Disturbed Th17/Treg balance in patients with rheumatoid arthritis

Qian Niu, Bei Cai, -chun , Yun-ying Shi & -lan Wang

Rheumatology International Clinical and Experimental Investigations

ISSN 0172-8172 Volume 32 Number 9

Rheumatol Int (2012) 32:2731-2736 DOI 10.1007/s00296-011-1984-x

1 23 Your article is protected by copyright and all rights are held exclusively by Springer- Verlag. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication.

1 23 Author's personal copy

Rheumatol Int (2012) 32:2731–2736 DOI 10.1007/s00296-011-1984-x

ORIGINAL ARTICLE

Disturbed Th17/Treg balance in patients with rheumatoid arthritis

Qian Niu • Bei Cai • Zhuo-chun Huang • Yun-ying Shi • Lan-lan Wang

Received: 7 September 2010 / Accepted: 3 July 2011 / Published online: 2 August 2011 Ó Springer-Verlag 2011

Abstract Proinflammatory Th17 cells and CD4?CD25? Introduction regulatory T (Treg) cells are two newly identified T lym- phocyte subsets, which have opposite effects on autoim- Rheumatoid arthritis (RA) is a chronic autoimmune disease munity and inflammation. To assess the Th17/Treg pattern with persistent inflammation of multiple synovial joints, and cytokine microenvironment in peripheral blood of which results in progressive tissue destruction of bone and patients with RA, we included 66 RA patients and 20 cartilage [1]. The pathogenesis of this destructive disease healthy volunteers. Of all these subjects, peripheral Th17 was classically viewed as involving two hierarchical sys- and Treg frequencies were analyzed by flow cytometry tems, governing inflammation and autoimmunity, respec- (FCM) and the plasma levels of interleukin (IL)-17, 23, 6, tively. The key players in this paradigm were the tumor necrosis factor (TNF)-a, transforming growth factor proinflammatory cytokine tumor necrosis factor (TNF)-a (TGF)-b1 were detected by ELISA. The results demon- and the Th1 subset of helper T cells [2, 3], suggesting that strated that RA patients revealed obvious increase in the Th1/Th2 imbalance characterized by enhanced Th1 peripheral Th17 frequencies and levels of Th17-related response played a crucial role in driving RA. cytokines (IL-17, IL-23, IL-6, TNF-a) while a significant Knowledge recently acquired in the field of immunopa- decrease in Treg frequencies and Treg-related cytokine thology has challenged the classical paradigm for RA. Th17 (TGF-b1) levels when compared with healthy people. Our cells and Foxp3?CD4?CD25? regulatory T cells are newly study indicated that development of RA is associated with described as two distinct T cell subsets from Th1 and Th2 peripheral Th17/Treg imbalance and characterized by a cells, having opposite effects on autoimmunity and inflam- proinflammatory cytokine microenvironment, which sup- mation. Th17 cells is a key effector in the immune response ports continuing generation of Th17 cells. and play critical roles in the development of autoimmunity by producing IL-17 and, to a lesser extent, TNF-a and IL-6 [4], Keywords Rheumatoid arthritis Á Th17 cells while Treg cells orchestrate the overall immune response and Regulatory T cells Á Autoimmunity Á Inflammation Á play a role in maintaining peripheral immune tolerance by Inflammatory cytokine microenvironment regulating the activity of the effector T cells [5]. Therefore, the Th17/Treg balance may control the development of autoimmunity and inflammation. In the present study, we aim Q. Niu Á B. Cai Á Z. Huang Á L. Wang (&) to evaluate the Th17/Treg balance pattern in patients with RA. Department of Laboratory Medicine, West China Hospital, Sichuan University, No. 37, Guo Xue , Chengdu 610041, The People’s Republic of China e-mail: [email protected] Methods

Y. Shi (&) Patients Department of Nephrology, West China Hospital, Sichuan University, No. 37, Guo Xue Xiang, Chengdu 610041, The People’s Republic of China This study was approved by the Ethics Committee of the e-mail: [email protected] Chinese Human Genome and the Ethics Committee of 123 Author's personal copy

