Differentiation in Rheumatoid Arthritis Th17 Fibroblast-Like Synoviocytes

The Journal of Immunology

Cyr61 Induces IL-6 Production by Fibroblast-like Synoviocytes Promoting Th17 Differentiation in Rheumatoid Arthritis

Jinpiao Lin,*,1 Zhou Zhou,*,1 Rongfen Huo,* Lianbo Xiao,† Guilin Ouyang,† Li Wang,* Yue Sun,* Baihua Shen,* Dangsheng Li,‡ and Ningli Li*

Cysteine-rich protein 61 (Cyr61)/CCN1 is a product of an immediate early gene and functions in mediating cell adhesion and inducing cell migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) promotes FLS proliferation and participates in RA pathogenesis with the IL-17–dependent pathway. However, whether Cyr61 in turn regulates Th17 cell differentiation and further enhances inflammation of RA remained unknown. In the current study, we explored the potential role of Cyr61 as a proinflammatory factor in RA pathogenesis. We found that Cyr61 treatment dramatically induced IL-6 production in FLS isolated from RA patients. Moreover, IL-6 production was attenuated by Cyr61 knockdown in FLS. Mechanistically, we showed that Cyr61 activated IL-6 production via the avb5/Akt/NF-kB signaling pathway. Further, using a coculture system consisting of purified CD4+ T cells and RA FLS, we found that RA FLS stimulated Th17 differentiation, and the pro-Th17 differentiation effect of RA FLS can be attenuated or stimulated by Cyr61 RNA inter- ference or addition of exogenous Cyr61, respectively. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the anti-Cyr61 mAb led to reduction of IL-6 levels, decrease of Th17 response, and attenuation of inflammation and disease progression in vivo. Taken together, our results reveal a novel role of Cyr61 in promoting Th17 development in RA via upregulation of IL-6 production by FLS, thus adding a new layer into the functional interplay between FLS and Th17 in RA pathogenesis. Our study also suggests that targeting of Cyr61 may represent a novel strategy in RA treatment. The Journal of Immunology, 2012, 188: 5776–5784.

uman rheumatoid arthritis (RA) is a systemic inflam- macrophages and fibroblasts, and the upregulation of RANK li- matory disease that involves hyperplasia of synovial tis- gand in osteoclasts, leading to stimulation of the activity of matrix H sues and structural damage to cartilage, bone, and liga- metalloproteinases, matrix catabolism, and bone resorption (6, 7). ments (1). The cause and pathogenesis of RA are still unclear. Elevated production of IL-17 has also been observed in the Th17 cells are now believed to play a crucial role in the lesions (2, collagen-induced arthritis (CIA) murine model (8). Mice deficient 3). Synovial macrophages have been found to be particularly ef- in IL-17 are protected from the development of CIA (9), and fective at promoting Th17 differentiation suggesting that optimal blockade of IL-17 with specific Abs can ameliorate symptoms in generation of Th17 cells is fueled by the inflammatory milieu CIA (10), supporting a critical role of IL-17 in the development of present in the local microenvironment of RA (4). Elevated pro- arthritis symptoms. duction of IL-17 has been observed in RA patients (5). IL-17 is IL-6 is abundantly present in the synovial fluid (SF) of RA reported to induce the production of proinflammatory cytokines, patients and is thought to mediate many of the local and systemic such as TNF-a and IL-1, in many local tissue cell types, such as effects of this disease (11, 12). Moreover, it is now clear that IL-6 is an essential cytokine involved in inducing the differentiation of Th-17 cells in both humans and mice (13, 14). In the presence of *Shanghai Institute of Immunology, Institute of Medical Sciences, Shanghai Jiao Tong IL-1b (in human) or TGF-b (in mouse), IL-6 activates the ex- University School of Medicine, Shanghai 200025, China; †Department of Orthopedics, pression of the critical Th17 transcription factor RORc (in human) ‡ Guanghua Rheumatology Hospital, Shanghai 200052, China; and Shanghai Institutes or RORgt (in mouse) through the Jak/STAT3 pathway in Th17 cell for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China development (13, 15). IL-6 is induced by many inflammatory 1J.L. and Z.Z. contributed equally to this work. cytokines, such as IL-1b, TNF-a, and IFN-g (16, 17). However, Received for publication November 9, 2011. Accepted for publication March 28, 2012. whether cysteine-rich protein 61 (Cyr61), a member of the CCN protein family that we have previously shown to be abundantly This work was supported by the National Basic Research Program of China (2010CB529103), the National Natural Science Foundation of China (81072468 produced by RA fibroblast-like synoviocytes (FLS) (18), plays any and 81172856), the Science and Technology Commission of Shanghai Municipality role