brambilla et al. 2013 - the effect of stroke on immune function

Molecular and Cellular Neuroscience 53 (2013) 2633

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Molecular and Cellular Neuroscience

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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 Statesb The Department of Pharmacology, University of Oxford, Oxford, UKc The Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

The authors have no conflict of interest. The authors would like to acknowledge financiaFoundation and the Danish Medical Research Counciland from The Miami Project to Cure Paralysis to Dr. Rob

Corresponding author at: Departmentof NeurobiologyMedicine, University of Southern Denmark, J.B. WinslwsDenmark.

E-mail address: [email protected] (K.L. La1 All three authors contributed equally to this work.

1044-7431/$ see front matter 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.mcn.2012.08.011

a b s t r a c t
a r t i c l e i n f o
Article history:Received 15 March 2012Accepted 22 August 2012Available online 30 August 2012

Keywords:StrokeImmune responsesAutonomic nervous systemHPA axis

Neurological disorders affect over one billion lives each year worldwide. With population aging, this number ison the rise, making neurological disorders amajor public health concern.Within this category, stroke representsthe second leading cause of death, ranking after heart disease, and is associated with long-term physical disabil-ities and impaired quality of life.In this review, we will focus our attention on examining the tight crosstalk between brain and immunesystem and how disruption of this mutual interaction is at the basis of stroke pathophysiology. We willalso explore the emerging literature in support of the use of immuno-modulatory molecules as potentialtherapeutic interventions in stroke. This article is part of a Special Issue entitled 'Neuroinflammation inneurodegeneration and neurodysfunction'.

2012 Elsevier Inc. All rights reserved.

Introduction

Ischemic stroke results in a multitude of CNS events characterizedultimately by neuronal and glial cell death, and is also marked bynumerous peripheral events including cardiovascular, endocrine andimmune dysregulation (Emsley et al., 2008; Stevens and Nyquist,2007). The contribution of the immune system to the developmentand 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 mostcommon, fatal, post-stroke complication is pneumonia (Aslanyan etal., 2004; Johnston et al., 1998; Katzan et al., 2003). Indeed, a recentmeta-analysis has revealed that infection after acute ischemia cancomplicate recovery in up to 30% of cases (Westendorp et al., 2011).While in the past the assumption was that post-stroke infections weredependent 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 toinfections (Emsley et al., 2008).

l support from the Lundbeckto Dr. Kate Lykke Lambertsenerta Brambilla.Research, Institute ofMolecularvej 21, st., DK-5000 Odense C,

mbertsen).

rights reserved.

Risk factors for developing stroke include co-morbid diseases suchas 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 evidencepoints at having a causative role in the development of these diseases(Hansson and Libby, 2006). Several clinical studies have reportedmore severe neurological deficits in stroke patients with precedinginfection (McColl et al., 2009). Furthermore, elevated systemic concen-trations of a number of inflammatory markers have been associatedwith stroke incidence (Rodriguez-Yanez et al., 2008), emphasizingthe 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 contributorsto stroke incidence and physiopathology, this review will specificallyfocus 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 theseevents 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 StuartAllan on the review dealing with the afferent pathways in stroke.

Stroke and central inflammation

Since the brain has a very high glucose and oxygen demand, distur-bances in the blood supply to the brain rapidly lead to the depletion ofthese substrates and the development of an ischemic infarct with ac-companying necrosis of neurons, glial cells and small vessels withinthe affected territory. Depletion of cellular energy supplies (such asadenosine triphosphate (ATP)) occurs within minutes, resulting in the
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27R. Brambilla et al. / Molecular and Cellular Neuroscience 53 (2013) 2633