2732 Rheumatol Int (2012) 32:2731–2736

? - ? ? Table 1 Demographics of RA patients and healthy controls and analyzed CD3 CD8 cells instead of CD3 CD4 Group n Male/female Age (years) P value cells.) For Treg analysis, 50 ll whole blood without stim- ulation was incubated with FITC/allophycoerythrin (APC)- Active RA 36 30/6 48 ± 12 [0.05 labeled anti-human CD4/CD25 cocktail (RPA-T4/BC96) Inactive RA 30 25/5 44 ± 9 (eBioscience, San Diego, California, USA) at 4°C for Healthy control 20 17/3 49 ± 10 30 min. After the surface staining, the whole blood were Values are expressed as mean ± SD or number stained with phycoerythrin (PE)-labeled anti-human IL-17A (SCPL1362) (eBioscience) for Th17 detection or PE-labeled anti-human Foxp3 (PCH101) (eBioscience) for Treg West China Hospital, and written informed consent was detection after fixation and permeabilization according to obtained from all participants. A total of 66 patients the manufacturer’s instructions (eBioscience). Isotype con- diagnosed as RA based on the 1987 criteria of the Amer- trols were given to enable correct compensation and confirm ican College of Rheumatology [6] and 20 healthy controls antibody specificity. Stained cells were run on a FACSCanto were included. Disease activity was assessed with the cytometer (BD Bioscience), and the data were analyzed 28-joint disease activity score (DAS28) [7]. None of the using FACSDiva software (BD Bioscience). patient had advanced liver diseases, renal failure, malig- nant diseases, infectious diseases or other inflammatory ELISA detection of plasma IL-17, IL-23, IL-6, TNF-a diseases (such as septicemia, pneumonia.). Demographic and TGF-b1 features of all the patients and healthy volunteers were provided in Table 1. The plasma concentrations of IL-17, IL-23, IL-6, TNF-a and TGF-b1 were measured by enzyme-linked immunosorbent Blood samples assay (ELISA), following the manufacturer’s instructions (all ELISA kits from Bender MedSystems, Burlingame, For all patients and control subjects, a 3-ml fasting blood USA). All samples were measured in duplicate. sample was drawn into a BD Vacutainer tube containing sodium heparin at 8:00–9:00 a.m. Whole blood was used Statistical analysis for flow cytometric analysis. Plasma was obtained after centrifugation and stored at -80°C for the measurement of Values are expressed as mean ± SD or median (range) in the cytokines. the tables and figures. Data were analyzed using SPSS 16.0 software (Chicago, IL). Firstly, the data were analyzed by Flow cytometric analysis of Th17 and Treg cells ANOVA or Kruskal–Wallis H test. If significance was found, then Student–Newman–Keuls or Mann–Whitney test Cell preparation was performed to detect the difference among groups. Spearman’s correlation was used as a test of correlation For analysis of Th17, 500 ll whole blood of every sample between two continuous variables. P Values less than 0.05 was cultured in complete culture medium (RPMI 1640 were considered significant. supplemented with 10% heat-inactivated fetal calf serum) for 5 h, in the presence of phorbol myristate acetate (PMA, 50 ng/ml) plus ionomycin (1 lg/ml) and monensin (1 lg/ Results ml). The incubators were set at 37°C under a 5% CO2 environment. For analysis of Treg, 50 ll whole blood of Basic clinical characteristics of patients every sample was aliquoted into a tube for further staining. There was no significant difference in age and gender Surface and intracellular staining among RA patients and healthy controls (Table 1).

For Th17 analysis, 50 ll stimulated whole blood was incu- Frequencies of circulating Th17 in patients with RA bated with PerCP-labeled anti-human CD3 (SK7) (BD Bioscience, San Diego, USA) and fluorescein isothiocyanate As shown in Fig. 1, the frequencies of Th17 (CD8-IL17?/ (FITC)-labeled anti-human CD8 (SK2) (BD Bioscience) at CD3?CD8- T) cells were remarkably higher in patients 4°C for 30 min. (Because CD4 positive cells would turn into with active RA (2.31 ± 1.38%) and inactive RA (1.91 ± CD4 negative cells after stimulation with PMA due to the 1.18%) than that of healthy subjects (0.71 ± 0.45%) endocytosis of CD4 molecules caused by PMA, so we used (P \ 0.01), while there was no obvious difference between anti-human CD3 and CD8 for the surface staining of Th17 two RA groups (P [ 0.05). 123 Author's personal copy

Rheumatol Int (2012) 32:2731–2736 2733

Fig. 1 Frequencies of circulating Th17 (CD8-IL17?) cells in methods. a Representative FACS pictures from a single subject in patients with RA. Whole blood from RA patients and healthy each group. b Collective analysis of results from all three groups. subjects were stimulated with PMA, ionomycin and monensin for 5 h, *P \ 0.01 versus healthy control and then stained with labeled anti-human antibodies as described in

