Molecular and Cellular Neuroscience 53 (2013) 2633

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Molecular and Cellular Neuroscience

journal homepage: www.elsevier.com/locate/ymcne

The effect of stroke on immune function,

Roberta Brambilla a, 1, Yvonne Couch b, 1, Kate Lykke Lambertsen c,, 1
a
b
c

The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, United States
The Department of Pharmacology, University of Oxford, Oxford, UK
The Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

a r t i c l e

i n f o

Article history:
Received 15 March 2012
Accepted 22 August 2012
Available online 30 August 2012
Keywords:
Stroke
Immune responses
Autonomic nervous system
HPA axis

a b s t r a c t
Neurological disorders affect over one billion lives each year worldwide. With population aging, this number is
on the rise, making neurological disorders a major public health concern. Within this category, stroke represents
the second leading cause of death, ranking after heart disease, and is associated with long-term physical disabilities and impaired quality of life.
In this review, we will focus our attention on examining the tight crosstalk between brain and immune
system and how disruption of this mutual interaction is at the basis of stroke pathophysiology. We will
also explore the emerging literature in support of the use of immuno-modulatory molecules as potential
therapeutic interventions in stroke. This article is part of a Special Issue entitled 'Neuroinammation in
neurodegeneration and neurodysfunction'.
2012 Elsevier Inc. All rights reserved.

Introduction
Ischemic stroke results in a multitude of CNS events characterized
ultimately by neuronal and glial cell death, and is also marked by
numerous peripheral events including cardiovascular, endocrine and
immune dysregulation (Emsley et al., 2008; Stevens and Nyquist,
2007). The contribution of the immune system to the development
and progression of cerebral infarcts is well established. However, recent evidence suggests that it may also contribute to recovery and repair in the long term after ischemic damage (Gelderblom et al., 2009;
Hug et al., 2009). Stroke patients face severe immunological challenges while still in intensive care units, so much so that the most
common, fatal, post-stroke complication is pneumonia (Aslanyan et
al., 2004; Johnston et al., 1998; Katzan et al., 2003). Indeed, a recent
meta-analysis has revealed that infection after acute ischemia can
complicate recovery in up to 30% of cases (Westendorp et al., 2011).
While in the past the assumption was that post-stroke infections were
dependent on pre-existing co-morbidities and mismanagement of patient care, it is now clear that post-stroke immunodepression represents an independent factor associated with increased susceptibility to
infections (Emsley et al., 2008).
The authors have no conict of interest.
The authors would like to acknowledge nancial support from the Lundbeck
Foundation and the Danish Medical Research Council to Dr. Kate Lykke Lambertsen
and from The Miami Project to Cure Paralysis to Dr. Roberta Brambilla.
Corresponding author at: Department of Neurobiology Research, Institute of Molecular
Medicine, University of Southern Denmark, J.B. Winslwsvej 21, st., DK-5000 Odense C,
Denmark.
E-mail address: klambertsen@health.sdu.dk (K.L. Lambertsen).
1
All three authors contributed equally to this work.
1044-7431/$ see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.mcn.2012.08.011

Risk factors for developing stroke include co-morbid diseases such

as atherosclerosis, obesity, diabetes, hypertension and peripheral infection (Emsley et al., 2008; Hankey, 2006). Common to all is the association with elevated systemic inammation, which increasing evidence
points at having a causative role in the development of these diseases
(Hansson and Libby, 2006). Several clinical studies have reported
more severe neurological decits in stroke patients with preceding
infection (McColl et al., 2009). Furthermore, elevated systemic concentrations of a number of inammatory markers have been associated
with stroke incidence (Rodriguez-Yanez et al., 2008), emphasizing
the role of inammatory events occurring outside the brain prior to,
during and after stroke, on stroke susceptibility and outcome.
Even though these pre-existing conditions are major contributors
to stroke incidence and physiopathology, this review will specically
focus on the direct effects of stroke on peripheral immune function,
since dysregulation of such immune response has clear negative implications on patient outcome, and a better understanding of these
events is critical in devising appropriate and comprehensive therapeutic strategies for the treatment of stroke patients. The effect of inammation on stroke outcome will be covered separately by Stuart
Allan on the review dealing with the afferent pathways in stroke.
Stroke and central inammation
Since the brain has a very high glucose and oxygen demand, disturbances in the blood supply to the brain rapidly lead to the depletion of
these substrates and the development of an ischemic infarct with accompanying necrosis of neurons, glial cells and small vessels within
the affected territory. Depletion of cellular energy supplies (such as
adenosine triphosphate (ATP)) occurs within minutes, resulting in the