accumulation of lactic acid within the tissue. Ischemia also leads to theformation of free radicals that can induce cell damage, and to increasedrelease of excitatory glutamate (for recent review see (Iadecola andAnrather, 2011)). ATP depletion and glutamate release result inuncontrolled calcium-ion influx into the cells leading to activation of in-tracellular lipases and proteolytic enzymes and, ultimately, destructionof the cell. The ability of glutamate to kill neurons by excessive activationof glutamate receptors is referred to as excitotoxicity (Mergenthaler etal., 2004). The early excitotoxicity induced by the local energy deficitcauses fast necrotic cell death in the core area of the infarct (Lipton,1999). The ischemic penumbra that surrounds the infarct core suffersmilder 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 capacityallowing a continued uptake of glutamate from the synaptic cleft inthe penumbral area (Marrif and Juurlink, 1999). It has been observedthat the penumbra has suppressed cortical protein synthesis, but pre-served ATP content (del Zoppo et al., 2011; Hossmann, 2006). Forthese 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 theischemic lesion. It is believed that, early on, endogenous signalssuch as damage-associated molecular patterns (DAMPs; i.e. heatshock 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 downstreamactivation 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 etal. (2009)). This triggers the synthesis of primarily microglia-derivedpro-inflammatory cytokines, such as interleukin (IL)-1, IL-6 andtumor 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 presentat high levels, can exacerbate cell death and cause break down of thebloodbrain barrier (BBB) (for recent review see (Iadecola andAnrather, 2011)). Cytokines and chemokines also induce theupregulation 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 complicationsadversely affecting the clinical outcome in stroke patients. Althoughsome infections may occur as a direct consequence of dysphagia caus-ing aspiration, or may be linked to the advanced age of the patient, itis increasingly apparent that stroke itself represents a risk factor forinfections 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 thatthe majority of post-stroke infections manifest within three days ofhospitalization is further indication that immunodepression is in-volved, and infections are not simply a secondary outcome relatedto patient care (Westendorp et al., 2011). A recent meta-analysis ofthe 87 clinical studies conducted thus far, where rates of post-strokeinfections were examined, has indicated that infection complicatedacute stroke in 30% of patients, with rates varying considerably be-tween 5 and 67% (Westendorp et al., 2011). Pneumonia and urinarytract infections each occurred in 10% of patients, with pneumonia sig-nificantly associated with death. It is also clear that the severity of

post-stroke infections is directly correlated with the magnitude ofthe stroke itself, and specifically with how extensive the infarct areais (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 reportsdating back over 40 years (Czlonkowska et al., 1979). Rapid reductionof lymphocyte counts and functional deactivation of monocytes andT helper type 1 cells have been observed in acute stroke patients, withmore pronounced immunodepression in patients with severe clinicaldeficit or large infarction (Haeusler et al., 2008). Lymphocytopeniadoes correlate with the occurrence of post-stroke infections (Haeusleret al., 2008;Hug et al., 2009). In some instances, rather than a generalizedreduction in total lymphocyte counts, only selective lymphocytopenia inthe NK cell subset was reported immediately after stroke (Hug et al.,2009).

The tight relationship between lymphocytopenia, size of infarctand the occurrence of post-stroke infections has been demonstratedalso in animal models of stroke. In mice, severely reduced lymphocytecounts (B cells and CD4+ T helpers, especially) were found in lym-phoid organs (spleen and thymus) within 12 h after stroke (Prass etal., 2003). This was paralleled by spontaneous bacteremia and pneu-monia (often leading to death), which were completely preventedby administration of a sympathetic blocker, but not by inhibition ofthe hypothalamic-pituitary-adrenal (HPA) axis (the paravetricularnucleus in the hypothalamus, the anterior pituitary gland and the cor-tex of the adrenal glands), suggesting that a catecholamine-mediateddefect in early lymphocyte activation is the key factor in the impairedantibacterial 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-strokeinfections (Wong et al., 2011). Modulation of hepatic iNKT throughblockade of noradrenergic neurotransmitters or directly with admin-istration of -galactosylceramide results in reduced infection and as-sociated lung injury after stroke, demonstrating that these cells act asconductor of immunity, meaning that their acute responses modulateand facilitate the adaptive immune response (Wong et al., 2011).

