Inhibition of acute lung inflammation by a neuroimmune circuit induced by vagal nerve stimulation

Abstract

Vagus nerve stimulation (VNS) has been shown to limit immune cell activity across several pathologies ranging from sepsis to auto-immune diseases. While stimulation of vagal efferent neurons is known to reduce maladaptive host responses during endotoxemia, only selective vagal afferent neuron stimulation inhibited TLR7-induced macrophage activation and neutrophil recruitment to the lung. These anti-inflammatory actions are dependent on adrenal gland-derived epinephrine, as adrenalectomy or inhibition of epinephrine production eliminated the protection afforded by VNS. Selective afferent VNS induced activation in the nucleus tractus solitarius and the rostral ventrolateral medulla. Inhibition of neuronal activity in this brain region that controls peripheral sympathetic nervous system activity rendered VNS ineffective. Activation of the β₂-adrenergic receptor (β₂AR) is critical for innate immune cell suppression, as the anti-inflammatory effects of VNS were eliminated in β₂AR knockout mice and with pharmacological inhibition of the β₂AR. These findings demonstrate a previously unidentified neuroimmune circuit elicited by VNS that can control acute lung inflammation.

Fig. 1.. Electrical stimulation of the vagus nerve reduces TLR7-induced neutrophil and macrophage activation.

(A) Experimental timeline. The vagus nerve was surgically isolated and electrically stimulated for 20 min. R848 (0.25 mg/kg) was instilled intranasally (i.n.) 10 min post-VNS, and 1 hour post-R848 challenge mice were euthanized. (B) mRNA expression of Tnfα, Cc/4, lfn/3, and Cxc/1 in the right lung cranial lobe by qRT-PCR. (C) TNFα protein levels in the serum by enzyme-linked immunosorbent assay (ELISA). Flow cytometric analysis of TNFα+ (D) alveolar macrophages, (E) neutrophils, and (F) interstitial macrophages post-total lung digestion. (G) Immunofluorescence staining of neutrophils (GR1), epithelial cells (CDH1), and cell nuclei (DAPI) in the left lung lobe. Scale bars, 50 μm. (H) Quantification of (G) as the percentage of GR1⁺ neutrophils in each lung section. Data represented as means ± SD. One-way analysis of variance (ANOVA) was used for statistical analysis followed by post hoc analysis with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.. Electrical stimulation of the vagus nerve reduced both TLR7- and TLR3-induced lung acute and chronic inflammation.

(A) Experimental timeline. High doses of R848 (2.5 mg/kg) was instilled intranasally three times with 3-hour rest periods in between. After resting overnight, mice underwent 20-min electrical VNS and intranasal instillation with R848 (2.5 mg/kg). (B) mRNA expression of Tnfα in the right lung cranial lobe by qRT-PCR. (C) TNFα protein levels in the serum by ELlSA. (D) Experimental timeline. Electrical VNS was performed for 20 min. Poly(I:C) (2.5 mg/kg) was instilled intranasally 10 min post-VNS, and 2 hours post-poly(I:C) challenge mice were euthanized. mRNA expression of (E) lfnλ and (F) lfnβ in the right lung cranial lobe by qRT-PCR. (G) Experi mental timeline. Poly(I:C) (2.5 mg/kg) was instilled intranasally, and mice underwent 20-min electrical VNS 1 hour post-poly(I:C) challenge. Mice were euthanized 1 hour post-VNS. (H) Blood oxygen saturation was measured at 0- and 140-min time points. mRNA expression of (I) lfnλ and (J) lfnβ in the right lung cranial lobe by qRT-PCR. Data represented as means ± SD. One-way ANOVA was used for statistical analysis followed by post hoc analysis with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. AP, area postrema.

Fig. 3.. Optogenetic stimulation of vagal afferent neurons ameliorates TLR7-induced lung inflammation.

