but lost suppressive functions. In this study, we observed increased frequency of IL-17-producing Tregs in RA synovial fluid, but there was a seriously loss of Treg suppressive activity in RA synovial fluid (figures 1E and 4B). It is likely that in peripheral blood, the pathogenic function of IL-17-producing Foxp3+ Treg cells is limited because of their relatively low frequency and retaining suppressive functions. However, in RA synovial tissues, proinflammatory cytokines such as TNFα, IL-1, IL-6, and so on, dramatically increased than in peripheral blood.26 ,27 IL-1 or IL-6 increase might promote the Treg plasticity toward Th17, leading to elevated IL-17-producing Treg frequency in synovial fluid. Meanwhile, TNFα overexpression disabled Treg suppression in synovial fluid by dephosphorylating Foxp3.28 Thus, in RA synovial sites, IL-17-producing Treg cells losing suppressive function may act more like Th17 cells, which will promote the joint inflammation due to heavily impaired suppressive function as well as their IL-17 production and increased frequency. The IL-17-producing Treg subset may remain suppressive in RA peripheral blood but contribute to autoimmunity in synovial sites.

    We detected significantly elevated levels of IL-6 and IL-23 in the peripheral blood of patients with RA compared with HCs. Importantly, IL-6 and IL-23 have been recognised as key contributors to Th17 cell differentiation. IL-6 is engaged in polarising Th17 cell differentiation, whereas IL-23 is critical for Th17 cell stabilisation and expansion.22 ,29 ,30 Therefore, there may be a Th17 cell polarising environment that consists of Th17-related proinflammatory cytokines in the periphery blood of patients with RA. In the present study, IL-6 alone or IL-6 and IL-23 potently promoted the production of IL-17 by purified CD4CD25+ Treg cells from RA and HC peripheral blood in vitro, suggesting that IL-17-producing Treg cells may be differentiated from Foxp3+ Treg cells in RA inflammatory conditions. Treg cells from HCs and RA patients showed similar plasticity toward Th17 (figure 3C), which suggested that there might be no significant intrinsic differences in Treg cell sensitivity to polarising cytokines between RA patients and HCs. The higher RA peripheral IL-17-producing Treg frequency might be attributed to the microenvironment with significantly elevated polarising cytokines in RA peripheral blood.

    In conclusion, our data identified increased IL-17-producing regulatory T cells in the presence of elevated IL-6 and IL-23 levels in patients with RA, providing evidence that Treg cells with the plasticity of IL-17 expression in autoimmune conditions could retain their suppressive capacity. The peripheral Treg cell population with increased Th17 plasticity may still play a role as negative regulator in RA autoimmunity, but they lose suppressive activity in synovial sites and may become pathogenic. Future investigations regarding this cell population and the development of Treg/Th17 plasticity will provide a better understanding of the immunopathogenesis of RA.

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    Supplementary materials

    • Supplementary Data

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    Footnotes

    • Handling editor Tore K Kvien

    • TW and XS contributed equally to this paper.

    • Contributors LL and Z-GL designed the research. TW and XS performed the research and analysed the data. TW, XS, LL and Z-GL wrote the manuscript. JZ, JZ, HZ, CL, NG, YJ, DX, F-PH and NL helped with sample collection, data analysis and manuscript writing.

    • Funding This work was supported by National Basic Research Program of China (973 program, 2010CB529104), National Natural Science Foundation of China (31000409), Major International Joint Research Project of NSFC (No:81120108020), Program of International Science & Technology Cooperation of MOST (No.2010DFB34000) and Chinese Medical Association Clinical Medicine Research Foundation (12040630363).

    • Competing interests None.

    • Patient consent Obtained.

    • Ethics approval The Ethics Committee of Peking University People's Hospital.

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