Table 2 Frequencies of circulating Foxp3?CD4?CD25? Treg, Foxp3-CD4?CD25? Teff cells in RA patients and healthy subjects Group n Foxp3?CD4?CD25? Foxp3-CD4?CD25? (2)/(1) ratio Treg cells (%) (1) Teff cells (%) (2)

Active RA 36 1.54 ± 0.23* 24.59 ± 10.86* 39.99 ± 17.94* Inactive RA 30 1.59 ± 0.24* 20.05 ± 8.14 21.02 ± 4.64 Healthy control 20 4.33 ± 0.35 16.02 ± 6.66 4.95 ± 0.66 Values are expressed as mean ± SD. * P \ 0.01 versus healthy control

Fig. 2 Frequencies of circulating Tregs in patients with RA. Whole intern box represented CD4?CD25? T cells. b Representative blood from RA patients and healthy subjects were stained with pictures of Foxp3 expression in CD4?CD25? T subsets from a single labeled anti-human antibodies as described in methods. subject in each group a CD4?CD25? T subsets were gated by flow cytometry. Plots in

Frequencies of circulating Treg and Teff in patients all these differences between two RA groups were not sta- with RA tistically significant (P [ 0.05) (Table 2, Fig. 2).

The frequencies of Treg (Foxp3?CD4?CD25?/CD4? T Plasma concentrations of cytokines in patients with RA cells) in both active RA (1.54 ± 0.23%) and inactive RA groups (1.59 ± 0.24%) decreased significantly when As shown in Table 3, the plasma concentrations of IL-17, compared with healthy control group (4.33 ± 0.35%) IL-23, IL-6 and TNF-a in patients with active RA [IL-17: (P \ 0.01), while the frequencies of Teff (Foxp3-CD4? 2.85(1.84-4.91) pg/ml; IL-23: 54.33(49.49–65.37) pg/ml; CD25?/CD4? T cells) and the Teff/Treg ratios were all IL-6: 32.98(8.90–176.88) pg/ml; TNF-a: 1,158.10(338.63– significantly higher in active RA group (24.59 ± 10.86% 2,303.12) pg/ml] and inactive RA [IL-17: 2.69(1.91–5.11) and 39.99 ± 7.94%) than those in inactive RA (20.05 ± pg/ml; IL-23: 65.84(51.88–76.69) pg/ml; IL-6: 14.59(3.83– 8.14% and 21.02 ± 4.64%) and healthy control groups 62.44) pg/ml; TNF-a: 1,417.81(707.15–2,289.46) pg/ml] (16.02 ± 6.66% and 4.95 ± 0.66%) (P \ 0.01). However, increased significantly when compared with healthy

123 Author's personal copy

2734 Rheumatol Int (2012) 32:2731–2736

Table 3 Plasma concentrations of cytokines in RA patients and healthy subjects Group n IL-17 (pg/ml) IL-23 (pg/ml) IL-6 (pg/ml) TNF-a (pg/ml) TGF-b1 (ng/ml)

Active RA 36 2.85 (1.84–4.91)* 54.33 (49.49–65.37)* 32.98 (8.90–176.88)* 1,158.10 (338.63–2,303.12)* 4.36 (2.70–7.09)*, # Inactive RA 30 2.69 (1.91–5.11)* 65.84 (51.88–76.69)* 14.59 (3.83–62.44)* 1,417.81 (707.15–2,289.46)* 6.90 (4.78–8.70)* Health 20 1.43 (0.96–2.57) 36.07 (32.99–48.82) 1.55 (1.27–1.78) 13.50 (9.73–19.08) 24.19 (16.58–27.07) control Values are expressed as median (range). * P \ 0.001 versus healthy control; # P \ 0.05 versus inactive RA

