Stroke Infeksi Imun

Int. J. Mol. Sci. 2016, 17(1), 64; doi:10.
3390/ijms17010064
Review
Ischemia, Immunosuppression and InfectionTackling the Predicaments of Post-Stroke
Complications
Raymond Shim and Connie H. Y. Wong *
Centre for Inflammatory Diseases, Department of Medicine, Monash Medical Centre, Monash
University, Clayton, VIC 3168, Australia
*
Correspondence: Tel.: +61-3-8572-2618
Academic Editor: Chris Sobey
Received: 23 November 2015 / Accepted: 24 December 2015 / Published: 5 January 2016
Abstract
: The incidence of stroke has risen over the past decade and will continue to be one of the leading
causes of death and disability worldwide. While a large portion of immediate death following
stroke is due to cerebral infarction and neurological complications, the most common medical
complication in stroke patients is infection. In fact, infections, such as pneumonia and urinary
tract infections, greatly worsen the clinical outcome of stroke patients. Recent evidence suggests
that the disrupted interplay between the central nervous system and immune system contributes
to the development of infection after stroke. The suppression of systemic immunity by the
nervous system is thought to protect the brain from further inflammatory insult, yet this comes at
the cost of increased susceptibility to infection after stroke. To improve patient outcome, there
have been attempts to lessen the stroke-associated bacterial burden through the prophylactic use
of broad-spectrum antibiotics. However, preventative antibiotic treatments have been
unsuccessful, and therefore have been discouraged. Additionally, with the ever-rising obstacle of
antibiotic-resistance, future therapeutic options to reverse immune impairment after stroke by
augmentation of host immunity may be a viable alternative option. However, cautionary steps are
required to ensure that collateral ischemic damage caused by cerebral inflammation remains
minimal.
Keywords:
stroke; immunosuppression; infection; antibiotics; clinical outcome
1. Introduction
Stroke is a devastating cerebrovascular event that occurs upon interruption of blood flow to areas
of the brain, due to blockage (ischemic stroke) or bursting/bleeding (hemorrhagic stroke) of a
cerebral blood vessel. This resulting brain damage contributes to long-lasting disabilities and
multiple functional impairments. In 2010, the global prevalence of stroke was 33 million, with an
incidence rate of 16.9 million [1]. Approximately a third of strokes are fatal, which accounts for
11% of total deaths around the world, making them one of the leading causes of death and
disability worldwide [1,2]. While mortality rate may be decreasing in recent times, stroke
incidence is rising and predicted to increase to 23 million by 2030 [3]. Because a majority of
long-term disability is due to stroke, it comes as no surprise that the global economic burden it
creates on the health care system and society is immense and predicted to rise due to our everaging population [4,5]. In order to lower the mortality, morbidity, and social and economic
strains of stroke, more effective treatments that target the underlying mechanisms of damage due
to stroke are needed. While immediate cell death and brain damage is directly due to deprivation
of nutrients to cells during stroke, exacerbation of neurological deficit is largely due to
inflammatory processes after stroke. Additionally, despite the local inflammatory immune
responses in the brain, stroke has been shown to alter systemic immunity to predispose patients
to predicaments such as immune suppression and infection. A greater understanding of the
pathophysiological processes that occur during and following stroke is required to shed light on
potential therapeutic targets to improve patient outcomes. Thus, this review will attempt to
describe the changes in local and systemic immunity after stroke, outline mechanisms of strokeinduced immune suppression and infection, and highlight potential therapeutic targets to reduce
post-stroke complications and improve patient health.
2. Local Immune Responses and Impairment after Stroke

Stroke is an ischemia/reperfusion injury that is caused when the blood supply to the brain is
insufficient. During occlusion, deprivation of oxygen and glucose can induce immediate,
localized neuronal cell death at the ischemic core via excitotoxic activity [6]. Normally,
glutamate is a major neurotransmitter which is released into the synapse by neurons to stimulate
glutamate receptors on the postsynaptic neuron to induce depolarization via influx of calcium
and sodium ions [7]. Glutamate is then internalized via active transport mechanisms that
sequester glutamate action. However, the low abundance of oxygen and glucose during ischemia
impairs ATP synthesis and thus clearance of glutamate does not occur. As a result, continual
stimulation of glutamate receptors allows for constant neuron depolarization, generation of
reactive oxygen species (ROS), and dysfunction of mitochondria to induce necrotic and
apoptotic pathways (reviewed in [6,8]).
Subsequent release of damage associated molecular patterns (DAMPS) by dying cells activates
resident microglial cells through stimulation of pathogen recognition receptors (PRRs), such as
toll-like receptor (TLR) 2 and TLR 4 [9,10,11]. This triggers downstream inflammatory signaling
cascadesinducing the activation of NF-B and mitogen-activated protein (MAP) kinase
pathways. Within minutes, local production of pro-inflammatory molecules occurs at the site of
occlusion [12]. Such molecules include cytokines, such as interleukin-(IL-) 1, IL-6 and tumor
necrosis factor-(TNF-) [13,14,15], chemokines and their receptors [16,17], integrins and
adhesion molecules, such as Very Late Antigen-4 (VLA-4), Intercellular Adhesion Molecule-1
(ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) [18].
Upon resolution of the vascular blockage, re-oxygenation of the brain promotes mitochondrialmediated production of ROS to cause further injury to cellular components and promote cell
death in surrounding neurons, glial cells and blood vessels [19]. The production of the
aforementioned cytokines, chemokines, adhesion molecules and ROS are all pro-inflammatory
and triggers the recruitment of peripheral immune cells in an attempt to initiate clearance of cell
debris and healing in the brain. However, excessive infiltration of leukocytes is found to be more
harmful. As the brain is encased within the skull, swelling, as a result of infiltrating immune
cells, can increase the intracranial pressure. High intracranial pressure has been shown to
increase fatality by up to 80% in stroke patients, and reducing the intracranial pressure in patients
within 48 h after stroke can reduce death and improve stroke outcomes [20,21].
Various immune cells have different roles in infarct development after stroke. Macrophages and
neutrophils have been found to infiltrate within 24 h following reperfusion [22]. Macrophages
are amongst the first to enter the brain during reperfusion and their role in infarct development is
under debate [22]. While some studies show that macrophages can have neuroprotective effects,
others suggest they can be neurodegenerative. Bone marrow-derived macrophages have been
reported to produce Transforming Growth Factor Beta (TGF-) and confer neuroprotection
following stroke by initiating healing and clearance of cell debris [23,24]. In contrast,
macrophages are also a major source of IL-1, TNF- and ROS, adding to further inflammatory
insult after stroke [25,26]. The controversial findings of macrophages in infarct development
may be explained by classical or alternative activation of macrophages, whereby classically
activated macrophages (M1 macrophages) exacerbate damage while alternatively activated
macrophages (M2 macrophages) assist in repair and neurogenesis [23]. In fact, macrophages in
the brain of post-stroke mice initially express M2 markers; however, a shift toward an M1
phenotype occurred following 5 days after ischemia which may result in the release of
inflammatory mediators to exacerbate damage in the brain [27].
Neutrophils are also known to rapidly infiltrate ischemic tissue within hours after stroke. While
the role of neutrophils is controversial, trends in literature suggest that neutrophils contribute
more toward detrimental outcomes in the brain following stroke [18]. Preventing the migration
of neutrophils into cerebral ischemic tissue could reduce infarct volume and improve
neurological outcomes in mice [22,28,29,30,31]. However, other recent studies do not support
the damaging role of neutrophils as depletion of neutrophils did not reduce brain damage after
stroke [32]. Interestingly, similar to macrophages, while an N1 phenotype in neutrophils was
observed in the brain of post-stroke mice, therapeutic activation of the peroxisome proliferatoractivated receptor- could polarize neutrophils toward an N2 phenotype to assist in
neuroprotection by clearance of neutrophils and resolution of inflammation [33].
Influx of lymphocytes usually occurs after 23 days following stroke. Most notably, T cells have
been found to contribute to infarct development through production of IL-1, interferon gamma
(IFN-) and macrophage inflammatory protein (MIP)-2 [34,35]. Mice deficient in T and B cells
had similar infarct sizes compared to wild-type mice, whereas reintroduction of T lymphocytes
increased infarct sizes post-stroke [36]. In contrast, B cells may be neuroprotective as B cell
deficient mice tended to have increased infarct volume, suggesting an immune regulatory role of
B cells in the brain after stroke, possibly in an IL-10 directed manner [37,38]. The somewhat
controversial roles of various immune cells in infarct development are complex and have been
more extensively reviewed elsewhere [39]. Interestingly, recent research into the role of the
regulatory T cells (Tregs) after stroke have revealed their contentious roles in infarct
development. On one hand, Treg depletion using CD25-specific monoclonal antibodies resulted
in exacerbated long-term brain damage and worsened functional outcome in experimental stroke,
potentially due to the lack of IL-10 production [40]. On the other hand, Treg depletion by
diphtheria toxin in Foxp3DTR (diphtheria toxin receptor) mice did not alter ischemic lesion
volume 3 days after stroke to argue against the neuroprotective role of Tregs [38].
Confoundingly, pre-stroke depletion of Tregs by diphtheria toxin actually reduced infarct

