Basic Sciences

Cholinergic Pathway Suppresses Pulmonary

Innate Immunity Facilitating Pneumonia After Stroke
Odilo Engel, DVM*; Levent Akyüz, MSc*; Andrey C. da Costa Goncalves, PhD;
Katarzyna Winek, MD; Claudia Dames, MSc; Mareike Thielke, MLS; Susanne Herold, MD;
Chotima Böttcher, PhD; Josef Priller, MD; Hans Dieter Volk, MD; Ulrich Dirnagl, MD;
Christian Meisel, MD*; Andreas Meisel, MD*

Background and Purpose—Temporary immunosuppression has been identified as a major risk factor for the development
of pneumonia after acute central nervous system injury. Although overactivation of the sympathetic nervous system
was previously shown to mediate suppression of systemic cellular immune responses after stroke, the role of the
parasympathetic cholinergic anti-inflammatory pathway in the antibacterial defense in lung remains largely elusive.
Methods—The middle cerebral artery occlusion model in mice was used to examine the influence of the parasympathetic
nervous system on poststroke immunosuppression. We used heart rate variability measurement by telemetry, vagotomy,
α7 nicotinic acetylcholine receptor–deficient mice, and parasympathomimetics (nicotine, PNU282987) to measure and
modulate parasympathetic activity.
Results—Here, we demonstrate a rapidly increased parasympathetic activity in mice after experimental stroke. Inhibition
of cholinergic signaling by either vagotomy or by using α7 nicotinic acetylcholine receptor–deficient mice reversed
pulmonary immune hyporesponsiveness and prevented pneumonia after stroke. In vivo and ex vivo studies on the role
of α7 nicotinic acetylcholine receptor on different lung cells using bone marrow chimeric mice and isolated primary
cells indicated that not only macrophages but also alveolar epithelial cells are a major cellular target of cholinergic anti-
inflammatory signaling in the lung.
Conclusions—Thus, cholinergic pathways play a pivotal role in the development of pulmonary infections after acute central
nervous system injury.   (Stroke. 2015;46:3232-3240. DOI: 10.1161/STROKEAHA.115.008989.)
Key Words: bone marrow chimeric mice ◼ cholinergic anti-inflammatory pathway ◼ parasympathetic nervous
system ◼ pneumonia ◼ stroke-induced immunodepression

T he management of medical complications is one of the

most critical factors in the therapy of stroke because 95%
of stroke patients experience at least one relevant medical
complication in the first 3 months after stroke.1 In both experi-
mental and clinical studies, infections are the most common
complication after stroke.2 Among these, pneumonia is the
most relevant poststroke infection,3 accounting for the highest
attributable mortality after stroke.4
To maintain body homeostasis in response to challenges
from the environment, the nervous and immune systems are
closely interconnected in an intense bidirectional communica-
tion.5 Recent experimental and clinical evidence indicates that
central nervous system injuries like stroke, spinal cord injury,
or traumatic brain injury disturb the normally well-balanced
interplay between these 2 supersystems, resulting in a profound
and long-lasting immunodepression.6,7 Overactivation of the
sympathetic nervous system after stroke was shown to mediate
suppression of peripheral cellular immune responses and to con-
tribute to development of poststroke bacterial lung infections.8–10
However, changes in the pulmonary immune compartment
and the underlying mechanisms of impaired antibacterial lung
immune responses after stroke are currently not understood.
Besides resident lung immune cells, such as alveolar
macrophages (MΦ), alveolar epithelial cells (AEC) express

