T helper type 1 and 17 cells determine efficacy of interferon-beta in multiple sclerosis and experimental encephalomyelitis

Abstract

Interferon-beta (IFN-beta) is the major treatment for multiple sclerosis. However, this treatment is not always effective. Here we have found congruence in outcome between responses to IFN-beta in experimental autoimmune encephalomyelitis (EAE) and relapsing-remitting multiple sclerosis (RRMS). IFN-beta was effective in reducing EAE symptoms induced by T helper type 1 (T(H)1) cells but exacerbated disease induced by T(H)17 cells. Effective treatment in T(H)1-induced EAE correlated with increased interleukin-10 (IL-10) production by splenocytes. In T(H)17-induced disease, the amount of IL-10 was unaltered by treatment, although, unexpectedly, IFN-beta treatment still reduced IL-17 production without benefit. Both inhibition of IL-17 and induction of IL-10 depended on IFN-gamma. In the absence of IFN-gamma signaling, IFN-beta therapy was ineffective in EAE. In RRMS patients, IFN-beta nonresponders had higher IL-17F concentrations in serum compared to responders. Nonresponders had worse disease with more steroid usage and more relapses than did responders. Hence, IFN-beta is proinflammatory in T(H)17-induced EAE. Moreover, a high IL-17F concentration in the serum of people with RRMS is associated with nonresponsiveness to therapy with IFN-beta.

Fig. 1

Effect of IFN-β on mouse T_H17 differentiation. a) IFN-β attenuates T_H17 differentiation. Spleen cells, depleted of CD8 T-cells, were cultured with and without IFN-β in non-polarizing, T_H1 and T_H17 conditions. CD4 T-cells were analyzed for IL-17 production by flow cytometry. b) IFN-β stimulation of naïve CD4 T-cells (CD62⁺) cultured in TGF-β and IL-6 with APCs, effector/memory CD4 T-cell (CD62⁻) cultured in TGF-β and IL-6 with APCs and effector/memory CD4 T-cell (CD62⁻) cultured in IL-23 and APCs. All CD4 T-cells were cultured with APCs at a ratio of 1:5. Cytokine secretion was analyzed by ELISA. Results are the mean ± SD of triplicates. Results are representative 3 experiments. *P<0.05. c) STAT1^−/;− spleen cells, depleted of CD8 T-cells, were cultured with and without IFN-β in T_H17 conditions. IL-17 production from CD4 T-cells was analyzed by flow cytometry. d) CD8-depleted spleen cells from C57BL/6 mice were cultured in T_H17 conditions with or without IFN-β in the presence and absence of neutralizing antibodies to IFN-γ, IL-10 or IL-27p28. e) Purified WT or IFNγR^−/;−CD4 T-cells were polarized in T_H17 conditions with WT or IFNγR^−/;− in the presence or absence of IFN-β and IL-17 production was assessed by ELISA. *P<0.01. f) Purified CD4 T-cells were stimulated with plate-bound anti-CD3 and anti-CD28 in T_H17 conditions in the presence or absence of IFN-β, IFN-γ or both; IL-17 production was assessed by ELISA. *P<0.01. Results are the mean ± SD of triplicates. Results represent 1 of 3 similar experiments.

Fig. 2

(a–c) Effect of IFN-β on mouse T_H1 differentiation. a) Spleen cells, depleted of CD8 T-cells, were cultured with and without IFN-β in non-polarizing, T_H1 and T_H17 conditions. CD4 T-cells were analyzed for IFN-γ production by flow cytometry. b) Purified WT or IFNγR^−/;− CD4 T-cells were stimulated in non-polarizing conditions with APCs from WT or IFNγR^−/;− in the presence or absence of IFN-β. IFN-γ was assessed by ELISA. * P<0.05. c) Purified WT or IFNγR^−/;− CD4 T-cells were stimulated in T_H1 conditions with APCs from WT or IFNγR^−/;− in the presence or absence of IFN-β. IFN-γ was assessed by ELISA. d) CD8 depleted spleen cells were stimulated with or without IFN-β in non-polarizing, T_H1, and T_H17 conditions and IL-10 expression in CD4 T-cells was analyzed by flow cytometry. e) CD8 depleted spleen cells were stimulated with or without IFN-β in non-polarizing conditions in the presence or absence of antibodies to IFN-γ or IL-10. f) IFN-β requires IFN-γ signaling in mouse CD4 T-cells and APCs to induce IL-10. Purified WT or IFNγR^−/;− CD4 T-cells were cultured in non-polarizing conditions with WT or IFNγR^−/;− in the presence or absence of IFN-β and IL-10 was assessed by ELISA. *P<0.01 and **P<0.005. g) Purified CD4 T-cells were stimulated with plate-bound anti-CD3 and anti-CD28 in non-polarizing conditions in the presence or absence of IFN-β.

