Opposing Functions of Microglial and Macrophagic TNFR2 in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

Abstract

In multiple sclerosis (MS), soluble tumor necrosis factor (TNF) is detrimental via activation of TNF receptor 1 (TNFR1), whereas transmembrane TNF is beneficial primarily by activating TNF receptor 2 (TNFR2). Here, we investigate the role of TNFR2 in microglia and monocytes/macrophages in experimental autoimmune encephalomyelitis (EAE), a model of MS, by cell-specific gene targeting. We show that TNFR2 ablation in microglia leads to early onset of EAE with increased leukocyte infiltration, T cell activation, and demyelination in the central nervous system (CNS). Conversely, TNFR2 ablation in monocytes/macrophages results in EAE suppression with impaired peripheral T cell activation and reduced CNS T cell infiltration and demyelination. Our work uncovers a dichotomy of function for TNFR2 in myeloid cells, with microglial TNFR2 providing protective signals to contain disease and monocyte/macrophagic TNFR2 driving immune activation and EAE initiation. This must be taken into account when targeting TNFR2 for therapeutic purposes in neuroinflammatory diseases.

Keywords: TNF signaling; cytokines; macrophages; microglia; multiple sclerosis; neuroinflammation; tumor necrosis factor.

Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Ablation of microglial TNFR2 in Cx3cr1CreER:Tnfrsf1bfl/fl mice leads to the early onset of EAE

A) Clinical course of EAE in Tnfrsf1bfl/fl and Cx3cr1CreER:Tnfrsf1bfl/fl mice; n=13–17/group from two experiments; **p ≤ 0.01, one-way ANOVA, Mann-Whitney test. (B–E) Flow cytometric analysis of microglia in the spinal cord at 17 dpi EAE. (C, D) Quantification of percentage (C) and number (D) of microglia, and percentage of MHCII+ activated microglia (E); n=5/group, *p ≤ 0.05, Student’s t test. (F) Representative flow plots of Ki67 expression in microglia at 17 dpi. (G) Quantification of Ki67+ microglia; n=4–5/group, *p ≤ 0.01, Student’s t test. (H) Representative flow plots of Annexin V expression in microglia at 17 dpi. (I) Quantification of Annexin V+PI− and Annexin V+PI− microglia; n=4–5/group. (J) Representative flow plots of CD4 and CD8 cells in the spinal cord at 17 dpi. (K, L) Percentages (K) and absolute numbers (L) of infiltrated immune cells. Mo/MΦ are defined as CD45hiCD11b+NK1.1−Ly6G−SSAlow; n=5/group, *p ≤ 0.05, **p ≤ 0.01, Student’s t test. (M) Representative flow plots of total and MHCII+ splenic B cells at 17 dpi. (N, O) Percentages (N) and absolute numbers (O) of splenic immune cells; n=6/group, *p ≤ 0.05, Student’s t test.

Figure 2. Ablation of microglial TNFR2 leads to exacerbated T cell effector function in the spinal cord after EAE

A, B, D, E, G, H) Representative flow plots of cytokine expression in CD4 and CD8 T cells isolated from spinal cord and spleen of Tnfrsf1bfl/fl and Cx3cr1CreER:Tnfrsf1bfl/fl mice at 17 dpi; n=4–5/group, *p ≤ 0.05, Student’s t test. (C, F, I) Quantification of cytokine expression in CD4 and CD8 T cells from spinal cord and spleen; n=4–5/group. (J) Western blot analysis of tight junction proteins in the spinal cord at 12 dpi EAE; n=4–9/group, **p ≤ 0.01, Student’s t test.

Figure 3. Microglial TNFR2 ablation leads to increased demyelination at chronic EAE

A) Assessment of demyelination in the spinal cord of Tnfrsf1bfl/fl and Cx3cr1CreER:Tnfrsf1bfl/fl mice by LFB staining. Left: contoured areas show regions of demyelinated white matter; scale bar = 200 µm. Right: 3D reconstructions of the demyelinated areas (red). (B) Quantification of the demyelinated white matter volume; n=3–5/group, *p ≤ 0.05, Student’s t test. (C, D) Quantification of Olig2+CC1+ cells (C) and Olig2+PDGFRα+ cells (D) in the spinal cord; n=3–5/group, *p ≤ 0.05, **p ≤ 0.01, Student’s t test. (E) Toluidine blue staining of spinal cord sections from naive and 40 dpi mice. Red arrows indicate degenerated collapsed axons; scale bar =10 µm. (F, G) Quantification of myelinated (F) and degenerated (G) axons; n=4–5/group.