in IL-6 production and the inflammation processes of RA has (10JC1408300), and Shanghai Municipal Education (J50207). not yet been explored. Address correspondence and reprint requests to Prof. Ningli Li, Shanghai Institute of Cyr61 is a product of an immediate early gene and is known to Immunology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China. E-mail address: function in mediating cell adhesion and inducing cell migration [email protected] (19). Cyr61 enhances growth factor-induced DNA synthesis in The online version of this article contains supplemental material. fibroblasts and endothelial cells (20, 21). As a secreted extracel- Abbreviations used in this article: CIA, collagen-induced arthritis; Cyr61, cysteine- lular matrix protein, Cyr61 could elicit diverse functions by rich protein 61; FLS, fibroblast-like synoviocyte; MRI, magnetic resonance imaging; binding to different types of integrin molecules such as avb3, OA, osteoarthritis; RA, rheumatoid arthritis; SF, synovial fluid. avb5, aIIbb3, and a6b1 on different cells (22–24). We reported Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 previously that the expression of Cyr61 is greatly enhanced in FLS www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103201 The Journal of Immunology 5777 from RA patients and that this increased expression of Cyr61 in NMR system (Varian) equipped with a 160-mm bore-diameter magnet, turn acts further to stimulate FLS proliferation (18). Notably, we with maximum 400 mT/m gradients. The mouse was positioned on its side also found that IL-17 produced by Th17 cells is a major signal that with its left hind paw held at a right angle on a horizontal plate, which was similar to the spontaneous posture of the treated mice. A rapid phased- is responsible for stimulating the production of Cyr61 by FLS. array surface coil was used for data acquisition, and the radio frequency These results not only reveal for the first time to our knowledge surface coil was positioned horizontally just above the mouse joint. The that Cyr61 contributes to hyperplasia of synovial lining cells but following parameters were used for optimized MRI imaging analysis: 3 2 also establish a new mechanism of the functional link between gradient echo sequence with fat suppression; FOV, 30 30 mm ; matrix size, 256 3 256; voxel size, 117 3 117 mm2; thickness, 0.8 mm; TR/TE, Th17 cells and FLS in RA, whereby the Th17 cells act to promote 195/5 ms; flip angle, 45˚; NEX, 4 (30). FLS proliferation through stimulating the expression of Cyr61 in these cells (18). Nevertheless, whether Cyr61 has any effect on Real-time PCR analysis Th17 differentiation and plays roles in the inflammation process of Total RNA extracted from cells and real-time PCR were performed as RA remains unknown. previously reported (18). The primers used in this study are shown in In this study, we found that Cyr61 stimulated IL-6 production by Supplemental Table I. FLS via the Cyr61/avb5/Akt/NF-kB signaling pathway. Increased Proliferation assay IL-6 in turn promoted Th17 differentiation. Blocking of Cyr61 action with an mAb (093G9) reduced IL-6 production by FLS, Proliferation assay was performed as described previously (31). Briefly, 3 5 attenuated Th17 response, and ameliorated disease progression in mouse mononuclear cells (1 10 per well) from spleens or lymph nodes treated with 093G9 or control IgG were incubated in the presence or ab- CIA mice. In conclusion, Cyr61 plays a critical role in stimulating sence of CII (20 mg/ml). Cultures were maintained at 37˚C in 5% CO2 and IL-6 expression by FLS in RA and contributes to Th17 cell dif- pulsed with 1 mCi [3H]thymidine 16 h before harvest. [3H]Thymidine in- ferentiation and thus is likely a key molecule involved in the in- corporation was measured as count pulse per minute. flammation process of RA. Targeting of Cyr61 may be a novel Western blot analysis therapeutic strategy for RA. Protein immune blotting was performed as described previously (18). In brief, tissue or cell lysates was separated by SDS-PAGE electrophoresis Materials and Methods followed by transfer to polyvinylidene fluoride membranes (Millipore) at Animals 30 V overnight. The expression of Cyr61 and the activation of STAT3, Jak1, Akt, and NF-kB was analyzed using specific Abs (Cell Signaling Six- to eight-week-old male DBA/1J mice were purchased from the Technology, Beverly, MA). After washing with PBS, the membranes were Shanghai Laboratory Animal Center, Chinese Academy of Science. Mice incubated with HRP-conjugated goat anti-mouse IgG at room temperature were maintained under pathogen-free conditions. All experiments were for 1 h followed by washing with PBS. The target proteins were examined performed according to the animal care and use committee guidelines. with an