Fig. 3 Spearman correlations between circulating Th17 cells and plasma cytokines. a Frequencies of circulating Th17 cells positively correlate with plasma IL-23 concentrations (r = 0.302, P \ 0.01). b Frequencies of circulating Th17 cells negatively correlate with plasma TGF-b1 concentrations (r =-0.436, P \ 0.001)

subjects [IL-17: 1.43(0.96–2.57) pg/ml; IL-23: 36.07 in patients with RA, we analyzed Th17/Treg on different (32.99–48.82) pg/ml; IL-6: 1.55(1.27–1.78) pg/ml; TNF-a: levels including cell frequencies and related cytokines 13.50(9.73–19.08) pg/ml] (P \ 0.001), while there was no secretion. The results demonstrated that balance of obvious difference between two RA groups (P [ 0.05). peripheral Th17/Treg was disturbed in RA patients, and the Plasma concentrations of TGF-b1 in active RA [4.36 Th17/Treg imbalance might act synergistically with micro- (2.70–7.09) ng/ml] and inactive RA groups [6.90(4.78– inflammation on the development of RA. 8.70) ng/ml] decreased significantly when compared with Th17 cells, an exciting newcomer to the T helper (TH) healthy control group [24.19(16.58–27.07) ng/ml] (P \ cell field in the last few years, distinct from Th1 and Th2 0.001), and the TGF-b1 concentrations in the active RA cells, have been shown to play a crucial role in several group was significantly lower than that in the inactive RA autoimmune and inflammatory diseases, such as inflam- groups. In addition, frequencies of Th17 cells were posi- matory bowel disease (IBD), childhood arthritis [juvenile tively correlated with plasma concentrations of IL-23 idiopathic arthritis (JIA)] and acute coronary syndrome (r = 0.302, P \ 0.01, Fig. 3a) and negatively correlated (ACS) [10–12]. Th17 cells are characterized by production with plasma concentrations of TGF-b1(r =-0.436, of IL-17, an important inflammatory cytokine which exerts P \ 0.001, Fig. 3b). robust proinflammatory effect by, alone and also in com- bination with TNF-a and IL-1, inducing the synthesis of proinflammatory cytokines (such as IL-6 and IL-8) and Discussion chemokines [such as macrophage chemoattractant protein (MCP)-1 and macrophage inflammatory chemokine-2 Although the Th1/Th2 imbalance paradigm allowed (MIP-2)], which mediate tissue infiltration and inflamma- researchers to achieve breakthroughs in the treatment of tion [13]. IL-17 has been found at high levels in synovial RA, it fails to explain some incontrovertible facts, such as fluid from RA patients [14], suggesting the potential role of the paradoxical effects of IFN-c, which alleviates inflam- IL-17 in the development of RA. The inflammatory mation in murine models of arthritis but promotes arthritis symptoms of collagen induced arthritis (CIA), the animal flares in healthy mice, and the lack of efficacy of mono- model for RA, were alleviated when blocked the endoge- clonal antibodies to IFN-c in most patients with RA [8, 9]. nous IL-17 [15]. In addition to IL-17, Th17 cells also Recent knowledge about the pathogenesis of RA has led to produce other proinflammatory cytokines to a lesser extent, a new paradigm for this destructive disease, in which the including IL-6 and TNF-a, which were two well-known Th17/Treg imbalance is the focus of concern in those inflammatory mediators and critical players in regulating studies. To explore the shift of Th17/Treg balance in detail tissue inflammation. Therefore, the roles of Th17 cells and

