IFN-γ-induced Th1-Treg polarization in inflamed brains limits exacerbation of experimental autoimmune encephalomyelitis

Abstract

Experimental autoimmune encephalomyelitis (EAE) is the most widely used rodent model for multiple sclerosis. Interferon-γ (IFN-γ) and regulatory T cells (Tregs) are individually well known to play beneficial roles in amelioration of EAE. However, little is known about the relationship between IFN-γ and Tregs during the disease. Here, we show that IFN-γ polarizes Tregs into T helper 1 (Th1)-type Tregs (Th1-Tregs) to recover from EAE. Single-cell RNA sequencing analysis revealed that brain Tregs showed signs of IFN-γ stimulation during EAE. Loss of IFN-γ signaling in Tregs and of T cell-derived IFN-γ impaired the Th1-Treg polarization and worsened the disease. Moreover, selective ablation of Th1-Tregs using an intersectional genetic method promoted proinflammatory features of macrophages in the inflamed brains and exacerbated the EAE. Taken together, our study highlights a critical role of T cell-derived IFN-γ for Th1-Treg polarization in inflamed brain to ameliorate EAE.

Keywords: EAE; IFN-γ; Th1-Treg; VeDTR.

Fig. 1.

Brain Tregs express IFN-γ-signature genes during EAE. (A–E) CD3⁺ cells from spleens and brains of EAE-induced WT mice (n = 3) at the peak of disease were subjected to scRNA-seq. t-SNE-plot (A) of clusters (see also SI Appendix, Fig. S1), (B) of CD4 expression (Left) and Foxp3 expression (Right), of origin tissues and (D) of annotation of Tconvs or Treg s from spleen or brain. (E) Volcano plot comparing gene expression between brain Tregs and brain Tconvs (Left) or splenic Tregs (Center), and Venn diagram of up-regulated DEGs [Fold change (FC) > 1, q-value < 0.05] in brain Tregs (Right). Overlapped 64 DEGs are listed in Dataset S1. (F) 64 DEGs in (E) were applied to GO term enrichment analysis using Metascape. Top 20 clusters with their representative enriched terms (Top), network of enriched terms (Middle), and terms in the top of cluster “defense response to protozoan” (Bottom). Data show pool replicates (n = 3 biologically independent mice).

Fig. 2.

Signatures of IFN-γ-stimulation in brain Tregs during EAE. (A) GFP⁺ CD4⁺ T cells were isolated from spleens and brains of EAE-induced FDG mice (n = 3) at the peak of disease. mRNA expression of the indicated genes was evaluated by quantitative PCR. (B) IFN-γ neutralizing antibody (α-IFN-γ; n = 7) or control IgG (ctrl IgG; n = 6) (1 mg each) were i.p. injected to FDG mice 1 d before EAE induction. GFP⁻ CD4⁺ T cells (Tconvs) and GFP⁺ CD4⁺ T cells (Tregs) were isolated from the brains of the EAE-induced FDG mice at the peak of the disease. mRNA expression of the indicated genes was evaluated by quantitative PCR. Data are means with SD. Statistical significance assessment: (A) unpaired two-tailed Student’s t test and (B) One-way ANOVA with a post Dunnett’s test. ***P < 0.001, **P < 0.01, *P < 0.05 and ns; not significant.

Fig. 3.

T cell-derived IFN-γ stimulates brain Tregs during EAE. (A–C) Single cell suspensions prepared from the brains of EAE-induced IFN-γ-reporter (YFP) mice (n = 4) were stimulated with PMA/ionomycin for 4 h. Then, YFP expression in the indicated cell populations was evaluated by flow cytometry. (A) Representative plot of the indicated cell populations. (B) Total results of the frequency of YFP⁺ cells in the indicated cell populations. (C) The breakdown of YFP⁺ cells. (D and E) FDG mice were induced EAE. (D) GFP expression and MOG_35-55 tetramer binding of brain CD4⁺ cells were measured by flow cytometry at the preonset and peak of disease. (E) Indicated cells were sorted based on plot of (D) and mRNA expression of Ifng gene was evaluated by quantitative PCR (n = 4 each). (F-H) CD3⁺ cells from the brains of EAE-induced Ifng^fl/fl mice and CD4-Cre/Ifng^fl/fl mice (n = 3 each) at the peak of disease were subjected to scRNA-seq. t-SNE plot (F) of indicating origin genotypes and (G) of Foxp3 expression. (H) Volcano plot comparing expression of DEGs in Fig. 1E in Foxp3⁺ cells from Ifng^fl/fl mice or CD4-Cre/Ifng^fl/fl mice during EAE. FC and q-value of the indicated 18 genes are described in Dataset S2. Data show pool replicates (n = 3 biologically independent Ifng^fl/fl mice and n = 3 biologically independent CD4-Cre/Ifng^fl/fl mice). Data of (C) and (D) are means with SD. Statistical significance assessment: (D) One-way ANOVA with a post Tukey’s test ***P < 0.001.

Fig. 4.

T cell-derived IFN-γ stimulates Tregs to ameliorate EAE. (A–C) EAE was induced in the indicated mice and the clinical scores were measured. (A) WT mice and IFN-γR1 KO mice (n = 9 each). (B) Ifng^fl/fl mice (n = 4) and CD4-Cre/Ifng^fl/fl mice (n = 6). (C) Ifngr1^fl/fl mice (n = 7) and Foxp3-Cre/Ifngr1^fl/fl mice (n = 6). Data are means with SEM. Statistical significance assessment: unpaired two-tailed Student’s t test. ***P < 0.001, **P < 0.01 and *P < 0.05.