ECL system (Millipore) and visualized with autoradiography film. Patients and specimens Confocal laser scanning fluorescence microscopy assay 6 A total of 48 RA patients (8 men and 40 women, aged 36–70 y, mean SD NF-kB nuclear translocation in FLS was studied with confocal laser scan- 53 6 9 y) were included in the study. The disease duration of RA patients 6 ning fluorescence microscopy (LSM510; Zeiss, Jena, Germany) technique was 14 9 y. The diagnosis of RA fulfilled the revised criteria by the as described previously (18). American College of Rheumatology (25). The control subjects were 35 osteoarthritis (OA) patients fulfilling the diagnosis criteria of OA proposed ELISA detection by Altman (26). Synovial tissues were obtained from patients and FLS were cultured and identified as reported previously (18). The FLS were Sera of mice, SF, and sera of RA and OA patients and supernatants of cell freshly isolated, and the cells were used for experiment at three to six culture were collected and diluted for the measurement of IL-6, IL-1b, and passages. SF, serum specimens, and supernatants were collected as re- TGF-b by ELISA (R&D Systems) according to the manufacturer’s rec- ported previously (18). All study protocols and consent forms were ap- ommendations. A standard curve was performed for each plate and used to proved by the Institutional Medical Ethics Review Board of the Shanghai calculate the absolute concentrations of the indicated cytokines. Jiao Tong University School of Medicine. Flow cytometric analysis CD4+ T cell cocultured with FLS Single-cell suspensions from CIA mice spleen and lymph nodes were CD4+ T cells (2 3 106), purified from healthy individual PBMC with isolated. CD4+ T cells cocultured with FLS were collected after 4 d. For Dynabeads Untouched Human CD4 T Cells kit (Invitrogen, Life Tech- surface markers staining, fluorescence conjugated CD4, CD130 Abs were nologies) according to the manufacturer’s instructions, were cocultured used. We performed intracellular staining for IL-4, IL-10, IL-17, and with 2 3 104 FLS plated in 24-well plates stimulated with 5 mg/ml Cyr61 IFN-g after stimulation with PMA (50 ng/ml) and ionomycin (1 mg/ml; protein (PeproTech, Rocky Hill, NJ) or the Cyr61 gene was knocked down Sigma) for 5 h in the presence of GolgiPlug (BD Biosciences) according in a Transwell system. Abs to CD3, CD28, and rhIL-1b, anti–IFN-g, and to the manufacturer’s protocol. Flow cytometry was performed using a anti–IL-4 Ab (BD Biosciences) were added into the coculture system FACSCalibur cytometer and analyzed using CellQuest software (BD according to previous reports for Th17 differentiation (27). Biosciences). Establishment and treatment of CIA Statistical analysis CIA was induced as described previously (28). Briefly, male DBA/1J All experiments were performed in triplicate. The difference among groups mice were injected intradermally with 150 mg chicken type II collagen was determined by ANOVA analysis, and comparison between two groups (Chondrex, Redmond, WA) in 0.05 M acetic acid emulsified in FCA. was analyzed by the t test using GraphPad Prism 4.0 (GraphPad Software, Booster injections were administered on day 21 with a total of 75 mg San Diego, CA). A value of p , 0.05 was considered statistically signif- collagen II in Freund’s incomplete adjuvant. Joint inflammation was icant. evaluated on a scale of 1–4 (29), with a maximum clinical score of 16 per mouse. Mice were treated with control IgG or anti-Cyr61 mAb 093G9 generated in our laboratory (200 mg/mouse) i.p. twice a week when the Results clinical score reached 2. Cyr61 induced IL-6 production by FLS of RA patients H&E staining and magnetic resonance imaging assay IL-6 is abundantly present in SF of RA patients, and a number of cell types are known to produce IL-6, including macrophages and Mice joints were removed from sacrificed CIA mice and fixed in 10% phosphate-buffered formalin, decalcified in 10% EDTA, embedded in fibroblasts (1, 11). We have previously shown that Cyr61, an ex- paraffin, and stained with H&E for light microscopy. The magnetic reso- tracellular matrix protein secreted by FLS, plays an important role nance imaging (MRI) experiments were carried out at 7 T on a Varian in promoting FLS proliferation (18). To explore further whether 5778 Cyr61-INDUCED IL-6 PRODUCTION PROMOTES Th17 DIFFERENTIATION

