The effect of stroke on immune function

Molecular and Cellular Neuroscience 53 (2013) 26–33

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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 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 'Neuroinflammation 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 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 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. Winsløwsvej 21, st., DK-5000 Odense C, Denmark. E-mail address: [email protected] (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 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 concentrations 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 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 inflammation on stroke outcome will be covered separately by Stuart Allan 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, 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

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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 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 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 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-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, 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 significantly associated with death. It is also clear that the severity of

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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 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 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 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 β-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 benefit was found with prophylactic administration of the fluoroquinolone 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, 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 moxifloxacin, a similar broad-spectrum antibiotic, administration significantly reduced 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 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 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

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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 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 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. Significant delays are known to exist between the central challenge and the peripheral inflammatory response (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 peripheral 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 peripheral immune system is via the HPA axis in concert with the autonomic 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 believed 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 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 significantly reduces 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 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 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 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 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 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 α7 subunit (nAChRα7), which is expressed on both neurons and resident immune cells such as macrophages 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

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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 sympathetic 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 increased 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 depress 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 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 specific types of injury. Such phenotypic 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 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 inflammatory diseases such as cerebral malaria pave the way for interesting 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

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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 significant contributing factor to bacterial translocation (Tascilar et al., 2010). Studies specifically 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 inflammatory events affect the mucosal epithelium is currently speculative. Splenic response to stroke While little is known about brain–gut 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 significantly 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-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 systemic 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 production of pro-inflammatory mediators and the deployment of proinflammatory monocytes into the blood, leading researchers to suggest splenectomy as a possible prophylactic intervention for cerebral ischemia (Izci, 2010). Indeed, splenectomized rats display a significantly 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 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, significantly reducing infarct size (Newcomb et al., 2006; Vendrame et al., 2004). Moreover, transfused HUCBC appeared to switch splenic cytokine expression from a proinflammatory (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 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 α and β 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-inflammatory 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 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 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 inflammatory cascade capable of eliciting a peripheral immune response. As mentioned previously, the peak central inflammatory period 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 lymphocytes, 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

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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 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. 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 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 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 specific population may be useful in the implementation of the appropriate immunomodulatory 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 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 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 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 finding, 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 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 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 specific subsets of peripheral immune cells after ischemic events of differing intensities.

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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 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 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 infiltrate 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 inflammatory 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 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 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 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

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