Because of the correlation between post-stroke infections andclinical outcome, the prophylactic use of antibiotics to prevent suchinfections and improve the outcome has been proposed. The dataemerging 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 acutestroke patients admitted within 24 h after symptom onset. The rateof stroke-related infections at 7 days was identical to the placebogroup, and levofloxacin administration was directly correlated withpoor 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 etal., 2008). This is in contrast with data obtained in a mouse modelof middle cerebral artery occlusion (MCAO), where moxifloxacin, asimilar broad-spectrum antibiotic, administration significantly re-duced infarct size (Bao et al., 2010). A possible explanation for thefailure of these molecules in human therapy is the neurotoxicity offluoroquinolones, 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-lactamaseinhibitor sulbactam decreased incidence and severity of fever withinthe first 34 days after stroke and was associated with a lower rateof post-stroke infection and improved long term outcome (Schwarzet al., 2008). Finally, minocycline, a semisynthetic second generationtetracycline, 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 thatminocycline treatment significantly improved the clinical outcome,even though it did not protect from post-stroke infections. Thisseems to suggest that the protective effect of minocycline in strokemay not be dependent on its antibacterial properties, but rather onits anti-inflammatory effect, which has been associated with a de-crease in microglial activation and nitric oxide production, inhibitionof matrix metalloproteinases (MMPs) (e.g. MMP2, MMP9), and inhi-bition of apoptotic cell death (Zemke and Majid, 2004). Recently, ina small exploratory trial minocycline was found to be safe and well tol-erated in intravenous doses, either given alone or in combination withtissue plasminogen activator (tPA) (Fagan et al., 2010), supporting therationale for a possible clinical application in stroke.

Studies in animal models have also underscored that post-strokeinfections may be effectively prevented with the use of immunomod-ulatory molecules (Prass et al., 2003; Wong et al., 2011), rather thanantibiotics, 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 elicita substantial immune response. This concept is relatively new andlargely unexplored, and the signaling pathways that mediate theseevents are mostly unknown. Significant delays are known to existbetween the central challenge and the peripheral inflammatory re-sponse (Blond et al., 2002). Interestingly, in ischemia, the peak centralinflammatory phase has been shown to be induced at approximately24 h post-ischemia, whereas the peak peripheral inflammatory phaseoccurs 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 thousandgenes 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-1 and IL-6 are pivotal in the cross talk between brain and immunesystem. Indeed, they activate the autonomic nervous system and theHPA axis, where release of catecholamines and glucocorticoids canmodulate 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 (Haddadet 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), whichin 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 glucocorticoidsfrom 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 tolymphopenia and lymphocyte dysfunction, particularly monocyte de-activation (Fig. 1) potentially resulting in bacteremia and pneumoniain 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 bothcatecholamines and acetylcholine (Ach) (Anne et al., 2007; Elenkovet al., 2000; Franceschini et al., 1994) (Fig. 1). The detrimental effectsof these neurotransmitters on the peripheral immune system isdiscussed in detail below.

Hepatic signaling pathwaysVery 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 beenshown 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 vagalnerve lesion (Ottani et al., 2009). This suggests that the brain isswitching 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 centraladrenergic and cholinergic circuits within the brain that protect neu-rons from the aftermath of CNS injury, and is therefore particularlyrelevant in stroke research (Feinstein et al., 2002; Galea et al., 2003).

The role of the autonomic nervous system in the communicationbetween brain and periphery has been described by a number ofstudies. 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-adrenergic agonist, shows protection in animal models ofexcitotoxicity, demonstrating both the anti-inflammatory potentialof the autonomic nervous system and the concept of neurogenicneuroprotection (Ryan et al., 2011). In addition, studies show thatchanges 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-sidedstroke 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-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, arereleased during stress, a common phenomenon after acute stroke(Anne et al., 2007; Oto et al., 2008). Therefore, it is not unreasonableto postulate that systemic release of cytokines after stroke might bemediated, in part, by the autonomic nervous system and catechol-amines (Marz et al., 1998). Both locally produced cytokines in thebrain, 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 thesestudies have shown that the activation of the SNS is capable of pro-found immunomodulation, there remains some confusion over theimpact this will have on outcome, somemaintain that inhibition of au-tonomic signaling can produce beneficial outcomes after stroke(Savitz et al., 2000). The direct pathways of communication betweenthe injury site and the SNS immediately post-stroke are currentlyspeculative. However, it is clear that some uncoupling occurs be-tween the CNS, SNS and their control of the immune system duringthe immediate post-stroke period.

