Sex, Stroke, and Inflammation: The potential for Estrogen-mediated immunoprotection in stroke

Abstract

Stroke is the third leading cause of death and the primary cause of disability in the developed world. Experimental and clinical data indicate that stroke is a sexually dimorphic disease, with males demonstrating an enhanced intrinsic sensitivity to ischemic damage throughout most of their lifespan. The neuroprotective role of estrogen in the female brain is well established, however, estrogen exposure can also be deleterious, especially in older women. The mechanisms for this remain unclear. Our current understanding is based on studies examining estrogen as it relates to neuronal injury, yet cerebral ischemia also induces a robust sterile inflammatory response involving local and systemic immune cells. Despite the potent anti-inflammatory effects of estrogen, few studies have investigated the contribution of estrogen to sex differences in the inflammatory response to stroke. This review examines the potential role for estrogen-mediated immunoprotection in ischemic injury.

Stroke

Stroke is now the third leading of cause of death after heart disease and cancer. Stroke affects 15 million people worldwide each year, and is the leading cause of long-term disability, affecting nearly 800,000 in US alone (Lloyd-Jones et al., 2009). Increasing life expectancy together with an expanding aging population will place a profound burden on the economy well beyond the current estimate of $69 billion annually (Lloyd-Jones et al., 2009). The continued inability to effectively treat stroke coupled with our aging population and the projected lack of health care resources will place patients at a higher risk of mortality and long-term disability than currently seen today.

Sex and Stroke

Analysis of the epidemiological data indicates that stroke is a sexually dimorphic disease (Bushnell, 2008; Reeves et al., 2008; Turtzo and McCullough, 2010). Although the overall life-time incidence of stroke in men is approximately 30% higher than in women, females have more severe strokes with poorer recovery and greater long-term disability (Appelros et al., 2009; Fukuda et al., 2009; Niewada et al., 2005; Roquer et al., 2003). Women now account for 61% of all stroke deaths (Rosamond et al., 2008). The risk for incident stroke at 65 years of age has decreased significantly from 19.5% to 14.5% in men but more modestly from 18.0% to 16.1% in women (Roger et al., 2011). The higher mortality in women is in part due to older age and the greater number of co-morbid conditions in women at the time of stroke (Devries et al., 2011). Men experience a higher risk of stroke over most of the lifespan, with several notable exceptions. There is a stroke surge in women between the ages of 19 and 30 years, associated with the peripartum (Pathan and Kittner, 2003; Putaala et al., 2009; Rasura et al., 2006) and a second increase in risk in women between 45 and 54 years of age, in the peri-menopause (Towfighi et al., 2007). While it appears that transitioning to a lower estrogenic state predisposes females to higher stroke risk, it is unlikely that sex hormones fully account for these sex differences. For example, after the age of 50 years, the risk of stroke for both sexes rises dramatically, but the incidence in women does not surpass that of men until after the age of 85, well past menopause (Lloyd-Jones et al., 2009). In addition, childhood ischemic stroke is more common in boys than in girls, despite relatively equivalent hormone levels, possibly due to a greater influence of sex chromosomes (Golomb et al., 2009). However, an emerging contribution from testosterone has added complexity to studies of sex differences in stroke. Exposure to testosterone may contribute to the higher incidence and severity of stroke in boys, as elevated levels correlated with stroke risk (Normann et al., 2009; Siegel et al.; Vannucci and Hurn, 2009), and recent studies in elderly men showed an increased risk in men treated with exogenous testosterone replacement (Basaria et al., 2010).

The concept that genetic sex may also contribute to ischemic sensitivity independently of the actions of gonadal steroids is becoming increasing investigated. Expression of both autosomal and sex chromosome-linked genes in the mouse brain displays regional sexual dimorphism, as well as sex-specific imprinting from the parent of origin (Gregg et al., 2010). Females inherit one X-chromosome from each parent, whereas males inherit a single, maternal X-chromosome. While one of the X chromosomes in females is randomly inactivated during early development, up to 20% of X-linked genes escape X inactivation in humans (Carrel and Willard, 1999). X inactivation can also become unstable with aging (Hatakeyama et al., 2004; Smrt et al., 2011) allowing for re-expression on genes silenced earlier in development. This may be an important contributor to the immunological response to stroke, as clinical data demonstrate that men and women differ in their humoral, innate, and cell-mediated responses to other immunological challenges such as viral vaccines (Klein et al., 2010). However, as both androgen and estrogen response elements are present in the promoters of several innate immunity genes, hormone receptor concentration, and duration of sex steroid exposure may also lead to sex differences in the innate immune responses.