Received March 11, 2015; final revision received August 19, 2015; accepted August 24, 2015.
From the Department of Experimental Neurology (O.E., K.W., M.T., U.D., A.M.), Department of Neurology (U.D., A.M.), NeuroCure Clinical Research
(U.D., A.M.), Institute for Medical Immunology (L.A., C.D., H.D.V., C.M.), BCRT Berlin Brandenburg Centre for Regenerative Medicine (L.A., H.D.V.),
Department of Neuropsychiatry and Laboratory of Molecular Psychiatry (C.B., J.P.), and Center for Stroke Research Berlin (O.E., K.W., M.T., U.D., A.M.),
Charité University Medicine Berlin, Berlin, Germany; German Center for Neurodegeneration Research (DZNE), partner site Berlin, Germany (J.P., U.D.);
Department of Internal Medicine II, Justus-Liebig-University, Universities Giessen and Marburg Lung Center, Member of the German Center for Lung
Research (DZL) (S.H.); and Max Delbrück Center for Molecular Medicine, Berlin, Germany (A.C.d.C.G.).
Guest Editor for this article was Malcolm R. Macleod, PhD.
*O. Engel, L. Akyüz, Drs C. Meisel, and A. Meisel contributed equally.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
115.008989/-/DC1.
Correspondence to Andreas Meisel, MD, NeuroCure Clinical Research Center, Charité–Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin,
Germany. E-mail andreas.meisel@charite.de
© 2015 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.115.008989

