Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis

Abstract

Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4(+) T cells secreting Th1 cytokines, while recovery from disease is associated with expression of Th2 cytokines. Investigations into the role of individual cytokines in disease induction have yielded contradictory results. Here we used animals with targeted deletion of the STAT4 or STAT6 genes to determine the role of these signaling molecules in EAE. The STAT4 pathway controls the differentiation of cells into a Th1 phenotype, while the STAT6 pathway controls the differentiation of cells into a Th2 phenotype. We found that mice deficient in STAT4 are resistant to the induction of EAE, with minimal inflammatory infiltrates in the central nervous system. In contrast, STAT6-deficient mice, which have a predominantly Th1 phenotype, experience a more severe clinical course of EAE as compared with wild-type or STAT4 knockout mice. In addition, adoptive transfer studies confirm the regulatory functions of a Th2 environment in vivo. These novel data indicate that STAT4 and STAT6 genes play a critical role in regulating the autoimmune response in EAE.

Figure 1

EAE disease course in STAT4^–/–, STAT6^–/–, and wild-type mice. A representative experiment showing disease induction in wild-type (triangles), STAT4^–/– (circles), and STAT6^–/– (squares) mice. Mice were immunized with MOG 35-55 and graded for disease daily. The mean daily disease grade for each group (n = 5 mice per group) is shown.

Figure 2

Quantitation of inflammatory cell infiltrates in the CNS of wild-type (WT), STAT4^–/– (STAT4KO), and STAT6^–/– (STAT6KO) mice. Quantitation of immunohistochemically stained spinal cord sections for CD4 and CD8 T cells and macrophages from C57BL/6 wild-type mice, STAT4^–/– mice, and STAT6^–/– mice. Spinal cord sections from three mice from each group were harvested on day 16 (a) and day 50 (b) and stained for CD4 (white bars), CD8 (dark gray bars), macrophages (black bars), and granulocytes (light gray bars). At least three sections from different levels of the cord were evaluated. Number of cells staining positive for the given marker were counted in 10 high-power fields (×40) per section. The results for one section were totaled, and the results between sections were averaged. Error bars represent standard deviation (SD). Statistical analysis was done using the Student’s t test. Results from a, day 16, show sections from STAT4^–/– mice displayed significantly less CD4⁺ (P = 0.05) and CD8⁺ cells (P = 0.0005), macrophages (P = 0.02), and granulocytes (P = 0.004) than did wild-type mice. Sections from STAT6^–/– mice consistently displayed more inflammatory cells than wild-type mice (however the P value was not significant), and significantly more than STAT4^–/– mice (P < 0.05). Results from b, day 50, show significantly lower numbers of CD4⁺ cells in the STAT4^–/– group compared with wild-type (P = 0.0024). There were no significant differences in inflammatory infiltrates between the STAT6^–/– and wild-type mice at day 50.

Figure 3

Proliferative response of splenocytes isolated from wild-type, STAT4^–/–, and STAT6^–/– mice to MOG 35-55 in vitro. Splenocytes were obtained on day 14 postimmunization from wild-type (white bars), STAT4^–/– (gray bars), and STAT6^–/– (black bars) mice. The cells were cultured in the presence of MOG 35-55 at 1, 10, and 100 μg/ml (on the x axis). The cpm is indicated on the y axis (± SD). Proliferation to 10 μg/ml of MOG was significantly more in cultures from STAT4^–/– (P = 0.0009) and STAT6^–/– (P = 0.0001) mice compared with wild-type.

Figure 4

IFN-γ production of splenocytes from wild-type, STAT4^–/–, and STAT6^–/– mice in response to MOG peptide in vitro. (a) IFN-γ production was measured by ELISA (± SD) in the supernatants of splenocytes harvested on day 14 from C57BL/6 (white bars), STAT4^–/– (gray bars), or STAT6^–/– mice (black bars) after 48 hours of culture with MOG 35-55 at concentrations of 1, 10, or 100 μg/ml. IFN-γ production was significantly greater in the cultures from wild-type mice compared with STAT4^–/– mice at all concentrations of MOG 35-55 (P < 0.005). (b) MOG 35-55–specific IFN-γ–producing cells (± SD) were measured by ELISPOT assay in cultures of splenocytes from C57BL/6 wild-type (white bars), or STAT4^–/– (gray bars), or STAT6^–/– mice (black bars). The y axis represents the number of positive cells per 2 × 10⁵ cells plated. The frequency of IFN-γ–producing cells was significantly higher in wild-type than STAT4^–/– (P < 0.005) and STAT6^–/– mice (P < 0.01) at all concentrations of MOG peptide.