Fig. 5.

Accumulation of Th1-Tregs in inflamed brain area during EAE. (A and B) Brain CD45⁺ CD4⁺ T cells from EAE-induced WT FDG mice and IFN-γR1 KO FDG mice at the peak of disease were analyzed by flow cytometry. (A) CXCR3 expression in GFP⁺CD4⁺T cells and GFP⁻CD4⁺ T cells. Representative plot (Left) and total results (Right) (WT FDG: n = 6, IFN-γR1 KO FDG: n = 5). (B) T-bet expression in GFP⁺CD4⁺T cells. Representative plot of fluorescence minus one (FMO) (Left) and T-bet (Center) and total results (Right) (FDG: n = 3, FDG IFN-γR1 KO: n = 3). Data are mean with SD. (C) YFP expression in CD4⁺ T cells of the spleen and brain in naïve and EAE-induced Foxp3-Cre/Tbx21-Flp/VeDTR mice at the peak of disease. (D) Foxp3 and T-bet expression in YFP⁻CD4⁺ cells and YFP⁺ CD4⁺ cells of the brain in EAE-induced Foxp3-Cre/Tbx21-Flp/VeDTR mice at the peak of disease. Unstained Ctrl of Foxp3 and T-bet (Left) and Foxp3 and T-bet stained (Right). (E and F) YFP expression in CD4⁺ T cells of the spleen and brain in naïve (n = 3) and EAE-induced (n = 4) Foxp3-Cre/Tbx21-Flp/VeDTR mice at the peak of disease. (E) total results of frequency of YFP⁺ in CD4⁺ T cells in each tissue. (F) Cell number of YFP⁺ CD4⁺ cells in brains. Data are mean with SD. (G) Immunofluorescence imaging of series sections from the cerebellum of EAE-induced Foxp3-Cre/Tbx21-Flp/VeDTR mice at the peak of disease that were stained with anti-DTR (green) for visualization of Th1-Tregs, anti-CD31(orange) for visualization of vasculatures, fluoromyelin (FM; section#1 gray) for visualization of myelin, anti-CD4 (section#2 red) for visualization of CD4⁺ T cells and DAPI (blue). The demyelinated areas are delineated with yellow lines. (H) Frequency of YFP⁺ cells in brain CD4⁺ T cells of EAE-induced WT Foxp3-Cre/Tbx21-Flp/VeDTR mice and IFN-γR1 KO Foxp3-Cre/Tbx21-Flp/VeDTR mice (n = 6 each) at the peak of disease were evaluated by flow cytometry. Representative plot (Top) and total results of cell number of YFP⁻ cells (Left) and YFP+ cells (Center) and the percentage of YFP⁺ in CD4⁺ cells (Right). Data are mean with SD. Statistical significance assessment: (A, B, D, E, and G) unpaired two-tailed Student’s t test. ***P < 0.001, **P < 0.01, *P < 0.05 and ns; not significant.

Fig. 6.

Deletion of Th1-Tregs exacerbates EAE. (A–D) EAE was induced in Foxp3-Cre/Tbx21-Flp/VeDTR mice. PBS or DT (500 ng) was i.p. administered daily from day 17 to day 20. (A) The frequency of YFP⁺ cells in brain CD4⁺ T cells at day 21 was evaluated by flow cytometry. Representative plot (Left) and total results (Right) (PBS: n = 4, DT: n = 4). (B) EAE clinical scores were measured. (PBS: n = 9, DT: n = 9). (C) Single cell suspensions prepared from the brains (n = 4) at day 21 were stimulated with PMA/ionomycin for 4 h in the presence of Goldistop. Then, IL-17A and GM–CSF expression in DTR⁻CD4⁺ cells was evaluated by flow cytometry. (D) Immunofluorescence images of sections from PBS- or DT-treated mice that were stained with fluoromyelin (FM; red) and anti-CD11b (light blue). CD11b⁺ area in FM⁺ area was regarded as a lesion area in WM of the cerebellum at day 21 after EAE induction. Representative image (Left) and total results of percentage of lesion area (Naïve: n = 3, EAE-induced/PBS: n = 4, EAE-induced/DT: n = 4). Data of (A), (C), and (D) are means with SD. Data of (B) are means with SEM. Statistical significance assessment: (A and B) unpaired two-tailed Student’s t test and (C) One-way ANOVA with a post Tukey’s test. ***P < 0.001, **P < 0.01 and *P < 0.05.

Fig. 7.

Ablation of Th1-Tregs increases proinflammatory bias of infiltrated myeloid cells. (A–C) EAE was induced in Foxp3-Cre/Tbx21-Flp/VeDTR mice were. PBS or DT (500 ng was administered daily from day 17 to day 20. Brain CD45⁺ CD11b⁺ cells from PBS-treated or DT-treated mice at day 21 were analyzed by flow cytometry. (A) Gating strategy for discriminating the indicated cell populations (Left) and cell number of each population (Right). (PBS: n = 6, DT: n = 7). (B) Expression of the FMO and indicated proteins in microglia (MG; Left) and macrophages/monocytes (Mf/Mo; Right). Representative plot (Top) and total results of MFI (Bottom) (PBS: n = 6, DT: n = 7). (C) Mf/Mo were sorted and measured mRNA expression of indicated gene by quantitative PCR. (PBS: n = 6, DT: n = 6). Data are means with SD. Statistical significance assessment: unpaired two-tailed Student’s t test. ***P < 0.001, **P < 0.01, *P < 0.05 and ns; not significant.