The main target of PNS pathways, such as output from the vagus,is the nicotinic acetylcholine receptor 7 subunit (nAChR7), whichis expressed on both neurons and resident immune cells such as mac-rophages andmicroglia (Shytle et al., 2004). This affects inflammationvia the nuclear factor kappa B (NF-B) pathway (Borovikova et al.,2000). Stimulation of the peripheral immune system of vagotomizedmice has lead to the conclusion that expression of nAChR7 onKupffer cells enables the hepatic branch of the vagus to suppressthe production of reactive oxygen species in these cells (Hiramotoet al., 2008). This top down immune suppression has been confirmedin nAChR7 knockout mice, which show significantly elevated levels

Fig. 1. Schematic representation of the communication pathways between the stroke-injured brain and the peripheral immune system. Pro-inflammatory cytokines stimulate neurons inthe 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 adrenalcortex 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, givingway to the predominance of Th2 anti-inflammatory activities. Alsoactivation 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 vagusnerve, 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-inflammatorycytokines. 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 in-creased damage, suggesting that this receptor is responsible notonly for the peripheral inflammatory response, but also for regulatinginflammatory output within the CNS (Egea et al., 2007). These datasuggest that central activation of the PNS by stroke will largely de-press the immune system, whereas activation of the SNS will largelyactivate it.

One potential, and novel, mechanism of inflammatory output fromthe CNS is the microparticles (MPs). These are membrane-derivedvesicles produced as a result of cell stress and can be released throughblebbing 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 thusinjury in the CNS would be able to use them as a mechanism of com-municating with the periphery. Recent advances have allowed MPs tobe phenotyped, showing not only the cellular origin of the MPs butalso distinct populations after specific types of injury. Such phenotyp-ic differences may suggest a mechanism of discreet communicationbetween the CNS and the peripheral immune system. Studies in

central inflammatory diseases such as cerebral malaria have shownthat MPs can be used as markers of cerebral dysfunction (PankouiMfonkeu et al., 2010). However, data from acute stroke patientshave been less promising so far (Williams et al., 2007) since no differ-ences were found in MP characteristics. While these studies could notdistinguish between stroke and stroke-mimic patients (those whopresent the symptoms of a stroke but do not show any underlyingvascular anomalies), in other instances the phenotype of MPs hasbeen successfully used to discriminate between cerebrovascularevents (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 centralinflammatory diseases such as cerebral malaria pave the way for inter-esting future work.

Braingut axisThe gut is composed of three key immunological components: the

gut epithelium (the first point of contact for foreign bodies), themucosalimmune system (a particularly sensitive site high in IgA-positive cells),and the gut microflora (commensal bacteria that live in a symbiotic


relationship with the host). Within the mucosal immune system,lymph drainage occurs at Peyer's patches, sites where high numbersof immune cells gather. Interestingly, studies using the permanentMCAO model of stroke in rats have demonstrated stark alterationsin the gut mucosa post-ischemia (Tascilar et al., 2010). The heightand depth of the intestinal villi, a critical factor critical for mucosalintegrity, were shown to be damaged by permanent MCAO, poten-tially allowing indigenous gut bacteria to migrate into otherwisesterile 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 possiblythe HPA axis, is a significant contributing factor to bacterial transloca-tion (Tascilar et al., 2010).