Figure 5

IL-4 and IL-5 cytokine production of splenocytes from wild-type, STAT4^–/–, and STAT6^–/– mice in response to MOG peptide in vitro. (a) IL-5 production was measured by ELISA (± SD) in the supernatants of splenocytes harvested on day 14 postimmunization with MOG p35-55 from C57BL/6 wild-type (white bars), STAT4^–/– (gray bars), or STAT6^–/– mice (black bars), after 48 hours of culture with three different concentrations of MOG p35-55. Measurable IL-5 production was significantly greater in the cultures from STAT4^–/– mice at all concentrations of MOG p35-55 (P < 0.05 compared to wild-type and STAT6^–/–). (b) MOG p35-55–specific IL-5–producing cells (± SD) were measured by ELISPOT in cultures of splenocytes from C57BL/6 wild-type (white bars), STAT4^–/– (gray bars), or STAT6^–/– mice (black bars) taken 14 days postimmunization with MOG p35-55. The y axis represents the number of positive cells per 2 × 10⁵ cells plated. The frequency of IL-5–producing cells was significantly higher in STAT4^–/– compared with wild-type or STAT6^–/– cultures at all concentrations of MOG (P < 0.005). (c) MOG p35-55–specific IL-4–producing cells (± SD) were measured by ELISPOT in cultures of splenocytes from C57BL/6 wild-type (white bars), STAT4^–/– (gray bars), or STAT6^–/– mice (black bars) taken from mice 14 days postimmunization with MOG p35-55. The y axis represents the number of positive cells per 2 × 10⁵ cells plated. The frequency of IL-4–producing cells was significantly higher in STAT4^–/– and wild-type compared with STAT6^–/– cultures at all concentrations of MOG (P < 0.01).

Figure 6

Anti-MOG Ab quantitation in STAT4^–/–, STAT6^–/–, and wild-type mice. The relative titers of anti-MOG total IgG (a), IgG1 (b), and IgG2a (c) in serum samples from STAT4^–/– (open circles), STAT6^–/– (filled triangles), and wild-type mice (filled squares) at four time points. Titer was determined by comparison to a naive serum sample. Serial 1:2 dilutions were made. The reciprocal of the titer (1/Y), at which the sample was the same as naive, was plotted on a log₂ scale. Statistical analysis by the Mann-Whitney test shows no differences in total IgG titers among the three groups. IgG2a titers were not significantly different in either the STAT4^–/– or STAT6^–/– groups compared with wild-type mice. As anticipated, IgG1 titers were significantly lower in the STAT6^–/– (P = 0.028), but not STAT4^–/– (P = NS), mice compared with wild-type.

Figure 7

Adoptive transfer studies. (a) Adoptive transfer of wild-type cells into TCR-αβ^–/– mice. Splenocytes from wild-type mice were harvested 12 days postimmunization with MOG peptide. The cells were resuspended in PBS and wild-type splenocytes at concentrations of either 100 × 10⁶ (open circles), 50 × 10⁶ (filled triangles), or 25 × 10⁶ (filled squares) were injected intraperitoneally into recipient TCR-αβ^–/– mice. The recipients were immunized with MOG peptide 1 day after transfer. Five recipient mice per group were used. (b) Adoptive transfer of splenocytes from STAT4^–/– or STAT6^–/– mice into TCR-αβ^–/– mice. Splenocytes from either wild-type STAT4^–/– or STAT6^–/– mice were harvested 12 days after immunization with MOG peptide. The cells were resuspended in PBS and 50 × 10⁶ splenocytes from STAT4^–/– mice (filled circles) or STAT6^–/– mice (filled squares) were injected intraperitoneally into recipient TCR-αβ^–/– mice. The recipients were immunized with MOG peptide 1 day after transfer. Five recipient mice per group were used.

Figure 8

Adoptive transfer of wild-type splenocytes into a STAT4^–/– host results in less disease than transfer of STAT6^–/– splenocytes. Splenocytes (50 × 10⁶) from wild-type (triangles) or STAT6^–/– (squares) mice were injected intraperitoneally into recipient STAT4^–/– mice that were then immunized as above. A nontransfer STAT4^–/– group (circles) is also shown. Five recipient mice per group were used.

Figure 9

Cytokine production after adoptive transfer of T cells into STAT4^–/– recipients. Splenocytes were harvested on day 35 after immunization from STAT4^–/– and from STAT4^–/– recipients of wild-type and STAT6^–/– cells. Splenocytes were cultured in vitro with various concentrations of MOG peptide. (a) IFN-γ production (± SD) was measured by ELISA in splenocytes harvested from control STAT4^–/– mice (white bars), STAT4^–/– recipients of either wild-type C57BL/6 splenocytes (gray bars), or STAT6^–/– splenocytes (black bars) on day 35 postimmunization. IFN-γ production in STAT4^–/– recipients of STAT6^–/– splenocytes was greater than STAT4^–/– recipients of wild-type splenocytes, which was similar to levels of IFN-γ produced by STAT4^–/– mice immunized with MOG peptide with no transfer (P < 0.0001 at MOG 10 μg /ml). (b) IL-5 production (± SD) was measured by ELISA in splenocytes harvested from control STAT4^–/– mice (white bars), STAT4^–/– recipients of either wild-type C57BL/6 splenocytes (gray bars), or STAT6^–/– splenocytes (black bars) on day 35 postimmunization. IL-5 production in STAT4^–/– recipients of wild-type splenocytes was greater than STAT4^–/– recipients of STAT6^–/– splenocytes and STAT4^–/– mice with no transfer (P < 0.05 at MOG 10 μg/ml and P < 0.001 at MOG 100 μg/ml).