3232
 Engel et al   Cholinergic Pathway in Stroke-Associated Pneumonia    3233
a variety of receptors recognizing pathogens or their prod-
ucts.11 Rapid activation of MΦ and airway epithelium are of
crucial importance for effective host defense during bacterial
lung infections.12 Both MΦ and lung epithelial cells have been
shown to express the α7 nicotinergic acetylcholine receptor
(α7nAChR),13 which has been identified as a crucial down-
stream element of the so-called cholinergic anti-inflammatory
pathway.14 This neuronal pathway, involving the parasym-
pathetic vagus nerve and the neurotransmitter acetylcholine
(ACh), was shown to act as a negative feedback loop to prevent
a potentially harmful overreaction of the immune system dur-
ing inflammatory conditions, such as bacterial infections, by
suppressing the production of proinflammatory cytokines by
activated macrophages on binding of ACh to the α7nAChR.15
Here we demonstrate that stroke directly activates cholinergic
anti-inflammatory pathways, which are crucially involved in
the development of poststroke lung infections.
Materials and Methods
A detailed description of all materials and methods can be found in
the online-only Data Supplement.
Animals and Housing
Male specific pathogen free C57Bl6/J mice (Charles River
Laboratories, Sulzfeld, Germany), sex-mixed α7nAChR knockout
(KO) mice,16 and wild-type (WT) littermates, respectively (B6.129S7-
Chrna7tm1Bay/J; The Jackson Laboratory, Bar Harbor) were housed in
cages lined with chip bedding and environmental enrichment (mouse
tunnel and igloo) on a 12 h light/dark cycle with ad libitum access to
food and water. Experiments were performed using 11- to 14-week-
old mice in a­ ccordance with the European directive on the protec-
tion of animals used for scientific purposes and all other applicable
regulations and approved by the relevant authority, Landesamt für
Gesundheit und Soziales, Berlin, Germany.
Experimental Stroke
Under general isoflurane anaethesia, a small silicon-coated filament
was temporarily introduced via the left common carotid artery into
the circle of Willis blocking the origin of the middle cerebral artery
for 60 min (middle cerebral artery occlusion [MCAo])17 following the
standard operating procedures of our laboratory. Success of MCAo
was verified by Bederson score, and animals without neurological
deficit after operation were excluded from the study.
Vagotomy
Five days before MCAo surgery and under isoflurane anesthesia, the
left cervical vagus nerve was carefully and bluntly dissected from the
common carotid artery and, in the vagotomy but not sham-operated
group, transected.
Broncho-Alveolar Lavage and Measurement of
Bacterial Burden
Under anaethesia with midazolam (5 mg/kg body weight) and me-
detomidin (0.5 mg/kg body weight) IP (antagonization with flumaze-
nil 0.5 mg/kg body weight and atipamezol 5 mg/kg body weight, if
needed), mice were intubated with a 22G peripheral venous catheter.
Afterward, 0.4 mL of saline plus 0.2 mL air were applied over the
tubus and immediately withdrawn.18 For the determination of colo-
ny-forming units, broncho-alveolar lavage (BAL) fluid was serially
diluted, plated on LB agar plates, and incubated at 37°C for 18 h,
and bacterial colonies were counted.8 The cut-off value (<104 colony-
forming units/mL) has been determined by investigating BAL fluid
of naïve mice.
Drug Preparation and Administration
Nicotine (Sigma Aldrich) was given via drinking water in a concen-
tration of 100 μg/mL. The effects on heart rate (HR) were monitored
by pulse oximetry (MouseOx; STARR life sciences Corp.).
Creation of Bone Marrow Chimeric Mice
For generation of bone marrow (BM) chimeric mice, BM cells
were isolated under sterile conditions from the tibias and femurs
of donor mice. 5×106 BM cells were IV injected into 6- to 8-week-
old lethally irradiated recipient mice 24 h after total body irra-
diation (1100 cGy) using a 137Caesium Gammacell 40 Exactor
(Theratronics).
Telemetry
Blood pressure (BP) and HR were measured by telemetry combined
with fast Fourier transform analysis of BP and HR, as described
elsewhere.19 In mice aged 8 to 10 weeks, transmitters (TA11PA-C10
BP; Data Sciences International) were implanted in a subcutaneous
pocket along the right flank with the pressure-sensing catheter in the
abdominal aorta. After 7 days recovery, baseline was recorded us-
ing DATAQUEST software (A.R.T. 2.1, Data Sciences International),
and sympathetic and parasympathetic activities were verified phar-
macologically by applying metoprolol (4 mg/kg) and atropine (2 mg/
kg), respectively. Parasympathetic activity was assessed via spectral
analysis using PV-wave software (Visual Numerics). The power spec-
tra of systolic BP, pulse interval time series, and the cross spectra
were calculated using fast Fourier transformation. Low-frequency
components of pulse intervals spectrum (LF), root mean square of
successive differences between adjacent normal pulse intervals, total
power, and the baroreflex sensitivity (BRS-LF) were calculated. The
baroreceptor HR reflex was investigated using sequence technique
and spectral function analysis of spontaneous changes in systolic
BP and HR, and BRS was defined as the mean magnitude value of
transfer function between systolic BP and R-R interval in the low-
frequency band (BRS-LF).
Flow Cytometry
Cell phenotyping was performed on an LSRII flow cytometer us-
ing FACSDiva software (BD Biosciences) and FlowJo software
(Tree Star Inc.) with the following anti-mouse monoclonal antibod-
ies: CD45-PE-Cy7, CD3-APC, CD4-A700, CD8-PB, NK1.1-PE,
CD11b-APC-Cy7, B220-FITC, SiglecF-PE, F4/80-A700, CD11c-
FITC, CD11b-PE-TR, Gr1-PB, and MHCII-APC-Cy7 (Biolegend).
Fluorescence-activated cell sorter–based cell sorting was performed
on a FACSAria III cell sorter (BD Biosciences).
Isolation of Primary Alveolar Macrophages and
Alveolar Epithelial Cells
MΦ were obtained by repeated gentle flushing of tracheotomized
lungs from naïve C57Bl6/J mice with NaCl, centrifugation (300g,
10 min), and resuspension in Roswell Park Memorial Institute
medium. For purification of AEC,20 lungs were flushed after per-
fusion and BAL with 1500 μL dispase followed by intratracheal
injection of 500 μL of 1% low melting temperature agarose. Lung
single-cell suspension (homogenization in DMEM) underwent
magnetic cell separation for depletion of nonepithelial cells using
anti-CD45, anti-CD31, and anti-CD16/CD32 mAb (BioLegend).
Purity of macrophages and epithelial cells was confirmed by flow
cytometry (>95% and >90%, respectively).
Analysis of Ex Vivo Cytokine Production
