Regulation of experimental autoimmune encephalomyelitis by TPL-2 kinase

Abstract

Tumor progression locus 2 (TPL-2) expression is required for efficient polarization of naive T cells to Th1 effector cells in vitro, as well as for Th1-mediated immune responses. In the present study, we investigated the potential role of TPL-2 in Th17 cells. TPL-2 was found to be dispensable for Th17 cell differentiation in vitro, and for the initial priming of Th17 cells in experimental autoimmune encephalomyelitis (EAE), a Th17 cell-mediated disease model for multiple sclerosis. Nevertheless, TPL-2-deficient mice were protected from EAE, which correlated with reduced immune cell infiltration, demyelination, and axonal damage in the CNS. Adoptive transfer experiments demonstrated that there was no T cell-intrinsic function for TPL-2 in EAE, and that TPL-2 signaling was not required in radiation-sensitive hematopoietic cells. Rather, TPL-2 signaling in radiation-resistant stromal cells promoted the effector phase of the disease. Importantly, using a newly generated mouse strain expressing a kinase-inactive form of TPL-2, we demonstrated that stimulation of EAE was dependent on the catalytic activity of TPL-2 and not its adaptor function to stabilize the associated ubiquitin-binding protein ABIN-2. Our data therefore raise the possibility that small molecule inhibitors of TPL-2 may be beneficial in multiple sclerosis therapy.

Figure 1. TPL-2 is not required for in vitro differentiation of Th17 cells

(A - C) CD25⁻ CD44^hiCD4⁺ T cells (naïve CD4⁺ T cells) were isolated from WT (Map3k8^+/+) and Map3k8^−/− mice by FACS sorting, and cultured in triplicates for 4 days with anti-CD3/anti-CD28 plus the indicated Th17 polarizing cytokines and the AhR ligand FICZ. (A and B) IFNγ and IL-17A expression in CD4⁺ T cells was determined by intracellular staining and flow cytometry (mean ± s.e.m.). (C) ELISA of IL-17A production by CD4⁺ T cells (mean + s.e.m.). (D) Expression of the indicated mRNAs in Th17-polarized cell populations was determined by qRT-PCR, presented relative to Hprt1 mRNA (mean ± s.e.m.). Data are representative for three independent experiments.

Figure 2. TPL-2 regulates the onset and severity of EAE

(A) Mean clinical scores of WT and Map3k8^−/− mice (n = 19/WT; n=18/Map3k8^−/−) at various times after immunization with MOG_33-55/CFA. Data presented are combined from two independent experiments (n = 9-10/genotype/experiment). (B) Immune cell infiltration in the spinal cords of WT and Map3k8^−/− mice (n = 8/genotype) at the peak of disease was determined by flow cytometry using the indicated markers (mean ± s.e.m.) (C) Intracellular staining for infiltrated IL-17A-, IFNγ– and GM-CSF-expressing CD4⁺ T cells in the spinal cords of WT and Map3k8^−/− mice (n=8/genotype) on d12 after MOG_33-55/CFA immunization. In D and E, EAE was induced in WT and Map3k8^−/− mice, and spinal cords removed at the peak of disease. (D) Spinal cord sections were stained with luxol fast blue (LFB) to monitor demyelination (scale bar represents 200μm in the first panel, and 50μm in the lower panels). Immune cell infiltration was revealed by antibody staining: CD3 for T cells, Mac-3 for macrophages and B220 for B cells. Axonal damage was visualized by staining for amyloid precursor protein (APP). (E) Quantitation of demyelination, immune infiltration and axonal damage (mean ± s.e.m.). Data in B and C are compiled from 2 independent experiments (n=4/genotype/experiment). Data in D and E are representative of two independent experiments. * p ≤ 0.05; ** p≤0.01; *** p≤0.001.

Figure 3. TPL-2 is not required for T cell priming in EAE

WT and Map3k8^−/− mice were immunized with MOG_33-55/CFA, and draining LN isolated on d9. (A) CD4⁺ T cell numbers were determined by flow cytometry (mean ± s.e.m.; n = 3). (B) Flow cytometric analysis of the proliferation of CFSE-labelled CD4⁺ T cells cultured for 3d with MOG_33-55 peptide (mean ± s.e.m.; n = 3). (C) Frequencies of IL-17A- and IFNγ–expressing CD4⁺ T cells were determined by flow cytometry, after restimulation with PdBu/ionomycin or MOG_33-55 peptide (mean ± s.e.m.; n = 3). (D) ELISA of IFNγ and IL-17A production by LN cells restimulated with MOG_33-55 peptide (mean ± s.e.m.; n = 3). Data are representative of at least three independent experiments.

