Higher susceptibility to experimental autoimmune encephalomyelitis in Muc1-deficient mice is associated with increased Th1/Th17 responses

Abstract

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system in which dendritic cells (DC) play an important role in the development of inflammatory responses. Recently it has been shown that Muc1, a membrane tethered glycoprotein, has an ability to suppress inflammatory responses in cultured DC. The objective of this study was to investigate the possible involvement of Muc1 in the development of MS using experimental autoimmune encephalomyelitis (EAE) in mice, a widely used animal model of MS. Our results showed that: (1) Muc1(-/-) mice developed greater EAE severity compared with wild type (wt) mice, which correlated with increased numbers of Th1 and Th17 cells infiltrating into the CNS; (2) upon stimulation, splenic DC from Muc1(-/-) mice produced greater amounts of IL-1β, IL-6, and IL-12 but less amounts of IL-10 compared with those from wt mice; and (3) the ability of splenic DC to differentiate antigen-specific CD4+ T cells into Th1 and Th17 cells was greater in Muc1(-/-) mice compared with wt mice. We conclude that Muc1 plays an anti-inflammatory role in EAE. This is the first report demonstrating the possible involvement of Muc1 in the development of MS and might provide a potential target for immunotherapy.

Fig. 1

Deficiency in Muc1 exacerbates EAE. C57BL/6 wt mice (n = 10) and Muc1^−/− mice (n = 10) were immunized with MOG_35-55 as described in Materials and Methods. The EAE mice were followed for clinical scores for 30 days. Data represent mean ± SE. Statistical significance was determined as: * p<0.05, ** p<0.01 and *** p<0.001.

Fig. 2

Increased Th1 and Th17 infiltration in the CNS of Muc1-deficient EAE mice. C57BL/6 wt mice (n = 8) and Muc1^−/− mice (n = 8) were immunized with MOG_35-55 for EAE induction. (A) Clinical scores were assessed on day 12. (B) Mononuclear cells were isolated from spinal cord and brain on day 12 post-immunization and the numbers of CD4+ T cells were determined. (C) and (D) Percentage and numbers of intracellular IFNγ+ CD4 T cells determined by FACS. (E) and (F) Percentage and numbers of intracellular IL-17+ CD4 T cells determined by FACS.

Fig. 3

Increased Th1 and Th17 differentiation in the spleens of Muc1-deficient EAE mice. C57BL/6 wt mice (n = 3) and Muc1^−/− mice (n = 4) were immunized with MOG_35-55. On day 7 post-immunization, cells isolated from spleens (A), and axillary and brachial lymph nodes (B) were subjected to mRNA extraction followed by real-time RT-PCR for IFNγ and IL-17 expression. (C) and (D) Recall response. Splenocytes were restimulated ex vivo with 25 μg/ml of MOG_35-55. Three days later, supernatants were collected and analyzed for IL-17 and IFNγ production. Data represent the combined results from wt EAE (n=3) and Muc1^−/− EAE mice (n = 4).

Fig. 4

Muc1 deficiency in splenocytes promotes Th1 and Th17 differentiation. Splenocytes (2×10⁶ cells/ml) from wt and Muc1^−/− mice were stimulated under non-polarizing condition, i.e. soluble anti-CD3 (3 μg/ml) or soluble anti-CD3 (3 μg/ml) plus anti-CD28 (2 μg/ml), Th1 polarizing conditions in the presence of IL-12 (10 ng/ml), or Th17 polarizing conditions in the presence of IL-6 (20 ng/ml), TGF-β (2.5 ng/ml), anti-IL-4 (10 μg/ml) and anti-IFNγ (10 μg/ml). Intracellular expression of T-bet (A) and RORγt (D) were determined by FACS at 24 h. Supernatants were analyzed at 72 h for IFNγ (B and C) and IL-17 (E and F) by ELISA. One representative experiment of three is shown.

Fig. 5

Muc1 deficiency in CD4+ T cells does not promote Th1 and Th17 differentiation. CD4⁺ T cells (2×10⁶ cells/ml) were purified from the spleens of wt and Muc1^−/− mice. (A) CD4⁺ T cells were cultured for 72 h in the presence of plate-bound anti-CD3 antibody (5 μg/ml), soluble anti-CD28 antibody (1 μg/ml) plus IL-12 for Th1 differentiation, or with IL-6, TGF-β, and anti-IL-4 and anti-IFNγ antibodies for Th17 differentiation. Supernatants were collected and IFNγ (A) and IL-17 (B) production was determined by ELISA. Data represent mean ± SE (n = 4). One representative experiment of three is shown.

Fig. 6

Muc1 deficiency in splenic DC augments Th1 and Th17 differentiation. CD11c+ cells were purified from the spleens of wt and Muc1^−/− mice, stimulated with LPS (1 μg/ml) for 14 h and pulsed with MOG (50 μg/ml; specific antigen) or PLP (50 μg/ml; nonspecific antigen-control) for 3 h. After extensive washing, CD11c+ cells were cocultured with MOG-specific naïve CD4+ T cells purified from the spleens of 2D2 mice (2×10⁶cells/ml, DC:T cell=1:10). After 72 h, cells were subjected to FACS for intracellular IFNγ and IL-17 staining (A). Supernatants were collected for IL-17 ELISA after 72 h and 96 h coculture (B). Proliferation of CFSE-labeled MOG-specific CD4+ T cells was measured at 72 h or 96 h (C). Splenocytes from wt or Muc1^−/− mice were harvested and treated with LPS for 24 h. Cells were subjected to surface staining for CD11c, CD40, CD80, CD86 and MHCII. One representative experiment of three (A-C) or of two (D) is shown.

Fig. 7

Muc1 deficiency enhances the expression and production of IL-1β, IL-6 and IL-12 and diminishes the production of IL-10 in splenocytes. Splenocytes from wt or Muc1^−/− mice were harvested and treated with LPS. After 3 h, mRNA expression of IL-1β, IL-6, IL-12p35 and IL-12p40 was measured by real-time RT-PCR (A and C). The production of IL-1β, IL-6, IL-12p70, IL-12p40 and IL-10 was measured by ELISA at 24 h after LPS stimulation (B, D and E). Data are representative of two independent experiments.

Fig. 8

Muc1 deficiency in splenic DC results in overexpression of IL-1β, IL-6 and IL-12 but underexpression of IL-10. CD11c+ cells were purified from the spleens of wt and Muc1^−/− mice and treated with LPS in the presence or absence of IFNγ (100 ng/ml). After 3 h, mRNA expression of IL-1β and IL-6 was measured by real-time RT-PCR (A). Production of IL-1β, IL-6, IL-12p70 and IL-10 was measured by ELISA at 24 h, and of IL-12p40 at 36 h after LPS stimulation (B-D). Data are representative of two independent experiments.

Fig. 9

Model for the role of Muc1 in EAE. Lack of Muc1 in DC results in stronger activation of T cells through increased expression of costimulatory molecules, and in the upregulation of proinflammatory cytokines leading to preferential differentiation of proinflammatory subsets of effector Th1 and Th17 cells. The generation of higher numbers of pathogenic Th1 and Th17 cells in the absence of Muc1 leads to EAE disease exacerbation.