development and functional outcomes within 24 h following transient mid-cerebral artery
occlusion (tMCAO) model [41]. In fact, it was observed that Tregs disrupt microvascular
function in the brain via lymphocyte function-associated antigen 1 (LFA-1)/ICAM-1 action to
worsen brain damage [41]. As the role of Tregs remains under dispute, it is therefore vital that
the controversial role of Tregs in infarct development is elucidated before translating findings
into stroke patients. There are other promising neuroprotective therapies that aim to reduce
inflammation by exploiting activation of suppressor cells and preventing immune cell
recruitment by blocking adhesion molecule action in experimental models [27,29,33]. However,
as of yet, these have not been effective in humans [42]. In contrast, drugs currently in use to treat
multiple sclerosis, including natalizumab and fingolimod, and may have potential in attenuating
infarct development [43]. Natalizumab is a monoclonal antibody that blocks 4-integrin (a
subunit of VLA-4) to prevent the infiltration of lymphocytes into the brain [18]. The efficacy of
natalizumab is currently under dispute as experimental studies have shown either a
neuroprotective [18] or benign [44] effect of natalizumab in reducing brain damage. Despite this,
a preclinical phase III trial is in progress and may shed light on the effectiveness of natalizumab
in improving stroke outcomes [45]. Similarly, fingolimod is an agonist of the sphingosine-1phosphate (S1P) receptors, S1P1, S1P3, S1P4 and S1P5, which limits the migration of immune
cells out of lymph nodes and into the central nervous system (CNS) [46]. Promisingly,
fingolimod treatment after stroke has been found to effectively reduce brain infarct development
in both experimental [47,48] and clinical stroke [49].
The excess infiltration of leukocytes can be largely explained by increased permeability of the
blood brain barrier (BBB) [50]. Normally, the immune privileged status of the brain is regulated
by the BBB; however, disruption of BBB integrity can allow for unregulated entry of immune
cells [51,52]. Many molecules that are released after stroke, such as ROS, can increase the
permeability of the BBB and disrupt its homeostasis [51,52]. In addition, uncontrolled
macromolecule infiltration can cause movement of fluid into the brain, resulting in edema
[51,52]. Furthermore, permeabilization of the BBB also induces the exposure of CNS antigens to
the periphery, potentially leading to acquired autoimmunity [53]. Notably, loss of natural killer
(NK) cell tolerance to cell bodies and axons was shown to promote lesion development in poststroke mice [54]. Additionally, immune responses against myelin basic protein (MBP) and glial
fibrillary acidic protein (GFAP) have been associated with larger cerebral infarctions and
increased the likelihood of worse outcome in stroke patients [53,55]. Therefore, stroke disrupts
the normally tightly regulated homeostasis of the immune system and result in further collateral
brain damage following the initial ischemia event. However, stroke also has systemic
repercussions that leave patients in a worsened state of health.
3. Infections after Stroke

There is no doubt that sequences of highly inflammatory events add to cerebral insult after stroke
and leads to detrimental outcomes, however, despite all the neurological damage that occurs after
stroke, a major clinical complication that stroke survivors encounter is infection. It has been
reported that between 23% and 65% of patients acquire infection after stroke, with pneumonia
and urinary tract infections (UTI) being most common [56,57]. Infections contribute to
worsening of clinical outcomes, increased risk of recurrent stroke and death [58,59]. In fact,
stroke-associated infections can account for approximately 30% of fatality in stroke patients
[20,60,61]. As a result of an immune response to post-stroke infection, levels of inflammatory
molecules such as IL-6 and C-reactive protein are elevated and are associated with poor patient
outcomes [62,63].
Diagnosis of infection after stroke can be challenging as the criteria of diagnosis are inconsistent
between studies. Hallmark signs of infection in clinics, such as fever and inflammation, can be
presented by patients as a consequence of neurological damage that disrupts homeostatic
regulation of body temperature [64]. On the other hand, use of aspirin and paracetamol can veil
infection by reducing fever. Other symptoms, such as delirium, neurological deterioration and
dehydration, are indicative but are not specific for infection [65]. Chest radiographs are largely
relied upon, though with only a 65% sensitivity. Additionally, the detection of pneumonia with
this method may only be useful in later stages of infection due to a lack of infiltrates [66]. Thus,
there is a need for improved and universal diagnostic methods and markers to more accurately
identify infections after stroke. Interestingly, magnetic resonance imaging (MRI) was used to
measure severity of lung infection in post-stroke mice [67]. This may be a potential translational
opportunity to rapidly detect and diagnose chest infection in a clinical setting.
Identifying whether patients will succumb to infection could allow for early interventions to
reduce post-stroke bacterial burden and improve clinical outcome, however it can be difficult to
predict. One study identified a range of independent risk factors that could predict pneumonia
after stroke with 76% sensitivity and 88% specificity [59]. These factors included dysphagia,
National Institute of Health Stroke Scale (NIHSS) of 10 and non-lacunar basal-ganglia
infarction [68]. Similarly, a 12-point scoring system was devised to predict stroke-associated
pneumonia based on commonly tested variables such as age, blood pressure and leukocyte
levels, which had 77.6% sensitivity and 84% specificity [69]. Loss of lymphocytes may also be a
predictor of infection after stroke. Indeed, while leukocyte numbers increase, lymphopenia
occurs within 6 h and last for at least 6 days after stroke in patients [70]. Consequently, stroke
patients that exhibit signs of lymphopenia also acquired infections strongly emphasizing the
critical role of lymphocytes in host bacterial defense after stroke. In addition, decreased TNF-
production and reduced HLA-DR expression in monocytes were reported and may be viable
predictive markers to infection [70,71]. However, similar attributes in stroke patients without
infection were also found and this puts the reliability of these markers into question when it
comes to predicting infection onset after stroke. Similar findings were recently reported, where
total leukocyte counts were increased in the infected cohort of stroke patients [72]. Intriguingly,
recovery of circulating lymphocyte numbers was seen one day after stroke in non-infected
patients, whereas infected patients demonstrated persistent lymphopenia [72]. These findings
further support the notion of lymphopenia as a predictor of post-stroke infection, and future
therapeutics may require boosting lymphocyte levels in order to reduce infection. Importantly,
though, immunological changes seen in these studies and others allude to the phenomenon that is
stroke-induced immune suppression.
4. Stroke-Induced Immune Suppression