123 Author's personal copy

Rheumatol Int (2012) 32:2731–2736 2735 their proinflammatory cytokines in mediating autoimmune interrelated, and reciprocally influenced by cytokines, such and inflammation are worthy of deeper research. as TGF-b1, IL-6 and IL-23 [22–24]. TGF-b1 and IL-6 are In this study, results demonstrated that patients with RA sufficient for the differentiation of murine Th17 cells in exhibited a significant increase in peripheral Th17 fre- contrast to TGF-b1 alone, which promotes the develop- quency as compared with healthy controls, and this fre- ment of Treg cells that express Foxp3, a transcription quency was higher in active RA group than that in inactive factor important for maintaining the regulatory function of RA group, suggesting that Th17 cells may be the key Treg [25]. However, human Th17 cells generation appears player in the pathogenesis and progression of RA. Due to to be driven by IL-6 or IL-1 and enhanced by IL-23, but not the increase of Th17 cells, plasma concentrations of Th17- TGF-b1[26]. Therefore, the cytokine milieu of the local related cytokines (IL-17, IL-6 and TNF-a) increased environment plays a pivotal role in the differentiation from accordingly, which may play crucial roles in the inflam- naive CD4? T cells to Treg or Th17 cells. matory process of RA. Upregulated IL-17 and TNF-a can In the present study, results showed that the concentra- exert synergistic effects in stimulating synovial fibroblasts tions of IL-6 and IL-23, both of which promote the dif- and epithelial cells to secrete IL-6, IL-8, and PGE2, which ferentiation of Th17 cells, were all significantly higher in contribute to the formation of local tissue inflammation, RA patients, and IL-23 concentrations were positively and also inducing the receptor activator of NF-jB ligand correlated with Th17 cells frequencies. While, TGF-b1 (RANKL), which regulate osteoclastogenesis and the bone concentrations were obvious lower in RA patients and resorption process [13, 16]. negatively correlated with Th17 cells frequencies. These Contrary to Th17 cells, Foxp3?CD4?CD25? Treg cells suggested that the proinflammatory cytokine microenvi- have been identified as a key part of the immune system’s ronment, characterized by elevated IL-6 and IL-23 levels apparatus for controlling inflammatory processes [17]. Treg and decreased TGF-b1 concentrations, could potentially cells exert their regulatory function, in part, by secretion of support the continued generation of Th17 cells but mean- anti-inflammatory cytokines including TGF-b1, and so they while suppress the development of Treg cells, which led to are important in the induction and maintenance of immune the Th17/Treg numerical imbalance in RA patients. This homeostasis. Defects in Treg function or reduced numbers numerical imbalance might consequently result in the have been put forward as a cause of failed tolerance in functional imbalance of Th17/Treg which contributed to several human autoimmune diseases, including RA, multi- enhancing the formation of the inflammatory cytokine ple sclerosis (MS) and IBD [10, 18, 19]. In a series of murine microenvironment, and eventually formed a positive experiments, ablation of Treg led to multiple organ auto- feedback mechanism to amplify proinflammatory immune immunity, which could be reversed by adoptive transfer of responses. these Foxp3?CD4?CD25? T cells [20, 21]. Our results verified that peripheral Foxp3?CD4?CD25? Treg frequency and Treg-related cytokine (TGF-b1) all Conclusions significantly decreased in patients with RA, suggesting again that Treg cells and the anti-inflammatory cytokine Our results demonstrated that numerical and functional TGF-b1 have a potentially protective effect in the pro- imbalance of Th17/Treg existed in the peripheral blood of gression and stability of RA. While, the frequencies of patients with RA. Inflammatory cytokine microenviron- peripheral Foxp3-CD4?CD25? Teff cells increased ment led to the enhancement of Th17 cell differentiation at markedly in active RA patients, which in combination with the expense of Tregs, and these combined changes con- the decrease of Treg cells directly led to the increase of tributed to triggering autoimmunity and inducing inflam- peripheral Teff/Treg ratios in active RA patients. Results mation. All these suggested a potential role for Th17/Treg suggested that the imbalance between immune regulators imbalance in the pathogenesis and progression of RA. and responsive T cells might be the major cause of failed Therefore, blocking critical cytokines in vivo, notably IL-6 peripheral tolerance, which contributed to autoimmune and IL-23, or giving exogenous TGF-b1, may promote inflammation in RA patients. Meanwhile, our data showed recovery of RA patients though restoring the Th17/Treg a reciprocal relationship between peripheral Th17 and Treg balance. cells numbers, as well as between Th17 and Treg-related cytokines, indicating that the peripheral Th17/Treg balance Acknowledgments We thank the patients for donation of samples was disturbed in RA patients and the numerical and func- to the study, as well as the hospital staff who made this study possible. tional imbalance of Th17/Treg might have a potential We also thank and Bing for helping process patient impact on the disease progression. samples. We are very grateful to Prof. B. Ying for critical reading of the manuscript. This research was sponsored by National Natural Several studies have independently demonstrated that Science Foundation of China (No. 30670819, 30772051 and the development of Th17 and Treg cells may be 30950010). 123 Author's personal copy

