Cholinergic control of Th17 cell pathogenicity in experimental autoimmune encephalomyelitis

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS) in which Th17 cells have a crucial but unclear function. Here we show that choline acetyltransferase (ChAT), which synthesizes acetylcholine (ACh), is a critical driver of pathogenicity in EAE. Mice with ChAT-deficient Th17 cells resist disease progression and show reduced brain-infiltrating immune cells. ChAT expression in Th17 cells is linked to strong TCR signaling, expression of the transcription factor Bhlhe40, and increased Il2, Il17, Il22, and Il23r mRNA levels. ChAT expression in Th17 cells is independent of IL21r signaling but dampened by TGFβ, implicating ChAT in controlling the dichotomous nature of Th17 cells. Our study establishes a cholinergic program in which ACh signaling primes chronic activation of Th17 cells, and thereby constitutes a pathogenic determinant of EAE. Our work may point to novel targets for therapeutic immunomodulation in MS.

Fig. 1. T cell-specific ChAT deficiency reduces EAE severity.

A EAE clinical scores of Chat^fl/fl (n = 7) and Chat^fl/flCD4^cre (n = 5) mice immunized with an emulsion containing MOG_35–55 plus Complete Freund’s Adjuvant (CFA) and pertussis toxin (PTX). DPI, days post-immunization. Mean clinical score: 0, no disease; 1, decreased tail tone or mild balance defects; 2, hind limb weakness, partial paralysis or severe balance defects that caused spontaneous falling over; 3, complete hind limb paralysis or very severe balance defects that prevented walking; 4, front and hind limb paralysis or inability to move the body into a different position; 5, moribund state. B Quantitation of absolute numbers of viable brain-infiltrating immune cells per brain from the Chat^fl/fl and Chat^fl/flCD4^cre mice in A at day 30 post-EAE induction as determined by flow cytometry. C Quantitation of absolute numbers of brain-infiltrating total Th cells (CD3⁺CD4⁺) in the brain suspensions in B. D EAE clinical scores of Chat^fl/fl (n = 18) and Chat^fl/flIl17a^cre (n = 18) mice immunized as in A. E Histological analyses of the brains of the mice in D on day 30 post-EAE induction. Cross-sections of the same brain areas were stained with anti-CD3 antibody (to detect T cells) or anti-Mac-1 (CD11b) antibody (macrophages, activated microglia). Scale bars, 200 μm. Results shown are for one mouse/genotype representative of three mice/group. F Representative flow cytometric analysis of Chat^GFP mice or B6 wild type (WT) control animals (n = 4/group) that were immunized as in A and analyzed on day 21 post-immunization to detect ChAT-GFP⁺ Th cells. Corresponding clinical scores are indicated. For all applicable panels, data are the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For A, D, significance was determined by regression analysis with two-way analysis of variance (ANOVA) followed by Bonferroni post hoc multiple comparison test; (B, C) Student’s t-test (two-sided). Data are representative of three (A, F) or two (D) independent experiments.

Fig. 2. Bhlhe40-associated Th17 gene expression pattern drives ChAT expression.

A Heat map showing the top 20 up- and down-regulated genes derived from comparative RNA-seq analysis of splenic ChAT-GFP⁺ and ChAT-GFP^– CD4⁺ T cells. B Venn diagram showing numbers of up-regulated genes (compared to ChAT-GFP^- counterparts) shared among ChAT-GFP⁺ splenic CD4⁺ T cells (T-GFP⁺), follicular B cells (FoB-GFP⁺), and marginal zone B cells (MZB-GFP⁺) at steady-state as identified by comparative RNA-seq analysis. The four core genes shared by all these ChAT-GFP⁺ subsets are indicated. C Representative flow cytometric analysis of ChAT-GFP expression in vitro-differentiated Th17 cells derived from Chat^GFPBhlhe40^fl/fl and Chat^GFPBhlhe40^fl/flCD4^cre mice. Cells were stimulated for 6d with plate-bound anti-CD3 plus anti-CD28 in the presence of IL-1β + IL-6+IL-23 (PM). B6, WT control. D Quantitation of the ChAT-GFP⁺ Th17 cells in C. *P < 0.05, by Student’s t-test (two-sided). Relative chromatin enrichment as percentages of input in in vitro-differentiated Th17 cells from the mice in C that showed direct binding of Bhlhe40 to BS1 (E) or BS2 (F) in the murine Chat promoter region as revealed by ChIP. *P < 0.05, determined by regression analysis with two-way analysis of variance (ANOVA) followed by Sidak post hoc multiple comparison test. Data in B, and D are compiled from two independent experiments. Data in E, F are from one experiment.