Lung single-cell suspensions (2×106/mL), isolated primary MΦ
(1×105/mL), or primary AEC (1×10E5/mL) were stimulated with li-
popolysaccharide (LPS) (1 μg/mL; Sigma) or Pam3CSK4 (200 ng/
mL; Invivogen) in the absence or presence of different concentrations
of nicotine or PNU282987 (both from Sigma) for 12 h at 37°C.
3234  Stroke  November 2015
Analysis of Cytokines in BAL and Cell Culture
Supernatants
Cytokines in BAL and cell culture supernatant were measured by
magnetic bead–based assay on a Bio-Plex 200 System (Luminex
xMAP Technology) with Bio-Plex Manager 6.0 software (Bio-Rad)
according to the manufacturer’s recommendations.
Quantitative Reverse Transcriptase Polymerase
Chain Reaction
RNA was extracted from fluorescence-activated cell–sorted or
Dynabead-purified lung cells using the NucleoSpin RNA II Kit
(Macherey-Nagel). cDNA synthesis was done using the QuantiTect
kit (Qiagen) and measured by real-time quantitative reverse transcrip-
tase polymerase chain reaction (7500 Sequence Detection System,
Applied Biosystems) with β-Actin as housekeeping gene.
Statistics
Treatment allocation was performed randomly and remained com-
pletely blinded for the examiners. Appropriate 2-sided statistical tests
were used to compare groups for significant differences (P<0.05; SPSS
Statistics 18.0, IBM). Closed testing procedure was applied in telem-
etry experiments, putting the comparisons at different time points in
a hierarchical order. A priori sample size calculation (G*Power3.021)
assumed a change in bacterial burden by 100-fold as relevant or a dou-
bling of HR variability (HRV) parameters, respectively. Prespecified
inclusion and exclusion criteria and detailed report on flow of ani-
mals through the experiments are reported in the online-only Data
Supplement (Tables I and II in the online-only Data Supplement).
Results
Middle Cerebral Artery Occlusion Reduces HR and
Increases Blood Pressure
To obtain unbiased data on physiological responses after exper-
imental stroke in mice, we established a telemetric approach
to measure HR and BP in a quiet, undisturbed environment. In
MCAo mice, HR was decreased during the first 3 days after
surgery compared with baseline and sham-operated animals.
BP rose in both groups, but remained significantly elevated
for 5 days in MCAo animals compared with sham controls
(Figure IA and IB in the online-only Data Supplement).
Physical activity was slightly reduced in both groups com-
pared with baseline with no group differences in the first 2
weeks (Figure IC in the online-only Data Supplement).
Increased HRV and Baroreflex Sensitivity Indicates
Parasympathetic Activation After Experimental
Stroke
In contrast to sham-operated animals, we observed a strong
increase in the LF power component of HRV reflecting
increased parasympathetic activity in MCAo-treated mice on
day 1, 3, and 5 after surgery (Figure 1A). These findings were
corroborated by changes in 2 time domain parameters describ-
ing parasympathetic activity in HRV, the standard deviation of
RR intervals (Figure 1B), and the root mean square of succes-
sive RR differences (Figure 1C).
BRS is the ability to adapt the sinus frequency in response
to BP changes. Cross spectral analysis of the BRS-LF, calcu-
lated as mean value of the transfer function between systemic
BP and pulse intervals in the LF band, revealed a significant
increase within the first 5 days after experimental stroke
(Figure 1D). One, 3, and 5 days after experimental stroke, the
adaptation time for up slopes and down slopes of systemic
BP were increased compared with baseline and sham controls
(Figure II in the online-only Data Supplement).
Unilateral Vagotomy Reduces Bacterial Burden by
Restoring Pulmonary Immune Function
MCAo animals develop spontaneous bacterial pneumonia
between day 1 and day 3 after experimental cerebral isch-
emia.8 To investigate the influence of parasympathetic acti-
vation on poststroke infections, we first performed unilateral
cervical vagotomy. In mice that underwent vagotomy 5 days
before MCAo, we observed a significant reduction in bacte-
rial load on day 3 after MCAo compared with sham vagoto-
mized stroke animals (Figure 2A). Importantly, administration
of nicotine in drinking water reversed the beneficial effect of
vagotomy on bacterial pneumonia after stroke (Figure 2A).
Infarct volumes and HR did not significantly differ between
the groups (Figure III in the online-only Data Supplement).
Assessment of ex vivo cytokine secretion by lung cells iso-
lated 1 day after MCAo revealed reduced release of proinflam-
matory cytokines interleukin (IL)-6 and tumor necrosis factor
(TNF)-α after stimulation with the Toll-like receptor 4 (TLR4)
agonist LPS (Figure 2B and 2C) and the TLR1/2 agonist
Pam3CSK4 (Figure IV in the online-only Data Supplement) in
MCAo mice compared with sham controls. In contrast, lung
cells isolated from mice vagotomized 5 days before MCAo
released similar amounts of IL-6 and TNF-α after Pam3CSK4
and LPS stimulation as lung cells from sham-operated controls.
Treatment of MCAo animals with nicotine in drinking water
decreased ex vivo proinflammatory cytokine production after
TLR stimulation of lung cells isolated from vagotomized mice
to levels observed in non- or sham-vagotomized animals (Figure
2B and 2C; Figure IV in the online-only Data Supplement).
Role of α7nAChR in Diminished Antibacterial
Defense in Lung After Experimental Stroke
The α7nAChR receptor has been described as mediator of
cholinergic anti-inflammatory effects on macrophages.14
Similar to the effects of vagotomy in WT animals, mice lack-
ing the α7nAChR showed significantly less bacterial load in
lungs after MCAo compared with WT littermates (Figure 3A).
Nicotine did not further increase lung bacterial burden in WT
mice. In α7nAChR KO mice, bacterial loads in lung increased
only slightly by nicotine treatment but remained at lower lev-
els compared with WT animals. Infarct volumes and HR did
not significantly differ between the groups (Figure 3B and
3C). The rather small number of male compared with female
animals per group does not allow a definitive conclusion on
sex-related differences of α7nAChR KO mice in response to
stroke. However, subgroup analysis of infarct volumes and
bacterial burden in lung does not suggest sex-specific differ-
ences (data not shown).
Cholinergic Signaling Attenuates Antibacterial
Defense of Lung Myeloid and Epithelial Cells After
Experimental Stroke
To differentiate between cholinergic effects on BM-derived
immune cells and resident lung cells contributing to poststroke
 Engel et al   Cholinergic Pathway in Stroke-Associated Pneumonia    3235