Cyr61 may also stimulate IL-6 production by FLS, we set up an TGF-b expression did not show any change (Fig. 1E). The re- in vitro cell culture system using FLS isolated from RA patients. duction of IL-6 production by FLS upon Cyr61 knockdown was We first analyzed the cytokine profile of these patients (SF and also confirmed by ELISA measurement of IL-6 protein levels in serum samples from RA and OA patients) and found that the culture supernatant (Fig. 1F). These data indicate that Cyr61 could levels of IL-1b and IL-6 were higher in RA SF than in OA SF, but induce IL-6 expression by FLS of RA patients, which may con- the serum levels of both cytokines were very low in both RA and tribute to the higher concentration of IL-6 observed in RA SF. OA patients (Fig. 1A), consistent with other reports (11, 32). a b k However, TGF-b levels were higher in sera of RA and OA patients Cyr61 induced IL-6 production in FLS via v 5/Akt/NF- B than those in SF (Fig. 1A). After confirming that RA SF contained signaling pathway higher levels of IL-6 and IL-1b, we next tested the potential effect Previous studies have identified several integrins as the cell surface of Cyr61 on the expression of these two cytokines by FLS of RA receptors for Cyr61 in different cell types (20, 22–24). We thus patients. The results showed that Cyr61 significantly stimulated examined the mRNA expression profile of the integrin family in IL-6 and IL-1b mRNA expression in FLS in a dose-dependent RA FLS and found high levels of expression for integrin av, b1, manner and reached a peak at 2 h (Fig. 1B, 1C), whereas the and b5 (Fig. 2A). However, only the anti-avb5 Ab could block expression of TGF-b was not enhanced in response to Cyr61 stim- Cyr61-induced IL-6 production (Fig. 2B and data not shown), ulation (Fig. 1B). Consistent with these observations, ELISA assay suggesting that avb5 is likely the major receptor on FLS that showed that concentration of IL-6 in FLS culture supernatant mediates the effect of Cyr61, which is consistently with our pre- was significantly increased upon addition of exogenous Cyr61, vious reports (18). To probe further the downstream signaling whereas the level of TGF-b did not show much alteration. Nota- pathway(s), we used known inhibitors of several pathways, in- bly, although IL-1b mRNA expression in FLS was moderately cluding PDTC (inhibitor of NF-kB activation), SB203580 (in- increased by Cyr61 treatment, the concentration of IL-1b in FLS hibitor of p38 MAPK), and PD98059 (inhibitor of ERK-1/2). The culture supernatant was too low to be detected (Fig. 1D), sug- results showed that Cyr61-stimulated IL-6 mRNA and protein gesting that FLS might not be a significant source of IL-1b in RA expression in FLS was markedly decreased in the presence of the SF (11). To examine further the autocrine role of Cyr61 in the NF-kB inhibitor. In contrast, inhibition of p38 MAPK and ERK1/ regulation of IL-6 expression by FLS, we used a specific small 2 activities showed no effect on Cyr61-induced IL-6 production interfering RNA (18) to knock down Cyr61 expression in FLS. (Fig. 2C). Further analysis showed that Cyr61 treatment led to The results showed that both IL-6 and IL-1b mRNA expression a dramatic increase in the phosphorylation level of the NF-kB p65 were remarkably reduced in Cyr61-knockdown FLS, whereas subunit in FLS (Fig. 2D) and enhanced NF-kB nuclear translo-

FIGURE 1. Cyr61 promoted IL-6 production in RA FLS. (A) IL-6, IL-1b, and TGF-b levels in SF and serum from RA patients (n = 20) and OA patients (n = 15) detected by ELISA. OA-SF (n), RA-SF (:), OA-sera (▼), and RA-sera (♦). (B) IL-6, IL-1b, and TGF-b mRNA expression in RA FLS stimulated by Cyr61 (0.62, 1.25, 2.5, 5, 10 mg/ml) determined by real-time PCR using a housekeeping gene as an endogenous control. (C) IL-6, IL-1b, and TGF-b mRNA expression in RA FLS stimulated by Cyr61 (0, 2, 4, 6, 8 h) determined by real-time PCR. (D) IL-6 (s), IL-1b (*), and TGF-b (d) protein levels in supernatant were evaluated by ELISA. FLS were seeded into 12-well plates (1 3 105 per well). The FLS were stimulated with Cyr61 protein (10, 5, 2.5, 1.25 mg/ml) for 48 h. (E) IL-6, IL-1b, and TGF-b mRNA expression in Cyr61-knockdown FLS was detected by real-time PCR. (F) The protein levels of IL-6, IL-1*, and TGF-b in FLS culture supernatant treated with SiCyr61 (small interfering RNA against Cyr61, black bar) or SiNC (small RNA of negative control, open bar) were measured by ELISA. Data represent the mean 6 SEM of at least three independent experiments. **p , 0.01. The Journal of Immunology 5779

