Increased osteopontin expression in dendritic cells amplifies IL-17 production by CD4+ T cells in experimental autoimmune encephalomyelitis and in multiple sclerosis

Abstract

Osteopontin (Opn) is a broadly expressed pleiotropic cytokine, and has been shown to play an important role in various autoimmune diseases, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). It is reported that Opn exacerbates EAE by skewing T cell differentiation toward IFN-gamma-producing Th1 cells. Opn expression in dendritic cells (DCs) and its role in IL-17 induction from T cells during EAE or MS are unknown. We found that during EAE, Opn expression is elevated in DCs both in the periphery and in the CNS. There was increased expression of Opn receptor on T cells, and Opn induced IL-17 production by CD4(+) T cells via the beta(3) integrin receptor and Opn inhibited IL-10 production via the CD44 receptor. Furthermore, anti-Opn treatment reduced clinical severity of EAE by reducing IL-17 production. Anti-Opn was also effective in reducing clinical severity of EAE when given after the appearance of clinical symptoms. Analogous to EAE, in subjects with MS, we found increased expression of Opn in DCs and increased expression of the Opn receptors CD44, beta(3), and alpha(v) on T cells. Furthermore, Opn-stimulated CD4(+) T cells from MS patients produced significantly higher amounts of IL-17. Our results demonstrate a role for DC-produced Opn both in EAE and MS that is linked to the production of IL-17.

Figure 1

Elevated Opn expression in DCs during EAE. Increased Opn mRNA and protein expression from CD11c⁺ DCs in EAE mice. (A) Opn mRNA was determined by quantitative real-time PCR analysis in CD11c⁺ DCs from spleen, LN and CNS from naïve, onset (EAE score 0.5–1, n=5–6 per group) and peak (EAE score 3, n=5–6 per group) C57BL/6 mice. Expression of Opn was normalized to GAPDH. (B) Opn protein expression was determined by Western blot analysis using anti-Opn antibodies. 20 μg of whole cell extract from CD11c⁺ DCs from indicated organs and timepoints were resolved on a 10% acrylamide gel. α-tubulin was used for loading control. (n=5–6 per group) (C) Increased infiltration of CD11c⁺ DCs in the CNS during disease- was assayed during naïve, onset and peak state of EAE. The mononuclear fraction from CNS was stained with antibodies to CD11c. Data are representative of three independent experiments.

Figure 2

Opn production from DC induces IL-17 production from T cells. Total CD4⁺ T cells isolated from 2D2 mice were cocultured with CD11c⁺ DC isolated from naïve and MOG35-55 immunized WT and Opn^−/− mice. (A) In the last 16h, cells were pulsed with thymidine and assayed for proliferation (cpm). Error bars represent s.e.m. between triplicates. Supernatants from parallel cultures were harvested 60 h after initiation of cultures and assayed by ELISA for (B) IFN-γ (C) IL-17 and (D) IL-10. (E) Splenic DCs isolated from WT and Opn^−/− mice were analyzed by real-time RT-PCR for the expression of IL6, IL-1β and IL-23. (F) DC expressed Opn induces IL-17 production from T cells. Total CD4⁺ T cells isolated from WT and Opn^−/− mice were cocultured with CD11c⁺ DCs isolated from WT and Opn^−/− mice. Supernatants from cultures were harvested 72 h after initiation of cultures and assayed by ELISA for IL-17.

Figure 3

Differential regulation of IL-17, IFN-γ and IL-10 by Opn. CD4⁺ T cells were activated with anti-CD3 and anti-CD28 mAb (0.3 μg/ml) in the presence or absence of 1 μg/ml of mouse rOPN. Supernatants from cultures were harvested 60 h after initiation of cultures and analyzed for cytokines (A) IL-17 (B) IFN-γ (C) IL-10. Similarly stimulated cells were harvested 24hrs post stimulation for mRNA and analyzed for (D) T-bet and (E) RORγT by quantitative real-time PCR. Data are representative of 3 independent experiments. (F–H) Opn induces IL-17 and IFN-γ while inhibiting IL-10 from CD8⁺ T cells. CD8⁺ T cells were activated with anti-CD3 and anti-CD28 mAb (0.3 μg/ml) in the presence or absence of 1 μg/ml of mouse rOPN. Supernatants from cultures were harvested 60 h after initiation of cultures and analyzed for cytokines (F) IL-17 (G) IFN-γ (H) IL-10.