Figure 1. 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 anal-
ysis as the standard deviation of RR intervals (SD-RRI, B), and the root mean square of successive RR differences (RMSSD, C). Barore-
flex 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).

immunodepression, we investigated the development of post-
stroke pneumonia in mice responding to α7nAChR-mediated
cholinergic signaling either in BM-derived cells (WT→KO)
or resident lung cells (KO→WT). For control, we transplanted
BM cells from WT into WT animals (WT→WT) and from
KO into KO animals (KO→KO). After experimental stroke,
bacterial burden in lung was significantly reduced in KO→KO
mice compared with WT→WT mice (Figure 4A), corroborat-
ing findings observed in α7nAChR KO and WT mice (com-
pare Figure 3A). Both KO→WT and WT→KO chimeric mice
showed an intermediate phenotype regarding susceptibility to
pneumonia compared with syngeneic controls (Figure 4A).
Lung cells isolated 3 days after MCAo from ischemic
WT→WT mice produced significantly lower TNF-α levels after
LPS stimulation than lung cells from KO→KO mice, whereas
lung cells from WT→KO and KO→WT chimeric mice again
exhibited an intermediate phenotype compared with KO→KO
and WT→WT mice (Figure 4B).
expression was not observed in endothelial cells, NK-, B-, and
T-lymphocytes, dendritic cells, or granulocytes (Figure 5).
Diminished Cytokine Production in AEC and MΦ
upon α7nAChR Stimulation
To investigate cell type–dependent α7nAChR-mediated
effects, we assessed TLR ligand–induced IL-6 release in pri-
mary AEC and MΦ. Nicotine diminished LPS-induced IL-6
production in AEC isolated from WT but not α7nAChR KO
mice in a dose–dependent manner (Figure 6A). Likewise,
α7nAChR-specific agonist PNU282987 suppressed IL-6
release in LPS-stimulated AEC from WT but not α7nAChR
KO mice (Figure 6C). In contrast, nicotine and PNU282987
dose-dependently reduced LPS-induced IL-6 secretion in
MΦ from WT and to lesser extent also in α7nAChR KO mice
(Figure 6B and 6D). These data suggest that cholinergic con-
trol of TLR-induced proinflammatory cytokine release by MΦ
is partly independent from α7nAChR.