Although initial systemic inflammation, characterized by circulating IL-6 and IFN- production,
may peak within 6 h following stroke [73], it has become increasingly evident that systemic
immune suppression takes place as a compensatory mechanism against brain damage [74]. As
previously discussed, despite an increase in granulocyte numbers, infection still occurs in
patients within days after stroke [70,72]. This may suggest impairments in innate immunity
contribute to infection development in the acute stages after stroke, however this has not been as
comprehensively explored. Neutrophils are well-known to rapidly respond and migrate to sites of
infections where they elicit protection against pathogens via expression of soluble inflammatory
mediators and superoxide generation [75]. Patients with hemorrhagic stroke had impaired
neutrophil respiratory burst, which suggests that future therapeutics may target
immunomodulatory pathways with an aim to restore neutrophil function [76]. This coincides
with a recent study that also reported impairment of respiratory burst by circulating granulocytes
and monocytes in ischemic stroke patients. Interestingly, the stroke-induced neutrophil
impairment was attributed by the granulocyte and monocytes responses to catecholamines [77].
Monocytes and macrophages are phagocytes that can engulf pathogens and infected cells to alert
and prime the rest of the immune system [78]. Splenic macrophages/monocytes have been shown
to lack VLA-4 which prevented the migration of these cells into other tissues [79]. Most
importantly, macrophages have a large role in bridging the innate and adaptive immunity by
interacting with T cells to initiate specific immune responses [80]. In order to trigger a sustained,
specific and effective immune response against a pathogen, antigen presenting cells (APCs), like
the macrophage, must recognize and present foreign peptide antigens through major
histocompatibility complexes (MHC) and present costimulatory molecules to T lymphocytes
[81,82]. This, in turn, initiates other parts of the immune system for an anti-pathogen response.
Previous studies have shown that expression of MHC class II (or HLA-DR in humans) is
reduced [72,83], costimulation is less efficient [84], and production of pro-inflammatory
cytokines by monocytes is decreased after stroke [71]. Collectively, these studies suggest that
stroke interferes with the normal functions of the immune system to the extent that the
bactericidal immunity is impaired to leave the host susceptible to infections.
Another indicator of immune suppression is the macroscopic shrinking of immune organs,
shown to occur within 12 h following stroke in mice [85]. Most notably, size reduction of spleen
and thymus may be partly due to apoptotic death of splenocytes [85,86]. The stroke-induced
death of splenocytes resulted in reduced production of T cell mitogenic factors and thus prevents
T cell proliferation and inflammatory cytokine production [79,85]. Additionally, migration of
cells out of the spleen and into the parenchyma in the brain may also contribute to the reduction
of spleen size [86]. Moreover, induction of Tregs and loss of B cells in the spleen was thought to
further compromise host pathogen defenses [79].
Adaptive immune changes can largely predispose to chronic susceptibility to infection [87].
Particularly, in T cells, a shift of from a cell-mediated inflammatory T-helper 1 (TH1) type
response to a humoral-mediated anti-inflammatory T-helper 2 (TH2) type response is suggested to
occur to protect the brain from further inflammatory damage and promote tissue repair and
neuronal regeneration [88,89]. However, this shift occurs to the extent that the hosts systemic
immune system is suppressed and becomes ineffective in fighting pathogens, resulting in
increased susceptibility to infections. The systemic cytokine milieu plays an important role in
determining immune suppression and the type of T-helper response that predominates. The
production of immunosuppressive and TH2-associated cytokines mediates the immune shift away
from a TH1 response and toward a TH2 response after stroke. In particular, IL-10 secretion by
monocytes, dendritic cells, and Tregs is greatly increased after stroke in both mice and humans
[79,90], and can act upon many immune cell types to avert from a pro-inflammatory response.
The production of IFN- by TH1 cells, proliferation of T cells and cytokine responses, TNF-
production by macrophages and cytokine production by monocytes are all inhibited by IL-10 to
further emphasize its role in the immune shift toward a TH2 response and immune suppression
after stroke [91]. Decreased levels of serum IFN- and TNF-, well known TH1 cytokines, were
seen 12 h after stroke in mice [85]. In fact, reduced serum IFN- level is critical for strokeinduced susceptibility to infection as adoptive transfer of IFN--producing splenocytes resulted
in a significant reduction of bacteria in the blood and lungs after stroke [85].
Although immune suppression is apparent following stroke, the mechanism in which this occurs
is not well understood. One explanation is that an early, sustained inflammatory response may
exhaust the immune system and ultimately lead to immune suppression [79,92]. Another
explanation may be that the brain attempts to suppress the immune system in order to prevent
further cerebral inflammation [79]. Indeed, neural control of immunity, possibly by the
hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS), is an
emerging concept and is thought to play the biggest role in the systemic immune shift after
stroke. Briefly, the HPA axis is the collaboration of the central nervous system (CNS) and
endocrine system that regulate bodily functions whereby corticotrophin releasing hormone
(CRH) is released by the hypothalamus in response to stress [93]. CRH then stimulate the
production of adrenocorticotropic hormone (ACTH) by the anterior pituitary gland which can
then travel to the adrenal gland to induce glucocorticoid production as an end product [94].
Production of IL-1, IL-6, IL-10 and TNF- as a consequence of cerebral insult from stroke can
be sensed by the hypothalamus to activate the HPA axis for excessive glucocorticoid release [92].
Similarly, stroke-induced activation of the SNS results in the secretion of catecholamines by the
adrenal medulla and nerve terminal [94]. Immune cells express receptors that can be stimulated
by glucocorticoids and catecholamines to promote an immune suppressive response [95].
Catecholamines signaling through -adrenergic receptors on immune cells have been shown to
reduce TNF- while promoting IL-10 production. Stimulation of -adrenergic receptors can also
inhibit cytotoxic T-lymphocyte-associated protein (CTLA)-4 expression on T cells [96]. On the
other hand, glucocorticoids can act upon T cells, to reduce IFN- production and induce
apoptotic death, and monocytes to promote IL-10 secretion [85]. However, administration of
propranolol, a -adrenergic receptor blocker, but not RU486, a glucocorticoid receptor blocker,
into post-stroke mice have been shown to reduce bacterial complications and mortality after
stroke, strongly suggesting a role catecholamines in inducing immune suppression post-stroke
[85]. Additionally, the apoptotic effect of catecholamine and glucocorticoid release after stroke
may account for the lymphopenia observed following stroke [85]. In fact, the concentration of
catecholamines in plasma has been shown to correlate with the amount of post-stroke immune
suppression in humans. A recent study in our laboratory found that hepatic invariant natural killer
(iNKT) cells are in a prime position to detect and respond to distant brain damage in a murine
model of stroke [97]. In that study, we showed that the behavior of iNKT cells are impaired by adrenergic signaling after stroke [97]. Furthermore, blocking of -adrenergic receptors with
propranolol resulted in increased IFN- production and reduced the bacterial burden in poststroke mice [97]. However, this protection conferred by propranolol administration was not seen
in mice deficient of iNKT cell, demonstrating the role of iNKT cells in catecholamine-mediated
immune modulation after stroke. Others have also investigated the impact of catecholamines and
glucocorticoids on immune suppression after stroke [98] but overall, these mechanisms work
together to dampen the inflammatory immune responses to minimize further immune pathology
in the brain, although this comes at the cost of susceptibility to infections in the host [97] (Figure
1).
Figure 1. Immunological processes and mechanisms for infection after stroke. During a stroke,
interrupted blood flow into the brain deprive cells of vital nutrients and thus undergo cell death.
This in turn activates local immune cells to produce pro-inflammatory factors, which can have
deleterious effects on the brain. Additionally, restoration of blood flow into the brain allows for
reperfusion, resulting in ROS production and BBB breakdown. This allows for excessive
recruitment and infiltration of circulating immune cells into the brain to promote swelling and
increased intracranial pressure to exacerbate brain damage. In an attempt to reduce damage and
resolve local inflammation, the brain responds by activating the SNS and HPA-axis in order to
produce catecholamines and glucocorticoids. Catecholamines and glucocorticoids can act upon
iNKT cells and T cells, respectively. Meanwhile, APC and neutrophils are also impaired to the
paint where their bactericidal activities are insufficient, all of which resulting in systemic
immune suppression and therefore leaving stroke sufferers more susceptible to infections. The
brain and stroke are represented by a cloud and a bolt, respectively. Arrows denote activate or
induce; blunted arrows denote inhibit or prevent. Dashed lines denote activities locally in
the brain versus systemically in the periphery.
5. Other Causes of Infection after Stroke

Pneumonia and UTIs are the main types of infection after stroke. Pneumonia is seen to be the
most common with an incidence rate of up to 57% [99], while UTIs can occur between 11% and
27% of stroke patients [100]. The occurrence of stroke-associated pneumonia has been shown to
worsen clinical and neurological outcomes, and therefore studies have recently investigated the
mechanisms in acquiring pneumonia after stroke. The most common cause for stroke-associated
pneumonia was thought to be solely aspiration and dysphagia that result from impaired
swallowing function [101]. Defects in the swallowing mechanisms may be partially explained by
depletion of substance P following stroke [102]. Substance P has roles in the coughing and
swallowing reflex. After stroke, dopamine production by the corpus nigrostriate is reduced and
thus substance P secretion by the glossopharyngeal nerve is downregulated [101]. This promotes
aspiration by allowing entry of gastric contents into the lungs to result in pneumonia [103]. In
addition, while the lack of substance P has been shown to enhance aspiration and dysphagia
following stroke, the pathophysiology of aspiration and dysphagia should be noted to be complex
and involve a variety of aspects, including decreased level of consciousness, body positioning in
bed, mechanical ventilation, and patient immobility [104]. However, it is now known that
aspiration and dysphagia only partially explain the high frequency and susceptibility of stroke
patients to pneumonia. There is evidence in experimental and clinical studies that show that
aspiration is not the only cause of pneumonia after stroke. In fact, intranasal administration of
only 200 colony-forming units (CFU) of Streptococcus pneumoniae was necessary to cause
pneumonia and bacteremia in post-stroke mice, whereas sham-surgery mice needed 1000-fold
greater amount of bacteria for a similar severity of pneumonia [105]. In a clinical setting, the
incidence of pneumonia in stroke patients who were fed via gastric tube to prevent aspiration
was 44% to further suggest alternative mechanisms in pneumonia susceptibility [106].
Nonetheless, later on it was found that gastric tube feeding could predispose to infection [107].
However, aspiration has been shown to occur in healthy individuals at a similar extent as in
stroke patients, though pneumonia does not develop [108]. Stroke-induced immunosuppression
is now more universally acknowledged to be the main explanation for susceptibility to infection
after stroke, with aspiration and dysphagia being a contributor rather than the sole cause.
While infections are common after stroke, the causative agent remains elusive and may be
contributed by a number of pathogens. Types of bacteria that have been commonly found in the
sputum and urine of stroke patients include Streptococcus pneumoniae, Staphylococcus aureus,
Klebsiella pneumoniae and Psuedomonas aeruginosa [109]. Surprisingly, reports of common gut
bacteria have been reported with Escherichia coli and Enterobacter cloacae being two of the most
frequently detected [110]. Indeed, more than 95% of bacterial cultures of blood and lungs of
stroke mice were found to be made up of Escherichia coli [85]. However, a number of studies
could not identify the causative agent of infection in a large portion of their stroke patient cohorts
[68,107,111]. Reasons for this may be due to low yields of sputum/aspirate, as neurological
deficit makes it difficult for patients to produce samples, and challenges in culturing the
causative agent as some microbes require extremely specific culture conditions to grow [112].
Overall, understanding or identifying the causative agents of infection can allow for more
targeted treatments to reduce post-stroke bacterial complications.
It may appear that we have evolved to react to stroke injury with an apparently maladaptive
response (post-stroke infection). During stroke, self-epitopes, which are normally shielded from
the systemic immune system by a number of mechanisms, may become exposed to adaptive
immunity. This may educate the immune system to react to self-antigens in the CNS, and,
ultimately, lead to autoimmunity or autoaggression. According to this concept, by globally downregulating innate and adaptive immunity, stroke-induced immune suppression may help to
prevent post-injury autoimmunity. Indeed, brain-specific antigens can be measured in the blood
plasma after stroke [113]. At present, there is no epidemiological evidence that patients with
stroke have an increased incidence of autoimmune CNS disorders (such as multiple sclerosis).
This might point to effective control of autoimmunity under injury conditions, which would
come at the price of an increased susceptibility to infection. Interestingly, infections after stroke
have been shown recently to predispose patients to autoimmunity against brain antigens [114].
Systemic inflammation as a response to infection is thought to induce an immune response
against infection-associated antigens, but immune responses against self-antigens may occur as a
collateral result. To mimic post-stroke infections in an experimental model, post-stroke rats,
administered with lipopolysaccharide (LPS) to induce a systemic inflammatory response, had a
greater TH1-mediated immune response toward MBP [115] and increased mortality rates [116].
Additionally, immune responses against MBP and GFAP were found to be more common in
stroke patients with acquired infection and had worsened outcomes compared to stroke patients
without infection [53]. This may explain why infection is independently associated with poor
outcomes and therefore emphasises the importance in reducing the incidence of infections after
stroke.
6. Treatments for Stroke-Associated Infections

