IL-12Rβ2 has a protective role in relapsing-remitting experimental autoimmune encephalomyelitis

Abstract

IL-12Rβ2 is a common receptor subunit of heterodimeric receptors for IL-12 and IL-35, two cytokines that are implicated in immunopathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We evaluated the role of IL-12Rβ2 in relapsing-remitting EAE (RR-EAE). IL-12Rβ2-deficient SJL/J mice developed markedly more severe clinical EAE, and had greater mortality and more severe relapses compared with wild-type controls. IL-12Rβ2-deficient EAE mice also had more infiltrating mononuclear cells in the CNS, as well as higher T cell proliferative capacity and decreased IFN-γ production at the periphery. These findings demonstrate a protective role of IL-12Rβ2 in RR-EAE.

Keywords: Experimental autoimmune encephalomyelitis (EAE); IL-12; IL-12Rβ2; IL-35; Relapse.

Figure 1. IL-12Rβ2^−/− SJL/J mice were more susceptible to EAE

A) IL-12Rβ2^−/− (n=10) and WT mice (n=10) were immunized with 100 µg of PLP_139–151 in CFA containing 3 mg/ml M. tuberculosis H37Ra. Pertussis toxin (200 ng) was given i.p. on days 0 and 2 p.i. Data represent the mean clinical scores ± SEM. One of two experiments is shown. B) IL-12Rβ2^−/− (n=7) and WT mice (n=7) were immunized with 100 µg of PLP_139–151 in CFA containing 3 mg/ml M. tuberculosis H37Ra. Pertussis toxin (200 ng) was given i.p. on days 0 and 2 p.i. Data represent the mean clinical scores ± SEM. C) IL-12Rβ2^−/− (n=8) and WT mice (n=8) were immunized with 50 µg of PLP_139–151 in CFA containing 2 mg/ml M. tuberculosis H37Ra. Pertussis toxin (100 ng) was given i.p. on days 0 and 2 p.i. Data represent the mean clinical scores ± SEM. One of two experiments is shown. D) IL-12Rβ2^−/− and WT mice were immunized with 100 µg of PLP_139–151 in CFA containing 3 mg/ml M. tuberculosis H37Ra. Pertussis toxin (200 ng) was given i.p. on days 0 and 2 p.i. Groups of mice (n=5–10, depending on the time point) were sacrificed at the time p.i. indicated in the figure. Spinal cords and brains from mice in the same group were pooled, MNCs isolated and their numbers determined by counting under the microscope.

Figure 2. IL-12Rβ2^−/− mice had a higher proportion of Th17 cells and lower proportion of Th1 cells among CD4⁺ T cells isolated from the CNS during EAE

IL-12Rβ2^−/− and WT mice were immunized with 100 µg of PLP_139–151 in CFA containing 3 mg/ml M. tuberculosis H37Ra. Pertussis toxin (200 ng) was given i.p. on days 0 and 2 p.i. Groups of mice (n=5–10, depending on the time point) were sacrificed at the time p.i. indicated in the figure. Spinal cords and brains from mice in the same group were pooled, MNCs isolated, incubated with PMA, ionomycin and GolgiPlug, stained, and gated CD4⁺ cells analyzed by flow cytometry. Data shown are representative of 4 experiments.

Figure 3. Splenocytes of immunized IL-12Rβ2^−/− EAE mice produced less IFN-γ than WT splenocytes

Splenocytes harvested from PLP_139–151-immunized mice on days 7, 11, 21, 28, 50 p.i. were cultured for 72 h with or without 20 µg/ml PLP_139–151. Concentrations of IL-17A and IFN-γ in splenic culture supernatants were measured by ELISA. Data shown are rom 3 independent experiments.

Figure 4. IL-12Rβ2^−/− EAE mice developed more Treg cells in spleen

Splenocytes from PLP_139–151-immunized mice on day 7, 11, 21, 28 p.i. were isolated and stained. Expression of CD25 and Foxp3 in gated CD4⁺ T cells were analyzed by flow cytometry. Data shown originate from 3 independent experiments.

Figure 5. IL-12Rβ2^−/− splenocytes had higher proliferative capacity

Splenocytes harvested from PLP_139–151-immunized mice on days 7, 11, 21, 28, 50 p.i. were cultured (4×10⁵ cells/well) in triplicates for 72 h with 20 µg/ml PLP_139–151 or 1 µg/ml anti-CD3/CD28 antibodies. After 60 h, cells were pulsed for 12 h with 1µCi of [³H] methylthymidine. Data shown are from 3 independent experiments.

Figure 6. IL-12Rβ2^−/− mice had a higher proportion of CD11b⁺Gr1⁺ cells in the CNS and spleen

A) MNCs from the CNS of EAE mice on days 11, 21, 28 and 50 p.i. were isolated and stained for Gr1 and CD11b. B) Splenocytes from EAE mice on days 7, 11, 21, 28 and 50 p.i. were isolated, stained for Gr1 and CD11b and analyzed by flow cytometry. One representative of three experiments is shown.