2736 Rheumatol Int (2012) 32:2731–2736

Conflict of interest The authors declare that they have no conflict 14. Kim KW, Cho ML, Park MK et al (2005) Increased interleukin- of interest. 17 production via a phosphoinositide 3-kinase/Akt and nuclear factor jB-dependent pathway in patients with rheumatoid arthritis. Arthritis Res Ther 7:R139–R148 15. Berg WB, Miossec P (2009) IL-17 as a future therapeutic target for rheumatoid arthritis. Nat Rev Rheumatol 5:549–553 References 16. Lubberts E (2008) IL-17/Th17 targeting: on the road to prevent chronic destructive arthritis? Cytokine 41:84–91 1. Firestein GS (2003) Evolving concepts of rheumatoid arthritis. 17. Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T Nature 423:356–361 cell development by the transcription factor Foxp3. Science 2. McInnes IB, Schett G (2007) Cytokines in the pathogenesis of 299:1057–1061 rheumatoid arthritis. Nat Rev Immunol 7:429–442 18. Abdulahad WH, Stegeman CA, van der Geld YM et al (2007) 3. Falgarone G, Duclos M, Boissier MC (2007) TNF-a antagonists Functional defect of circulating regulatory CD4 T cells in patients in rheumatoid arthritis patients seen in everyday practice. Joint with Wegener’s granulomatosis in remission. Arthritis Rheum Bone Spine 74:523–526 56:2080–2091 4. Bettelli E, Oukka M, Kuchroo VK (2007) T(H)-17 cells in the 19. Venken K, Hellings N, Liblau R et al (2010) Disturbed regulatory circle of immunity and autoimmunity. Nat Immunol 8:345–350 T cell homeostasis in multiple sclerosis. Trends Mol Med 5. Marie-Christophe B, Eric A, Geraldine F et al (2008) Shifting the 16:58–68 imbalance from Th1/Th2 to Th17/treg: The changing rheumatoid 20. Wright GP, Notley CA, Xue SA et al (2009) Adoptive therapy arthritis paradigm. Joint Bone Spine 75:373–375 with redirected primary regulatory T cells results in antigen- 6. Arnett FC, Edworthy SM, Bloch DA et al (1988) The American specific suppression of arthritis. Proc Natl Acad Sci USA Rheumatism Association 1987 revised criteria for the classifica- 106:19078–19083 tion of rheumatoid arthritis. Arthritis Rheum 31:315–324 21. Morgan ME, Flierman R, van Duivenvoorde LM et al (2005) 7. Prevoo ML, van’t Hof MA, Kuper HH et al (1995) Modified Effective treatment of collagen-induced arthritis by adoptive disease activity scores that include twenty-eight-joint counts. transfer of CD25? regulatory T cells. Arthritis Rheum Development and validation in a prospective longitudinal study 52:2212–2221 of patients with rheumatoid arthritis. Arthritis Rheum 38:44–48 22. Bettelli E, Carrier Y, Gao W et al (2006) Reciprocal develop- 8. Manoury-Schwartz B, Chiocchia G, Bessis N et al (1997) High mental pathways for the generation of pathogenic effector TH17 susceptibility to collagen-induced arthritis in mice lacking IFN- and regulatory T cells. Nature 441:235–238 gamma receptors. J Immunol 158:5501–5506 23. Kurts C (2008) Th17 cells: a third subset of CD4? T effector 9. Boissier MC, Chiocchia G, Bessis N et al (1995) Biphasic effect cells involved in organ-specific autoimmunity. Nephrol Dial of interferongamma in murine collagen-induced arthritis. Eur J Transplant 23:816–819 Immunol 25:1184–1190 24. Wilson NJ, Boniface K, Chan JR et al (2007) Development, 10. Eastaff-Leung N, Mabarrack N, Barbour A et al (2010) Foxp3? cytokine profile and function of human interleukin 17-producing regulatory T cells, Th17 effector cells, and cytokine environment helper T cells. Nat Immunol 8:950–957 in inflammatory bowel disease. J Clin Immunol 30:80–89 25. Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs 11. Nistala K, Moncrieffe H, Newton KR et al (2008) Interleukin- the development and function of CD4? CD25? regulatory T 17—producing T cells are enriched in the joints of children with cells. Nat Immunol 4:330–336 arthritis, but have a reciprocal relationship to regulatory T cell 26. Acosta-Rodriguez EV, Napoletani G, Lanzavecchia A et al numbers. Arthritis Rheum 58:875–887 (2007) Interleukins 1b and 6 but not transforming growth factor-b 12. Chengl X, X, Ding Y-J et al (2008) The Th17/Treg imbalance are essential for the differentiation of interleukin 17-producing in patients with acute coronary syndrome. Clin Immunol 127: human T helper cells. Nat Immunol 8:942 89–97 13. Paradowska A, Mas´´ski W, Grzybowska-Kowalczyk A et al (2007) The function of interleukin 17 in the pathogenesis of rheumatoid arthritis. Arch Immunol Ther Exp 55:329–334

123