Studies specifically investigating the intestinal immune systemhave shown decreased levels of lymphocytes in Peyer's patches fol-lowing cerebral ischemia (Schulte-Herbruggen et al., 2009). Thesedata are in line with numerous studies reporting changes in T and Bcell numbers and functionality after stroke, which will be discussedbelow. The exact role of lymph drainage from the intestines has yetto be elucidated (Newberry, 2008), but current data suggest that itlikely provides a tolerant immune barrier between gut bacteria andthe systemic circulation, as well as protection against pathogenic in-vaders. Thus far, there are no data regarding the exact mechanismsof communication between the brain and the gut immune system,but it seems reasonable to assume that changes, at least in circulatinglymphoid 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 braingut 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 ofimmune cells, spleen alterations after ischemic events have the poten-tial to induce severe perturbation of the immune function. The initialobservation of reduced cellularity in the spleens of rats subjected tocerebral ischemia (transient MCAO) was made by Gendron et al.(2002), who reported that in ischemic animals the total number ofspleen leukocytes was significantly decreased compared to sham oper-ated controls from day 2 to day 28 post-ischemia. A similar pattern ofsplenocyte loss was also observed in mice subjected to transientMCAO, and attributed to increased apoptotic cell death of all lympho-cyte populations (Prass et al., 2003). Interestingly, later studies byOffner and colleagues painted amore complex picture of the splenic re-sponse to stroke (Offner et al., 2006a,b). They found the acute phaseafter stroke (1 to 22 h) to be characterized by sustained splenocyte ac-tivation with increased expression of pro-inflammatory cytokines andchemokines (e.g. TNF, IL-6, IL-2, interferon gamma (IFN), CXCL2,CXCL10),mild apoptotic death of splenocytes and limited spleenweightloss (Offner et al., 2006a). This could help explain the increased system-ic inflammation observed immediately after stroke, which correlateswith the development of increased secondary damage at the infarctsite in the brain. At a later post-injury time (96 h) a drastic reductionin spleen weight as a consequence of reduced cellularity has beenobserved, and this was associated with massive apoptotic death ofsplenocytes (Shimizu et al., 1999), mostly B cells and CD4+ T effectorsubsets.

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 cerebralischemia (Izci, 2010). Indeed, splenectomized rats display a signifi-cantly reduced infarct volume after permanent MCAO compared tonon-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 theshort term, questions remain as to whether this could lead todetrimental effects in the long term, given the role of the spleen innormal immune function, especially in stroke patients who areintrinsically immuno-suppressed and less equipped to fight infec-tious complications.

Rather than ablation of the spleen, a strategy adopted by othersconsisted in replenishing the spleen with human umbilical cordblood cells (HUCBC) transfused after MCAO. HUCBC localize to thespleen counteracting MCAO-induced spleen atrophy, and migrate tothe site of injury in the brain, significantly reducing infarct size(Newcomb et al., 2006; Vendrame et al., 2004). Moreover, transfusedHUCBC appeared to switch splenic cytokine expression from a pro-inflammatory (TNF, IL-1) to an anti-inflammatory (IL-10) profile(Vendrame et al., 2004), which could be the key to the therapeuticeffect 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 intothe circulation from the adrenal medulla as described above, MCAOalso 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 and adrenergic recep-tors, but not spleen denervation, prevented spleen atrophy and re-duced infarct volume, suggesting that it is the increased systemiccatecholamine level resulting from adrenal release that regulatesthe splenic response after stroke.

Collectively, these studies point at a key crosstalk between spleenand ischemic brain beginning at the very early stages of injury. Acutely,the spleen acts as a reservoir of pro-inflammatory immune cellsready 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, strategiesaimed at containing the splenic response may be beneficial. On theother hand, long-term suppression of the splenic response mayhamper physiologic immune function and limit the ability to fightinfections, compromising the chances of recovery of stroke patients.In conclusion, it appears clear that strategies aimed at modulatingthe splenic response to stroke may be extremely effective in con-taining the propagation of secondary damage within the CNS, aslong as they are delivered in a timely fashion.

