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. 2016 Nov 4:6:36002.
doi: 10.1038/srep36002.

IL-25 attenuates rheumatoid arthritis through suppression of Th17 immune responses in an IL-13-dependent manner

Dan Liu  1   2   3 , Tuanping Cao  2 , Na Wang  1 , Chengfei Liu  1 , Ning Ma  1 , Ran Tu  4 , Xiaoyun Min  1
Affiliations

IL-25 attenuates rheumatoid arthritis through suppression of Th17 immune responses in an IL-13-dependent manner

Dan Liu et al. Sci Rep. .

Abstract

IL-25, a new member of the IL-17 cytokine family, is involved in type 2 immunity initiation and has been associated with the pathogenesis of rheumatoid arthritis (RA). However, its exact role remains unclear. Here, we aimed to analyse IL-25 expression in the serum and synovial fluid of RA patients and evaluated the correlations between serum IL-25 levels, clinical and laboratory values and inflammation cytokines. Additionally, we investigated whether IL-25 can suppress Th1/Th17 responses involved in RA pathogenesis. We further determined whether IL-25 can alleviate collagen-induced arthritis (CIA) development in mice and the underlying mechanisms using in vitro and in vivo experiments. Our results showed that IL-25 was upregulated in the serum and synovial fluid of RA patients. Increased serum IL-25 levels were associated with disease severity and inflammatory response in RA patients. Furthermore, IL-25 inhibited CD4+ T-cell activation and differentiation into Th17 cells, without affecting Th1 cells in human RA and CIA models. Administration of IL-25 could attenuate CIA development by Th17 suppression in an IL-13-dependent manner. Our findings indicate that IL-25 plays a potent immunosuppressive role in the pathogenesis of RA and CIA by downregulating Th17 cell response, and thus, may be a potential therapeutic agent for RA.

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Figures

Figure 1
Figure 1. IL-25 is upregulated in serum and synovial fluid in RA patients.
(A,B) Serum and synovial fluid IL-25 levels of patients with RA or OA or HCs by enzyme-linked immunosorbent assay (ELISA). (C,D) Serum and synovial fluid IL-25 levels in RA patients distributed according to disease activity (active, n = 26; inactive, n = 22) by ELISA. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
Figure 2. Increased IL-25 levels are associated with disease severity and inflammation response in RA patients.
Correlation analysis between serum IL-25 levels and clinical and laboratory values as well as serum inflammation cytokines levels in RA patients. DAS28, 28-joint Disease Activity Score; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; RF, rheumatoid factor; anti-CCP, anti-cyclic citrullinated peptide antibodies. Spearman’s correlation test was used. The r value indicates the calculated regression coefficient.
Figure 3
Figure 3. IL-25 inhibits Th17 cells without affecting Th1 cells in RA.
CD4+ T cells from PBMCs of RA patients or HCs were isolated and stimulated with or without anti-CD3 (5 μg/mL) plus anti-CD28 (2 μg/mL) in the presence or absence of rhIL-25 (100 ng/mL) for 24 h. IL-17A (A), IFN-γ (B), and IL-4 (C) levels in supernatant were assessed by ELISA. The mRNA levels of IL-17A (D), IFN-γ (E), and IL-4 (F) were detected by real-time PCR. The mRNA levels of the master transcription factors ROR-γt (G), T-bet (H), and GATA3 (I) were detected by real-time PCR. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
Figure 4. Administration of IL-25 attenuates CIA development in mice.
Male DBA/1 mice were immunized with CII to induce arthritis, and received rmIL-25 (1 μg/mice) or PBS for 5 consecutive days beginning on day 1 after the second immunization with CII. Mice were killed on day 36 for experimental analysis. Incidence (A) and mean clinical scores (B) of CIA in mice treated with rmIL-25 or PBS (n = 10 per group). (C) Representative H&E staining of knee joints of rmIL-25 or PBS treated mice with CIA. Scale bars, 500 μm. (D) Evaluation results for synovitis, pannus, and erosion of bone and cartilage in the knee joint sections of rmIL-25 or PBS treated mice with CIA. (E,F) Serum levels of CII-specific immunoglobulin G2a (IgG1) and IgG2a antibodies were measured by ELISA. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5. IL-25 suppresses Th17 cell responses in CIA mice.
Serum (A) and synovial fluid (B) cytokines (IL-1β, IL-6, IL-17A, TNF-α, and IFN-γ) from CIA mice treated with rmIL-25 or PBS were measured by ELISA. (C) mRNA expression of IL-17A, and ROR-γt in synovial tissues of CIA mice treated with rmIL-25 or PBS were determined by realtime PCR. Spleen CD4+ T cells were isolated from CIA mice and stimulated with or without plate-bound anti-CD3 (5 μg/mL) plus anti-CD28 (2 μg/mL) in the presence or absence of rm IL-25 (1, 10, 100 ng/mL) for 24 h. IL-17A (D) levels in supernatant were assessed by ELISA. The mRNA levels of IL-17A (E), and ROR-γt (F) were detected by real-time PCR. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6
Figure 6. IL-25-mediated protection from CIA and Th17 suppression is IL-13 dependent.
Male DBA/1 mice were immunized with CII to induce arthritis, and received rmIL-25 (1 μg/mice) or PBS for 5 consecutive days beginning on day 1 after the second immunization with CII, The mice were injected with anti-IL-4, IL-5 or IL-13 blocking monoclonal antibody (mAb) on day 3 before (−3d) and day 3 (+3d) after the second immunization with CII. Incidence of CIA in mice treated with anti-IL-4 (A), anti-IL-5 (B), and anti-IL-13 (C) (n = 8 per group). Spleen CD4+ T cells were isolated from CIA mice and stimulated with plate-bound anti-CD3 (5 μg/mL) plus anti-CD28 (2 μg/mL) in the presence or absence of rm IL-25 (100 ng/mL) for 24 h. anti-IL-4 mAb (10 μg/mL), anti-IL-5 mAb (10 μg/mL), anti-IL-13 mAb (10 μg/mL) or rmIL-13 (100 ng/ml) were added at the start of the culture. IL-17A (D) levels in supernatant were assessed by ELISA. CD4+ T cells from PBMCs of RA patients were isolated and stimulated with anti-CD3 (5 μg/mL) plus anti-CD28 (2 μg/mL) in the presence or absence of rhIL-25 (100 ng/mL) for 24 h. anti-IL-13 mAb (10 μg/mL) or anti-IL-4 mAb (10 μg/mL) was added at the start of the culture. IL-17A (E) levels in supernatant were assessed by ELISA. The mRNA levels of ROR-γt (F) was detected by real-time PCR. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
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