Control of IL-17 receptor signaling and tissue inflammation by the p38α-MKP-1 signaling axis in a mouse model of multiple sclerosis

Abstract

T helper 17 (T(H)17) cells, a subset of CD4+ T cells that secrete the proinflammatory cytokine interleukin-17 (IL-17), play a key pathogenic role in autoimmune diseases. Through inducible and tissue-specific deletion systems, we described the time- and tissue-specific roles of the mitogen-activated protein kinase (MAPK) p38α in mediating T(H)17 cell-induced tissue inflammation. Inducible deletion of Mapk14 (which encodes p38α) after the onset of experimental autoimmune encephalomyelitis (EAE), a murine model for human multiple sclerosis, protected mice from inflammation. Furthermore, the severity of EAE was markedly reduced in mice with specific loss of p38α in neuroectoderm-derived cells, including astrocytes, an effect that was associated with defective production of chemokines and decreased infiltration of the target tissue by immune cells. p38α linked IL-17 receptor (IL-17R) signaling to the expression of genes encoding proinflammatory chemokines and cytokines. Mice that lacked MAPK phosphatase 1 (MKP-1), an inhibitor of p38α, had exacerbated EAE and enhanced expression of IL-17R-dependent genes. Our results suggest that the p38α-MKP-1 signaling axis links IL-17R signaling in tissue-resident cells to autoimmune inflammation dependent on infiltrating T(H)17 cells.

Fig. 1. p38α is required for the effector stage of EAE

(A) Spinal cord cells from unimmunized mice (naïve) and from mice immunized to develop EAE (day 15) were analyzed by Western blotting to determine the relative abundances of total and phosphorylated p38 proteins. β-actin was used as a loading control. Bottom: Relative band intensities for the indicated proteins from three independent experiments were determined by densitometry and are shown as means ± SEM. Samples from naïve mice are in filled bars, whereas those from EAE mice are in empty bars. (B) EAE disease course of MOG-immunized wild-type (WT) and p38α^CreER mice that were treated with tamoxifen at the onset of disease (day 12 or 13). Data are means ± SEM of five to seven mice per group. (C to E) WT and p38α^CreER mice treated with tamoxifen before they were injected with in vitro–derived T_H17 cells were (C) monitored to determine EAE disease course, (D) subjected to histopathological analysis of spinal cords, and (E) given histology scores. Data in (C) and (E) are means ± SEM of four to five mice per group. (D) Images are 10× original magnification. Data are representative of two to three independent experiments. *P < 0.05.

Fig 2. Loss of p38α in CNS-resident cells delays EAE progression and impairs the infiltration of inflammatory cells into the CNS

(A) EAE disease course of MOG-immunized WT and p38α^NesCre mice. Data are means ± SEM of 8 to 11 mice per group. (B to D) WT and p38α^NesCre EAE mice were analyzed on day 16 after immunization. (B) H&E staining of the indicated regions of the spinal cord. (C) Histology scores of the indicated regions of the spinal cord. (D) Staining of thoracic spinal cords with anti-CD3 antibody (α-CD3), α-Iba1, and Luxol fast blue. Images are 10× original magnification. Data are from five mice per group. (E) Flow cytometric analysis of cells from the spinal cords of WT and p38α^NesCre EAE mice on day 16 after immunization. Data are representative of 10 mice per group. (F) Analysis of the relative numbers and percentages of the indicated cell types in the spinal cords of the mice analyzed in (E). *P < 0.05; **P < 0.01; ***P < 0.001. Data are representative of two to three independent experiments.