mRNA Expression Pattern of α7nAChR in Alveolar Discussion

Macrophages and Alveolar Epithelial Cells
To determine the cellular expression pattern of α7nAChR in
lung tissue, we sorted single cell suspensions from WT mice
by magnetic cell sorting. α7nAChR was ≈50-fold higher
expressed in AEC compared with MΦ. In contrast, α7nAChR
Besides neurological deficits leading to dysphagia and aspira-
tion, suppression of peripheral immune responses because of an
overactivation of stress pathways has been recognized as a criti-
cal risk factor for the development of infectious complications
after stroke. Previous studies in experimental stroke models have
3236  Stroke  November 2015

Figure 2. 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, treat-
ment 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 compari-
son (*indicates P<0.05, **P<0.01, and ***P<0.001; pooled data from 2 independent experiments).

provided evidence that an increased activity of the sympathetic
nervous system after cerebral ischemia leads to rapid impair-
ment of peripheral cellular immune responses, in particular natu-
ral killer cell and T cell responses. These experimental findings
were corroborated by clinical studies showing increased levels
of catecholamines and reduced T cell counts in peripheral blood,
as well as impaired ex vivo T cell function in stroke patients
before the onset of infectious complications.6,10 Thus, although
pathophysiological responses in human diseases and respective
mouse models may markedly differ as recently shown for the
genomic response in acute inflammatory conditions,22 the MCAo
model in mice has proven to be clinically relevant to elucidate

Figure 3. 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 experi-
mental stroke. α7nAChR showed significantly lower bacterial burden compared with WT animals. Treatment of animals with nicotine in drink-
ing 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.)
 Engel et al   Cholinergic Pathway in Stroke-Associated Pneumonia    3237

Figure 4. α7 nicotinic acetylcholine receptor (α7nAChR) on bone marrow–derived and resident lung epithelial cells is involved in cho-
linergic 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 iso-
lated 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).