(A) Experimental timeline. The no dose ganglion is carefully exposed (confirmed by existence of fluorescent reporter) and stimulated at 465 nm for 20 min. R848 was instilled 10 min into 465-nm stimulation, and mice were euthanized 1 hour post-R848 challenge. (B) Fluorescent YFP reporter localized in right nodose ganglion in wild-type (C57BL/6J) versus transgenic optogenetic (VGlut2.Cre⁺ CHR2^YFP) mice. Scale bar, 150 μm. (C) Serum TNFα protein detected by ELISA in VGlut2.Cre⁺ CHR2^YFP mice. mRNA expression of (D) Tnfα, lfnβ, Ccl4, and Cxcl1 from right lung cranial lobe in VGlut2.Cre CHR2^YFP (experimental) mice by qRT-PCR. (E) Activated (cFOS⁺ DAPI⁺) neurons in the NTS, the brain region where vagal afferent neurons terminate, in VGlut2.Cre CHR2^YFP mice subject to 465 nm or control stimulation. Scale bars, 100 μm. Data represented as means ± SD. One-way ANOVA was used for statistical analysis followed by post hoc analysis with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.. Vagus nerve activation induces adrenal gland–derived epinephrine release.

Epinephrine in BALF and serum 5 min post (A) electrical VNS and (B) optogenetic activation of vagal afferent neurons detected by ELISA. (C) Right lung cranial lobe mRNA expression of Tnfα and lfnβ by qRT-PCR in mice challenged with intraperitoneal delivery of water (vehicle) or PNMT inhibitor (LY78335) 1 hour before VNS. (D) Right lung cranial lobe mRNA expression of Tnfα in mice exposed to sham surgery (right) or ADX (left) 20 min before VNS. (E) Right lung cranial lobe mRNA expression of Tnfα in mice exposed to sham surgery (right) or ADX (left) 20 min before optogenetic stimulation of vagal afferent neurons. (F) Confocal image and (G) quantification of the RVLM in VGlut2.Cre⁺ CHR2YFP mice subject to 465-nm optogenetic or control stimulation. Neuronal activation was determined by colocalization of cFOS and DAPI. A separate cohort of mice was used for this experiment. (H) TNFα mRNA expression form the lung of mice with stereotactic injection of vehicle or KYNA into the RVLM before receiving vehicle or R848 ± VNS stimulation. (I) illustration of the circuit analysis performed in (H) [Created in BioRender (K. Murray, 2025); https://BioRender.com/jbqgzxx]. Scale bar, 100 μm. Data represented as means ± SD. Student’s t test or one-way ANOVA was used for statistical analysis followed by post hoc analysis with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

Fig. 5.. VNS requires β₂AR activation for anti-inflammatory effects in lung.

(A) Serum TNFα protein detected by ELISA and (B) lung mRNA expression of Tnfα by qRT-PCR in WT (C57BL/6N) and β₂AR KO mice subjected to electrical VNS. (C) Serum TNFα protein detected by ELISA and (D) lung mRNA expression of Tnfα in mice subjected to intravenous administration of vehicle (water) or β₂AR antagonist (ICI 118 551) before challenge with R848 and electrical VNS. (E) Uniform manifold approximation and projection (UMAP) plot of all lung cells that express the adrb2, with the highest expression indicated by yellow. Expression of adrb1, adbr2, and adrb3 and percent of cells that expressed these genes of interest were evaluated. Populations with higher expression of the β₂AR are indicated by larger circles and deeper coloring. (F) mRNA expression of β₁AR (left) and β₂AR (right) by qRT-PCR from CD45⁺-enriched BALF cells in WT (C57BL/6N) and β₂AR KO mice. (G) mRNA expression of splenic Tnfα by qRT-PCR. Frequency of TNF⁺ (H) splenic marginal metallophilic macrophages (MMMɸ), (I) red pulp macrophages (RPMɸ), (J) marginal zone macrophages (MZMɸ), and (K) pDCs by flow cytometry analysis. (L) mRNA expression of the β₂AR on CD45⁺-enriched BALF cells and FACS-sorted splenic pDCs by qRT-PCR. (M) mRNA expression of right lung cranial lobe Tnfα in mice treated with vehicle (water) or the β₂AR agonist salbutamol (Sal, 1 mg/kg, i.v.) before challenge with water or R848. (N) Frequency of TNF⁺ alveolar macrophages treated in vitro with R848^+/− salbutamol, isolated from the BALF of WT (C57BL/6N) or β₂AR KO mice. Data represented as means ± SD. One-way ANOVA was used for statistical analysis followed by post hoc analysis with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.