Fig. 3

IFN-β treatment blocks T_H1 induced EAE but exacerbates T_H17 induced EAE. (a and b) Clinical scores from mice with passive EAE induced by adoptive transfer of (a) T_H1 and (b) T_H17 cells that were treated with rmIFN-β or PBS every second day from day 0 to 10 post transfer (n=9 to 11 mice per group). *P<0.05. c) Histology of spinal cord sections from T_H1 and T_H17 induced EAE treated with IFN-β or PBS. Sections of spinal cord were obtained 45 days after transfer and stained with H&E and Luxol fast blue. Scale bars, 50 μm. (d–f) Frequency of CD4⁺ lymphocytes expressing IFN-γ (d), IL-17 (e) and IL-10 (f) in the spinal cords 45 day post transfer. The mean percentage ± standard deviation (N=3 experiments) of cytokine positive cells is given. Each experiment is a pool from 2–3 mice per group. (g–i) Concentration of IFN-γ (g), IL-17 (h) and IL-10 (i) from supernatants of MOGp35–55 stimulated spleens taken from mice 45 days post transfer. Data represent mean and standard deviation of 3–4 mice per group. *P<0.05.

Fig. 4

IFN-β treatment requires IFN-γ signaling to suppress EAE symptoms. (a and b) Clinical scores from active EAE in (a) C57BL/6 and (b) IFNγR^−/;− mice that were treated with Rebif® or PBS daily from day 7 to day 17 post EAE induction (n=7 to 9 mice per group). (c and d) Clinical scores from active EAE in (c) C57BL/6 and (d) IFNγR^−/;− mice that were treated with Rebif® or PBS daily from day 0 to day 6 post EAE induction (n=4 to 5 mice per group). e) Clinical scores from IFNγR^−/;− mice with passive EAE induced by adoptive transfer of WT T_H1 and treated with IFN-β or PBS every second day from day 0 to 10 post transfer (n=6). Treatment doses indicated with arrows. *P<0.05.

Fig. 5

Effect of IFN-β on Human T_H differentiation. Naïve human CD4 T-cells were cultured in non-polarizing, T_H1-polarizing (IL-12) and T_H17-polarizing (IL-23) conditions for 5 days (a–c) or 11 days (d–f) and IFNγ, IL-17A and IL-10 were assessed by ELISA. Day 11 cultures were further reactivated in the presence of beads coated with antibodies to CD3, CD28 and CD2 Abs for 48 hrs prior to analysis. *P<0.01, **P<0.02, and ***P<0.05.

Fig. 6

Pre-Treatment cytokine profiles in serum of IFN-β responder and non-responder MS patients. a) Relative cytokine levels in responder and non-responder MS patients. Relative cytokine levels in serum from responder and non-responder MS patients are depicted as the difference in relation to healthy controls. Samples were analyzed by hierarchical clustering, and displayed as a heat map where red represents increased levels, black represents similar levels and green represents decreased levels of cytokine compared to healthy controls. b) Concentration of IL-17F in subsets of pre-treatment MS patients and healthy control. Cytokine concentrations were calculated from a standard linear regression of known quantities of IL-17F. c) Concentration of IFN-β in subsets of pre-treatment MS patients and healthy control. Cytokine concentrations were calculated from a standard linear regression of known quantities of IFN-β. *P<0.001 and **P<0.002.