R. Brambilla et al. / Molecular and Cellular Neuroscience 53 (2013) 2633

accumulation of lactic acid within the tissue. Ischemia also leads to the
formation of free radicals that can induce cell damage, and to increased
release of excitatory glutamate (for recent review see (Iadecola and
Anrather, 2011)). ATP depletion and glutamate release result in
uncontrolled calcium-ion inux into the cells leading to activation of intracellular lipases and proteolytic enzymes and, ultimately, destruction
of the cell. The ability of glutamate to kill neurons by excessive activation
of glutamate receptors is referred to as excitotoxicity (Mergenthaler et
al., 2004). The early excitotoxicity induced by the local energy decit
causes fast necrotic cell death in the core area of the infarct (Lipton,
1999). The ischemic penumbra that surrounds the infarct core suffers
milder damage, partly due the numerous collaterals and anastomoses,
which supply the neurons within the penumbra (Astrup et al., 1981).
This area is characterized by compromised blood ow, impaired neuronal functionality, but preserved structural integrity (Astrup et al., 1981).
In addition, astrocytes are more resistant to cerebral ischemia than neurons and react to hypoxia by upregulating their glycolytic capacity
allowing a continued uptake of glutamate from the synaptic cleft in
the penumbral area (Marrif and Juurlink, 1999). It has been observed
that the penumbra has suppressed cortical protein synthesis, but preserved ATP content (del Zoppo et al., 2011; Hossmann, 2006). For
these reasons the penumbral area is still potentially salvageable and,
thus far, has been the target of stroke therapy (del Zoppo et al., 2011).
After ischemia, resident cells, including microglia and astrocytes,
are quickly activated and circulating leukocytes are recruited to the
ischemic lesion. It is believed that, early on, endogenous signals
such as damage-associated molecular patterns (DAMPs; i.e. heat
shock protein (HSP)60, HSP70 and high-mobility-group box-1
(HMGB1)) are released from stressed and dying cells and subsequently bind to toll-like receptors (TLRs), especially TLR2 and TLR4,
located on resident microglia and astrocytes, resulting in downstream
activation of MyD88- and/or TRIF-dependent pathways leading to activation of nuclear factor kappa B- and/or IRF3-dependent gene transcription (for a thorough review on this topic, please refer to Marsh et
al. (2009)). This triggers the synthesis of primarily microglia-derived
pro-inammatory cytokines, such as interleukin (IL)-1, IL-6 and
tumor necrosis factor (TNF) (reviewed in (Lambertsen et al., 2012)),
chemokines (CC and CXC chemokines) (Mirabelli-Badenier et al.,
2011), nitric oxide and reactive oxygen species, which, when present
at high levels, can exacerbate cell death and cause break down of the
bloodbrain barrier (BBB) (for recent review see (Iadecola and
Anrather, 2011)). Cytokines and chemokines also induce the
upregulation of adhesion molecules on the vascular endothelium, favoring diapedesis of circulating leukocytes that may further contribute to brain injury.
Stroke-associated infection
Post-stroke infections represent one of the principal complications
adversely affecting the clinical outcome in stroke patients. Although
some infections may occur as a direct consequence of dysphagia causing aspiration, or may be linked to the advanced age of the patient, it
is increasingly apparent that stroke itself represents a risk factor for
infections due to the induction of a so-called post-stroke immunodepression syndrome, which occurs immediately after stroke
(Chamorro et al., 2007; Vermeij et al., 2009). Indeed, the fact that
the majority of post-stroke infections manifest within three days of
hospitalization is further indication that immunodepression is involved, and infections are not simply a secondary outcome related
to patient care (Westendorp et al., 2011). A recent meta-analysis of
the 87 clinical studies conducted thus far, where rates of post-stroke
infections were examined, has indicated that infection complicated
acute stroke in 30% of patients, with rates varying considerably between 5 and 67% (Westendorp et al., 2011). Pneumonia and urinary
tract infections each occurred in 10% of patients, with pneumonia signicantly associated with death. It is also clear that the severity of

