Constitutive Vagus Nerve Activation Modulates Immune Suppression in Sepsis Survivors

Abstract

Patients surviving a septic episode exhibit persistent immune impairment and increased mortality due to enhanced vulnerability to infections. In the present study, using the cecal ligation and puncture (CLP) model of polymicrobial sepsis, we addressed the hypothesis that altered vagus nerve activity contributes to immune impairment in sepsis survivors. CLP-surviving mice exhibited less TNFα in serum following administration of LPS, a surrogate for an infectious challenge, than control-operated (control) mice. To evaluate the role of the vagus nerve in the diminished response to LPS, mice were subjected to bilateral subdiaphragmatic vagotomy at 2 weeks post-CLP. CLP-surviving vagotomized mice exhibited increased serum and tissue TNFα levels in response to LPS-challenge compared to CLP-surviving, non-vagotomized mice. Moreover, vagus nerve stimulation in control mice diminished the LPS-induced TNFα responses while having no effect in CLP mice, suggesting constitutive activation of vagus nerve signaling in CLP-survivors. The percentage of splenic CD4⁺ ChAT-EGFP⁺ T cells that relay vagus signals to macrophages was increased in CLP-survivors compared to control mice, and vagotomy in CLP-survivors resulted in a reduced percentage of ChAT-EGFP⁺ cells. Moreover, CD4 knockout CLP-surviving mice exhibited an enhanced LPS-induced TNFα response compared to wild-type mice, supporting a functional role for CD4⁺ ChAT⁺ T cells in mediating inhibition of LPS-induced TNFα responses in CLP-survivors. Blockade of the cholinergic anti-inflammatory pathway with methyllcaconitine, an α7 nicotinic acetylcholine receptor antagonist, restored LPS-induced TNFα responses in CLP-survivors. Our study demonstrates that the vagus nerve is constitutively active in CLP-survivors and contributes to the immune impairment.

Keywords: CD4+ ChAT+ T cell; TNFα; innate immune response; sepsis survivors; vagus tonic activity.

Figure 1

Vagotomy restores LPS-induced TNFα response in CLP-survivors. To assess the in vivo immune response of CLP-surviving mice to subsequent LPS challenge, BALB/c mice were subjected to control or CLP surgery, allowed to recover for 4 weeks, and then administered LPS (4 mg/kg) by intraperitoneal injection. Vagotomy (VGX) was performed 2 weeks post-CLP or control surgery. Blood and spleens were harvested 90 min after LPS administration. (A) Lower TNFα levels in serum of sepsis survivors in response to LPS challenge in vivo. (n = 8 mice/group). Results are the mean ± SEM from two independent experiments, Control + LPS vs. CLP + LPS ^***p < 0.001 (Tukey's post hoc test). (B) Restoration of LPS-induced TNFα responses following bilateral subdiaphragmatic vagotomy in sepsis survivors. (n = 8–19 mice/group). Results are the mean ± SEM from three independent experiments, CLP VGX⁻ vs. CLP VGX^{+ *}p < 0.05 and Control VGX⁻ vs. CLP VGX^{− ***}p < 0.001 (Tukey's post hoc test). (C) Anatomical localization of splenic LPS-induced TNFα in sepsis survivors. (n = 5–7 mice/group). Results are the mean ± SEM from two independent experiments, CLP VGX⁻ + LPS vs. CLP VGX⁺ + LPS ^*p < 0.05; Control without LPS vs. Control + LPS or Control + LPS vs. CLP VGX⁻ + LPS ^***p < 0.001 (Tukey's post hoc test). Magnification: (20X; scale bar = 50 μm). (D) Mice were subjected to with or without vagus nerve stimulation (VNS; 1 min) followed by LPS injection. Serum TNFα was measured by ELISA. (n = 11–16 mice/group). Results are the mean ± SEM from two independent experiments, Control VNS⁻ vs. Control VNS^{+ **}p < 0.01; CLP VNS⁻ vs. CLP VNS⁺ ns = not significant (Tukey's post hoc test).