Figure 4. RNA-seq analysis of the microglial transcriptome after EAE

A) Heat map of differentially expressed genes in spinal cord microglia sorted from Cx3cr1CreER:Tnfrsf1bfl/fl and Tnfrsf1bfl/fl mice at 17 dpi; n=4/group. Changes are expressed as Log2 values. Red: upregulated genes; blue: downregulated genes. (B) DAVID pathway analysis of differentially expressed genes in Cx3cr1CreER:Tnfrsf1bfl/fl vs Tnfrsf1bfl/fl mice. Red: upregulated pathways; blue: downregulated pathways. P values are shown next to the bars. (C-F, H) Expression profiles of select genes from the RNA-seq analysis shown as Fragments Per Kilobase per million Mapped reads (FPKM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, Student’s t test. (G) Expression of microglial sensome genes in Cx3cr1CreER:Tnfrsf1bfl/fl vs Tnfrsf1bfl/fl mice; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, EdgeR analysis. (I, J) Expression of purinergic (I) and Siglec (J) receptors obtained from the RNA-seq analysis; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student’s t test. (K, L) Quantification of microglial phagocytosis in vitro. Fluorescent beads incorporated by primary microglia from WT and Tnfrsf1b−/− mice (K) were quantified in unstimulated conditions or after LPS treatment (L); n=6/group, ***p ≤ 0.001, one-way ANOVA, Tukey test; ^p ≤ 0.05, Student’s t test. (M, N) Expression of Trem2 (M) and P2ry12 (N) in cultured microglia; n=6/group, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, one-way ANOVA, Tukey test; ^p ≤ 0.05, Student’s t test. See also Fig. S4.

Figure 5. Ablation of TNFR2 in LysMCre:Tnfrsf1bfl/fl mice results in suppression of EAE

(A) Clinical course of EAE in Tnfrsf1bfl/fl and LysMCre:Tnfrsf1bfl/fl mice; n=12–13/group from two experiments; ***p ≤ 0.001, one-way ANOVA, Mann-Whitney test. (B, C) Clinical course of EAE in bone marrow-transplanted chimeric mice with TNFR2 ablation in Mo/MΦ [LysMCre:Tnfrsf1bfl/fl→ CD45.1] and corresponding controls [Tnfrsf1bfl/fl → CD45.1] (B), or in microglia [CD45.1 → LysMCre:Tnfrsf1bfl/fl] and corresponding controls [CD45.1 → Tnfrsf1bfl/fl] (C); n=6–7/group, *p ≤ 0.05, one-way ANOVA, Mann-Whitney test. (D, F) Flow cytometric analysis of the frequency (D) and absolute numbers (F) of infiltrated leukocytes at 20 dpi; n=5/group, *p ≤ 0.05, Student’s t test. (E, G) Flow cytometric analysis of the frequency (D) and absolute numbers (F) of splenic leukocytes at 20 dpi. Mo/MΦ are defined as CD45hiCD11b+NK1.1−Ly6G−SSAlow; n=8–11/group, *p ≤ 0.05, Student’s t test. (H) Representative flow plots of spinal cord microglia at 20 dpi. (I-K) Quantification of percentage (I) and number (J) of microglia, and percentage of MHCII+ activated microglia (K); n=5–6/group, *p ≤ 0.05, Student’s t test.

Figure 6. Ablation of TNFR2 in LysMCre:Tnfrsf1bfl/fl mice results in impaired T cell proliferation and effector function in the spleen after EAE

A, B) Representative flow plots of Ki67 expression in CD4 (A) and CD8 (B) T cells in Tnfrsf1bfl/fl and LysMCre:Tnfrsf1bfl/fl mice at 12 dpi. (C) Quantification of Ki67+ splenic lymphocytes; n=4–5/group, *p ≤ 0.05, Student’s t test. (D) Representative flow plots of cytokine expression in CD4 T cells from spleen and spinal cord at 20 dpi. (E-H) Quantification of cytokine expression in CD4 and CD8 T cells; n=3–6/group, *p ≤ 0.05, **p ≤ 0.01, Student’s t test. (I) Representative flow plots of splenic Tregs in Tnfrsf1bfl/fl and LysMCre:Tnfrsf1bfl/fl mice at 20 dpi. (J) Quantification of splenic Treg frequency; n=4–5/group, *p ≤ 0.05, Student’s t test. See also Fig. S6.

Figure 7. Ablation of TNFR2 in LysMCre:Tnfrsf1bfl/fl mice results in reduced demyelination and improved remyelination and neuroprotection in EAE

A) Assessment of demyelination in the spinal cord of Tnfrsf1bfl/fl and LysMCre:Tnfrsf1bfl/fl mice by LFB staining. Left: contoured areas show regions of demyelinated white matter; scale bar = 200 µm. Right: 3D reconstructions of the demyelinated areas (red). (B) Quantification of the demyelinated white matter volume; n=3–5/group, **p ≤ 0.01, Student’s t test. (C) Quantification of Olig2+PDGFRα+ OPCs in the spinal cord; n=3–5/group, **p ≤ 0.01, Student’s t test. (D) Representative electron micrographs of the spinal cord at 50 dpi showing remyelinated axons (red arrows); scale bar: 1 µm. (E) Quantification of remyelinated axons; n=4–5/group, *p ≤ 0.05, Student’s t test. (F) Toluidine blue staining of spinal cord sections from naive and 50 dpi mice. Red arrows indicate degenerated axons; scale bar: 10 µm. (G, H) Quantification of myelinated (G) and degenerated (H) axons; n=4–5/group, *p ≤ 0.05, *p ≤ 0.01, Student’s t test.