Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis (original) (raw)
Clinical disease expression in STAT4–/– and STAT6–/– mice. First we tested the effects of targeted deletion of STAT4 versus STAT6 on the induction of EAE. Our data show that STAT4–/– mice are resistant to the development of clinical EAE, while STAT6–/– mice experience a more severe form of disease as compared with STAT4–/– or wild-type mice (Figure 1). Composite data from three experiments are shown in Table 1. Mice were followed for up to 50 days. STAT4–/– mice have a lower incidence of disease (<50%) compared with wild-type and STAT6–/– mice (100%; P = 0.0008 for either). The STAT4–/– mice that develop EAE experience a similar day of onset of disease as wild-type mice, but show a very mild clinical course (mean maximal disease grade of 0.46 ± 0.2). In contrast, STAT6–/– mice have an earlier onset of disease compared with wild-type (P < 0.01) mice and attain a higher disease grade compared with wild-type (P < 0.005) and to STAT4–/– (P < 0.0001) mice.
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.
Disease incidence, mean maximal grade, and day of onset in STAT4 or STAT6 knockout compared with wild-type
Immunopathology of the CNS after EAE induction. Pathologic examination of the CNS from STAT4–/–, STAT6–/–, and wild-type mice taken on day 16 post immunization showed minimal infiltrates in the CNS of STAT4–/– mice, while there were more infiltrates in the other two groups of mice (Figure 2a). Quantitation of CD4+ and CD8+ T cells, macrophages, and granulocytes after immunohistological staining showed significantly fewer infiltrating cells in STAT4–/– mice as compared with wild-type and STAT6–/– mice (Figure 2a). There were significantly fewer CD4+ cells in spinal cord sections from STAT4–/– compared with wild-type mice taken on day 50 postimmunization (Figure 2b).
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.
Cytokine production by primed T cells from STAT4–/– and STAT6–/– mice. We examined the proliferation of in vivo primed splenocytes isolated from STAT4–/–, STAT6–/–, and wild-type mice. T cells of STAT4–/– and STAT6–/– mice proliferated well to MOG peptide in vitro (Figure 3), indicating that they are primed. We examined Th1 and Th2 cytokine production by ELISA assay and quantified the number of cytokine-secreting cells by ELISPOT assay. Interestingly, IFN-γ production in culture supernatants was significantly higher in splenocytes of wild-type mice than in STAT6–/– mice after stimulation with MOG peptide (Figure 4a). This finding was confirmed by the ELISPOT assay data showing the highest frequency of IFN-γ–producing cells in the wild-type cultures (Figure 4b). As expected, STAT4–/– mice had the lowest IFN-γ production as determined by ELISA and the lowest frequency as determined by the ELISPOT assay (Figure 4, a and b). IL-4 is difficult to measure in culture supernatants using ELISA due to consumption by cells; thus we measured IL-5 in culture supernatants as a marker of Th2 cells. IL-5 production was highest in culture supernatants from STAT4–/– mice (Figure 5a). This was associated with a higher frequency of IL-5–producing cells as measured by ELISPOT assay in these mice (Figure 5b). The frequency of IL-4–producing cells as determined by ELISPOT assay was similar in the STAT4–/– and wild-type groups and significantly lower in the STAT6–/– mice (Figure 5c).
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.
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.
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).
Anti-MOG Ab titers in STAT4–/–, STAT6–/–, and wild-type mice. Anti-MOG Ab’s were reported to augment disease severity in a MOG EAE rat model (25), although B cell–deficient C57BL/6 mice develop EAE (26). A decreased level of IgG1 and IgE, typically associated with a Th2 phenotype, have been reported in STAT6–/– mice (16). IgG2a, typically associated with a Th1 phenotype, has been reported to be either elevated (27) or normal (16, 17) in STAT6–/– animals. We examined the relative titers of anti-MOG total IgG (Figure 6a), IgG1 (Figure 6b), and IgG2a (Figure 6c) from serum samples isolated from STAT4–/–, STAT6–/–, and wild-type mice at four different time points postimmunization: day 12, 16, 22, and 50. Three to five samples per group per time point were tested. Using Mann-Whitney analysis, no significant differences were found in total IgG titers between 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.
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.
Adoptive transfer studies into TCR-αβ–/– recipients. To further investigate the encephalitogenicity of Th1 and Th2 cells, we used an adoptive transfer model where primed splenocytes from mice immunized previously were transferred directly without in vitro stimulation into TCR-αβ–/– animals. The TCR-αβ–/– recipients were then immunized in order to activate the primed T cells in vivo. This method allows us to activate the donor cells in the cytokine environment being tested (see below), rather than activating the cells ex vivo and then transferring them into the recipient. Post immunization with MOG peptide, the recipients were observed for the development of clinical disease. TCR-αβ–/– mice do not develop EAE postimmunization, but adoptive transfer of primed splenocytes from wild-type animals induced moderately severe EAE. The optimal number of wild-type cells for use in adoptive transfer was determined in dose-response experiments (Figure 7a) and 50 × 106 cells were found to be optimal (incidence, six of six; mean maximal grade, 3.5 ± 0.7; mean day of onset, 14.4 ± 3.4). Therefore, we used this adoptive transfer model system to study the putative encephalitogenic functions of Th1 versus Th2 cells in vivo. When primed splenocytes from STAT6–/– mice were adoptively transferred to TCR-αβ–/– recipients, they induced moderate disease (incidence, five of five; mean maximal grade, 1.8 ± 1.2; mean day of onset, 16.6 ± 2.2), while adoptive transfer of splenocytes from STAT4–/– mice failed to cause EAE (incidence, zero of five; mean maximal grade, 0; mean day of onset, NA) (Figure 7b). These data confirm our active immunization studies showing that T cells from STAT6–/– animals (Th1 biased) are encephalitogenic, while T cells from STAT4–/– (Th2 biased) are not.
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.
Adoptive transfer of T cells into STAT4–/– recipients. To investigate the putative regulatory function of a Th2 environment on disease expression, we transferred 50 × 106 splenocytes from STAT6–/– or wild-type mice into STAT4–/– recipients that were subsequently immunized with MOG. Splenocytes harvested from STAT6–/– mice resulted in worse disease than wild-type splenocytes when transferred into STAT4–/– recipients (Figure 8). This may be due to the ability of the Th2 environment to better regulate uncommitted wild-type T cells than STAT6–/– cells, which lack the IL-4–regulated Th2 differentiation pathway and may only form Th1 cells. In vitro cytokine production confirmed this and showed high levels of IFN-γ production in the supernatants of cultures of primed cells harvested from STAT4–/– recipients of STAT6–/– splenocytes (Figure 9a). IFN-γ production in cultures of adoptively transferred wild-type cells were similar to STAT4–/– cell cultures with very little IFN-γ production (Figure 9a). IL-5 levels were similar between STAT4–/– recipients of STAT6–/– cells or wild-type cells (Figure 9b).
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.
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).