Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit

Abstract

Neural circuits regulate cytokine production to prevent potentially damaging inflammation. A prototypical vagus nerve circuit, the inflammatory reflex, inhibits tumor necrosis factor-α production in spleen by a mechanism requiring acetylcholine signaling through the α7 nicotinic acetylcholine receptor expressed on cytokine-producing macrophages. Nerve fibers in spleen lack the enzymatic machinery necessary for acetylcholine production; therefore, how does this neural circuit terminate in cholinergic signaling? We identified an acetylcholine-producing, memory phenotype T cell population in mice that is integral to the inflammatory reflex. These acetylcholine-producing T cells are required for inhibition of cytokine production by vagus nerve stimulation. Thus, action potentials originating in the vagus nerve regulate T cells, which in turn produce the neurotransmitter, acetylcholine, required to control innate immune responses.

Fig. 1

Vagus nerve stimulation increases acetylcholine levels in the spleen and requires T lymphocytes to attenuate TNF-α in endotoxemia. (A) BALB/c mice (n = 5) were subjected to vagus nerve stimulation (5 min), and spleen acetylcholine levels were determined in dialysate samples by mass spectrometry. Results are expressed as a percentage of the average levels of three consecutive samples ± SEM obtained before vagus nerve stimulation (basal). P < 0.05 at 20 min compared with basal [repeated measures analysis of variance (ANOVA) and the Dunnett post hoc test]. VNS, vagus nerve stimulation. (B) Acetylcholine was measured by mass spectrometry in supernatants of nonadherent spleen cells in the presence or absence of norepinephrine at the indicated concentrations. Data were obtained from pooled cells stimulated in duplicate. Results are expressed as the mean of two experiments. *P < 0.05 compared with unstimulated cells (two-tailed t test). (C) BALB/c mice (four or five per group) and (D) BALB/c nude mice (five per group) were subjected to sham surgery or vagus nerve stimulation followed by endotoxin injection. Serum was obtained 90 min after endotoxin administration, and TNF-α was measured by enzyme-linked immunosorbent assay (ELISA). Results are means ± SEM. *P < 0.05 compared with the sham group (two-tailed t test).

Fig. 2

Spleen acetylcholine-synthesizing T cells express a memory T cell phenotype. (A) ChAT-EGFP expression in spleen CD3⁺ T cells of wild-type and ChAT(BAC)-EGFP mice. (B) Expression of ChAT-EGFP in CD4⁺ and CD8⁺ spleen T cells. (C) CD44 and CD62L expression in spleen CD4⁺ cells (left), and ChAT-EGFP expression in CD4⁺ CD44^high CD62L^low and CD4⁺ CD44^low CD62L^high spleen cells (middle and right, respectively). (D) Percentage of ChAT-EGFP⁻ and ChAT-EGFP⁺ cells among spleen CD4⁺ CD44^high CD62L^low cells, n = 5. (E) Spleen CD4⁺ CD44^high CD62L^low ChAT-EGFP⁻ and CD4⁺ CD44^high CD62L^low ChAT-EGFP⁺ cells were obtained by cell sorting. (F) Acetylcholine concentration was determined in supernatants of cells under resting conditions. Data were obtained from pooled cells cultured in duplicate. Results are the means of two experiments. *P < 0.05 compared with CD4⁺ CD44^high CD62L^low ChAT-EGFP⁻ cells (two-tailed t test).

Fig. 3

Acetylcholine-synthesizing T cells in spleen are located in the proximity of catecholaminergic nerve endings. (A and B) Immunofluorescent micrographs of ChAT-EGFP (green) expression by T cells in spleen white pulp (CD3, red). (C) Immunofluorescent micrographs of ChAT-EGFP⁺ cells (green) and nerve fibers stained with tyrosine hydroxylase (TH, red). (D) Fluorescent micrographs of splenic nerve endings (synaptophysin, red) juxtaposed (arrows) to ChAT-EGFP⁺ (green) cells in the white pulp. CA, central artery. (A) ×400 magnification, (B) ×630 magnification, (C) ×400 magnification, (D) ×630 magnification. Images are representative of spleen sections (n = 3 to 5) from five experiments.

Fig. 4

Vagus nerve stimulation requires acetylcholine-synthesizing T cells to attenuate TNF-α in endotoxemia. Indicated groups of mice were subjected to sham surgery or vagus nerve stimulation followed by endotoxin injection. Serum was obtained 90 min after endotoxin administration, and TNF-α was measured by ELISA. (A) BALB/c nude mice receiving spleen CD4⁺ CD44^high CD62L^low ChAT-EGFP⁺ cells, eight mice per group. (B) BALB/c nude mice receiving spleen CD4⁺ CD44^high CD62L^low ChAT-EGFP⁻ cells, six or seven mice per group. (C) BALB/c nude mice receiving spleen CD4⁺ cells transfected with control scrambled siRNA, five or six mice per group. (D) BALB/c nude mice receiving spleen CD4⁺ cells transfected with ChAT siRNA, six or seven mice per group. Results are means ± SEM. *P < 0.05 compared with the respective sham group (two-tailed t test).