Cholinergic Pathway Suppresses Pulmonary Innate Immunity Facilitating Pneumonia After Stroke by Odilo Engel, Levent Akyüz, Andrey C. da Costa Goncalves, Katarzyna Winek, Claudia Dames, Mareike Thielke, Susanne Herold, Chotima Böttcher, Josef Priller, Hans Dieter Volk, Ulrich Dirnagl, Christian Meisel, and Andreas Meisel Stroke Volume 46(11):3232-3240 October 26, 2015 Copyright © American Heart Association, Inc. All rights reserved.

Markers of parasympathetic activity in heart rate (HR) variability are increased during the first days after experimental stroke. Markers of parasympathetic activity in heart rate (HR) variability are increased during the first days after experimental stroke. Heart rate variability was assessed by frequency domain analysis (low-frequency HR variability oscillation [LF-HRV], A), time domain analysis as the standard deviation of RR intervals (SD-RRI, B), and the root mean square of successive RR differences (RMSSD, C). Baroreflex sensitivity reflects the ability of the body to adapt the heart rate to changes of blood pressure, and less sensitive after middle cerebral artery occlusion (MCAo) in low-frequency oscillations (BRS-LF, D). Data from 2 independent experiments with n=14 (baseline), n=11 (d1), n=11 (d3), n=9 (d5) to n=7 (d14) animals; *indicates P<0.05 (Mann–Whitney U test in closed testing procedure). Odilo Engel et al. Stroke. 2015;46:3232-3240 Copyright © American Heart Association, Inc. All rights reserved.

A, Vagotomy prevents poststroke pneumonia. A, Vagotomy prevents poststroke pneumonia. In contrast to sham vagotomy, vagotomy 5 days before middle cerebral artery occlusion (MCAo) significantly reduced bacterial load in the lung at day 3 after experimental stroke. This effect is abrogated if animals were treated before MCAo with the acetylcholine receptor agonist nicotine in drinking water. Dashed line: bacterial load in broncho-alveolar lavage fluid from naïve mice (n=32; winsorized; Kruskal–Wallis test P=0.026; *indicating P<0.05 in Dunn’s post hoc test; pooled data from 2 independent experiments). B and C, Vagotomy improves ex vivo cytokine release by lung cells after stroke. Vagotomy (+) or sham vagotomy (Sham) was performed 5 days before MCAo, and nonvagotomzied animals (−) served as controls. Lung cells (2×106/mL) were isolated on day 1 after MCAo or Sham surgery and stimulated ex vivo for 12 h with lipopolysaccharide (LPS). Vagotomy significantly increased Toll-like receptor (TLR)-induced interleukin (IL)-6 (B) and tumor necrosis factor (TNF)-α (C) production after MCAo. Again, treatment of animals with nicotine in drinking water reversed the beneficial effect of vagotomy on ex vivo TLR-induced cytokine release. Bar graphs show mean±SD; n=39; 1-way ANOVA with Dunnett’s post hoc test using vagotomy MCAo without Nicotine as group of comparison (*indicates P<0.05, **P<0.01, and ***P<0.001; pooled data from 2 independent experiments). Odilo Engel et al. Stroke. 2015;46:3232-3240 Copyright © American Heart Association, Inc. All rights reserved.

α7 nicotinic acetylcholine receptor (α7nAChR) mediates susceptibility to poststroke pneumonia. α7 nicotinic acetylcholine receptor (α7nAChR) mediates susceptibility to poststroke pneumonia. A, Wild-type (WT) and α7nAChR knockout mice were subjected to middle cerebral artery occlusion (MCAo), and bacterial load in lungs was determined 3 days after experimental stroke. α7nAChR showed significantly lower bacterial burden compared with WT animals. Treatment of animals with nicotine in drinking water did not significantly increase bacterial load α7nAChR knockout mice. The dashed line indicates bacterial load in broncho-alveolar lavage fluid from naïve mice. Neither heart rate (B) nor infarct volume (C) differed significantly between WT and α7 nAChR knockout mice. (n=56; pooled data from 3 independent experiments; Kruskal–Wallis test P=0.001 with *P<0.05, **P<0.01, and ***P<0.001 in Dunn’s post hoc test.)‏ Odilo Engel et al. Stroke. 2015;46:3232-3240 Copyright © American Heart Association, Inc. All rights reserved.

