TPL2 mediates autoimmune inflammation through activation of the TAK1 axis of IL-17 signaling

Abstract

Development of autoimmune diseases, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), involves the inflammatory action of Th1 and Th17 cells, but the underlying signaling mechanism is incompletely understood. We show that the kinase TPL2 is a crucial mediator of EAE and is required for the pathological action of Th17 cells. TPL2 serves as a master kinase mediating the activation of multiple downstream pathways stimulated by the Th17 signature cytokine IL-17. TPL2 acts by linking the IL-17 receptor signal to the activation of TAK1, which involves a dynamic mechanism of TPL2-TAK1 interaction and TPL2-mediated phosphorylation and catalytic activation of TAK1. These results suggest that TPL2 mediates TAK1 axis of IL-17 signaling, thereby promoting autoimmune neuroinflammation.

Figure 1.

Tpl2-KO mice are resistant to CNS inflammation. (A) Mean clinical scores of age- and sex-matched WT and Tpl2-KO mice subjected to MOG_35-55-induced EAE (n = 6 mice per group). (B) H&E and Luxol Fast Blue (LFB) staining of spinal cord sections from MOG_35-55-immunized WT and Tpl2-KO EAE mice for visualizing immune cell infiltration and demyelination, respectively (arrows). Bars, 100 µm. (C and D) Flow cytometric analyses of CD4⁺ and CD8⁺ T cells, CD11b⁺ monocytes, and Gr-1⁺ neutrophils among the CD45⁺ immune cells infiltrating to the CNS (brain and spinal cord) of MOG_35-55-immunized WT and Tpl2-KO mice (n = 5 mice per group, day 15 after immunization). Data are presented as a representative plot (C) and summary graph of the absolute cell numbers (D). (E and F) Infiltrating immune cells isolated from CNS (brain and spinal cord) of MOG_35-55-immunized WT and Tpl2-KO mice (n = 5 mice per group, day 15 after immunization) were fixed and permeabilized, and CD4⁺ T cells were analyzed by flow cytometry for intracellular IFN-γ and IL-17. Data are presented as a representative plot (E) and summary graph of the absolute cell numbers (F). (G) QPCR analysis to determine the relative mRNA expression level of proinflammatory genes in spinal cords of unimmunized (naive) and MOG_35-55-immunized (EAE) WT and Tpl2-KO mice (n = 4 mice per group, day 15 after immunization). Data were normalized to a reference gene, Actb. *, P < 0.05; **, P < 0.01. Data are representative of three or more independent experiments. Error bars are mean ± SD values.

Figure 2.

TPL2 deficiency does not compromise T cell differentiation or recall responses. (A–C) Naive splenic CD4⁺ T cells (CD44^loCD62L^hi) isolated from WT and Tpl2-KO mice were stimulated for 4 d with 5 µg/ml of plate-bound anti-CD3 and 1 µg/ml anti-CD28 under Th0, Th1, Th17, or T reg conditions as described in Materials and methods. Flow cytometry was used to measure the frequency of IFN-γ– and IL-17–producing cells, and Foxp3⁺ cells, showing a representative plot (A and B) and a summary graph (C). (D and E) Flow cytometry analysis of IFN-γ– and IL-17–producing cells in the draining LNs of day 15 MOG_35-55-immunized WT and Tpl2-KO mice, showing a representative plot (D) and a summary graph (E). Data are representative of 4 animals. (F and G) Flow cytometry analysis of CD4⁺Foxp3⁺ T reg cells in the CNS, spleen, and draining LNs of day 15 MOG_35-55-immunized WT and Tpl2-KO mice, showing a representative plot of T reg cell frequency (F) and a summary graph of T reg cell number (G). Data are representative of 4 animals per group. (H and I) Draining LN cells or splenocytes isolated from day 15 MOG_35–55-immunized WT or Tpl2-KO mice were restimulated in vitro with the MOG peptide at the indicated concentrations and time periods. Supernatants were subjected to ELISA of the indicated cytokines (H), and cell proliferation was measured based on ³H-thymidine incorporation. *, P < 0.05. Data are representative of two (A–C, F, and G) or at least three (D, E, H, and I) independent experiments. Error bars are mean ± SD values.

Figure 3.

Tpl2 is dispensable in hematopoietic cells for EAE pathogenesis. (A) Mean clinical score after EAE induction in lethally irradiated WT recipient mice adoptively transferred with WT or Tpl2-KO BM cells (n = 5 mice per group). (B and C) Flow cytometry analysis of CNS-infiltrating mononuclear cells of day 15 MOG_35-55-immunized chimeric mice described in A, showing a representative plot (B) and a summary graph (C). n = 4 mice per group. (D) Mean clinical scores of age- and sex-matched Rag1-KO mice, adoptively transferred with WT and Tpl2-KO CD4⁺ T cells and then subjected to MOG_35-55-mediated EAE induction (n = 5 mice per group). Data are representative of two independent experiments for all panels. Error bars are mean ± SD values.

Figure 4.