FIGURE 2. Signaling pathways involved in Cyr61 regulation of IL-6 production in RA FLS. (A) Integrins involved in IL-6 production regulated by Cyr61. Integrin expression profiles in RA FLS treated with Cyr61 or BSA were evaluated by real-time PCR. (B) Inhibition of Cyr61-stimulated IL-6 expression by anti-avb5 mAb assessed by real-time PCR. The FLS were pretreated with anti-avb5 Ab (20 mg/ml) (Millipore, Billerica, MA) or control IgG for 1 h. After that, Cyr61 protein (5 mg/ml) was added to the culture system for another 2 h. The FLS were harvested to detect the expression of IL-6 by real-time PCR. (C) Effect of inhibitors of signaling pathways on Cyr61-induced IL-6 expression. RA FLS were treated with 4 mM PDTC, 1 mM PD98059, or 10 mM SB203580 in combination with Cyr61 (5 mg/ml) for 4 h (shaded bars). Cyr61 expression was evaluated by real-time PCR. Control (open bars); Cyr61 (no inhibitors, black bars). (D) Phosphorylation of NF-kB was detected by Western blot. Lane 1: unstimulated FLS. Lane 2 and lane 3, stimulated with Cyr61 (5 mg/ml) for 10 min and 30 min, respectively. (E) Nuclear translocation of NF-kB was monitored by confocal fluorescence microscopy. Top panels, Unstimulated FLS. Bottom panels, Stimulated with Cyr61 (5 mg/ml) for 60 min. NF-kB was detected by FITC–anti-p65 (green). Nuclei were stained with DAPI (blue). Merged picture shows NF-kB translocation into the nucleus. Original magnification 3600. (F) Phosphorylation of Akt was detected by Western blot. The data represent one of the three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. cation as shown by laser scanning confocal immunofluorescence IL-22 in the cocultured CD4+ T cells (Fig. 3B). We further ex- microcopy (Fig. 2E). Together, these results indicate that NF-kB amined the proportion of IL-17+ cells generated in this coculture activation plays an important role in Cyr61-induced IL-6 expres- system by intracellular cytokine staining. The results showed that sion in FLS. Previous studies in breast cancer cells implicated that Cyr61-stimulated FLS significantly enhanced the differentiation of Cyr61 could induce NF-kB activation via the PI3K/Akt pathway IL-17–producing Th17 cells, whereas knockdown of Cyr61 in (20, 33). We thus checked whether this pathway was also activated FLS or blocking of IL-6 by IL-6 neutralizing Ab reduced the in FLS upon Cyr61 stimulation. Indeed, we found that the phos- stimulatory effect of FLS on Th17 differentiation (Fig. 3C, 3D and phorylated (activated) form of Akt was strongly enhanced in re- Supplemental Fig. 1). These data indicate that Cyr61-stimulated sponse to Cyr61 treatment in FLS (Fig. 2F). On the basis of these FLS facilitated Th17 differentiation in vitro. results, we suggest that Cyr61 induced IL-6 production in FLS via Blocking of Cyr61 ameliorated inflammation of CIA mice and the avb5/Akt/NF-kB signaling pathway. downregulated Th17 population in vivo Cyr61-stimulated FLS promoted Th17 differentiation in vitro As we found that Cyr61-stimulated FLS promoted Th17 differ- Considering that IL-6 is critical for Th17 differentiation and its entiation in vitro, we asked whether Cyr61 indeed plays a role in expression was elevated in RA FLS stimulated by Cyr61, we Th17 cell differentiation in vivo. We treated CIA mice with an anti- hypothesized that this Cyr61-stimulated production of IL-6 by Cyr61 mAb (named 093G9), which was generated by our group FLS might in turn promote Th17 differentiation. To this end, we (35) and shown to block the effect of Cyr61 on the proliferation of cocultured RA FLS with purified CD4+ T cells in a Transwell and IL-6 production by FLS (Fig. 4A, 4B). As shown in Fig. 5A, system. The results showed that the expression of RORc, a critical the inflammatory score of CIA mice treated with 093G9 was transcription factor of human Th17, was significantly increased by significantly lower than that of CIA mice treated with control IgG. coculturing with Cyr61-stimulated FLS compared with that in Consistently, histological examination showed that the joints of control BSA-treated FLS, as was Th17-associated cytokine IL-17 mice treated with control IgG were infiltrated with leukocytes and (Fig. 3A). The expression of IL-22 (34) was also increased. To exhibited synovial hyperplasia, pannus formation, cartilage de- corroborate this result further, we performed a similar coculture struction, and bone erosion, which are characteristic features of experiment using Cyr61-knockdown FLS, and results showed that CIA. In contrast, these features were ameliorated by mAb 093G9 knockdown of Cyr61 reduced the expression of RORc, IL-17, and treatment. MRI analysis showed that in control IgG-treated mice, 5780 Cyr61-INDUCED IL-6 PRODUCTION PROMOTES Th17 DIFFERENTIATION