α7 nicotinic acetylcholine receptor (α7nAChR) on bone marrow–derived and resident lung epithelial cells is involved in cholinergic anti-inflammatory effects after stroke. α7 nicotinic acetylcholine receptor (α7nAChR) on bone marrow–derived and resident lung epithelial cells is involved in cholinergic anti-inflammatory effects after stroke. To assess the relative contribution of α7nAChR expression on bone marrow–derived and resident lung epithelial cells in antibacterial defense after middle cerebral artery occlusion (MCAo), we generated bone marrow chimeras between α7nAChR knockout mice (α7 KO) and wild-type (WT) littermates, as described in Materials and Methods. A, Bacterial burden in lung was significantly reduced only if α7nAChR was absent on both bone marrow–derived and epithelial cells (n=52; Kruskal–Wallis test P=0.01 with * indicating P<0.05 in Dunn’s post hoc test, pooled data from 2 independent experiments). B, Lung cells (2×106/mL) were isolated at day 3 after MCAo and stimulated ex vivo with lipopolysaccharide (1 μg/mL). Tumor necrosis factor (TNF)-α concentration in cell culture supernatants was measured 12 h after stimulation. TNF-α release was significantly increased only if α7nAChR was absent on both bone marrow–derived and epithelial cells (n=21; Kruskal–Wallis test P=0.01 with * indicating P<0.05 in Dunn’s post hoc test). Odilo Engel et al. Stroke. 2015;46:3232-3240 Copyright © American Heart Association, Inc. All rights reserved.

α7 nicotinic acetylcholine receptor (α7nAChR) mRNA is expressed only in lung epithelial cells and alveolar macrophages. α7 nicotinic acetylcholine receptor (α7nAChR) mRNA is expressed only in lung epithelial cells and alveolar macrophages. Single lung cell suspensions from naïve wild-type animals were sorted by flow cytometry into alveolar epithelial cells (AEC), endothelial cells, and different leukocyte subsets, and α7nAChR mRNA levels was determined by reverse transcriptase polymerase chain reaction (n=3 independent fluorescence-activated cell sorter (FACS) sort experiments, n.d. not detectable). Odilo Engel et al. Stroke. 2015;46:3232-3240 Copyright © American Heart Association, Inc. All rights reserved.

Lipopolysaccharide (LPS)-induced release of interleukin (IL)-6 from alveolar epithelium cells (AEC) as well as macrophages (MF) is diminished by cholinergic stimulation. Lipopolysaccharide (LPS)-induced release of interleukin (IL)-6 from alveolar epithelium cells (AEC) as well as macrophages (MF) is diminished by cholinergic stimulation. Isolated primary AEC and macrophages (MΦ) from wild-type (WT; white bars) or α7 nicotinic acetylcholine receptor (α7nAChR) knockout (KO) mice (gray bars) were stimulated ex vivo with LPS (1 μg/mL) for 12 h in the absence or presence of different concentrations of the nonselective nAChR agonists nicotine (A and B) or α7nAChR-selective agonist PNU282987 (C and D). Both nicotine (A) and PNU282987 (C) suppressed LPS-induced IL-6 production in AEC from WT but not α7nAChR KO mice in a dose-dependant manner. In contrast, nicotine (B) reduced the LPS-induced IL-6 secretion in MF isolated from both WT and α7nAChR mice, whereas PNU282987 (D) at lower concentrations selectively suppressed IL-6 secretion in MF from WT, but not α7nAChR KO mice. Data are presented as mean±SD with *P<0.05 and **P<0.01 (2-way ANOVA and Bonferroni post hoc test, n=4–12 per group, pooled data from 2 independent experiments). Odilo Engel et al. Stroke. 2015;46:3232-3240 Copyright © American Heart Association, Inc. All rights reserved.