Molecular and Cellular Neuroscience 53 (2013) 26–33

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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
The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, United States
b
The Department of Pharmacology, University of Oxford, Oxford, UK
c
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 a b s t r a c t

Article history: Neurological disorders affect over one billion lives each year worldwide. With population aging, this number is
Received 15 March 2012 on the rise, making neurological disorders a major public health concern. Within this category, stroke represents
Accepted 22 August 2012 the second leading cause of death, ranking after heart disease, and is associated with long-term physical disabil-
Available online 30 August 2012
ities and impaired quality of life.
In this review, we will focus our attention on examining the tight crosstalk between brain and immune
Keywords:
Stroke
system and how disruption of this mutual interaction is at the basis of stroke pathophysiology. We will
Immune responses also explore the emerging literature in support of the use of immuno-modulatory molecules as potential
Autonomic nervous system therapeutic interventions in stroke. This article is part of a Special Issue entitled 'Neuroinflammation in
HPA axis 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, re-
cent evidence suggests that it may also contribute to recovery and re-
pair in the long term after ischemic damage (Gelderblom et al., 2009;
Hug et al., 2009). Stroke patients face severe immunological chal-
lenges 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 pa-
tient care, it is now clear that post-stroke immunodepression repre-
sents an independent factor associated with increased susceptibility to
infections (Emsley et al., 2008).
Risk factors for developing stroke include co-morbid diseases such
as atherosclerosis, obesity, diabetes, hypertension and peripheral infec-
tion (Emsley et al., 2008; Hankey, 2006). Common to all is the associa-
tion with elevated systemic inflammation, 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 deficits in stroke patients with preceding
infection (McColl et al., 2009). Furthermore, elevated systemic concen-
trations of a number of inflammatory markers have been associated
with stroke incidence (Rodriguez-Yanez et al., 2008), emphasizing
the role of inflammatory 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 specifically
focus on the direct effects of stroke on peripheral immune function,
since dysregulation of such immune response has clear negative im-
plications on patient outcome, and a better understanding of these
events is critical in devising appropriate and comprehensive thera-
peutic strategies for the treatment of stroke patients. The effect of in-
flammation on stroke outcome will be covered separately by Stuart
Allan on the review dealing with the afferent pathways in stroke.

Stroke and central inflammation

☆ The authors have no conflict of interest.
☆☆ The authors would like to acknowledge financial support from the Lundbeck
Foundation and the Danish Medical Research Council to Dr. Kate Lykke Lambertsen Since the brain has a very high glucose and oxygen demand, distur-
and from The Miami Project to Cure Paralysis to Dr. Roberta Brambilla. bances in the blood supply to the brain rapidly lead to the depletion of
⁎ Corresponding author at: Department of Neurobiology Research, Institute of Molecular these substrates and the development of an ischemic infarct with ac-
Medicine, University of Southern Denmark, J.B. Winsløwsvej 21, st., DK-5000 Odense C,
Denmark.
companying necrosis of neurons, glial cells and small vessels within
E-mail address: klambertsen@health.sdu.dk (K.L. Lambertsen). the affected territory. Depletion of cellular energy supplies (such as
1
All three authors contributed equally to this work. adenosine triphosphate (ATP)) occurs within minutes, resulting in the

1044-7431/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.mcn.2012.08.011
 R. Brambilla et al. / Molecular and Cellular Neuroscience 53 (2013) 26–33
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 influx into the cells leading to activation of in-
tracellular 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 deficit
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 flow, impaired neuro-
nal functionality, but preserved structural integrity (Astrup et al., 1981).