Post-ischemia immune cell function

As discussed above, the potential for the immune system to havedeleterious 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 havelong-lasting effects on peripheral immunity. This dysregulation causesa central inflammatory cascade capable of eliciting a peripheral immuneresponse. Asmentioned previously, the peak central inflammatory peri-od is approximately 24 h post-ischemia, whereas the peak peripheralinflammatory period is approximately 4 h post-ischemia (Chapman etal., 2009). Discrepancies between the peak response times may allowfor the mobilization of peripheral immune cells, which have beenshown to play a crucial role in infarct size (Gelderblom et al., 2009).During stroke, disruption of the BBB allows for the influx of circulatingperipheral 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 capableof blocking or limiting the influx of these cell populations into the CNSare effective in preventing the evolution of stroke damage in animalmodels. To this effect, administration of the sphingosine 1-phosphate(S1P) analog FTY720 (fingolimod), which induces depletion of


circulating lymphocytes by preventing their egress from the lymphnodes, can reduce lesion size and improve neurological outcome afterstroke in mice (Czech et al., 2009). This is due to a reduced invasion ofimmune cells into the lesion, in addition to a direct neuroprotective ef-fect. In similar fashion, other immuno-modulatory agents affecting cellentry in the CNS have shown effectiveness. Administration of anti-CD11b antibody, for example, was shown to prevent the infiltration ofpolymorphonucleate 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 (endotheliumsurface) or its receptor CD18 (leukocyte surface), whose interaction isessential for leukocyte binding to the endothelium and subsequenttransendothelial migration, showed effectiveness in limiting strokedamage and promoting functional recovery (Bowes et al., 1993; Clarket al., 1991a,b; Jiang et al., 1994, 1995; Zhang et al., 1995a,b). Theseencouraging data in experimental models prompted the initiationof a clinical trial in stroke patients with the anti-ICAM-1 antibodyenlimomab which, contrary to expectations, resulted in significantworsening of the clinical outcome (Enlimomab Acute Stroke TrialInvestigators, 2001). This demonstrates the complex role of immunecells in the development of stroke, and underscores that not only dothey have pro-inflammatory functions potentially detrimental to recov-ery, but also protective functions, particularly against stroke-relatedinfections (as discussed above), and indiscriminate ablation of suchcellsmaynot represent the best course of action. Additionally, a better un-derstanding of the temporal patterns of entry of each specific populationmay be useful in the implementation of the appropriate immuno-modulatory strategy.

In spite of the influx of peripheral immune cells into the brain there islittle evidence of damaging autoimmunity after stroke. The presentationof CNS antigens to T and B cells should induce the production ofautoantigens, 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 ofan adaptive immune response will reduce infarct size suggesting thatT and B-cells do contribute to lesion volume (Offner et al., 2009). Howev-er, the re-establishment of the BBB post stroke means that autoreactiveimmune cells will have little or no opportunity to interact with CNSantigens during the recovery period. Interestingly, this has shown to beworsened in animal models using a peripheral inflammogen such asLPS (Becker et al., 2005).

Despite previous lateralization studies,work in both humans (NilupulPerera et al., 2006) and animal models (Dirnagl et al., 2007) has demon-strated that peripheral immune response post-ischemia is dependentnot 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 etal., 2011). Experimentally, this presents a problem, since both transientand permanent MCAOmodels tend to produce large infarct volumes, af-fecting both cortical and subcortical regions. Importantly, infarct size alsoseems to be reflected by concentrations of CNS antigens myelin basicprotein (MBP), creatine kinase-BB, neuron-specific enolase, S100beta,neurofilaments and portions of N-methyl-D-aspartate receptor in serumsamples from stroke patients (Bornstein et al., 2001; Dambinova et al.,2003; Jauch et al., 2006). Lymphocytes from these patients show morereactivity againstMBP than lymphocytes frommultiple sclerosis patients(McQuillan et al., 2011; Mfonkeu et al., 2010). Since it is acknowledgedthat an ischemic attack is a risk factor for developing dementia, this un-derpins the hypothesis that autoimmune responses to the brain in strokepatients might contribute to the cognitive decline and progression ofwhite matter disease seen in some stroke patients (Pendlebury andRothwell, 2009). Thus, any attempt at immunomodulatory therapy in an-imals or humans, must be approached with caution given the paucity ofknowledge regarding the dysregulation of specific subsets of peripheralimmune cells after ischemic events of differing intensities.