6.1. Antibiotics
The obvious strategy and current gold-standard treatment to combat infections is the use of
antibiotics upon diagnosis of infection [67]. However, treatment at this stage could decrease the
rate of recovery, worsens the functional outcome of patients, increases the likelihood of recurring
stroke, and extends the amount of suffering for the patient [117]. A main focus in recent years
has been prophylactic administration of broad-spectrum antibiotics to avoid the clinical problems
that are associated with infections. Experimentally, post-stroke mice that were on preventive
antibiotic treatment (PAT) with fluoroquinolones had improved survival rates and neurological
outcomes compared to mice either with antibiotic treatment upon diagnosis of infection or
without antibiotic treatment at all [67,118]. However, clinical studies on the effectiveness of PAT
are controversial and not practical. In fact, a number of studies do not support this strategy to
reduce infection after stroke [88,107,119,120].
A follow-up study with moxifloxacin, a fourth generation fluoroquinolone, was conducted in the
Preventive Antibiotic Therapy in Ischemic Stroke (PANTHERIS) clinical study [108]. With perprotocol (PP) analysis (n = 66), it was found that prophylactic treatment with moxifloxacin
reduced infections by 24.8% (p = 0.032), though this treatment did not seem to be beneficial
using with intention-to-treat (ITT) analysis (n = 79; p = 0.114), which included all recruited
patients. ITT analysis may be more viable to evaluate the effectiveness of this treatment as
excluding some patients for PP analysis may dramatically impact results due to the small group
size in this study (n = 31 in placebo group and n = 35 in treatment group). Overall, the main
finding in the PANTHERIS study was that patients did not have improved clinical outcomes or
reduced mortality after 6 months despite reduced infection rates and thus discourages
prophylactic use of moxifloxacin as a preferential treatment over current treatment guidelines to
reduce post-stroke infection.
Similar results were found for the most recent studies that evaluated the effectiveness of PAT.
The preventive antibiotics in stroke study (PASS) used ceftriaxone, a third generation
cephalosporin, to determine whether treatment could reduce infection to improve functional
outcomes after stroke [121]. Previous studies have shown that ceftriaxone had neuroprotective
qualities by reducing excessive excitatory mechanisms and thus has potential to reduce
neurological deficit after stroke [7,122]. Patients were administered ceftriaxone within 24 h after
onset of stroke symptoms, which was continued once daily for 4 days. Functional outcomes,
defined by the modified Rankin Scale (mRS), were assessed at 3 months. The main finding was
that infections could be reduced after ceftriaxone treatment, although improvement of
neurological and functional outcome was not observed. Interestingly, the study found that
prophylactic treatment with ceftriaxone could significantly reduce rate of UTI but not of
pneumonia between the treatment group and the placebo group. Despite this, it should be noted
the proportion of patients with infection in this study was relatively low, whereby study
populations of previous trials were often limited to patients with severe strokes. In the PASS
study, patients with mild strokes were included, and the median NIHSS was 5. The fact that
functional outcome does not improve despite absence of infection is perplexing as multiple
studies have shown the association of infection and worsened outcomes [61,111,123]. However,
there may be some explanations for this. One explanation may be that infection may merely be
marker or a symptom of the extent of poor functional outcome. This may be feasible as other
studies that showed prophylactic antibiotic interventions could reduce infection rates but not
improve functional outcomes; however, this requires further investigation for confirmation
[112,123,124]. Another explanation stems from the finding that ceftriaxone did not significantly
reduce incidence of pneumonia, which suggests that pneumonia is the greater cause for worsened
outcomes after stroke. Indeed, this would explain why, despite the decreased infection rates, the
functional outcomes of the stroke patients did not improve [121].
In the STROKE-INF study, prophylactic use of clarithromycin in combination with either

amoxicillin or co-amoxiclav was given within 48 h after stroke for 7 days in dysphagic patients
where occurrence of pneumonia and patient functional outcomes at 90 days was assessed
[111,125]. Interestingly, treatment did not reduce the incidence of pneumonia or mortality
compared to the untreated group and actually lengthened time of the patients hospital stay.
Again, these studies further suggest prophylactic antibiotic treatment is not better than standard
treatments.
Lastly, another possible reason that antibiotic treatment is not effective is the unsuitable use of
particular antibiotics due to antibiotic resistant bacteria. One study took sputum samples from
patients with stroke-associated pneumonia, cultured for bacteria and tested their drug sensitivity
against a range common antibiotics including penicillin, tetracycline, ceftriaxone and
moxifloxacin [109]. The prevalent strains of bacteria that were found in the sputum were
Psuedomonas aeruginosa (23.92%), Staphylococcus aureus (15.32%) and Escherichia coli
(14.25%). The study found that drug resistance against moxifloxacin, used in the PANTHERIS
trial [108], was 52.63% in S. aureus [109]. Strikingly, resistance rates against ceftriaxone, the
antibiotic of choice used in the PASS trial [107], was 100% for both Escherichia coli and
Psuedomonas aeruginosa [109] which may offer an explanation to why pneumonia was not
reduced in ceftriaxone-treated stroke patients. Importantly, resistance rates will differ between
hospitals and therefore future studies should assess for antibiotic resistance in the patient cohort.
Indeed, infections should be prevented by using selective antibiotics that take into consideration
the results of antibiotic testing [109]. However, the rise in antibiotic resistant pathogens implies
that it is high time for antibiotic-independent therapies to more effectively reduce infections after
stroke.
6.2. Immune Modulation
The impact that stroke has on the immune system is complex and, as discussed, innervation of
the sympathetic nervous system and HPA axis can result in an immune shift towards a TH2
response after stroke to leave the host susceptible to infections. Targeting these pathways to
prevent the immune shift following stroke may offer a viable alternative to antibiotics to reduce
infections. Catecholamines are produced as a result of cerebral ischemia and can act upon adrenoreceptors on immune cells to induce apoptosis and immune suppressive pathways.
Therapeutic blocking of the -adrenergic receptor after stroke has been shown to reduce
infections in experimental models of stroke by counteracting the immunosuppressive properties
of catecholamines [85,97]. Intriguingly, the occurrence of pneumonia in post-stroke patients that
were on -blockers, prior to ictus and during hospitalization, was less than that of patients who
did not receive -blockers. In addition, -blocker therapy significantly reduced 30-day mortality
in stroke patients [126]. A recent study that examined 5212 stroke patients from a Virtual
International Stroke Trials Archive also found that on-stroke -blocker therapy was associated
with reduced mortality and that both pre-stroke and on-stroke treatment with -blocker reduced
pneumonia frequency [127]. In contrast, the most recent clinical trial saw that, while using blocker treatment reduced the occurrence of UTI, it did not provide adequate protection against
pneumonia and the treatment group actually had a greater mortality after 30 days compared to
patients not on -blockers [128]. Taken together, it is clear that the use of -blocker therapy in
post-stroke patients requires further study in order to elucidate its effectiveness in reducing
stroke-associated infections.
We recently showed that the stroke impairs iNKT cell function within hours after stroke to
mediate immune suppressive pathways and result in increased host susceptibility to infections in
an experimental setting [97]. Interestingly, specific activation of iNKT cells with galactosylceramide (-GalCer), a glycolipid that is presented via CD1d, could significantly
increase systemic IFN- production and reduce bacteria in all of the tissue examined post-stroke,
while brain damage was not exacerbated. This study showed that iNKT cell is one of the major
mediators of the immune shift after stroke and modulating their responses using -GalCer was
efficient in reducing infections after stroke [97]. This merits further study in the design of novel
-GalCer analogues that can skew or bias iNKT cell responses in altering host immunity after
stroke to reduce infectious complications.
Additional studies in the mechanisms of brain-immune system axis are required to reveal
potential alternative therapeutic options. However, cautionary steps should be taken with
immunomodulatory treatments. Suppression of the immune system after stroke is thought to be a
brain-protective mechanism as the majority of penumbral damage is governed by inflammatory
events [14,89]. Therefore, boosting the immune system with immunomodulatory therapy to
reduce stroke-associated infection may promote further collateral damage in the brain. An
optimal immunomodulatory regime should reduce infection burden without exacerbating brain
damage. Future research may also choose to consider a combination therapy to minimize
immediate cerebral insult and prevent infections during the post-acute phrase of stroke for better
patient outcomes.
7. Conclusions
Damage to the brain after stroke is now known to be a largely immune mediated event. As a
compensatory response to the highly inflamed environment within the skull, the activation of the
sympathetic nervous system and hypothalamic-pituitary-adrenal axis results in the production
immune suppressive molecules including catecholamines and glucocorticoids, respectively. The
brain-immunity axis is a recently identified and complex pathway that induces immune
suppression after stroke. This is believed to be a defensive mechanism of the brain with an
attempt to lessen further cerebral damage, however rendering the host more susceptible to
infections. Infections such as pneumonia and UTI are extremely common in stroke patients and
have been associated with poorer functional outcomes and increased mortality. Therefore, a
recent focus has been to reduce infection to improve patient health. Much attention has been
drawn to preventive antibiotic treatment, though the majority of clinical studies have swayed
against prophylactic antibiotic administration due to a failure in reducing pneumonia and
improving clinical outcomes. Importantly, recent studies reveal an underlying issue in the
inconsistency of pneumonia diagnosis across clinics. Additionally, with the growing emergence
of antibiotic-resistant pathogens, alternative therapies to reduce infection are required.
Immunomodulation therapies that augment the immune system to combat infections is an
attractive avenue, however care is needed to ensure further cerebral injury is avoided. Future
studies may want to focus on the precise mechanisms of immune suppression after stroke in
order to unveil alternative therapeutic targets to reduce stroke-associated infections.
Acknowledgments
The work is supported by the Australia Research Council (ARC) and National Health and
Medical Research Council (NHMRC, Australia).
Author Contributions
Raymond Shim and Connie H. Y. Wong wrote the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Review
Innate Immunity and Inflammation Post-Stroke: An 7-Nicotinic Agonist Perspective
Silke Neumann 1,2, Nicholas J. Shields 1, Thomas Balle 3, Mary Chebib 3 and Andrew N. Clarkson
2,3,
*
1
Department of Pathology, University of Otago, Dunedin 9054, New Zealand

2
Department of Anatomy, Brain Health Research Centre and Brain Research New Zealand,
University of Otago, P.O. Box 913, Dunedin 9054, New Zealand
3
Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia

*
Correspondence: Tel.: +64-3-479-7326; Fax: +64-3-479-7254
Academic Editors: Chris Sobey and Hyun Ah Kim
Received: 14 October 2015 / Accepted: 25 November 2015 / Published: 4 December 2015
Abstract
: Stroke is one of the leading causes of death and long-term disability, with limited treatment
options available. Inflammation contributes to damage tissue in the central nervous system
across a broad range of neuropathologies, including Alzheimers disease, pain, Schizophrenia,
and stroke. While the immune system plays an important role in contributing to brain damage
produced by ischemia, the damaged brain, in turn, can exert a powerful immune-suppressive
effect that promotes infections and threatens the survival of stroke patients. Recently the
cholinergic anti-inflammatory pathway, in particular its modulation using 7-nicotinic
acetylcholine receptor (7-nAChR) ligands, has shown potential as a strategy to dampen the
inflammatory response and facilitate functional recovery in stroke patients. Here we discuss the
current literature on stroke-induced inflammation and the effects of 7-nAChR modulators on
innate immune cells.
Keywords:
stroke; inflammation; nicotinic; nicotinic acetylcholine receptor agonist; immune response;
myeloid cells
1. Introduction
Stroke is one of the leading causes of death and morbidity in developed nations. Restricted blood
supply following a stroke leads to a localised depletion in energy and oxygen, ultimately
resulting in cell death within the affected area [1]. These dying cells secrete danger signals that
stimulate an inflammatory response intended to support healing but which is, in most cases,
excessive and exacerbates neural damage to impede recovery. This post-stroke inflammatory
response is mediated by local innate immune cells in the brain (resident microglia) and by other
immune cells that later enter the brain from the bloodstream. As a consequence of excess
inflammation, recovery following a stroke can be severely hampered due to ineffective nerve cell
repair.
The inflammatory process is stimulated by danger signals, which include fragments of cells
undergoing cell death and factors secreted by damaged cells, such as adenosine triphosphate
(ATP), uric acid, and reactive oxygen species (ROS) [2,3]. Even though danger signals are
diverse in nature, they converge on similar pathways in local immune cells to stimulate the
production and secretion of pro-inflammatory cytokines. These pro-inflammatory cytokines
activate local microglial cells, induce the migration of blood-borne immune cells to the infarct
area, and influence systemic immune responses. Paradoxically, the excessive inflammatory
response in the brain induces a state of immune-suppression in the periphery, putting stroke
patients at risk of fatal secondary infections [4]. The mechanistic relationship between the initial
inflammatory response in the brain and the immune-suppression in the periphery observed in the
weeks after stroke has long been unclear. Only recent evidence suggests that pro-inflammatory
cytokines released in the brain stimulate the expansion of an immune-suppressive cell population
that suppresses both innate and adaptive immune responses [5].
Inflammatory reactions are usually self-limiting and resolve, driven by the secretion of inhibitory
molecules within the immune system. Interestingly, recent research has shown that the
autonomic nervous system also controls inflammation via neural circuits that affect immune cells
[6]. One of these brain-immune connections is the cholinergic anti-inflammatory pathway, which
senses inflammation via peripheral nerves and suppresses the production of pro-inflammatory
cytokines [7]. Remarkably, this interaction between the brain and the immune system relies on
neurons sensing the presence of cytokines (immune-modulating agents). Upon sensing the
presence of pro-inflammatory cytokines, such as interleukin (IL)-1 and tumour necrosis factor
(TNF)-, neurons release the neurotransmitter acetylcholine (ACh) which binds to nicotinic
acetylcholine receptors (nAChR) on peripheral immune cells. Notably, activation of the 7nAChR has recently been shown to improve functional recovery after stroke by limiting harmful
post-stroke inflammation [8].
2. Overview of Nicotinic Acetylcholine Receptors

(nAChRs) with a Focus on the 7-Subunit
Neuronal nAChRs are allosteric transmembrane proteins that are assembled from one or more subunits (110) either alone or together with one or more -subunits (14) [9,10,11]. Each
conformation of the nAChRs has a unique function, pharmacological profile, and expression
pattern, which makes it possible for subtype-selective compounds to have distinct therapeutic
applications with a restricted set of side effects [12,13]. nAChRs are widely distributed
throughout the central nervous system, where they participate in a variety of physiological
responses, like learning, memory, locomotion, and attention, among others. For the purpose of
this review, we will focus on the 7-nAChR, which is one of the most abundantly-expressed and
widely-distributed nAChRs throughout the brain [11,13,14]. In the brain, 7-nAChRs are
expressed on neurons and non-neuronal cells, including astrocytes, microglia, oligodendrocytes,
and endothelial cells [15,16,17]. Previous studies have highlighted that 7-nAChR modulators
can minimise the extent of cell death [18,19] and enhance synaptic plasticity [20], making 7nAChRs an ideal therapeutic target for several neurological diseases including depression,
Parkinsons disease, schizophrenia and Alzheimers disease [21]. Furthermore, positive 7nAChR modulators are potent inhibitors of inflammation [22] and have been suggested as
promising candidates for the treatment of inflammatory diseases, such as inflammatory bowel
disease, arthritis, asthma, and obesity. 7-nAChR agonists have the advantage of selectively
stimulating the 7-subunit, circumventing side effects caused through general activation of
nicotinic receptors. This has led to the establishment of several clinical trials investigating the
effectiveness of 7-nAChR agonists, predominantly for the treatment of cognitive disorders,
such as dementia and schizophrenia [23,24]. Even though the side effect profile of selective 7nAChRs is favourable in comparison to non-selective agonists, such as nicotine, it remains to be
determined if the observed pharmacological effects are as potent. The mechanisms by which 7nAChR agonists inhibit inflammation and promote an anti-inflammatory environment will be
discussed in more detail herein.
3. An Inflammatory Reflex Shapes Immune Responses

Post-Stroke via Activation of 7-nAChRs
The vagus nerve (cranial nerve X) is the longest of the cranial nerves and its main function is to
regulate the parasympathetic nervous system. Previous studies have shown that stimulation of
the vagus nerve can reduce inflammation, both in peripheral lymphoid organs as well as in the
brain [6,25]. The complex interactions between the nervous system and the immune system that
constitutes this inflammatory reflex are still not completely understood [26]. However, it is
clear that the anti-inflammatory effects observed after vagal stimulation are mediated via the
activation of 7-nAChRs found on innate immune cells [27]. 7-nAChRs can be found in tissues
throughout the central nervous system (CNS) and are also abundantly expressed by immune cells
[9]. Activation of 7-nAChRs on immune cells has been shown to minimise the production of
pro-inflammatory cytokines [22], while, importantly, not abating the secretion of antiinflammatory cytokines that promote the resolution of inflammation [28]. Innate immune cells
are an attractive target for the treatment of stroke, as they are responsible for inducing sterile
inflammation (inflammation not caused by microorganisms). This is mediated through the
secretion of high amounts of pro-inflammatory cytokines, including IL-1, IL-6, IL-8, IL-12 and
TNF-, by these cells. Elucidating the complex crosstalk that exists between immune responses
and the nervous system may provide a broad new range of targets for novel therapies, aimed at
reducing inflammation, and improving recovery from stroke.
In the following sections, we will discuss the role of innate immune cells in the development of
sterile inflammation after stroke, focusing on how this impairs recovery and shapes the
generation of an immune-suppressive cell population that further compromises the health of
stroke patients. We will discuss the involvement of these recently-discovered immature myeloid
cells in suppressing immune responses during the sub-acute phase (days) after stroke and how
this cell population could potentially be targeted therapeutically. In addition, we will discuss the
potential of 7-nAChR agonists in affording neuroprotection, resolving inflammation and aiding
in functional recovery.
4. Inflammatory Response of the Immune System PostStroke