In addition, astrocytes are more resistant to cerebral ischemia than neu-
rons 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 pre-
served 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 subse-
quently 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 ac-
tivation of nuclear factor kappa B- and/or IRF3-dependent gene tran-
scription (for a thorough review on this topic, please refer to Marsh et
al. (2009)). This triggers the synthesis of primarily microglia-derived
pro-inflammatory cytokines, such as interleukin (IL)-1β, 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
blood–brain barrier (BBB) (for recent review see (Iadecola and
Anrather, 2011)). Cytokines and chemokines also induce the
upregulation of adhesion molecules on the vascular endothelium, fa-
voring diapedesis of circulating leukocytes that may further contrib-
ute 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 caus-
ing 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 immuno-
depression 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 in-
volved, 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 be-
tween 5 and 67% (Westendorp et al., 2011). Pneumonia and urinary
tract infections each occurred in 10% of patients, with pneumonia sig-
nificantly 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 specifically with how extensive the infarct area
is (Hug et al., 2009). The size of the infarct area also parallels the se-
verity of leukocytopenia, which directly correlates with stroke-
associated immunodepression. The occurrence of leukocytopenia fol-
lowing 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
deficit 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 lym-
phoid organs (spleen and thymus) within 12 h after stroke (Prass et
al., 2003). This was paralleled by spontaneous bacteremia and pneu-
monia (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 cor-
tex 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 re-
cent study by Wong and colleagues has highlighted the role of invari-
ant 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 admin-
istration of β-galactosylceramide results in reduced infection and as-
sociated 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 exam-
ple, no benefit was found with prophylactic administration of the flu-
oroquinolone levofloxacin, 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 levofloxacin administration was directly correlated with
poor clinical outcome (Chamorro et al., 2005). On the other hand,
prophylactic treatment with another fluoroquinolone, moxifloxacin,
resulted in the significant reduction of post-stroke infections, al-
though 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 moxifloxacin, a
similar broad-spectrum antibiotic, administration significantly re-
duced infarct size (Bao et al., 2010). A possible explanation for the
failure of these molecules in human therapy is the neurotoxicity of
fluoroquinolones, which could be offsetting their beneficial antimi-
crobial effect. In contrast, other classes of broad-spectrum antibiotics
(e.g. penicillins and tetracyclines) have shown protective effects. In-
deed, prophylactic administration of the broad spectrum semisyn-
thetic penicillin mezlocillin in combination with the beta-lactamase
inhibitor sulbactam decreased incidence and severity of fever within
the first 3–4 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
28 R. Brambilla et al. / Molecular and Cellular Neuroscience 53 (2013) 26–33
stroke (Hayakawa et al., 2008; Hewlett and Corbett, 2006; Liu et al.,
2007), an open-label placebo-controlled clinical trial found that
minocycline treatment significantly 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-inflammatory effect, which has been associated with a de-
crease in microglial activation and nitric oxide production, inhibition
of matrix metalloproteinases (MMPs) (e.g. MMP2, MMP9), and inhi-
bition of apoptotic cell death (Zemke and Majid, 2004). Recently, in
a small exploratory trial minocycline was found to be safe and well tol-
erated 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 immunomod-
ulatory 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 antimi-
crobial therapy, namely the high incidence of bacterial resistance.
Brain-periphery signaling post-stroke
The activation of the CNS after ischemia results in signaling to pe-
ripheral 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. Significant delays are known to exist
between the central challenge and the peripheral inflammatory re-
sponse (Blond et al., 2002). Interestingly, in ischemia, the peak central
inflammatory phase has been shown to be induced at approximately
24 h post-ischemia, whereas the peak peripheral inflammatory phase
occurs as early as 4 h post-ischemia (Chapman et al., 2009). This pe-
ripheral inflammation 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 pe-
ripheral immune system is via the HPA axis in concert with the auto-
nomic nervous system (Fig. 1). Interestingly, cytokines such as TNF,
IL-1β 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 be-
lieved that cytokines known to be significantly 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 re-
leasing 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 homeo-
stasis of this system caused by cerebral ischemia may result in activa-
tion of the HPA axis, causing increased production of glucocorticoids
from the adrenal glands (Krugers et al., 1995). Since immune cells ex-
press 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 de-
activation (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 re-
view those known to be involved in the activation of the HPA axis,
and the autonomic nervous system (both sympathetic and parasym-
pathetic) 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 neurotransmis-
sion. This CNS activation has been shown to be mediated by choliner-
gic 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 pe-
riphery. This relatively recent idea has been dubbed ‘central neuro-
genic neuroprotection’ and suggests the existence of both central
adrenergic and cholinergic circuits within the brain that protect neu-
rons 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 significantly re-
duces lesion volume in a rat model of stroke (Hayakawa et al.,
2008). As for the SNS, administration of clenbuterol, a brain penetrant
β-adrenergic agonist, shows protection in animal models of
excitotoxicity, demonstrating both the anti-inflammatory 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 deter-
mine 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 stimulat-
ing immune cells. Indeed, adrenergic receptors such as the