T and B cells post-strokeThe recent interest in lymphocyte function after stroke ismainly due

to the increased likelihood of encounters between lymphocytes andCNS antigens immediately after stroke. To date, research suggests thatimmunodepression is largely caused by lymphocytopenia, abnormallylow levels of circulating lymphocytes immediately after stroke. Howev-er, work has failed to suggest a mechanism for this phenomenon, anddata in human and animal models appear to be conflicting. Hug et al.have shown in both humans and animalmodels that T-cell proliferationand activation ex vivo are not affected by ischemia (Hug et al., 2011).Nevertheless, reduced T cell numbers after stroke, aswell as generalizedsplenic atrophy, have been demonstrated in a number of studies (Huget al., 2011; Offner et al., 2006b; Vogelgesang et al., 2010). It has beensuggested that this lymphocytopenia is due to increased apoptosiswithin all T-cell populations (Chamorro et al., 2007). Recent work onTregs has shown that they have potential to provide a degree ofimmunoprotection 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 bereduced 4 days after stroke, CD4+CD25+Foxp3+ T regulatory cells(Tregs) and CD11b+ monocytes in the blood were highly increased atthis time (Offner et al., 2006b), suggesting that splenic atrophy is notonly dependent upon splenocyte apoptosis but is also partly due to themigration of cells out of the spleen and into the blood to then travel tothe injured CNS. Indeed, CD11b+ monocytes can eventually infiltratethe brain parenchyma at the site of infarct and further extend the sec-ondary damage to the CNS tissue. The drastic reduction in splenicT and B cells must be a contributing factor to the immuno-suppressionobserved 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 immunosuppressingfactor in itself. Indeed, several studies now indicate that an excessiveTreg presence may impede immuno-surveillance against tumor cellsand 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 haddescribed the potent regulatory effects of B lymphocytes on inflam-matory responses (LeBien and Tedder, 2008) and depletion of Bcells worsened disease severity in models of multiple sclerosis(Matsushita et al., 2010). Ren and colleagues were the first to identifyIL-10-secreting B regulatory cells as a major protective cell type instroke. Indeed, B cell deficiency exacerbated stroke outcomes anddramatically 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 systemmay also be affectedby ischemic events in the CNS. Offner'swork 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 macrophagein the innate immune response to infection the increased susceptibilityto infection post-stroke seems rather counter-intuitive. Other datashow that the expression profile of genes on the surface of macrophagesand neutrophils is altered after stroke (Tang et al., 2006), suggesting thepossibility of immunomodulation, rather than immunodepression.

Conclusion

The nature of the post-ischemic immune response is multifacetedand complex, resulting in immunomodulation as well as immuno-depression. Factors such as current immune status and infarct volumeare capable of directly affecting the function of the immune system im-mediately after stroke and therefore, indirectly, affecting recovery and


repair from stroke. The window of opportunity for outside immuno-modulatory therapy after stroke is relatively narrow and the currentpaucity of knowledge regarding the consequences of such therapiesmeans that they are often unwise. However, current work is aiming tofurther our knowledge of the immune system post-stroke and shouldprovide us with ample opportunity to improve clinical outcomes inthe future.

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The effect of stroke on immune functionIntroductionStroke and central inflammationStroke-associated infectionBrain-periphery signaling post-strokeHepatic signaling pathwaysBraingut axisSplenic response to strokePost-ischemia immune cell functionT and B cells post-strokeConclusionReferences

brambilla et al. 2013 - the effect of stroke on immune function

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