FIGURE 3. CD4+ T skewed into Th17 when cocultured with FLS. (A) Profile of Th17 differentiation relevant factors, RORc, IL-17, IL-21, IL-22, and IL- 23 expression in CD4+ T cells cocultured with Cyr61-provoked FLS. (B) Profile of genes indicated as in (A) in CD4+ T cells cocultured with Cyr61- knockdown FLS. (C) The proportion of CD4+IL-17+ cells detected by intracellular staining among CD4+ T cells cocultured with Cyr61-stimulated FLS or Cyr61-knockdown FLS or no FLS. (D) Percentage of CD4+IL-17+ cells. Data represent the mean 6 SEM of at least three independent experiments. *p , 0.05, **p , 0.01. the surrounding tissues were squeezed by joint inflammation and 5D). Flow cytometry analysis confirmed a specific reduction in the edema, and these symptoms were improved in 093G9-treated Th17 population in spleens and lymph nodes of 093G9-treated mice (Fig. 5A). Furthermore, the proliferation of T cells isolated CIA mice compared with that in the control IgG-treated mice from 093G9-treated mice in response to in vitro CII Ag challenge (Fig. 5E). We also noticed an apparent increase in IL-10–pro- was significantly inhibited compared with that of the control IgG- ducing CD4+ T cell population upon anti-Cyr61 Ab treatment; treated group (Fig. 5B). Thus, treatment with 093G9 significantly however, this change did not reach statistical significance (Fig. reduced the inflammation phenotype of the CIA mice. 5E). Together, these results indicate that blocking of Cyr61 with Given that Cyr61 production by FLS constitutes a positive- the specific mAb ameliorated the inflammatory reaction of the feedback loop in which its production further stimulates FLS CIA mice and led to the downregulation of the Th17 population proliferation (18) and that anti-Cyr61 treatment significantly im- in vivo. proved the inflammation phenotype of CIA mice, we next examined Cyr61 expression in local synovial tissues of these treated Blocking of Cyr61 attenuated IL-6/IL-6R signaling pathway mice. The results showed that Cyr61 expression was significantly in vivo decreased at both mRNA and protein levels after 093G9 treatment As we had observed that Cyr61 was able to stimulate IL-6 pro- (Fig. 5C). Analyses of inflammatory cytokine profiles of these duction in FLS (Fig. 1) and facilitate Th17 cell differentiation mice showed that levels of TNF-a and IL-17 were reduced in in vitro (Fig. 3), and that blocking of Cyr61 with a specific mAb spleens and lymph nodes of 093G9-treated mice (Fig. 5D). Con- led to downregulation of the Th17 population in CIA mice in vivo sistent with this, the expression of RORgt was also reduced (Fig. (Fig. 5), we asked whether the effect of anti-Cyr61 on Th17 differentiation in vivo was due to the reduction of IL-6 levels. Indeed, IL-6 expression in synovial tissues was dramatically decreased in 093G9-treated CIA mice compared with that of control IgG- treated mice, whereas the expression of TGF-b was not altered (Fig. 6A). Moreover, the concentration of IL-6 in sera of 093G9- treated mice was also reduced compared with that of control mice (Fig. 6B). We next examined whether Cyr61 blockage impaired the IL-6 signaling pathway in T cells of 093G9-treated mice. The results showed that the expression of IL-6Ra and IL-6Rb was reduced significantly in T cells of 093G9-treated CIA mice compared with that of control mice (Fig. 6C). Consistently, the + + FIGURE 4. Anti-Cyr61 mAb 093G9 inhibits proliferation of FLS and proportion of CD4 gp130 T cells in 093G9-treated CIA mice was IL-6 production by FLS of RA patients in vitro. (A) The proliferation of also dramatically decreased as shown by the FACS assay (Fig. FLS was inhibited by anti-Cyr61 Ab 093G9 compare with control IgG. 6D). We also examined the Jak1/STAT3 signaling pathway (B) Cyr61-stimulated production of IL-6 by FLS was inhibited by 093G9. downstream of IL-6 receptor activation (36, 37) and found that the *p , 0.05, **p , 0.01. ***p , 0.001. phosphorylation of Jak1 and STAT3 was inhibited (Fig. 6E) in The Journal of Immunology 5781

FIGURE 5. Blocking of Cyr61 ameliorated inflammation of CIA mice by downregulated Th17 population in vivo. (A) Clinical score and severity of CIA mice treated with anti-Cyr61 mAb 093G9. Left panel, Mice were monitored and scored as described in Materials and Methods. Control (s), 093G9 (d). Data are representative of three independent experiments. The other two experimental results are shown in Supplemental Fig. 2. Right panel, Histopa- thology of joint tissue sections of CIA mice treated with 093G9 or control by H&E staining (upper panels) and MRI (lower panels), respectively. Original magnification 3200. Quantitative analysis of infiltrated leukocytes and T1 signal intensity is shown in Supplemental Fig. 3. (B) T cell in 093G9-treated (black bar) and control mice (open bar) response was measured as CII-induced T cell proliferation in vitro. (C) Cyr61 expression in synovial tissues of control IgG (open bar) or 093G9 (black bar) treatment, respectively. Left panel, Cyr61 mRNA relative expression evaluated by real-time PCR. Right panel, Cyr61 protein level examined by Western blotting. (D) Cytokines and transcriptional factors expression in splenocytes or lymph node cells from 093G9- treated mice (solid bars) or control (open bars). (E) Th subsets identified with intracellular staining technique isolated from spleen and lymph node of 093G9-treated or control mice. Data are representative of at least three independent experiments. *p , 0.05, **p , 0.01.