Innate immune cells are considered the bodys first line of defence, providing protection from
potentially harmful pathogens. They also have a crucial role in both initiating and advancing
repair processes through the induction of inflammation. Whether this role is associated with
improved recovery or further tissue damage in stroke patients depends partly on the size of the
initial infarct and the ensuing inflammatory response. Post-ischemic inflammatory cascades are
initially prompted by a lack of oxygen, with the resulting metabolic failure leading to
uncontrolled cell death [29]. The membrane of cells undergoing uncontrolled cell death typically
disintegrates and releases cellular content and organelles into the surrounding tissue [30]. The
released cellular contents are then recognised as danger signals by the innate immune system,
namely specialised phagocytic cells, and are crucial for initiating inflammation [31].
The CNS has its own phagocytic innate immune cells, called microglia, that share similarities to
peripheral macrophages. These long-lived microglial cells, which develop from haematopoietic
progenitors that take up residency in the CNS during early embryogenesis, can self-renew, and
proliferate in response to stimuli [32]. Microglia are the first immune cells to detect and
immediately respond to cellular damage caused by stroke, supporting the clean-up of the infarct
area [33]. This is possible due to a wide variety of pattern recognition receptors (PRRs), which
are expressed ubiquitously on the surface and in intracellular compartments of innate immune
cells [34]. PRRs bind pathogens and danger signals, also known as damage-associated molecular
patterns (DAMPs), with high affinity and allow for the rapid detection of changes in the tissue
environment [35]. The binding of DAMPs to PRRs and the subsequent activation of intracellular
signalling pathways within microglia increase the expression of co-stimulatory cell surface
molecules and, finally, the production of pro-inflammatory cytokines by these cells [36]. Both of
these processes are necessary to attract and stimulate adaptive immune cells, called lymphocytes,
which are capable of recognising antigens and forming memory cell populations. Long-lived
memory B- and T-lymphocytes provide long-lasting immune protection and respond rapidly
upon secondary encounter with their cognate antigen [37,38]. However, in the case of brain
injury where adaptive immunity is not required, the secretion of pro-inflammatory cytokines may
tip the balance of the immune response from tissue healing and resolution to destruction. In
contrast to innate immune cells in the periphery, microglia remain chronically activated even in
the absence of initial DAMPs [39,40]. This is associated with the continued secretion of proinflammatory factors that drive chronic neuroinflammation and, thus, contributes to delayed
recovery in stroke patients [41]. Thus, microglia are damaging in the sub-acute and recovery
phase, whereas the initial inflammatory response mediated by these cells is vital in the acute
phase following stroke. This illustrates the biphasic dynamics of inflammatory signalling poststroke, which can be protective or deleterious depending on the context. For example, the
secretion of growth factors and matrix metalloproteinases (MMPs) are vital for tissue reconstruction and vascularisation processes in the sub-acute and recovery phase of stroke, while
being detrimental in the acute phase [42]. In addition to these observations, many other studies
[29] emphasise the biphasic role of inflammation in stroke recovery, highlighting the need to
carefully tailor anti-inflammatory strategies in order to maximise stroke recovery.
In addition to the local response, pro-inflammatory cytokines secreted by microglia reach the
blood stream and attract innate immune cells from within the circulatory system to infiltrate the
damaged tissue, which is facilitated by the breakdown of the blood-brain barrier [43]. Whilst
blood-derived innate immune cells can help with the clearance of damaged tissue in the area of
infarction, they can also exacerbate tissue damage by producing more pro-inflammatory
cytokines and ROS [36].
Consistent with this, recent findings have shown that inflammation impairs sensory learning and
cortical plasticity after stroke [44]. In addition, the depletion of T-cells, especially of CD4+ Tcells, results in increased neurogenesis and accelerates functional recovery [45]. As both synaptic
plasticity and neurogenesis are crucial for recovery after stroke [46,47], specific targeting of
these processes may yield effective therapies. Notably, positive 7-nAChR modulators appear to
be ideal candidates as they can both enhance synaptic plasticity [20] and neurogenesis [48].
However, further studies are required to validate 7-nAChR modulators as an effective treatment
option for stroke before they can be tested in a clinical setting.
4.1. Early Innate Immune Responses Post-Stroke
The activation of microglia through DAMPs and the release of pro-inflammatory cytokines leads
to the expansion of the local microglial cell population and attraction of blood-borne innate
immune cells to the site of injury [49]. The infiltrating cells are generally thought to be
macrophages of myeloid origin based on their high expression of CD45 (lymphocyte common
antigen) and CD11b (cluster of differentiation molecule 11b), whereas microglia exhibit a low
expression of CD45 and can be distinguished from macrophages on this basis [50]. Furthermore,
in parasymbiosis experiments where two mice are surgically joined in order to share blood,
researchers have been able to distinguish infiltrating blood-derived immune cells from local
microglial populations [33,51]. These experiments demonstrated that circulating cells are able to
enter the CNS post-stroke and that resident microglia are the main cell population that expands
in response to injury [51]. Further studies have elucidated the importance of resident microglia in
the immediate response following stroke, but less is known regarding the role of infiltrating
myeloid cells in this context [50,52].
4.1.1. Myeloid Cells
Myeloid cells (monocytes, macrophages, dendritic cells, granulocytes, and erythrocytes) and
lymphoid cells (T- and B-cells, natural killer cells) arise from multipotent haematopoietic stem
cells in the bone marrow. The differentiation of these cells into specialised subsets depends on
the environmental stimuli they are exposed to (e.g., growth factors and cytokines) and/or their
lineage origin [53,54]. Myeloid cells are vital for the response to and clearing of infections,
wound healing, and tissue homeostasis. They respond rapidly to tissue damage and can attract
adaptive immune cells, such as T- and B-cells, to the site of injury. In damaged brain tissue
following stroke, phagocytic myeloid cells play a key part in tissue repair and re-organisation,
facilitating the clearance of necrotic cells and debris. However, despite their crucial roles in the
healing process, myeloid cells can also amplify the inflammatory response and contribute to
secondary tissue damage [55].
Profiling the temporal dynamics of infiltrating immune cells has revealed a massive influx of
phagocytic cells, such as neutrophils, monocytes, dendritic cells (DCs, CD11c+ CD11b+) and
macrophages (CD11c CD11b+), into the CNS around day three, post-stroke [43]. Depending on
environmental stimuli, such as cytokines and inflammatory factors, monocytes circulating in the
blood can migrate to injured tissues and give rise to tissue-resident macrophages and DCs. As
different populations of monocytic cells share similarities in the expression of cell surface
markers, the identification of specific monocytic cell populations has been challenging [56]. For
example, the commonly-investigated cell surface markers CD11c and CD11b are
interchangeably expressed by macrophages and DCs, as well as immature progenitor cells that
egress from the bone marrow after trauma, injury, or infection [57].
Understanding what factors activate myeloid cells and disposes their function from pro-repair to
pro-inflammatory and, hence, is detrimental, requires further investigation. The field of flow
cytometry has advanced rapidly over the last decade and offers tremendous power for the
identification of cell populations using a variety of molecular markers. The analysis of several
molecular markers at once enables greater understanding of the phenotypic and functional
characteristics of specific cell populations, providing the ability to better define their role in
stroke pathology. With an improved understanding of the changes in cell populations, it may be
possible to target myeloid cells using specific modulators to improve functional recovery in
patients.
4.1.2. Immature Myeloid Cells
The role of immature myeloid progenitor cells in stroke has only just started to be investigated
and remains somewhat poorly understood. These cells originate from the bone marrow, where
their development is inhibited in response to an inflammatory stimulus, such as those occurring
in stroke, cancer, trauma, sepsis, and autoimmune diseases. In these circumstances, proinflammatory cytokine release arrests myeloid stem cell differentiation, leading to the expansion
of immature myeloid cells which then migrate into the circulation [57]. While mature
macrophages and DCs initiate and promote innate and adaptive immune responses, these
immature counterparts have been found to have opposing roles and are immune-suppressive
[57]. As a result, they have been termed myeloid-derived suppressor cells (MDSCs) [58]. Liesz
and colleagues demonstrated that immature myeloid progenitor cells egress from the bone
marrow in response to DAMPs, such as high-mobility group box 1 (HMGB1), secreted as a
consequence of stroke [5]. This release of MDSCs from the bone marrow is beneficial following
acute trauma, as they restrain excessive and potentially harmful immune responses in healthy
individuals [59] and promote repair mechanisms, such as angiogenesis [60]. In other diseases,
such as cancer where a potent immune response is beneficial, MDSCs can be detrimental and
facilitate disease progression by suppressing anti-cancer immune responses [61].
Recently, two major subsets of MDSCs have been identified; granulocytic and monocytic
MDSCs, which differentiate from a granulocytic and a monocytic precursors, respectively, in
bone marrow [62,63]. The granulocytic subset shows phenotypic and morphological similarities
to neutrophils and can be identified by high expression of CD11b and lymphocyte antigen Ly6G,
whereas monocytic MDSCs are commonly characterised by high CD11b and Ly6C expression
[63,64]. Both MDSC subsets are able to suppress T-cell responses, impair lymphocyte
trafficking, induce regulatory T-cells, and skew innate immune cells towards an antiinflammatory phenotype [61].
Of significance for stroke recovery, MDSCs promote vascularisation by secreting MMP-9 and 13
[60,65]. The biology and function of MDSCs is too broad and complex for the scope of this
review, however, we refer the reader to these reviews for further details [57,61,65]. It should be
noted that cell surface markers commonly used to study the phenotype of myeloid cells
infiltrating the CNS post-stroke, such as macrophages, neutrophils, DCs and monocytes, are also
expressed by MDSCs. Consequently, there has most likely been incorrect identification of
infiltrating myeloid cells in the past. Therefore, detailed analysis and identification of cell
populations using extensive flow cytometric markers should be employed to further elucidate the
true phenotypic characteristics of infiltrating myeloid cells.
4.2. Infection Post-Stroke
Ischemic stroke is confounded by conditions such as atherosclerosis, diabetes, and infection, all
of which alter peripheral inflammatory processes with a concomitant impact on stroke outcome.
Furthermore, stroke commonly results in peripheral infections that can impede recovery. It has
been reported that 23%65% of patients who have a stroke will subsequently suffer from an
infection, which is also associated with increased mortality and poor patient outcome [66].
Bacteria that cause these infections further stimulate the immune system to produce proinflammatory cytokines, which in turn contribute to secondary brain damage [29]. The rapid
development of inflammation is one of the key elements of stroke pathophysiology and recent
studies have demonstrated that modulation of early inflammatory responses improves recovery
and outcomes after stroke [67,68]. Furthermore, the suppression of inflammation post-stroke as a
therapeutic approach offers the unique advantage of prolonging the therapeutic window and
enables the treatment of patients who do not qualify for thrombolysis. However, the
pharmacological manipulation of inflammatory responses has to be carefully balanced as it may
compromise tissue repair mechanisms. One novel and selective mechanism for dampening the
pro-inflammatory cytokine response during early post-ischemic stroke is the activation of the 7nAChR [68].
Taking into account the aforementioned features of MDSCs, it is possible that they represent a
physiological response that functions to control and dampen inflammation post-stroke (Figure 1).
MDSCs are highly phagocytic [69], they prevent adaptive immune responses [61], support tissue
re-organisation via MMP-mediated angiogenesis [70] and, in general, secrete anti-inflammatory
factors that break the destructive cycle of chronic inflammation. Unfortunately, the actions of
MDSCs are not limited to the CNS where they are desirable, and as a consequence, they can also
affect immune responses occurring in the periphery.
Infections acquired in the days to weeks following a stroke are the major cause for mortality in
stroke patients [71], as they are unable to resolve usually non-life-threatening infections, such as
respiratory and urinary tract infections. Liesz and colleagues have provided what appears to be
the first evidence that MDSCs might be responsible for inducing immune-suppression poststroke [5]. Further, evidence to support the theory that MDSCs are responsible for the observed
immune-suppression after stroke comes from the analysis of gene expression in circulating
neutrophils and monocytes in humans [72]. The expression of arginase 1 (expressed by
monocytic MDSCs), S100A9 (induces generation and expansion of MDSCs), and MMP-9
(secreted by MDSCs), were found to be amongst the genes that showed the highest increase in
expression post-stroke [72]. These findings suggest that MDSCs may circulate in the
bloodstream after stroke and shape systemic inflammatory responses.
4.3. Pattern Recognition Receptors in Stroke
Even though innate immune cells in the brain and periphery have different phenotypes, they all
sense DAMPs through PRRs. In the context of stroke, the toll-like receptors (TLRs) have been
the most extensively studied PRRs due to their ability to activate microglia and peripheral innate
immune cells. TLRs are a family of transmembrane proteins that are located primarily on the cell
surface (TLR 1, 2, 4, 5, 6 and 11) or in intracellular compartments (TLR 3, 7, 8 and 9) [34]. The
downstream effects of TLR-signalling include activation of the adapter molecules myeloiddifferentiation factor 88 (MyD88) or TIR-domain-containing adapter-inducing interferon-
(TRIF) [73]. Stimulation of these pathways results in the activation of pro-inflammatory
signalling cascades that mainly involve the transcription factor nuclear factor (NF)-B. Upon
stimulation of PRRs and downstream adaptor proteins, NF-B translocates to the nucleus and
induces the transcription of genes that code for pro-inflammatory cytokines. Even though
stimulation of some PRRs (such as the TLR4), and the subsequent production of proinflammatory cytokines has been linked to increased infarct size and worse clinical outcome in
stroke patients, the role of NF-B activation as a consequence of stroke remains controversial
[74,75]. Unexpectedly, recent studies suggest that activation of TLR4 is an important aspect of
microglial phagocytosis and correlates with increased neurogenesis after stroke [76,77].
Figure 1. Immature myeloid cells mediate immune-suppression post-stroke. (1) Stroke is