Fig. 3. Antigen affinity determines ChAT expression in Th17 cells.

A Representative flow cytometric analysis of the kinetics over 5 days of ChAT-GFP expression in naïve CD4⁺ T cells that were polarized towards the Th17 cell fate through stimulation with plate-bound anti-CD3 plus soluble anti-CD28 in the presence of PM. B Representative flow cytometric analysis of ChAT-GFP expression by CD4⁺ T cells among total splenocytes that were isolated from mice of the indicated genotypes and cultured for 3 days in the presence of PM plus either MOG or NFM peptide (0.4 mM). C Quantitation of the data shown in B. Each data point represents a technical replicate, with a total of 4 biological replicates, from a combination of two independent experiments. Data in A and B are representative of two independent experiments.

Fig. 4. ChAT is a key determinant of Th17 cell pathogenicity.

A Representative flow cytometric analysis of ChAT-GFP expression by CD4⁺ splenocytes polarized towards the Th17 cell fate through stimulation with plate-bound anti-CD3 plus anti-CD28 in the presence of either HM (TGFβ + IL-6), HM + IL-23, PM, or PM + TGFβ. Data are representative of at least two independent experiments. B. Quantitation of the data shown in A. Each data point represents one biological replicate (mean of three technical replicates). C EAE clinical scores of Rag1-deficient recipients transplanted with in vitro-generated and sorted 2D2 ChAT-GFP⁺ (green, n = 8) or 2D2 ChAT-GFP^- (black, n = 7) Th17 cells. Disease severity was scored daily. Data are the mean score ± s.e.m. and are representative of three experiments. D Percent survival of the mice in C. E qPCR determination of mRNA levels (relative to Actin) of the indicated genes in ChAT-GFP⁺ and ChAT-GFP^- splenic T cells polarized towards the Th17 fate by incubation in PM for 6 days. Data are the mean ± s.e.m. of 5 biological replicates and representative of 2 experiments.

Fig. 5. AChR antagonists impair brain infiltration by Th cells in EAE-induced mice.

A Clinical scores of Chat^GFP mice that were immunized to induce EAE and either left untreated or treated daily with mecamylamine or oxybutynin starting on day 3 (n = 5 mice/group). B Quantitation of absolute numbers of total viable brain-infiltrating immune cells per brain from the Chat^GFP mice in A at day 17 post-EAE induction as determined by flow cytometry. C Quantitation of absolute numbers of brain-infiltrating Th cells (CD3⁺CD4⁺) in the brain suspensions in B. D Quantitation of absolute numbers of brain-infiltrating ChAT-expressing Th cells (ChAT-GFP⁺CD3⁺CD4⁺) in the brain suspensions in C. For B–D, n = 4 mice/group. For A significance was determined by regression analysis with two-way analysis of variance (ANOVA) followed by Bonferroni post hoc multiple comparison test; for B–D, significance was determined using ordinary one-way ANOVA followed by False Discovery Rate (FDR) post hoc multiple comparisons correction. The FDR was controlled by employing the two-stage step-up method of Benjamini, Krieger, and Yekutieli. Data are from one experiment.