T cells of 093G9-treated mice. Consistent with the observed effect Recently, it is known that FLS do not only provide the structural of Cyr61 in vitro (Fig. 3), blocking of Cyr61 in vivo using 093G9 basis of organ composition and function as structure-building cells: also led to reduction in the expression of IL-21 (Fig. 6F). We thus under disease conditions such as inflammation, fibroblasts are conclude that Cyr61 blockage attenuated IL-6/IL-6R signaling critical switches that regulate the response to tissue injury, con- in vivo. Together, Cyr61 produced by RA FLS can initiate a novel tribute to the resolution or chronicity of the organ-specific pa- cross-talk between FLS and Th17 cells. Cyr61 acts to stimulate thology, and determine the consequences of disease. In RA, FLS IL-6 production by FLS, and the increased IL-6 then acts to are a key part of the local immune system and, through the inte- promote Th17 differentiation (Fig. 7). gration of signals from different sources, contribute to both disease initiation and perpetuation (1). Nevertheless, whether FLS- Discussion produced Cyr61 has in turn any effect on Th17 differentiation, as CCN1/Cyr61 was the first identified member of the CCN family. It well as the mechanisms of Cyr61 in the inflammation process of is a cysteine-rich protein of ∼40 kDa encoded by a growth factor- RA, remain unclear. inducible immediate-early gene (19, 24). Numerous studies have In this study, we found that apart from its function to stimulate shown that Cyr61 regulates cell adhesion and cell migration. For FLS proliferation, Cyr61 is able to induce IL-6 expression in these instance, Cyr61 was shown to promote proliferation of endothelial cells. Knockdown of endogenous Cyr61 led to reduced IL-6 ex- cells and angiogenesis in vitro and in vivo (20, 38, 39). We recently pression in FLS, further substantiating the autocrine role of Cyr61 reported that Cyr61 is overexpressed by FLS of RA patients and in inducing IL-6 expression by FLS. As IL-6 is a critical cytokine stimulates FLS proliferation in an autocrine manner (18). Notably, that functions in promoting Th17 differentiation (13, 40), we went the high expression of Cyr61 in RA FLS appears to be stimulated on to test whether RA FLS could stimulate Th17 differentiation by IL-17. These results not only revealed for the first time to our in a coculture system and found that it was indeed the case. knowledge that Cyr61 contributes to hyperplasia of synovial lining Moreover, addition of exogenous Cyr61 or knockdown of endoge- cells but also established a new link between Th17 cells and FLS nous Cyr61 enhanced or reduced the stimulatory effect of FLS on in RA, whereby the Th17 cells act to promote FLS proliferation Th17 differentiation. Importantly, administration of a specific anti- through stimulating the expression of Cyr61 in these cells. Cyr61 Ab in CIA mice not only ameliorated the inflammation 5782 Cyr61-INDUCED IL-6 PRODUCTION PROMOTES Th17 DIFFERENTIATION

FIGURE 6. Blocking Cyr61 resulted in attenuation of IL-6/IL-6R signaling pathway. (A) IL-6 and TGF-b mRNA in synovial tissue, spleen, and lymph node detected by real-time PCR. (B) The IL-6 protein level in sera from 093G9 (black bar) or control (open bar) mice examined by ELISA. (C) IL-6Ra and IL-6Rb (gp130/CD130) expression pattern in spleens and lymph node examined using real-time PCR. Black bar: 093G9-treated mice. Open bar: control mice. (D) CD4+ and CD130+ cells in splenocytes and lymph node cells from 093G9-treated or control mice analyzed using flow cytometry. (E) Phos- phorylated and total Jak1 and STAT3 profiles in splenocytes and lymph node cells derived from 093G9-treated or control mice. (F) The expression of IL-21, IL-22, and IL-23 in splenocytes and lymph node cells isolated from 093G9-treated (black bar) or control (open bar) CIA mice analyzed with real-time PCR using b-actin as a reference. Data are representative of at least three independent experiments. *p , 0.05, **p , 0.01. phenotype of these animals but also led to downregulation of the the current study, we failed to detect variation of IL-23 production Th17 population in vivo. Mechanistically, we show that this was both in vitro and in vivo. Collectively, the results suggest that accompanied by a reduction of the IL-6/IL-6R signaling pathway Cyr61 produced by RA FLS can initiate a novel cross-talk be- in T cells of the anti-Cyr61–treated mice. Recently, it is observed tween FLS and Th17 cells, whereby Cyr61 acts to stimulate IL-6 that IL-21 is both a product and inducer of Th17 cells, providing production by FLS, and the increased IL-6 then acts to promote a critical autocrine feedback loop in mice. However, human Th17 Th17 differentiation (Fig. 7). This new cross-talk, together