followed by (2) the production of pro-inflammatory cytokines, which reach the bone marrow via
the blood-stream (3) where they stimulate the expansion of immature myeloid cells and partially
block their maturation into granulocytes, macrophages, and DCs. The immature myeloid cells
(also known as MDSCs) migrate along a chemokine/cytokine gradient to secondary lymphoid

organs and to the infarction area where they exert anti-inflammatory effects on other cell
populations. () Immature myeloid cells are able to suppress innate and adaptive immune
responses in the periphery, exposing stroke patients to an increased risk of acquiring infections,
such as pneumonia and urinary tract infections (UTIs), which impede recovery and can
potentially be life-threatening. (+) We hypothesise that immature myeloid cells travelling from
the bone marrow to the brain exert protective effects there. Immature myeloid cells are highly
phagocytic and would help clearing the infarct area of dead cells, and they are known to dampen
inflammation through the secretion of cytokines that skew innate immune cells towards an antiinflammatory phenotype. Furthermore, they are able to induce angiogenesis through the
secretion of MMPs, which facilitates re-vascularisation. All of these effects would be beneficial,
acting to limit excessive inflammation and promote healing. MMPs, matrix metalloproteinases.
TLRs are activated by a multitude of DAMPs, such as heat shock proteins, nucleic acids [78],
purine metabolites (e.g., ATP [2], uric acid [3]), and HMGB1 [79]. In addition, activated
microglia are able to further propagate inflammatory responses by secreting ligands for the
TLR4. One of these recently identified endogenous ligands, carbohydrate-binding lectin galectin3, activates neighbouring immune cells in a paracrine manner [80]. Depending on their nature,
DAMPs bind to specific TLRs, which in turn, influences the type of cytokines secreted. For
example, signalling involving the adapter molecule TRIF, through the binding of double-stranded
RNA to TLR3, stimulates the secretion of type I interferons [81]. In stroke, administration of an
interferon (IFN-) neutralising antibody has been shown to decrease infarct size, as well as the
number of CD3+ cells that infiltrate the infarct [82]. The ambivalence of TLR signalling in stroke
needs further elucidation to clearly identify molecular targets that convey neuroprotection and/or
enhance recovery when modulated at a delay post-stroke.
Instead of targeting individual TLRs to prevent the synthesis of pro-inflammatory cytokines, a
more elegant solution might be to inhibit the common endpoint for all TLRs; the activation of
NF-kB. Inhibiting NF-kB is one of the mechanisms by which 7-nAChR agonists reduce
inflammation [83]. It has been demonstrated that activation of 7-nAChRs on neural microglia,
peripheral macrophages, and monocytes lowers the secretion of pro-inflammatory cytokines,
resulting in reduced neuroinflammation (Figure 2) and improved outcomes in experimental
models of stroke [84,85,86]. Additionally, 7-nAChR agonists stimulate the Janus kinase 2Signal transducer and activator of transcription 3 (JAK2-STAT3) pathway and, in turn, the
secretion of anti-inflammatory cytokines, such as IL-10 [87,88].
Figure 2. Activation of 7-nAChRs reduces inflammation. Damaged and dying cells in the brain
release DAMPs after a stroke. DAMPs can bind to several receptors, including TLRs, cytokine
receptors, and the receptor for advanced glycation end products (RAGE). Ligation of these
receptors activates NF-B to translocate to the nucleus (bold arrow) where it induces the
transcription of genes that code for pro-inflammatory cytokines, such as IL-6, IL-8, TNF- and
HMGB1. In addition, activation of NF-B stimulates the production of the immature forms of
IL-1 and IL-18 (pro-IL-1 and pro-IL-18, respectively). These biologically-inactive cytokines
are cleaved to their active form by inflammasomes and are subsequently secreted from the cell
(dashed arrows). These intracellular multi-protein complexes assemble in response to NF-B
stimulation and require a second stimulus (such as ATP) to become activated. 7-nAChR
agonists interfere with these pathways and prevent the secretion of pro-inflammatory cytokines
by inhibiting NF-B and inflammasome activation (bar-headed red lines). 7-nAChRs, 7nicotinic acetylcholine receptor; DAMPs, damage-associated molecular patterns; TLRs, toll-like
receptors; NF-B, nuclear factor kappa B; IL, interleukin; TNF-, tumour necrosis factor-;
HMGB1, high-mobility group box 1; ATP, adenosine triphosphate.
The release of the cytokine-like protein, HMGB1, from necrotic cells in the infarct area activates
microglia to a pro-inflammatory state, inducing the synthesis of cytokines and other
inflammatory molecules that contribute to further tissue damage [79,89]. Although a reduction of
HMGB1 production in the acute phase improves stroke recovery [90], it is vital for the induction
of repair processes in the sub-acute phase. This involves the promotion of angiogenesis through a
VEGF-dependent mechanism [91], the recruitment of stem cells [92] and the support of
neurovascular remodelling processes mediated by activated astrocytes [93]. Studies have also
reported that HMGB1 is inhibited in the presence of 7-nAChR agonists [83].
Interestingly, recent evidence indicates that inflammation can down-regulate the expression of
7-nAChRs in the brain, which is associated with the accumulation of -amyloid and the
development of Alzheimers disease [94]. This evidence could help explain the increased
prevalence of stroke patients developing dementia and also suggests that inflammation is one of
the primary causes for dementia [94,95]. Nicotine, through its binding to 7-nAChRs, has been
shown to prevent and reverse the accumulation of -amyloid in the brain, highlighting a potential
new therapeutic target for preventing the development of post-stroke dementia [96].
These findings highlight the fact that mediators of inflammation may have biphasic roles in
stroke recovery and even though counteracting inflammatory processes in the acute phase is
beneficial, it may be detrimental later on. Therapeutic approaches using 7-nAChR agonists, for
example, should be carefully tailored to support not only the dampening of inflammatory
cascades but to also allow the enhancement of tissue repair processes.
This seems a particularly promising approach as nAChR agonists up-regulate the cell surface
expression of the receptors, whereas most drug therapies either cause the receptors to become
down-regulated or internalised following sustained exposure to their ligands [97]. Therefore, the
activation of 7-nAChRs may present a unique system to decrease inflammation in the acute
phase after stroke and to also prevent secondary damage.
4.4. Purinergic Signalling in Stroke
nAChR agonists can modulate the signalling of other important endogenous DAMPs, such as
ATP. ATP acts as a universal stress signal, detected by purinergic receptors on a multitude of
immune and non-immune cells [98]. ATP is recognised by G-protein coupled P2Y and P2X
receptors, which are ligand-gated ion channels that regulate the influx and efflux of cations [99].
Interestingly, ATP not only acts as a DAMP but, when secreted at low concentrations, can induce
the controlled digestion of dying cells, which is important to promote the clearance of injured
tissue without the induction of chronic inflammation [100]. In contrast, the sudden secretion of
high concentrations of ATP by dying cells after a stroke triggers a pro-inflammatory response
[101,102]. High ATP concentrations activate intracellular PRRs, which are part of multi-protein
complexes termed inflammasomes. Inflammasomes contain an intracellular PRR, such as the
NOD-like receptor (NLRs), which recognise DAMPs [103]. DAMPs are taken up by
phagocytosis or enter the cytosol via channels in the cell membrane initiated by pore-forming
molecules [104]. ATP activates the inflammasome via binding to P2X receptors, which results in
the efflux of potassium ions [105]. Independent of the type of DAMP, an initial priming step is
necessary to induce the transcription of pro-IL-1 and the assembly of the inflammasome
complex (Figure 3). Molecules that provide the first signal include ligands for TLRs, NLRs,
RIG-1-like receptors (RLRs), and certain cytokine receptors [105].
So far, several nAChR agonists have been identified that inhibit the ATP-dependent activation of
inflammasomes. These molecules are mainly endogenous ligands, such as acetylcholine, choline,
and modifications thereof [106,107]. It has been proposed that binding of the endogenous ligand
acetylcholine modulates ATP-induced changes resulting in inhibition of inflammasome
activation. Lu and colleagues demonstrated that acetylcholine could prevent the ATP-induced
mitochondrial perturbations and thus inflammasome activation by binding to the 7-nAChR in
mitochondria [107]. Furthermore, Hecker and colleagues demonstrated that acetylcholine
interrupted ATP-induced ion currents and thereby prevented inflammasome activation [106].
Both of these results indicate that nAChR ligands interfere with ATP signalling. It remains to be
determined if inflammasome activation can also be blocked if induced by different DAMPs.
Figure 3. Induction of inflammasome activity and pyroptosis after stroke. Signalling through
PRRs results in NF-B activation, which is associated with up-regulated transcription of
inflammasome components (inflammasome proteins, pro-caspase-1, and ASC) and the immature
cytokines pro-IL-1 and pro-IL-18, as well as an increased production of pro-inflammatory
cytokines, such as IL-6, IL-8, and TNF- (signal 1, bold arrow). A second signal (signal 2, bold
arrow) leads to oligomerisation of the inflammasome components and activates the