Fig. 3. Ablation of p38α in CNS-resident cells inhibits expression of genes encoding chemokines and inflammatory factors and represses T_H17 cell–induced EAE

(A) Spinal cord cells from WT and p38α^NesCre EAE mice on day 11 after immunization were subjected to real-time PCR analysis to determine the relative abundances of the indicated mRNAs. Data are means ± SEM of five to seven mice per group. (B to D) Cells were isolated from (B) the spleens, (C) DLNs, and (D) spinal cords of MOG-immunized WT and p38α^NesCre mice (on day 11), stimulated with PMA and ionomycin in vitro, and then analyzed by flow cytometry to determine the percentages of cells positive for IL-17 and IFN-γ among CD4⁺ T cells. Data are means ± SEM of five to seven mice per group. (D) The total numbers of CD4⁺ T cells that infiltrated the spinal cords are indicated above the flow cytometry plots. (E) EAE disease course of WT and p38α^NesCre mice (CD45.2⁺) that received in vitro–derived T_H17 cells from CD45.1 mice. Data are means ± SEM of 10 mice per group. (F and G) Analysis of the numbers of (F) donor-derived CD4⁺ (CD45.1⁺) T cells and (G) host-derived (CD45.2⁺CD11b⁺) myeloid cells isolated from the spinal cords of WT and p38α^NesCre mice on day 11 after adoptive transfer of cells. Data are means ± SEM of four mice per group. (H) Left: Cells were isolated from the spinal cords of recipient WT and p38α^NesCre mice on day 11 after transfer, stimulated with PMA and ionomycin in vitro, and then analyzed by flow cytometry to determine the percentages of cells positive for IL-17 and IFN-γ among donor-derived CD4⁺ (CD45.1⁺) T cells. Right: Data are means ± SEM of four mice per group. *P < 0.05; **P < 0.01; ***P < 0.001. Data are representative of two to three independent experiments.

Fig. 4. Analysis of IL-17–induced proinflammatory gene expression and MK2 activation in p38α-deficient MEFs and astrocytes

(A) MEFs isolated from WT or p38α^CreER mice were treated with 4-OHT, and were left untreated or treated with IL-17 or TNF-α alone or in combination for 24 hours. Cell culture medium was then analyzed to determine the amounts of CXCL1 protein secreted by the cells. Data are means ± SEM of six mice per group. (B) Astrocytes isolated from WT or p38α^CreER mice were treated with 4-OHT, and were then incubated in the absence or presence of IL-17 for 5 hours. Cells were subjected to real-time PCR analysis to determine the relative abundances of the indicated mRNAs. Data are means ± SEM of three to seven mice per group. (C) Left: Astrocytes isolated from WT or p38α^CreER mice were treated with 4-OHT, and were then incubated in the absence or presence of IL-17 for the indicated times. Samples were then analyzed by Western blotting with antibodies specific for the indicated proteins. Blots are representative of five mice per group from two independent experiments. Right: Relative band intensities were determined by densitometry and are shown as means ± SEM of five mice per group. (D) Astrocytes from WT mice were treated with vehicle or an MK2 inhibitor before being stimulated with IL-17 for 5 hours. Cells were analyzed by real-time PCR to determine the relative abundances of the indicated mRNAs. Data are means ± SEM of seven mice per group. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Data are representative of two to three independent experiments.

Fig 5. Loss of MKP-1 in nonhematopoietic cells exacerbates the development of EAE

(A to C) Bone marrow cells isolated from CD45.1⁺ mice were transferred into lethally irradiated WT or MKP-1^−/− mice to generate bone marrow chimeric mice. After reconstitution, the WT and MKP-1^−/− bone marrow chimeric mice were immunized with MOG and CFA before being (A) monitored to determine EAE disease course (data are means ± SEM of five mice per group), (B) subjected to histopathological analysis of spinal cords, and (C) given histology scores (data are means ± SEM of six or seven mice per group). Images are 10× original magnification. (D) MEFs from WT or MKP-1^−/− mice were left untreated or were treated with IL-17 or TNF-α alone or in combination for 24 hours. Cell culture medium was then analyzed to determine the amounts of CXCL1 and IL-6 proteins secreted by the cells. Data are means ± SEM of 10 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001. Data are representative of two to three independent experiments.