27

post-stroke infections is directly correlated with the magnitude of

the stroke itself, and specically with how extensive the infarct area
is (Hug et al., 2009). The size of the infarct area also parallels the severity of leukocytopenia, which directly correlates with strokeassociated immunodepression. The occurrence of leukocytopenia following stroke has been well documented in patients, with reports
dating back over 40 years (Czlonkowska et al., 1979). Rapid reduction
of lymphocyte counts and functional deactivation of monocytes and
T helper type 1 cells have been observed in acute stroke patients, with
more pronounced immunodepression in patients with severe clinical
decit or large infarction (Haeusler et al., 2008). Lymphocytopenia
does correlate with the occurrence of post-stroke infections (Haeusler
et al., 2008; Hug et al., 2009). In some instances, rather than a generalized
reduction in total lymphocyte counts, only selective lymphocytopenia in
the NK cell subset was reported immediately after stroke (Hug et al.,
2009).
The tight relationship between lymphocytopenia, size of infarct
and the occurrence of post-stroke infections has been demonstrated
also in animal models of stroke. In mice, severely reduced lymphocyte
counts (B cells and CD4 + T helpers, especially) were found in lymphoid organs (spleen and thymus) within 12 h after stroke (Prass et
al., 2003). This was paralleled by spontaneous bacteremia and pneumonia (often leading to death), which were completely prevented
by administration of a sympathetic blocker, but not by inhibition of
the hypothalamic-pituitary-adrenal (HPA) axis (the paravetricular
nucleus in the hypothalamus, the anterior pituitary gland and the cortex of the adrenal glands), suggesting that a catecholamine-mediated
defect in early lymphocyte activation is the key factor in the impaired
antibacterial immune response after stroke (Prass et al., 2003). A recent study by Wong and colleagues has highlighted the role of invariant natural killer T (iNKT) cells in the defense against post-stroke
infections (Wong et al., 2011). Modulation of hepatic iNKT through
blockade of noradrenergic neurotransmitters or directly with administration of -galactosylceramide results in reduced infection and associated lung injury after stroke, demonstrating that these cells act as
conductor of immunity, meaning that their acute responses modulate
and facilitate the adaptive immune response (Wong et al., 2011).
Because of the correlation between post-stroke infections and
clinical outcome, the prophylactic use of antibiotics to prevent such
infections and improve the outcome has been proposed. The data
emerging from clinical studies, however, are contradictory. For example, no benet was found with prophylactic administration of the uoroquinolone levooxacin, a broad-spectrum antibiotic, in acute
stroke patients admitted within 24 h after symptom onset. The rate
of stroke-related infections at 7 days was identical to the placebo
group, and levooxacin administration was directly correlated with
poor clinical outcome (Chamorro et al., 2005). On the other hand,
prophylactic treatment with another uoroquinolone, moxioxacin,
resulted in the signicant reduction of post-stroke infections, although not in the improvement of the clinical outcome (Harms et
al., 2008). This is in contrast with data obtained in a mouse model
of middle cerebral artery occlusion (MCAO), where moxioxacin, a
similar broad-spectrum antibiotic, administration signicantly reduced infarct size (Bao et al., 2010). A possible explanation for the
failure of these molecules in human therapy is the neurotoxicity of
uoroquinolones, which could be offsetting their benecial antimicrobial effect. In contrast, other classes of broad-spectrum antibiotics
(e.g. penicillins and tetracyclines) have shown protective effects. Indeed, prophylactic administration of the broad spectrum semisynthetic penicillin mezlocillin in combination with the beta-lactamase
inhibitor sulbactam decreased incidence and severity of fever within
the rst 34 days after stroke and was associated with a lower rate
of post-stroke infection and improved long term outcome (Schwarz
et al., 2008). Finally, minocycline, a semisynthetic second generation
tetracycline, is the antibiotic that perhaps holds the highest promise.
After being proven effective in numerous experimental models of

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stroke (Hayakawa et al., 2008; Hewlett and Corbett, 2006; Liu et al.,
2007), an open-label placebo-controlled clinical trial found that
minocycline treatment signicantly improved the clinical outcome,
even though it did not protect from post-stroke infections. This
seems to suggest that the protective effect of minocycline in stroke
may not be dependent on its antibacterial properties, but rather on
its anti-inammatory effect, which has been associated with a decrease in microglial activation and nitric oxide production, inhibition
of matrix metalloproteinases (MMPs) (e.g. MMP2, MMP9), and inhibition of apoptotic cell death (Zemke and Majid, 2004). Recently, in
a small exploratory trial minocycline was found to be safe and well tolerated in intravenous doses, either given alone or in combination with
tissue plasminogen activator (tPA) (Fagan et al., 2010), supporting the
rationale for a possible clinical application in stroke.
Studies in animal models have also underscored that post-stroke
infections may be effectively prevented with the use of immunomodulatory molecules (Prass et al., 2003; Wong et al., 2011), rather than
antibiotics, and this approach could be explored in clinical studies,
as it would allow to bypass potential downfalls of prolonged antimicrobial therapy, namely the high incidence of bacterial resistance.