inflammasome complex. Signal 2 can, for example, occur through binding of ATP to its P2X7
receptor. As a result of inflammasome assembly and activation, caspase-1 gets activated and
cleaves the inactive cytokines pro-IL-1 and pro-IL-18 to their active form, which are released
from the cell (dashed arrows). Caspase-1 is also an important mediator of pyroptosis, a highlyinflammatory form of cell death, which results in the swelling and bursting of cells with the
subsequent release of cell content into the extracellular space. PRRs, pattern recognition
receptors; NF-B, nuclear factor kappa B; ASC, apoptosis-associated speck-like protein
containing a CARD; IL, interleukin; tumour necrosis factor-; ATP, adenosine triphosphate.
The assembly of inflammasomes is required for the processing and secretion of proinflammatory cytokines, such as IL-1, which has been shown to contribute to pathological
diseases such as cancer, diabetes, dementia, and stroke [108,109]. IL-1 has pleiotropic functions
in the inflammatory response, that include the stimulation of non-haematopoietic cells to secrete
chemokines that promote the infiltration of neutrophils, increasing the expression of adhesion
molecules, and induction of cyclooxygenase 2 (COX-2) [110] and inducible nitric oxide synthase
(iNOS) gene expression [111]. Together, these mechanisms trigger an influx of inflammatory
cells from the circulation into regions of tissue damage where they impair the processes
associated with tissue remodelling [112].
4.5. Role of Inflammasomes in Stroke
It has been known for some time that inhibition of IL-1 signalling, downstream of
inflammasome activation, is an effective means to decrease the size of infarction and improve
function after stroke in experimental animal models [109,113,114]. Results from randomised
human clinical trials look promising and indicate that administration of IL-1 antagonists in the
first 72 h post-stroke dampens circulating pro-inflammatory cytokine levels in both blood and
cerebrospinal fluidwhether this will translate into beneficial therapeutic outcomes has yet to be
confirmed [115,116].
The type of inflammasomes involved in the inflammatory response and, thus, in the production
of IL-1 and IL-18 after stroke is slowly being unravelled. The NLRP1 inflammasome was the
first inflammasome identified to form and be activated post-stroke [117]. The NLRP1 complex
consists of the cytoplasmic NACHT leucine-rich repeat protein 1 (NLRP1), the adaptor molecule
apoptosis-associated speck-like protein containing a CARD (ASC), and the pro-inflammatory
caspases-1 and 5 [118]. After injury to the brain, the NLRP1 inflammasome mainly assembles in
neurons, astrocytes, and microglia [117,119]. On the other hand, the NLRP3 inflammasome,
which is commonly associated with the sensing of necrotic cell death, is predominantly
expressed in microglia and endothelial cells [120]. Pharmacological targeting of either the
NLRP1 or NLRP3 inflammasomes has been shown to reduce innate immune responses and
reduce infarct size post-stroke [114,117,119,120]. However, a recent study implied several other
inflammasomes might also play a role in the pathology of stroke [121]. In addition, Denes and
colleagues highlighted the adaptor molecule, ASC, as being a crucial regulator in the
inflammatory response after stroke, whereas inhibition of the other components that form the
NLRP3 inflammasome complex did not improve stroke outcome [121]. In particular, it was
shown that deficiency in the NLRC4 (CARD domain containing 4) or AIM2 inflammasomes
(absent in melanoma 2) was beneficial in reducing inflammation-related tissue damages; both of
these inflammasomes require ASC to recruit and activate caspase-1, which cleaves the immature
cytokines into their biologically active form [121,122]. These recent reports demonstrate the
need to clearly identify if all inflammasomes containing an ASC contribute to tissue damage
post-stroke, as this could become a likely candidate for targeting future drug treatments.
The recent findings that several inflammasomes are involved in the inflammatory response poststroke are particularly interesting in the context of cell death (Figure 3). A recently identified
caspase-1-dependent form of cell death, also known as pyroptosis, is characterised by rapid cell
membrane rupture due to swelling, followed by the release of pro-inflammatory content into the
extracellular space [123]. Cell death due to pyroptosis is highly inflammatory and is associated
with an increase in cell size and the cleavage of genomic DNA [124]. Double-stranded host DNA
segments act as DAMPs and can directly bind to TLR9 and/or the AIM2 inflammasome, which
leads to further activation of caspase-1 and subsequent release of IL-1 and IL-18 [122,125].
The activation of inflammasomes through the sensing of DAMPs in microglia, endothelial cells,
astrocytes, and neurons, is responsible for this detrimental inflammatory cycle. The assembly of
inflammasomes leads to the activation of caspase-1 which, in turn, instigates pyroptotic cell
death. It seems inevitable that the release of pro-inflammatory content into the extracellular
space due to pyroptotic cell death triggers the activation of other inflammasomes, such as the
AIM2 inflammasome, that have been implicated in the inflammatory response post-stroke [121],
fuelling an excessive inflammatory response. As such a process propagates a cycle of cell death,
compounds that can halt this process would likely result in minimised cell death and smaller
infarct size. In accordance with this concept of caspase-1-dependent cell death is the finding that
caspase-1 deficient mice have been found to have smaller ischemic lesions [126]. Recent
evidence also suggests that inflammasome and caspase-1 activation in endothelial cells reduces
angiogenesis after stroke [127]. Angiogenesis is an important repair mechanism after strokeit
not only stimulates re-vascularisation of the damaged tissue but also attracts newly born neurons
to migrate to the site of injury where they co-localise with newly formed blood vessels [128].
5. Conclusions
Inflammatory responses in the acute phase after stroke impair functional recovery but contribute
to tissue repair and re-modelling processes in the sub-acute phase. The biphasic nature of
inflammation has become evident in recent years and this will help to optimise antiinflammatory stroke treatments. A variety of molecules have been shown to effectively inhibit
inflammatory signalling cascades at different levels, which may translate into a reduction of
immune-suppression observed in the weeks after stroke. Particularly interesting are the versatile
effects of 7-nAChR agonists on innate immune cells. The observation that they suppress
inflammation and potentially minimise the risk of stroke-associated morbidities, through the upregulation of 7-nAChRs, make them promising candidates for further study. In addition, the
more thorough characterisation of infiltrating immune cells after stroke will provide further
information on how this can be achieved.
The discovery that MDSCs are the cells that convey immune suppression after stroke will greatly
advance the field of stroke immunology. The phenotype, the mechanisms of action, and the role
of MDSCs in disease progression have been systematically studied in the context of many other
diseases, such as cancer and sepsis, which will aid our understanding of their role in stroke.
Similar to the inflammatory process itself, MDSCs may be shown to play a biphasic role in
stroke recovery. On the one hand, they limit inflammation and initiate vital tissue re-modelling
processes but they also mediate an immune-suppressive state in the periphery responsible for
stroke fatalities. In conclusion, it appears crucial to finely balance the inflammatory response to
sufficiently engage the immune system in tissue repair processes but to also prevent detrimental
effects of excessive inflammation after stroke.
Acknowledgments
This review was written during tenure of a New Zealand Lottery Health Postdoctoral Fellowship
(Silke Neumann, Dunedin, New Zealand) and funding from the Health Research Council of New
Zealand and Royal Society of New Zealand Marsden fund (Andrew N. Clarkson, Dunedin, New
Zealand) and the Australian National Health and Medical Research Council (Mary Chebib,
Thomas Balle, Andrew N. Clarkson, Sydney, Australia).
Author Contributions
Silke Neumann and Andrew N. Clarkson conceived the ideas and prepared the manuscript;
Nicholas J. Shields, Thomas Balle and Mary Chebib contributed to sections of the manuscript,
edited and revised the manuscript; Nicholas J. Shields created the figures; and all authors
reviewed and approved the final version of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Int. J. Mol. Sci. 2016, 17(1), 64; doi:10.

2. Local Immune Responses and Impairment after Stroke

Confoundingly, pre-stroke depletion of Tregs by diphtheria toxin actually reduced infarct

3. Infections after Stroke

4. Stroke-Induced Immune Suppression

5. Other Causes of Infection after Stroke

6. Treatments for Stroke-Associated Infections

In the STROKE-INF study, prophylactic use of clarithromycin in combination with either

glutamate transporter-1 (EAAT2) upregulation. Stroke 2007, 38, 177182. [Google

poststroke pneumonia in experimental stroke: A bed to bench approach. J. Cereb. Blood

nt. J. Mol. Sci. 2015, 16(12), 29029-29046; doi:10.3390/ijms161226141

Department of Pathology, University of Otago, Dunedin 9054, New Zealand

Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia

2. Overview of Nicotinic Acetylcholine Receptors

3. An Inflammatory Reflex Shapes Immune Responses

4. Inflammatory Response of the Immune System PostStroke

Figure 1. Immature myeloid cells mediate immune-suppression post-stroke. (1) Stroke is

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