Figure 2

Vagus nerve signaling increases the number of ChAT⁺ memory T cells in CLP-survivors. To determine the number of ChAT⁺ T cells, ChAT-EGFP mice were subjected to control or CLP surgery. Vagotomy was performed 2 weeks post control and CLP surgery. Splenocytes isolated from CLP or control mice at 4 weeks post-surgery were stained for CD4, CD44, and CD62L and analyzed by flow cytometry. (A) Representative dot plots depicting the gating strategy for splenic CD44 and CD62L expression in CD4⁺ T cells and ChAT-EGFP expression in CD4⁺ CD44^highCD62L^low cells. (B) The percentage of ChAT-EGFP⁺ cells among spleen CD4⁺ CD44^highCD62L^low cells. Results represent the mean ± SEM of 5–11 mice per group from two independent experiments; CLP VGX⁻ vs. CLP VGX^{+ *}p < 0.05; Control VGX⁻ vs. CLP VGX^{− ***}p < 0.001 and Control VGX⁻ vs. Control VGX⁺ ns = not significant (Tukey's post hoc test).

Figure 3

CD4 T cell-deficiency restores LPS-induced TNFα responses in CLP-survivors. To identify the role of CD4⁺ T cells in mediating inflammatory reflex, C57BL6/J WT and CD4 knockout mice were subjected to CLP surgery, allowed to recover for 4 weeks, and then challenged with LPS by intraperitoneal injection (6 mg/kg). Blood and spleens were harvested 90 min after LPS administration. Serum TNFα was measured by ELISA. (A) Enhanced LPS-induced serum levels of TNFα in endotoxemic CD4 T cell-deficient CLP-surviving mice. Results represent the mean ± SEM of 6–7 mice per group from one of two independent experiments; CLP WT + LPS vs. CLP CD4 KO + LPS ^*p < 0.05. (Mann-Whitney U-test). (B) TNFα staining in spleen sections of CLP WT and CLP CD4 KO. Magnification (20X; scale bar = 50 μm). Images are representative of spleen sections from two independent experiments (n = 7). CLP WT + LPS vs. CLP CD4 KO + LPS ^***p < 0.001. (Mann-Whitney U-test).

Figure 4

Methyllycaconitine treatment restores LPS-induced TNFα responses in CLP-survivors. BALB/c mice were subjected to control and CLP surgery, allowed to recover for 4 weeks, and then treated by intraperitoneal injection with methylcaconitine (MLA; 4 mg/kg) or vehicle (saline) 30 min prior to LPS-challenge (4 mg/kg). Blood and spleens were harvested 90 min after LPS administration. Serum TNFα was measured by ELISA. (A) Enhanced LPS-induced serum levels of TNFα in endotoxemic MLA-treated CLP-surviving mice. Results represent the mean ± SEM of 6–9 mice per group from two independent experiments; CLP + Saline + LPS vs. CLP + MLA + LPS ^**p < 0.01. (Mann-Whitney U-test). (B) TNFα staining in spleen sections of CLP + Saline + LPS and CLP + MLA + LPS. Magnification (20X; scale bar = 50 μm). Images are representative of spleen sections from two independent experiments (n = 6). CLP + Saline + LPS vs. CLP + MLA + LPS ^**p < 0.01. (Mann-Whitney U-test).

Figure 5

Enhanced vagus nerve signaling effects on LPS-induced TNFα in CLP-survivors. (A) Increased vagus nerve signaling leads to activation of the higher number of acetylcholine - producing T cells (ChAT⁺ T cells) in the spleen. Subsequent activation of α7 nicotinic acetylcholine receptor (α7AchR) on monocytes/macrophages leads to an inability to further suppress LPS-induced TNFα. (B) The cholinergic tone determines the immune balance, shifting between enhanced vagus nerve activation leading to an immunosuppressed phenotype and decreased vagus nerve activation leading to restored immunocompetence.