Brain-periphery signaling post-stroke

The activation of the CNS after ischemia results in signaling to peripheral organs, particularly the gut and the liver, which in turn elicit
a substantial immune response. This concept is relatively new and
largely unexplored, and the signaling pathways that mediate these
events are mostly unknown. Signicant delays are known to exist
between the central challenge and the peripheral inammatory response (Blond et al., 2002). Interestingly, in ischemia, the peak central
inammatory phase has been shown to be induced at approximately
24 h post-ischemia, whereas the peak peripheral inammatory phase
occurs as early as 4 h post-ischemia (Chapman et al., 2009). This peripheral inammation is characterized by an acute phase response
(APR) in the liver, where it is reported that close to one thousand
genes are switched on in response to CNS injury (Campbell et al.,
2007).
One mechanism by which the brain communicates with the peripheral immune system is via the HPA axis in concert with the autonomic nervous system (Fig. 1). Interestingly, cytokines such as TNF,
IL-1 and IL-6 are pivotal in the cross talk between brain and immune
system. Indeed, they activate the autonomic nervous system and the
HPA axis, where release of catecholamines and glucocorticoids can
modulate the immune function (Turnbull and Rivier, 1999). It is believed that cytokines known to be signicantly increased after stroke,
such as IL-1 (Clausen et al., 2005), act on the hypothalamus (Haddad
et al., 2002; Uehara et al., 1987) leading to release of corticotropin releasing hormone (CRH) into the hypophyseal portal blood supply.
Here CRH stimulates the secretion of adrenocorticotropic hormone
(ACTH) from the anterior pituitary gland (Anne et al., 2007), which
in turn circulates to the adrenal glands. Any imbalance in the homeostasis of this system caused by cerebral ischemia may result in activation of the HPA axis, causing increased production of glucocorticoids
from the adrenal glands (Krugers et al., 1995). Since immune cells express receptors for hormones and neurotransmitters (Heijnen, 2007),
it is believed that the HPA axis, via glucocorticoid release, can lead to
lymphopenia and lymphocyte dysfunction, particularly monocyte deactivation (Fig. 1) potentially resulting in bacteremia and pneumonia
in stroke patients (reviewed in (Prass et al., 2003)). Furthermore,
overstimulation of both the sympathetic (SNS) and parasympathetic
(PNS) nervous systems results in increased circulating levels of both
catecholamines and acetylcholine (Ach) (Anne et al., 2007; Elenkov
et al., 2000; Franceschini et al., 1994) (Fig. 1). The detrimental effects
of these neurotransmitters on the peripheral immune system is
discussed in detail below.

Hepatic signaling pathways

Very little is known about the pathways responsible for turning on
the genes of the APR in the liver after brain injury. Below, we will review those known to be involved in the activation of the HPA axis,
and the autonomic nervous system (both sympathetic and parasympathetic) as well as emerging putative mechanisms.
In terms of neuronal activation, CNS injuries have previously been
shown to switch-on neuronal pathways, i.e. initiate neurotransmission. This CNS activation has been shown to be mediated by cholinergic efferents to the liver from the brain and can be attenuated by vagal
nerve lesion (Ottani et al., 2009). This suggests that the brain is
switching on neurotransmission in order to signal injury to the periphery. This relatively recent idea has been dubbed central neurogenic neuroprotection and suggests the existence of both central
adrenergic and cholinergic circuits within the brain that protect neurons from the aftermath of CNS injury, and is therefore particularly
relevant in stroke research (Feinstein et al., 2002; Galea et al., 2003).
The role of the autonomic nervous system in the communication
between brain and periphery has been described by a number of
studies. As for the PNS, peripheral vagal stimulation signicantly reduces lesion volume in a rat model of stroke (Hayakawa et al.,
2008). As for the SNS, administration of clenbuterol, a brain penetrant
-adrenergic agonist, shows protection in animal models of
excitotoxicity, demonstrating both the anti-inammatory potential
of the autonomic nervous system and the concept of neurogenic
neuroprotection (Ryan et al., 2011). In addition, studies show that
changes in infusion rates from the left or right carotid arteries determine whether tachycardia (sympathetic activation) or bradycardia
(parasympathetic activation) will develop. Patients with right-sided
stroke affecting the insular cortex most frequently show tachycardia,
suggesting an increase in sympathetic activation (Colivicchi et al.,
2004; Tokgozoglu et al., 1999).
The autonomic nervous system is also capable of directly stimulating immune cells. Indeed, adrenergic receptors such as the
-adrenoceptor, are expressed on T helper type-1 (Th1) lymphocytes,
B cells and macrophages (Kohm and Sanders, 2001; Mackroth et al.,
2011). In the periphery, catecholamines, such as noradrenaline, are
released during stress, a common phenomenon after acute stroke
(Anne et al., 2007; Oto et al., 2008). Therefore, it is not unreasonable
to postulate that systemic release of cytokines after stroke might be
mediated, in part, by the autonomic nervous system and catecholamines (Marz et al., 1998). Both locally produced cytokines in the
brain, and direct brain stem irritation (by local cytokines or compression) can trigger strong sympathetic activation and release of catecholamines which, in turn, can lead to systemic release of cytokines
(Marz et al., 1998; Woiciechowsky et al., 1998, 1999). While these
studies have shown that the activation of the SNS is capable of profound immunomodulation, there remains some confusion over the
impact this will have on outcome, some maintain that inhibition of autonomic signaling can produce benecial outcomes after stroke
(Savitz et al., 2000). The direct pathways of communication between
the injury site and the SNS immediately post-stroke are currently
speculative. However, it is clear that some uncoupling occurs between the CNS, SNS and their control of the immune system during
the immediate post-stroke period.
The main target of PNS pathways, such as output from the vagus,
is the nicotinic acetylcholine receptor 7 subunit (nAChR7), which
is expressed on both neurons and resident immune cells such as macrophages and microglia (Shytle et al., 2004). This affects inammation
via the nuclear factor kappa B (NF-B) pathway (Borovikova et al.,
2000). Stimulation of the peripheral immune system of vagotomized
mice has lead to the conclusion that expression of nAChR7 on
Kupffer cells enables the hepatic branch of the vagus to suppress
the production of reactive oxygen species in these cells (Hiramoto
et al., 2008). This top down immune suppression has been conrmed
in nAChR7 knockout mice, which show signicantly elevated levels