mechanisms of increased susceptibility to infection and to
develop preventive treatment strategies.23 However, it remained
unclear whether cerebral ischemia affects resident lung cells,
which mediate first-line pulmonary immune responses during
respiratory infections. Our data suggest that lung innate immune
responses are suppressed after stroke by increased cholinergic
signaling, which is relayed by the parasympathetic vagus nerve
and the α7nAChR expressed on AEC and MΦ. Hence, both an
impaired early T/NK-cell dysfunction mediated by sympathetic
nervous system and an impaired innate immune response in the
lung mediated by the parasympathetic nervous system seem to
be important additive mechanisms leading to decreased anti-
bacterial defense and poststroke bacterial infections.
The cholinergic anti-inflammatory pathway has been
described as a protective mechanism that prevents potentially
Figure 5. 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 poly-
merase chain reaction (n=3 independent fluorescence-activated
cell sorter (FACS) sort experiments, n.d. not detectable).
harmful overactivation of the immune system in response to
infections or inflammation-induced tissue damage. Sensing
of peripheral inflammatory processes via vagal afferent fibers
was initially proposed to trigger an anti-inflammatory response
via release of acetylcholine from vagal efferent fibers, which
acts on α7nAChR on macrophages to suppress the release of
proinflammatory cytokines.14 Recent evidence suggests, how-
ever, that the neural reflex that is triggered by and dampens
systemic inflammatory responses may be more complex and
involve non-neural cholinergic and noradrenergic sympathetic
mechanisms.24 Nevertheless, vagus nerve stimulation by cen-
trally acting muscarinic agonists, such as CNI-1493, has been
shown to suppress peripheral inflammatory responses that can
be prevented by vagotomy, demonstrating that the vagus is
an essential efferent pathway of the central cholinergic anti-
inflammatory pathway.25
Assessment of HRV has been recognized to provide reli-
able, noninvasive information on the activity of the autonomic
nervous system, including its vagal and sympathetic compo-
nents. Using a telemetry system, we observed an immediate
and long-lasting activation of the parasympathetic nervous
system after cerebral ischemia, which is in line with previous
observations showing a similar increase in root mean square
of successive RR differences and LF-HRV after experimental
stroke in mice.26 Because HRV parameters have been shown
to differ between species with respect to the influence of para-
sympathetic and sympathetic outflow,27 we have verified the
specificity of selected HRV measures by pharmacological
blockade of sympathetic and parasympathetic input using the
selective antagonists metoprolol and atropine, respectively.19
According to the few clinical data, autonomic dysfunctions in
stroke patients occur in the acute phase with a predominance
of parasympathetic rather than sympathetic dysfunction.28–30
Although measurement of HRV reflects primarily auto-
nomic influences on the cardiovascular system, several
human studies have linked changes in HRV with markers of
3238  Stroke  November 2015
inflammation and immunity.31,32 An increase in sympathetic
and parasympathetic HRV parameters is associated with
worse outcome after stroke.9,33,34
In the present study, we demonstrated that increased
parasympathetic activity after cerebral ischemia plays a cru-
cial role in mediating increased susceptibility to bacterial
pneumonia after stroke. Although MCAo animals developed
spontaneous bacterial infections, either disruption of para-
sympathetic signaling in WT mice by vagotomy or inhibi-
tion of cholinergic effects by using α7nAChR-deficient mice
reduced bacterial burden in BAL fluid almost completely to
levels observed in naïve mice (<104 colony-forming units /
mL).35 Furthermore, vagotomy resulted in the restoration of
inflammatory responses triggered by TLR agonist in lung
cells in stroke animals. Moreover, treatment of MCAo animals
with the nonselective nAChR agonist nicotine prevented the
beneficial effect of vagotomy on lung bacterial burden and on
ex vivo cytokine secretion by lung cells in WT mice but only
slightly increased bacterial load in α7nAChR mice, indicating
that suppression of pulmonary antibacterial responses after
stroke by cholinergic signals involves α7nAChR. Because we
have not assessed all physiological parameters under para-
sympathetic control in these experiments, other than immune
mechanisms might be involved in poststroke pneumonia.
However, vagotomy resulted in the restoration of inflamma-
tory responses triggered by TLR agonist in lung cells in stroke
animals. Recently, Lafargue et al made the important obser-
vation that mice deficient in α7nAChR showed reduced lung
injury and mortality compared with WT mice in an aspiration-
induced Pseudomonas aeruginosa pneumonia model after
experimental stroke.36 However, the Pseudomonas aspiration
pneumonia model does not reflect the pathophysiological