TPL2 deficiency in radioresistant cells inhibits immune cell recruitment into the CNS and ameliorates EAE pathogenesis. (A) Lethally irradiated WT and Tpl2-KO mice were adoptively transferred with GFP⁺ WT bone marrow cells, and the generated chimeric mice were subjected to EAE induction by MOG_35-55. Graph represents the clinical scores of WT and Tpl2-KO GFP-chimeric mice. (B–F) The WT and Tpl2-KO GFP⁺ bone marrow–chimeric mice were either not immunized (B) or immunized with MOG_35-55 for EAE induction (for 15 d; C–F). Flow cytometry was performed to monitor the frequency of GFP⁺ cells in the spleen (B), total CNS-infiltrating cells (GFP⁺; C and D), and CNS-infiltrating (GFP⁺CD4⁺; E and F) CD4⁺ T cells. Data are presented as representative plots (B, C, and E) or summary graphs (D and F). (G and H) Flow cytometric analyses of the frequency (G) and absolute numbers (H) of IFN-γ– and IL-17–producing CD4⁺ T cells in the CNS (brain and spinal cord) of MOG_35-55-immunized WT and Tpl2-KO GFP-chimeric mice (n = 5 mice per group, day 15 after immunization). (I) Flow cytometric analyses to measure the number of GFP⁺CD4⁺ cells in the draining lymph nodes of MOG_35-55-immunized WT and Tpl2-KO GFP-chimeric mice. (J) QPCR analyses of the relative mRNA expression level of the indicated proinflammatory genes in the spinal cords of MOG_35-55-immunized WT and Tpl2-KO GFP-chimeric mice (n = 4 mice per group, day 15 after immunization). Data were normalized to a reference gene, Actb. *, P < 0.05. Data are representative of two independent experiments for all panels. Error bars are mean ± SD values.

Figure 5.

TPL2 is required for EAE induction by Th17 cells but not by Th1 cells. (A and B) EAE clinical scores of WT or Tpl2-KO mice adoptively transferred with MOG_35-55-specific Th17 T cells (donor) derived from MOG_35-55-immunized WT (A) or Tpl2-KO (B) mice. (C and D) Flow cytometry analysis of the indicated immune cell (CD45⁺) populations infiltrated into the CNS (brain and spinal cord) of adoptively transferred WT and Tpl2-KO mice described in A (n = 4 mice per group, day 15 after donor Th17 cell transfer). Data are presented as a representative plot (C) and summary graphs of the absolute cell numbers (D). (E and F) Absolute cell number of the transferred CD4⁺ T cells (CD45.1⁺) in CNS (brain and spinal cord, E) and spleen (F) of WT and Tpl2-KO mice adoptively transferred with MOG-specific CD45.1⁺ Th17 cells (derived from B6.SJL mice), determined by flow cytometry (n = 4 mice per group). (G) EAE clinical scores of WT and Tpl2-KO mice adoptively transferred with MOG_35-55-specific WT Th1 cells. (H) Absolute cell number of the indicated immune cell populations infiltrated into the CNS (brain and spinal cord) of adoptively transferred WT and Tpl2-KO mice described in G (n = 4 mice per group, day 15 after donor Th1 cell transfer). *, P < 0.05; **, P < 0.01. Data are representative of three independent experiments. Error bars are mean ± SD values.

Figure 6.

TPL2 mediates IL-17–stimulated gene expression. (A and B) Lethally irradiated WT and Tpl2-KO mice were adoptively transferred with WT bone marrow cells derived from B6.SJL mice, and the generated chimeric mice were subjected to EAE induction by MOG_35-55. The mice were also intravenously injected with control rabbit IgG or an anti–IL-17 antibody on days 5, 7, 9, and 11 after MOG immunization (n = 4 mice per group). The mice were monitored for mean clinical scores (A) and sacrificed on day 14 for QPCR analyses of the indicated genes using FACS-sorted CNS astrocytes (CD45.1⁻ASCA-1⁺) or endothelial cells (CD45.1⁻CD31⁺). (C and D) QPCR analysis of relative mRNA expression for the indicated genes in WT and Tpl2-KO astrocytes (C) or primary MEFs (D) that were either not treated (NT) or stimulated with IL-17 for 6 h. Data were normalized to a reference gene, Actb. (E) TPL2-deficient primary MEFs were reconstituted by retroviral infection with expression vectors encoding GFP, HA-TPL2, or a catalytically inactive HA-TPL2 mutant (K167M). QPCR was performed for analysis of the relative mRNA levels for the indicated genes, and an IB was performed to monitor TPL2 expression. (F) QPCR analysis of relative mRNA expression for the indicated cytokine and chemokine genes in WT and Tpl2-KO astrocytes stimulated with IL-1, TNF, or IL-1 plus TNF for 2 h. *, P < 0.05; **, P < 0.01. P-values were calculated based on 3 technical repeats, and data are representative of one (A and B), two (F), or at least three (C–E) independent experiments. Error bars are mean ± SD values.

Figure 7.