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29

Fig. 1. Schematic representation of the communication pathways between the stroke-injured brain and the peripheral immune system. Pro-inammatory cytokines stimulate neurons in
the paraventricular nucleus of the hypothalamus to secrete CRH, which then facilitates the release of ACTH from the anterior pituitary. This leads to the release of GCs from the adrenal
cortex resulting in suppression of the production of pro-inammatory mediators and facilitates the release of anti-inammatory mediators resulting in a classic negative feedback loop.
The SNS also plays an important role in the communication between the stroke injured brain and the peripheral immune system. Activation of the SNS causes the release of CAs from sympathetic nerve terminals and from the adrenal medulla resulting in inhibition of Th1 pro-inammatory activities, giving way to the predominance of Th2 anti-inammatory activities. Also
activation of the parasympathetic nervous system is believed to play an important role in the communication between the injured brain and the immune system. Activation via the vagus
nerve, the cholinergic anti-inammatory pathway, results in release of ACh acting on cholinergic receptors on macrophages leading to a decrease in the production of anti-inammatory
cytokines. ACTH, adrenocorticotrophic hormone; APR, acute phase response; BBB, blood brain barrier; CAs, catecholamines; CRH, corticoreleasing hormone; GCs, glucocorticoids; HPA,
hypothalamic-pituitary-adrenal; IL, interleukin; IL-1Ra, interleukin-1 receptor antagonist; M, macrophages; NA, noradrenaline; NK, natural killer; SNS, sympathetic nervous system;
TGF, transforming growth factor beta; Th, T helper cells; TNF, tumor necrosis factor.

of TNF and IL-6 in response to immune challenge (Fujii et al., 2007).

Oxygen deprivation in nAChR7 knockout animals results in increased damage, suggesting that this receptor is responsible not
only for the peripheral inammatory response, but also for regulating
inammatory output within the CNS (Egea et al., 2007). These data
suggest that central activation of the PNS by stroke will largely depress the immune system, whereas activation of the SNS will largely
activate it.
One potential, and novel, mechanism of inammatory output from
the CNS is the microparticles (MPs). These are membrane-derived
vesicles produced as a result of cell stress and can be released through
blebbing from a variety of different cell types including neutrophils,
endothelial cells and microglia. These microparticles would theoretically be small enough to cross an intact blood brain barrier and thus
injury in the CNS would be able to use them as a mechanism of communicating with the periphery. Recent advances have allowed MPs to
be phenotyped, showing not only the cellular origin of the MPs but
also distinct populations after specic types of injury. Such phenotypic differences may suggest a mechanism of discreet communication
between the CNS and the peripheral immune system. Studies in

central inammatory diseases such as cerebral malaria have shown

that MPs can be used as markers of cerebral dysfunction (Pankoui
Mfonkeu et al., 2010). However, data from acute stroke patients
have been less promising so far (Williams et al., 2007) since no differences were found in MP characteristics. While these studies could not
distinguish between stroke and stroke-mimic patients (those who
present the symptoms of a stroke but do not show any underlying
vascular anomalies), in other instances the phenotype of MPs has
been successfully used to discriminate between cerebrovascular
events (Haeusler et al., 2008). While work studying MPs and their potential as a communication pathway between the CNS and the periphery is currently in its infancy, the promising studies using central
inammatory diseases such as cerebral malaria pave the way for interesting future work.
Braingut axis
The gut is composed of three key immunological components: the
gut epithelium (the rst point of contact for foreign bodies), the mucosal
immune system (a particularly sensitive site high in IgA-positive cells),
and the gut microora (commensal bacteria that live in a symbiotic

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relationship with the host). Within the mucosal immune system,