Figure 4. TPL-2 signaling in T cells is not required for EAE development

(A) BM cells from WT and Map3k8^−/− mice were mixed in a 1:4 ratio with BM cells from Tcra^−/− mice, and transferred into Rag1^−/− hosts. After 8 weeks, EAE was induced by immunization with MOG_33-55/CFA, and clinical scores determined (mean ± s.e.m.; n = 6). (B) Draining LN cells from WT and Map3k8^−/−mice were isolated 9d after immunization with MOG_33-55/CFA, and restimulated in vitro with MOG_33-55 peptide in the presence of recombinant IL-1β and 1L-23 for 5d. These Th17-polarized cells were then transferred into naïve WT and Map3k8^−/− mice. Graph represents the average clinical score after Th17 cell transfer (mean ± s.e.m.; n = 5). Data in A and B are representative of two independent experiments.

Figure 5. TPL-2 regulates the effector phase of EAE

(A) MOG_33-55-specific Th17 cells were generated from CD45.1⁺ WT mice, and then transferred into CD45.2⁺ WT and Map3k8^−/− mice as in Figure 6C. Numbers of CD45.1⁺ CD4⁺ T cells in the spleen and brain were determined by flow cytometry at the indicated time points after Th17 cell transfer (mean ± s.e.m.; n = 3). Macrophage numbers in the brain after transfer were determined by F4/80 staining. (B) Intracellular staining for IL-17A- and IFNγ-expression in transferred Th17 polarized CD45.1⁺ CD4⁺ T cells in the spleen and brain on d14 after transfer (mean ± s.e.m., n=3). Data are representative of at least two independent experiments. * p ≤ 0.05

Figure 6. TPL-2 signaling in radiation-resistant cells promotes EAE

(A) BM cells from WT and Map3k8^−/− mice were adoptively transferred into lethally irradiated Rag1^−/− or Map3k8^−/−Rag1^−/− hosts, and EAE induced 8 weeks later. Mean clinical scores are shown (mean ± s.e.m; n = 4-5). (B and C) EAE was induced in chimeric mice generated as in A. (B) Total numbers of CD4⁺ and CD45⁺ cells in the spinal cords 12d after MOG_33-55 peptide plus CFA immunization were determined by flow cytometry (mean ± s.e.m., n=4). (C) Intracellular staining for IL-17A- and IFNγ–expressing CD4⁺ T cells in the spinal cord at peak of disease (mean ± s.e.m.; n = 4). Data are representative for three independent experiments. * p ≤ 0.05; ** p≤0.01.

Figure 7. TPL-2 regulates pro-inflammatory gene expression in primary microglia and astrocytes

Quantitative RT-PCR of mRNA expression in primary microglia (A, B) and astrocytes (C, D) from WT and Map3k8^−/− mice, normalized to Hprt mRNA (mean ± s.e.m.). Cells were either left untreated (nt) or stimulated with the indicated agonists for 6h. Data are compiled from three (A, C) or two (B, D) independent experiments. * p ≤ 0.05, ** p≤0.01.

Figure 8. TPL-2 is required for optimal MAP kinase activation in microglia and astrocytes

Lysates of microglia (A) and astrocytes (B), generated from WT and Map3k8^−/− mice and stimulated for the indicated time points with LPS or TNF, were immunoblotted. Phosphorylated protein bands were quantified by laser densitometry using the Quantity One software package, and are presented as relative density normalized to the respective total protein. Data are representative for 3 – 4 independent experiments with similar results.

Figure 9. TPL-2 kinase activity is required to induce EAE

(A) Lysates were prepared from splenocytes (left panel) and BMDM (right panel) generated from WT, Map3k8^−/−, Map3k8^D270A/D270A and TNIP2^−/− mice, and immunoblotted for the indicated antigens. (B) Mean clinical scores of WT and Tnip2^−/− mice (mean ± s.e.m.; n = 7 - 8). Data are compiled from 2 different experiments (n = 3-4/genotype/experiment). (C) Mean clinical scores of WT, Map3k8^D270A/D270A and Map3k8^−/− mice mice at various times after immunization with MOG_33-55/CFA (mean ± s.e.m.; n = 8 - 9). Data are compiled from 2 different experiments (n = 3-4/genotype/experiment). * p ≤ 0.05; *** p≤0.001. (D) Mean clinical scores of WT, Map3k8^−/−, Ifnar^−/− and Map3k8^−/−Ifnar^−/− mice after immunization with MOG_33-55/CFA (mean ± s.e.m.; n = 9 - 10). Data are compiled from 2 different experiments (n = 4-5/genotype/experiment).