β-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 catechol-
amines (Marz et al., 1998). Both locally produced cytokines in the
brain, and direct brain stem irritation (by local cytokines or compres-
sion) can trigger strong sympathetic activation and release of cate-
cholamines 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 pro-
found immunomodulation, there remains some confusion over the
impact this will have on outcome, some maintain that inhibition of au-
tonomic signaling can produce beneficial 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 be-
tween 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 α7 subunit (nAChRα7), which
is expressed on both neurons and resident immune cells such as mac-
rophages and microglia (Shytle et al., 2004). This affects inflammation
via the nuclear factor kappa B (NF-κB) pathway (Borovikova et al.,
2000). Stimulation of the peripheral immune system of vagotomized
mice has lead to the conclusion that expression of nAChRα7 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 confirmed
in nAChRα7 knockout mice, which show significantly elevated levels
 R. Brambilla et al. / Molecular and Cellular Neuroscience 53 (2013) 26–33 29

Fig. 1. Schematic representation of the communication pathways between the stroke-injured brain and the peripheral immune system. Pro-inflammatory 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-inflammatory mediators and facilitates the release of anti-inflammatory 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 sym-
pathetic nerve terminals and from the adrenal medulla resulting in inhibition of Th1 pro-inflammatory activities, giving way to the predominance of Th2 anti-inflammatory 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-inflammatory pathway, results in release of ACh acting on cholinergic receptors on macrophages leading to a decrease in the production of anti-inflammatory
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; Mф, macrophages; NA, noradrenaline; NK, natural killer; SNS, sympathetic nervous system;
TGFβ, 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 nAChRα7 knockout animals results in in-
creased damage, suggesting that this receptor is responsible not
only for the peripheral inflammatory response, but also for regulating
inflammatory output within the CNS (Egea et al., 2007). These data
suggest that central activation of the PNS by stroke will largely de-
press the immune system, whereas activation of the SNS will largely
activate it.
One potential, and novel, mechanism of inflammatory 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 theoreti-
cally 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 com-
municating 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 specific types of injury. Such phenotyp-
ic differences may suggest a mechanism of discreet communication
between the CNS and the peripheral immune system. Studies in
central inflammatory 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 differ-
ences 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 po-
tential as a communication pathway between the CNS and the periph-
ery is currently in its infancy, the promising studies using central
inflammatory diseases such as cerebral malaria pave the way for inter-
esting future work.
Brain–gut axis
The gut is composed of three key immunological components: the
gut epithelium (the first point of contact for foreign bodies), the mucosal
immune system (a particularly sensitive site high in IgA-positive cells),
and the gut microflora (commensal bacteria that live in a symbiotic
30 R. Brambilla et al. / Molecular and Cellular Neuroscience 53 (2013) 26–33
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, poten-
tially allowing indigenous gut bacteria to migrate into otherwise
sterile body cavities (Tascilar et al., 2010). This work has been corrobo-
rated 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 significant contributing factor to bacterial transloca-
tion (Tascilar et al., 2010).
Studies specifically investigating the intestinal immune system
have shown decreased levels of lymphocytes in Peyer's patches fol-
lowing 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 in-
vaders. 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 sys-
tems. However, the route by which central inflammatory events af-
fect the mucosal epithelium is currently speculative.
Splenic response to stroke
While little is known about brain–gut communication and the po-
tential for physiological failure in the immediate post-stroke period,
the data available regarding changes in the spleen are much more ro-
bust. Since the spleen is the main ‘storage facility’ for large numbers of
immune cells, spleen alterations after ischemic events have the poten-
tial 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 significantly decreased compared to sham oper-
ated 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 lympho-
cyte populations (Prass et al., 2003). Interestingly, later studies by
Offner and colleagues painted a more complex picture of the splenic re-
sponse to stroke (Offner et al., 2006a,b). They found the acute phase
after stroke (1 to 22 h) to be characterized by sustained splenocyte ac-
tivation with increased expression of pro-inflammatory cytokines and
chemokines (e.g. TNF, IL-6, IL-2, interferon gamma (IFNγ), CXCL2,
CXCL10), mild apoptotic death of splenocytes and limited spleen weight
loss (Offner et al., 2006a). This could help explain the increased system-
ic inflammation 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 pro-
duction of pro-inflammatory mediators and the deployment of pro-
inflammatory monocytes into the blood, leading researchers to sug-
gest splenectomy as a possible prophylactic intervention for cerebral
ischemia (Izci, 2010). Indeed, splenectomized rats display a signifi-
cantly reduced infarct volume after permanent MCAO compared to
non-splenectomized rats, and this is accompanied by a significant
reduction in activated microglia and infiltrating 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 fight infec-
tious 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, significantly reducing infarct size
(Newcomb et al., 2006; Vendrame et al., 2004). Moreover, transfused
HUCBC appeared to switch splenic cytokine expression from a pro-
inflammatory (TNF, IL-1β) to an anti-inflammatory (IL-10) profile
(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 splen-
ic activation and the release of macrophages from the spleen into cir-
culation 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 di-
rect splenic innervations (Young et al., 1983). Studies by Ajmo et al.