with cells produce large amounts of IL-22, although mouse Th17 cells the regulation of Cyr61 production in FLS by IL-17 that we had do not uniquely produce this cytokine. So, in our study, there was previously discovered, forms a novel feed-forward and malicious significant variation of IL-22 in human FLS treated with Cyr61, cycle that leads to mutual stimulation of FLS and Th17, two and the significant variation of IL-21 was observed in CIA mice important cell populations in RA via Cyr61 promotion of IL-6 treated with 093G9. IL-23 may be more important for the survival production. This malicious cycle may in turn play important and population expansion of Th17 cells than for Th17 lineage roles in RA pathogenesis. commitment (15, 41). In our study, Cyr61-induced IL-6 may in- How does Cyr61 induce IL-6 production in FLS? Our results fluence CD4+ T cell differentiation toward Th17 cells, but it may showed that among the candidate receptors for Cyr61, the levels of not affect the survival and population expansion of Th17 cells. In integrins av, b1, and b5 were high on RA FLS. Nevertheless, only

FIGURE 7. A schematic model for Cyr61-stimulated IL-6 production and its role in Th17 differentiation. Cyr61 secreted by FLS stimulates IL-6 production via avb5/Akt/NF-kB signaling pathway. Production of IL-6 activates Jak1/STAT3 phosphorylation, which triggers Th17 differentiation. In turn, IL-17 produced by Th17 cells enhances Cyr61 expression through p38 and NF-kB signaling pathways. Thus, Cyr61 might act as a novel proinflammatory factor contributing to the inflammation of RA via stimulation of Th17 differentiation. The Journal of Immunology 5783 the anti-avb5 Ab could block Cyr61-induced production of IL-6. 8. Lubberts, E., L. A. Joosten, B. Oppers, L. van den Bersselaar, C. J. Coenen-de Roo, J. K. Kolls, P. Schwarzenberger, F. A. van de Loo, and W. B. van den Berg. Further analysis showed that Cyr61 treatment of FLS led to strong 2001. IL-1-independent role of IL-17 in synovial inflammation and joint de- activation of Akt and NF-kB signaling. Inhibitor studies indicate struction during collagen-induced arthritis. J. Immunol. 167: 1004–1013. that NF-kB activation plays an important role in Cyr61-induced 9. Nakae, S., A. Nambu, K. Sudo, and Y. Iwakura. 2003. Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice. J. Immunol. 171: IL-6 production. Notably, previous studies in other cell types also 6173–6177. implicated that Cyr61 binding to integrin receptors could activate 10. Lubberts, E., M. I. Koenders, B. Oppers-Walgreen, L. van den Bersselaar, an Akt/NF-kB signaling pathway (20, 42, 43). On the basis of C. J. Coenen-de Roo, L. A. Joosten, and W. B. van den Berg. 2004. Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of these results, we propose that Cyr61 induces IL-6 production in collagen-induced arthritis reduces joint inflammation, cartilage destruction, and FLS via the avb5/Akt/NF-kB signaling pathway. bone erosion. Arthritis Rheum. 50: 650–659. An interesting observation in 093G9-treated mice was that the 11. Feldmann, M., F. M. Brennan, and R. N. Maini. 1996. Role of cytokines in + rheumatoid arthritis. Annu. Rev. Immunol. 14: 397–440. expression of IL-6R on CD4 T cells was decreased when these 12. Dayer, J. M., and E. Choy. 2010. Therapeutic targets in rheumatoid arthritis: the mice were treated with the anti-Cyr61 Ab. Similarly, the same interleukin-6 receptor. Rheumatology (Oxford) 49: 15–24. + 13. Stockinger, B., and M. Veldhoen. 2007. Differentiation and function of Th17 finding was observed on human CD4 T cells cocultured with FLS T cells. Curr. Opin. Immunol. 19: 281–286. in which the endogenous Cyr61 was knockdown (data not shown). 14. Zhu, J., and W. E. Paul. 2010. Heterogeneity and plasticity of T helper cells. Cell It is well known that a signaling cascade is triggered when IL-6 Res. 20: 4–12. 15. Laurence, A., and J. J. O’Shea. 2007. T(H)-17 differentiation: of mice and men. binds its receptor and gp130 homodimerization occurs (12, 44). Nat. Immunol. 8: 903–905. Our observation is consistent with the finding by others that IL-6R 16. Antonelli, A., C. Ferri, S. M. Ferrari, C. Mancusi, M. Colaci, M. Sebastiani, and signaling is responsible for the IL-6 effect on activation of its own P. Fallahi. 2011. IFN-g and TNF-a induce a different modulation of interleukin- 6 in systemic sclerosis fibroblasts compared to healthy controls. Scand. J. downstream transcription factors. Therapeutic benefit of blocking Rheumatol. 40: 453–456. of IL-6 activity with an anti–IL-6 receptor mAb in RA treat- 17. Eda, H., B. L. Burnette, H. Shimada, H. R. Hope, and J. B. Monahan. 2011. ment has been proved effectively (45). 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