TPL2 mediates IL-17–stimulated activation of multiple signaling pathways. (A and B) IB analyses of the indicated phosphorylated (P-) and total proteins in lysates from WT and Tpl2-KO astrocytes (A) or MEFs (B) stimulated for the indicated time periods with 100 ng/ml IL-17. (C) IB analysis of phosphorylated (P-) IKK and total IKKβ in IL-17-stimulated astrocytes. (D) IKK complex was isolated from WT and Tpl2-KO astrocyte by IP (using anti-IKKγ) and subjected to IKK kinase assay (KA) using 1 µg GST-IκBα (1–54) as a substrate or IKKβ IB (IB) assay. (E) WT and Tpl2-KO astrocyte and MEFs were stimulated with 100 ng/ml IL-17 for the indicated time periods, and nuclear extracts were subjected to NF-κB EMSAs. A Lamin B IB and an NF-Y EMSA were used as loading controls. (F) Flow cytometry analysis of IL-17R expression on WT and Tpl2-KO primary astrocytes. (G) IB analysis of the Act1 in the lysates of Tpl2-KO and WT control astrocyte stimulated with IL-17 for the indicated time periods. (H) IB analyses of the indicated phosphorylated (P-) and total proteins in lysates from WT and Tpl2-KO astrocytes stimulated for the indicated time periods with 20 ng/ml IL-1β. (I) P-ERK and total ERK bands in H were quantified by densitometry. Data are representative of two (F–H) or at least three (A–E) independent experiments. *, P < 0.05; **, P < 0.01. Error bars are mean ± SD values.

Figure 8.

TPL2 phosphorylates TAK1 and mediates IL-17–stimulated TAK1 activation. (A and B) IB analysis of phosphorylated (P-) and total TAK1, as well as the loading control HSP60, in lysates of WT and Tpl2-KO astrocytes (A) and MEFs (B), stimulated for the indicated time periods with 100 ng/ml IL-17). (C) TAK1 was isolated by IP from IL-17–stimulated WT and Tpl2-KO astrocytes and subjected to kinase assay (KA) using GST-MKK7 (1 µg) as substrate. The KA membrane was analyzed by IB to monitor TAK1 protein level. (D–F) TPL2 in vitro kinase assays using TPL2 isolated (by IP) from IL-17–stimulated astrocytes (D) or Tpl2-KO MEFs reconstituted with WT or K167M mutant of TPL2 (F) or using purified recombinant TPL2 (E). 1 µg GST-TAK1-K63R-V5-(1–292) (labeled GST-TAK1) was used as kinase assay substrate. The phosho-TAK1 (Thr187) antibody (α–P-TAK1) was used for phospho-IB (E). (G) HA-TPL2–reconstituted Tpl2-KO MEFs were stimulated with IL-17 (100 ng/ml) and subjected to the indicated IP assays, followed by detecting precipitated and coprecipitated proteins (labels on the right) by IB. (H) 293T cells were transfected with the expression vectors indicated on top of the figure. Whole-cell lysates were subjected to IP using anti-TPL2, followed by IB analysis of the precipitated and coprecipitated proteins. Lysates were also analyzed by direct IB. (I) IB of TAK1 and TPL2 in the TPL2 complex isolated by IP from IL-17–stimulated p105-KO MEFs reconstituted with WT or a degradation-resistant mutant (SS/AA) of p105. (J) p105-KO MEFs were reconstituted with WT p105 or a p105 mutant harboring serine to alanine substitutions at its phosphorylation site. The cells were stimulated with 100 ng/ml IL-17 for the indicated time periods, and whole-cell lysates were subjected to IB analysis of the indicated phosphorylated (P-) and total. (K) IB analysis of phosphorylated (P-) and total TAK1 in the lysates of IKKβ-KO and WT control MEFs stimulated with IL-17 for the indicated time periods. (L) A TPL2 kinase assay (KA) in IL-17–stimulated IKKβ-KO and WT control MEFs. The TPL2 KA membrane was subsequently subjected to IB using HRP-conjugated anti-TPL2. Data are representative of two (A–C, F, and G) or at least three (D, E, H, and I) independent experiments.

Figure 9.

TAK1 mediates IL-17–induced activation of JNK, p38, and IKK/NF-κB. (A) IKK was isolated by IP (using anti-IKKγ) from whole-cell lysates of IL-17–stimulated WT or Tak1-KO MEFs and subjected to kinase assays (KA) using 1 µg GST-IκBα (1–54) as a substrate, followed by IKKβ IB. (B) IB analysis of the indicated phosphorylated (P-) and total proteins in the whole-cell lysates from WT and Tak1-KO MEFs. (C) IB analysis of the indicated phosphorylated (P-) and total proteins in the whole-cell lysates from WT and Tak1-KO MEFs. (D) IB analyses of the indicated phosphorylated (P-) and total proteins in whole-cell lysates from WT and Tak1-KO primary astrocytes stimulated with 100 ng/ml IL-17. (E) Mean clinical scores of age- and sex-matched Tak1^+/+ Gfap-cre and Tak1^fl/fl Gfap-cre mice subjected to MOG_35-55-induced EAE (n = 5 mice per group). An IB, using astrocytes of unimmunized mice, was included to show the efficiency of TAK1 ablation. Data are representative of three independent experiments for all panels. *, P < 0.05. Error bars are mean ± SD values.