(2009) have shown that only blockade of α and β adrenergic recep-
tors, but not spleen denervation, prevented spleen atrophy and re-
duced 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-inflammatory immune cells
ready to be deployed into the blood immediately after stroke.
These cells ultimately reach the infarct area of the brain and contrib-
ute to the propagation of secondary damage. At this stage, strategies
aimed at containing the splenic response may be beneficial. On the
other hand, long-term suppression of the splenic response may
hamper physiologic immune function and limit the ability to fight
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 con-
taining 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 in-
dicate that dysregulation of CNS homeostasis by ischemia can have
long-lasting effects on peripheral immunity. This dysregulation causes
a central inflammatory cascade capable of eliciting a peripheral immune
response. As mentioned previously, the peak central inflammatory peri-
od is approximately 24 h post-ischemia, whereas the peak peripheral
inflammatory 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 influx of circulating
peripheral immune cells such as macrophages, neutrophils and lym-
phocytes, each with specific temporal patterns (Nilupul Perera et al.,
2006). There is evidence that immuno-modulatory molecules capable
of blocking or limiting the influx 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 (fingolimod), which induces depletion of
 R. Brambilla et al. / Molecular and Cellular Neuroscience 53 (2013) 26–33
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 ef-
fect. In similar fashion, other immuno-modulatory agents affecting cell
entry in the CNS have shown effectiveness. Administration of anti-
CD11b antibody, for example, was shown to prevent the infiltration of
polymorphonucleate cells (neutrophils and monocyte/macrophages)
into the ischemic tissue (Chen et al., 1994a, 1994b; Chopp et al.,
1994), significantly reducing infarct size and neurological deficit. Simi-
larly, 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 significant
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-inflammatory functions potentially detrimental to recov-
ery, 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 un-
derstanding of the temporal patterns of entry of each specific population
may be useful in the implementation of the appropriate immuno-
modulatory strategy.
In spite of the influx 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 num-
bers 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). Howev-
er, 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 inflammogen 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 demon-
strated that peripheral immune response post-ischemia is dependent
not on anatomical location, but on infarct size. Work by Hug and col-
leagues have corroborated this finding, further showing that infarct vol-
ume 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, af-
fecting both cortical and subcortical regions. Importantly, infarct size also
seems to be reflected by concentrations of CNS antigens myelin basic
protein (MBP), creatine kinase-BB, neuron-specific enolase, S100beta,
neurofilaments 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 un-
derpins 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 an-
imals or humans, must be approached with caution given the paucity of
knowledge regarding the dysregulation of specific 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. Howev-
er, work has failed to suggest a mechanism for this phenomenon, and
data in human and animal models appear to be conflicting. 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 detri-
mentally 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 infiltrate
the brain parenchyma at the site of infarct and further extend the sec-
ondary 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 sus-
ceptibility to infections of stroke patients (Harms et al., 2008). The abnor-
mal 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 experimen-
tal stroke has been uncovered (Ren et al., 2011). Previous studies had
described the potent regulatory effects of B lymphocytes on inflam-
matory 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 first to identify
IL-10-secreting B regulatory cells as a major protective cell type in
stroke. Indeed, B cell deficiency exacerbated stroke outcomes and
dramatically increased inflammatory cell invasion into the brain,
suggesting that enhancement of regulatory B cells could have thera-
peutic 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 atro-
phy also showed an increase in circulating monocytes (Offner et al.,
2006b). This has been suggested to be a ‘clean-up’ operation by the pe-
ripheral immune system (Manoonkitiwongsa et al., 2001), in order to re-
move 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 profile 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 immuno-
depression. Factors such as current immune status and infarct volume
are capable of directly affecting the function of the immune system im-
mediately after stroke and therefore, indirectly, affecting recovery and
32 R. Brambilla et al. / Molecular and Cellular Neuroscience 53 (2013) 26–33
repair from stroke. The window of opportunity for outside immuno-
modulatory 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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