Figure 4

Regulation of T cell cytokine expression by distinct Opn receptors. (A) Opn receptor expression on T cells from CNS and peripheral blood of naïve mice and mice with peak EAE. Cells were stained with fluorochrome conjugated CD4 and indicated integrin receptors. CD4⁺ T cells were gated and analyzed for expression of Opn receptors. (B–D) Opn induces secretion of IFN-γ and IL-17 while inhibiting IL-10 production from T cells. CD4⁺ T cells were activated with anti-CD3 and anti-CD28 mAb (0.3 μg/ml) in the presence or absence of 1 μg/ml of mouse rOPN. Blocking antibodies to integrin β3, CD44 or integrin β1 were added at 5μg/ml. Supernatants from cultures were harvested 60 h and analyzed for IFN-γ, IL-17 and IL-10. (E–G) T cells from WT, Itgb3^−/− and CD44^−/− mice were activated with anti-CD3 and anti-CD28 mAb (0.3 μg/ml) in the presence or absence of 1 μg/ml of mouse rOPN. Culture supernatants were harvested 60h later were analyzed for IFN-γ, IL-17 and IL-10. Data are representative of 2–4 independent experiments.

Figure 5

Anti-Osteopontin suppresses EAE. (A) Mean clinical scores of EAE mice treated with anti-Opn antibody or isotype control (n = 8 per group) on days 5, 7 and 9 post immunization (indicated by arrows on X-axis) (P < 0.0001) (B) Spleen cells from anti-Opn and control mice were activated in vitro MOG35-55 (20 μg/ml) for 60h. In the last 16h, cells were pulsed with thymidine and proliferation was represented as cpm. Data represent the s.e.m+/− of triplicate assays. (C–E) Cell-free supernatants from the above culture conditions were harvested at 60h and assayed for indicated cytokines. (F) CNS-T cells isolated from mice treated with anti-Opn or control antibody (n = 10 mice per group) were analyzed by real-time RT-PCR for the expression of IL-17, IFN-γ and IL-10. (G) Mice were treated with anti-Opn after onset of EAE (score of ≥1.5) on days 16, 17, 18 and 19 (post immunization) resulted in a rapid clinical recovery from EAE.

Figure 6

Opn induces IL-17 production from human CD4⁺ T cells. (A–C) Effect of Opn on T cell cytokine and transcription factors in healthy controls. Total CD4⁺ T cells from HC were stimulated with anti-CD3/CD28 in the presence or absence of rOPN (1μg/ml). Supernatants (at 60h) were harvested and assayed for indicated cytokines by ELISA. Parallel cultures were analyzed for real-time PCR analysis for human (A) T-bet and (B) RORC. (D) Opn stimulation of CD4⁺ T cells leads to increased expression of IL-17+ and IFN-γ+ T cells. Naïve CD4⁺ T cells from HC were stimulated with anti-CD3/CD28 in the presence or absence of rOPN (1μg/ml). Cells were also stimulated under the indicated Th17 polarizing conditions. Intracellular cytokine staining was performed after 5 days of culture.

Figure 7

Investigation of Opn in DCs and T cells in subjects with MS. (A) Elevated Opn in DC from MS patients. RNA from peripheral blood CD11c⁺ cells of MS patients (n=8) and healthy controls (HC) (n=8) was analyzed by real-time PCR and normalized to GAPDH. (B) Opn receptor expression is increased in patients with MS. Total CD4⁺ T cells isolated from MS patients (n=12) and HC (n=12) were analyzed for Opn receptor expression by real-time PCR and normalized to GAPDH. (C–E) CD4⁺ T cells from controls (n=5) or MS patients (n=5) were stimulated with anti-CD3/CD28 in the presence or absence of rOPN (1mg/ml). Supernatants were harvested and assayed for indicated cytokines by ELISA. All statistical analyses herein were performed using the unpaired t test. A value of P < 0.05 was considered significant.