course of lung infections in stroke patients because it requires
a high bacterial inoculum, which by itself may trigger (neu-
ral) mechanisms to perturb pulmonary immune defense other
than those induced by central activation of cholinergic anti-
inflammatory mechanisms after acute central nervous system
Figure 6. Lipopolysaccharide (LPS)-induced
release of interleukin (IL)-6 from alveolar epi-
thelium 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 stimu-
lated ex vivo with LPS (1 μg/mL) for 12 h in
the absence or presence of different concen-
trations of the nonselective nAChR agonists
nicotine (A and B) or α7nAChR-selective
agonist PNU282987 (C and D). Both nico-
tine (A) and PNU282987 (C) suppressed
LPS-induced IL-6 production in AEC from WT
but not α7nAChR KO mice in a dose-depen-
dant 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).
-injury.37 Our study provides direct evidence for the crucial
role of the cholinergic anti-inflammatory pathway in the
development of spontaneous pneumonia after MCAo in mice.
Both, MΦ and AEC play a crucial role in the first-line
defense against pulmonary infections.11,12 Cholinergic signals
transmitted via the α7nAChR have been previously shown to
limit inflammatory responses in these cells.38 Accordingly,
we examined whether resident lung immune competent cells
or BM-derived immune cell are predominantly targeted by
cholinergic anti-inflammatory signaling after stroke. Our
results using a model of BM chimerism between WT and
α7nAChR-deficient mice suggest that the inhibitory effects of
α7nAChR-mediated cholinergic signaling are exerted on both
lung-resident cells and BM-derived immune cells because
chimeric mice showed an intermediate phenotype in terms
of bacterial burden and ex vivo cytokine secretion capacity
of whole lung cells compared with syngeneic WT→WT and
KO→KO controls.
To further elucidate pulmonary cellular targets of cholin-
ergic anti-inflammatory signaling, we determined α7nAchR
expression in sorted cells isolated from lung. We observed
α7nAChR expression only in AEC and MΦ but not in endo-
thelial cells or other leukocyte subsets. Ex vivo functional
analysis revealed that the anti-inflammatory effects of cho-
linergic signaling on AEC were largely dependent on the
presence of the α7-subunit nAChR because suppression of
TLR-induced proinflammatory cytokine production by the
nonselective nAchR agonist nicotine and the selective α7
agonist PNU282987 was observed in primary AEC from
WT but not α7nAChR-deficient mice. In contrast, although
PNU282987 showed α7nAChR-dependent inhibitory effects
on MΦ, nicotine dose-dependently suppressed IL-6 produc-
tion in these cells also in the absence of α7nAChR. Although
α7nAChR was consistently detectable in MΦ, expression lev-
els were ≈100-fold lower compared with primary AEC. These
data suggest that nicotinic receptors other than α7nAchR may
be involved in cholinergic anti-inflammatory signaling in MΦ.
 Engel et al   Cholinergic Pathway in Stroke-Associated Pneumonia    3239
Several studies suggested that stimulation of the choliner-
gic anti-inflammatory pathway via vagus nerve and α7nAChR
may also be a neuroprotective strategy.39–41 However, our data
strongly suggest that inhibition of peripheral parasympathetic
actions is beneficial in terms of medical complications after
stroke, whereas an additional stimulation of the cholinergic
pathway might exacerbate the risk for life-threatening bacte-
rial infections. Thus, immunomodulatory therapeutic strategies
will have to consider and keep the balance between reversal of
the peripheral stroke-induced immunodepression and concur-
rent preservation of its putative neuroprotective effects.
In summary, although activation of cholinergic anti-
inflammatory mechanisms during local and systemic inflam-
matory responses may be a protective feedback mechanism to
limit potentially harmful effects of overt inflammation, central
nervous system injury–induced activation of such pathway in
the absence of an inflammatory stimulus may inappropriately
inhibit first-line antimicrobial responses and increase suscep-
tibility to infectious complications after acute central nervous
system injury.
Acknowledgments
We thank Sabine Kolodziej, Carena Teufelhart, Verena Wörtmann,
Yvonne Amoneit, Claudia Muselmann-Genschow (Charité), and
Ilona Kamer (MDC) for excellent technical assistance.
Sources of Funding
This study was supported by German Research Foundation (Exc257,
SFB-TRR84, SFB-TRR43, FOR1336, SFB1021 C05, Excellence
Cluster Cardio-Pulmonary System), German Federal Ministry of
Education and Research (01EO0801, German Center for Lung
Research), European Community FP7 (201024), and Hessen State
Ministry of Higher Education, Research and the Arts (Universities
Giessen & Marburg Lung Center).
Disclosures
A patent on preventive antibacterial therapy after stroke has been
filed to the European Patent Office (PCT/EP03/02246) by Drs Volk,
Dirnagl, C. Meisel, and A. Meisel. The other authors report no
conflicts.
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