lymph drainage occurs at Peyer's patches, sites where high numbers
of immune cells gather. Interestingly, studies using the permanent
MCAO model of stroke in rats have demonstrated stark alterations
in the gut mucosa post-ischemia (Tascilar et al., 2010). The height
and depth of the intestinal villi, a critical factor critical for mucosal
integrity, were shown to be damaged by permanent MCAO, potentially allowing indigenous gut bacteria to migrate into otherwise
sterile body cavities (Tascilar et al., 2010). This work has been corroborated in animal models (Maes et al., 2008; Schulte-Herbruggen et al.,
2009), where it has been suggested that stress, and therefore possibly
the HPA axis, is a signicant contributing factor to bacterial translocation (Tascilar et al., 2010).
Studies specically investigating the intestinal immune system
have shown decreased levels of lymphocytes in Peyer's patches following cerebral ischemia (Schulte-Herbruggen et al., 2009). These
data are in line with numerous studies reporting changes in T and B
cell numbers and functionality after stroke, which will be discussed
below. The exact role of lymph drainage from the intestines has yet
to be elucidated (Newberry, 2008), but current data suggest that it
likely provides a tolerant immune barrier between gut bacteria and
the systemic circulation, as well as protection against pathogenic invaders. Thus far, there are no data regarding the exact mechanisms
of communication between the brain and the gut immune system,
but it seems reasonable to assume that changes, at least in circulating
lymphoid cells, would be communicated to all systemic immune systems. However, the route by which central inammatory events affect the mucosal epithelium is currently speculative.
Splenic response to stroke
While little is known about braingut communication and the potential for physiological failure in the immediate post-stroke period,
the data available regarding changes in the spleen are much more robust. Since the spleen is the main storage facility for large numbers of
immune cells, spleen alterations after ischemic events have the potential to induce severe perturbation of the immune function. The initial
observation of reduced cellularity in the spleens of rats subjected to
cerebral ischemia (transient MCAO) was made by Gendron et al.
(2002), who reported that in ischemic animals the total number of
spleen leukocytes was signicantly decreased compared to sham operated controls from day 2 to day 28 post-ischemia. A similar pattern of
splenocyte loss was also observed in mice subjected to transient
MCAO, and attributed to increased apoptotic cell death of all lymphocyte populations (Prass et al., 2003). Interestingly, later studies by
Offner and colleagues painted a more complex picture of the splenic response to stroke (Offner et al., 2006a,b). They found the acute phase
after stroke (1 to 22 h) to be characterized by sustained splenocyte activation with increased expression of pro-inammatory cytokines and
chemokines (e.g. TNF, IL-6, IL-2, interferon gamma (IFN), CXCL2,
CXCL10), mild apoptotic death of splenocytes and limited spleen weight
loss (Offner et al., 2006a). This could help explain the increased systemic inammation observed immediately after stroke, which correlates
with the development of increased secondary damage at the infarct
site in the brain. At a later post-injury time (96 h) a drastic reduction
in spleen weight as a consequence of reduced cellularity has been
observed, and this was associated with massive apoptotic death of
splenocytes (Shimizu et al., 1999), mostly B cells and CD4+ T effector
subsets.
The early splenic response after stroke involves increased production of pro-inammatory mediators and the deployment of proinammatory monocytes into the blood, leading researchers to suggest splenectomy as a possible prophylactic intervention for cerebral
ischemia (Izci, 2010). Indeed, splenectomized rats display a signicantly reduced infarct volume after permanent MCAO compared to
non-splenectomized rats, and this is accompanied by a signicant

reduction in activated microglia and inltrating macrophages

(Ajmo et al., 2008). Although this approach may be useful in the
short term, questions remain as to whether this could lead to
detrimental effects in the long term, given the role of the spleen in
normal immune function, especially in stroke patients who are
intrinsically immuno-suppressed and less equipped to ght infectious complications.
Rather than ablation of the spleen, a strategy adopted by others
consisted in replenishing the spleen with human umbilical cord
blood cells (HUCBC) transfused after MCAO. HUCBC localize to the
spleen counteracting MCAO-induced spleen atrophy, and migrate to
the site of injury in the brain, signicantly reducing infarct size
(Newcomb et al., 2006; Vendrame et al., 2004). Moreover, transfused
HUCBC appeared to switch splenic cytokine expression from a proinammatory (TNF, IL-1) to an anti-inammatory (IL-10) prole
(Vendrame et al., 2004), which could be the key to the therapeutic
effect of HUCBC in these experimental models of stroke.
It has been proposed that one of the mechanisms sustaining splenic activation and the release of macrophages from the spleen into circulation after ischemic stroke is the activation of the SNS. Indeed,
besides the increase in systemic catecholamine levels released into
the circulation from the adrenal medulla as described above, MCAO
also results in elevated catecholamine levels in the spleen through direct splenic innervations (Young et al., 1983). Studies by Ajmo et al.
(2009) have shown that only blockade of and adrenergic receptors, but not spleen denervation, prevented spleen atrophy and reduced infarct volume, suggesting that it is the increased systemic
catecholamine level resulting from adrenal release that regulates
the splenic response after stroke.
Collectively, these studies point at a key crosstalk between spleen
and ischemic brain beginning at the very early stages of injury. Acutely,
the spleen acts as a reservoir of pro-inammatory immune cells
ready to be deployed into the blood immediately after stroke.
These cells ultimately reach the infarct area of the brain and contribute to the propagation of secondary damage. At this stage, strategies
aimed at containing the splenic response may be benecial. On the
other hand, long-term suppression of the splenic response may
hamper physiologic immune function and limit the ability to ght
infections, compromising the chances of recovery of stroke patients.
In conclusion, it appears clear that strategies aimed at modulating
the splenic response to stroke may be extremely effective in containing the propagation of secondary damage within the CNS, as
long as they are delivered in a timely fashion.
Post-ischemia immune cell function
As discussed above, the potential for the immune system to have
deleterious effects on infarct volume and stroke outcome is numerous.
However, recent studies in both mouse models and patient cohorts indicate that dysregulation of CNS homeostasis by ischemia can have
long-lasting effects on peripheral immunity. This dysregulation causes
a central inammatory cascade capable of eliciting a peripheral immune
response. As mentioned previously, the peak central inammatory period is approximately 24 h post-ischemia, whereas the peak peripheral
inammatory period is approximately 4 h post-ischemia (Chapman et
al., 2009). Discrepancies between the peak response times may allow
for the mobilization of peripheral immune cells, which have been
shown to play a crucial role in infarct size (Gelderblom et al., 2009).
During stroke, disruption of the BBB allows for the inux of circulating
peripheral immune cells such as macrophages, neutrophils and lymphocytes, each with specic temporal patterns (Nilupul Perera et al.,
2006). There is evidence that immuno-modulatory molecules capable
of blocking or limiting the inux of these cell populations into the CNS
are effective in preventing the evolution of stroke damage in animal
models. To this effect, administration of the sphingosine 1-phosphate
(S1P) analog FTY720 (ngolimod), which induces depletion of

R. Brambilla et al. / Molecular and Cellular Neuroscience 53 (2013) 2633

circulating lymphocytes by preventing their egress from the lymph

nodes, can reduce lesion size and improve neurological outcome after
stroke in mice (Czech et al., 2009). This is due to a reduced invasion of
immune cells into the lesion, in addition to a direct neuroprotective effect. In similar fashion, other immuno-modulatory agents affecting cell
entry in the CNS have shown effectiveness. Administration of antiCD11b antibody, for example, was shown to prevent the inltration of
polymorphonucleate cells (neutrophils and monocyte/macrophages)
into the ischemic tissue (Chen et al., 1994a, 1994b; Chopp et al.,
1994), signicantly reducing infarct size and neurological decit. Similarly, antibodies against the adhesion molecule ICAM-1 (endothelium
surface) or its receptor CD18 (leukocyte surface), whose interaction is
essential for leukocyte binding to the endothelium and subsequent
transendothelial migration, showed effectiveness in limiting stroke
damage and promoting functional recovery (Bowes et al., 1993; Clark
et al., 1991a,b; Jiang et al., 1994, 1995; Zhang et al., 1995a,b). These
encouraging data in experimental models prompted the initiation
of a clinical trial in stroke patients with the anti-ICAM-1 antibody
enlimomab which, contrary to expectations, resulted in signicant
worsening of the clinical outcome (Enlimomab Acute Stroke Trial
Investigators, 2001). This demonstrates the complex role of immune
cells in the development of stroke, and underscores that not only do
they have pro-inammatory functions potentially detrimental to recovery, but also protective functions, particularly against stroke-related
infections (as discussed above), and indiscriminate ablation of such
cells may not represent the best course of action. Additionally, a better understanding of the temporal patterns of entry of each specic population
may be useful in the implementation of the appropriate immunomodulatory strategy.
In spite of the inux of peripheral immune cells into the brain there is
little evidence of damaging autoimmunity after stroke. The presentation
of CNS antigens to T and B cells should induce the production of
autoantigens, and in fact in patients with a history of stroke, high numbers of T-cells reactive to CNS antigen can be found in the circulation
(Bornstein et al., 2001). Offner and colleagues have shown that lack of
an adaptive immune response will reduce infarct size suggesting that
T and B-cells do contribute to lesion volume (Offner et al., 2009). However, the re-establishment of the BBB post stroke means that autoreactive
immune cells will have little or no opportunity to interact with CNS
antigens during the recovery period. Interestingly, this has shown to be
worsened in animal models using a peripheral inammogen such as
LPS (Becker et al., 2005).
Despite previous lateralization studies, work in both humans (Nilupul
Perera et al., 2006) and animal models (Dirnagl et al., 2007) has demonstrated that peripheral immune response post-ischemia is dependent
not on anatomical location, but on infarct size. Work by Hug and colleagues have corroborated this nding, further showing that infarct volume directly correlates with post-stroke immune competence (Hug et
al., 2011). Experimentally, this presents a problem, since both transient
and permanent MCAO models tend to produce large infarct volumes, affecting both cortical and subcortical regions. Importantly, infarct size also
seems to be reected by concentrations of CNS antigens myelin basic
protein (MBP), creatine kinase-BB, neuron-specic enolase, S100beta,
neurolaments and portions of N-methyl-D-aspartate receptor in serum
samples from stroke patients (Bornstein et al., 2001; Dambinova et al.,
2003; Jauch et al., 2006). Lymphocytes from these patients show more
reactivity against MBP than lymphocytes from multiple sclerosis patients
(McQuillan et al., 2011; Mfonkeu et al., 2010). Since it is acknowledged
that an ischemic attack is a risk factor for developing dementia, this underpins the hypothesis that autoimmune responses to the brain in stroke
patients might contribute to the cognitive decline and progression of
white matter disease seen in some stroke patients (Pendlebury and
Rothwell, 2009). Thus, any attempt at immunomodulatory therapy in animals or humans, must be approached with caution given the paucity of
knowledge regarding the dysregulation of specic subsets of peripheral
immune cells after ischemic events of differing intensities.

31

T and B cells post-stroke

The recent interest in lymphocyte function after stroke is mainly due
to the increased likelihood of encounters between lymphocytes and
CNS antigens immediately after stroke. To date, research suggests that
immunodepression is largely caused by lymphocytopenia, abnormally
low levels of circulating lymphocytes immediately after stroke. However, work has failed to suggest a mechanism for this phenomenon, and
data in human and animal models appear to be conicting. Hug et al.
have shown in both humans and animal models that T-cell proliferation
and activation ex vivo are not affected by ischemia (Hug et al., 2011).
Nevertheless, reduced T cell numbers after stroke, as well as generalized
splenic atrophy, have been demonstrated in a number of studies (Hug
et al., 2011; Offner et al., 2006b; Vogelgesang et al., 2010). It has been
suggested that this lymphocytopenia is due to increased apoptosis
within all T-cell populations (Chamorro et al., 2007). Recent work on
Tregs has shown that they have potential to provide a degree of
immunoprotection but that their co-stimulatory activity may be detrimentally affected by stroke through mechanisms as of yet uncovered
(Hug et al., 2011).
While leukocyte populations in the spleen have been shown to be
reduced 4 days after stroke, CD4+CD25+Foxp3+ T regulatory cells
(Tregs) and CD11b+ monocytes in the blood were highly increased at
this time (Offner et al., 2006b), suggesting that splenic atrophy is not
only dependent upon splenocyte apoptosis but is also partly due to the
migration of cells out of the spleen and into the blood to then travel to
the injured CNS. Indeed, CD11b+ monocytes can eventually inltrate
the brain parenchyma at the site of infarct and further extend the secondary damage to the CNS tissue. The drastic reduction in splenic
T and B cells must be a contributing factor to the immuno-suppression
observed following stroke, which has been linked to the increased susceptibility to infections of stroke patients (Harms et al., 2008). The abnormal production of Tregs could also represent an immunosuppressing
factor in itself. Indeed, several studies now indicate that an excessive
Treg presence may impede immuno-surveillance against tumor cells
and may suppress the ability of CD4+ effector T cells to, for example,
eliminate parasites (Belkaid et al., 2002; Mackroth et al., 2011).
More recently, the contribution of regulatory B cells in experimental stroke has been uncovered (Ren et al., 2011). Previous studies had
described the potent regulatory effects of B lymphocytes on inammatory responses (LeBien and Tedder, 2008) and depletion of B
cells worsened disease severity in models of multiple sclerosis
(Matsushita et al., 2010). Ren and colleagues were the rst to identify
IL-10-secreting B regulatory cells as a major protective cell type in
stroke. Indeed, B cell deciency exacerbated stroke outcomes and
dramatically increased inammatory cell invasion into the brain,
suggesting that enhancement of regulatory B cells could have therapeutic applications in stroke (Ren et al., 2011).
Other aspects of the peripheral immune system may also be affected
by ischemic events in the CNS. Offner's work demonstrating splenic atrophy also showed an increase in circulating monocytes (Offner et al.,
2006b). This has been suggested to be a clean-up operation by the peripheral immune system (Manoonkitiwongsa et al., 2001), in order to remove necrotic tissue. However, considering the role of the macrophage
in the innate immune response to infection the increased susceptibility
to infection post-stroke seems rather counter-intuitive. Other data
show that the expression prole of genes on the surface of macrophages
and neutrophils is altered after stroke (Tang et al., 2006), suggesting the
possibility of immunomodulation, rather than immunodepression.
Conclusion
The nature of the post-ischemic immune response is multifaceted
and complex, resulting in immunomodulation as well as immunodepression. Factors such as current immune status and infarct volume
are capable of directly affecting the function of the immune system immediately after stroke and therefore, indirectly, affecting recovery and

32

R. Brambilla et al. / Molecular and Cellular Neuroscience 53 (2013) 2633

repair from stroke. The window of opportunity for outside immunomodulatory therapy after stroke is relatively narrow and the current
paucity of knowledge regarding the consequences of such therapies
means that they are often unwise. However, current work is aiming to
further our knowledge of the immune system post-stroke and should
provide us with ample opportunity to improve clinical outcomes in
the future.
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