The concept that intrinsic sex differences after ischemic injury exist between males and females also comes from emerging in vitro experiments using cells derived from male (XY) versus female (XX) animals. One critical advantage of studying cells in culture is that sex steroid hormones (and agents that may activate sex steroid responses, such as phenol red) can be eliminated from the cell culture medium. If sex-typed cells are used, any differences in behavior seen between male and female cells must result from inherent sex differences within the cells, from prenatal hormone exposure (ie. “organizational effects”), or a combination of the two. The two most common methods of mimicking an ischemic insult in cell culture are either to subject cells to oxygen-glucose deprivation (OGD) or to expose them to N-methyl-D-aspartic acid (NMDA). In cultures of hippocampal slices, slices from female P7 pups demonstrated intrinsic protection against OGD or NMDA exposure relative to those from male pups (Li et al., 2005). Similarly, primary rat hippocampal neuron male cultures were more sensitive to hypoxic insult than female cultures (Heyer et al., 2005). Sex differences in ischemic sensitivity have also been reported in cultured astrocytes (Liu et al., 2007a) and splenocytes (Du et al., 2004), demonstrating that sex dimorphism is present in multiple cell types (see Table 1). The reader is directed to several recent reviews on this topic (Herson et al., 2009; Manwani and McCullough, 2011).

Table 1.

The anti-inflammatory effects of estrogen on glial cells

Astrocytes
Author	Model	Drug/Treatment/mutation	Downstream Effect
(Santos-Galindo et al., 2011)	In vitro Mouse	LPS (sex effect)	Male: ↑IL-1b, IL-6, TNF Female: ↑TPSO, pregnenolone
(Liang et al., 2002)	In vitro Human (Alzheimer)	E2	↑GLT-1, GLAST
(Pawlak et al., 2005)	In vitro Mouse	E2	↑GLT-1, GLAST
(Barouk et al., 2011)	In vivo Rat	E2	↑VEGF
(Giraud et al., 2010)	In vivo / In Vitro Mouse	E2 + EAE, TNF stimulation	↓CCL2
Brain Edema
(Tomas-Camardiel et al., 2005)	In vivo Rat	E2 + LPS	↓AQ -4, BBB deterioration
(Rutkowsky et al., 2011)	In vitro Rat	E2 + hypoxia	↓AQ-4, swelling
(Liu et al., 2008b)	In vivo Mouse	AQ-4 deletion, MCAO	NO SEX DIFFERENCES in AQ-4 or edema
Microglia
Author	Model	Drug/Treatment/mutation	Downstream Effect
(Saijo et al., 2011)	In vitro - Mouse, Human	E2 + LPS	↓IL-1β, IL-6, IL-23p19, iNOS
(Liu et al., 2005)	In vitro - Rat	E2 + LPS	↑neuroprotection (neuronal co-culture) ↓TNF
(Dimayuga et al., 2005)	In vitro - Mouse	E2 + LPS	↑IL-10 ↓TNF, IFNγ, MHCI/II, CD40, CD86, CD152, Fas/L
(Bruce-Keller et al., 2001; Bruce-Keller et al., 2000)	In vitro - Rat, Mouse	E2 + LPS	↑MAPK-phosphorylation ↓iNOS
(Vegeto et al., 2001)	In vitro - Rat	E2 + LPS	↓iNOS, PGE-2, MMP-9
(Barreto et al., 2007)	In vivo - Rat	E2 + stab wound	↓MHCII
(Vegeto et al., 2006)	In vivo - Mouse	E2 + LPS	↓CCL2, MIP-2, TNF
(Zhang et al., 2004)	In vivo - Rat	E2 + hypoxia-ischemia	↑bfl -1

As epidemiological data and clinical observations suggested that exposure estrogen was responsible for the lower incidence of stroke in pre-menopausal women (Billeci et al., 2008), most early preclinical studies focused on estrogen as the primary mediator of sex differences in ischemic sensitivity. Estrogen’s neuroprotective actions have been well studied in the laboratory using experimental models of stroke in animals (see Table 1), most of which have utilized 17 beta-Estradiol, which binds equally well to both isoforms of the estrogen receptor. Of note, most clinical studies have utilized other estrogen formulations (i.e., Premarin), which may also contribute to conflicting experimental and clinical results. Young female animals are more resistant to ischemic damage than males, an effect that can be reversed in part by ovariectomy (OVX) and rescued with estrogen replacement (McCullough and Hurn, 2003). Estrogen also reduces damage after induced stroke in intact males implying a direct role for sex hormone-mediated neuroprotection. The epidemiology of stroke in aging is also well mirrored in animal models, with older reproductively senescent females showing greater infarct volume than ovary-intact young females and age-matched males (Liu et al., 2009), a reversal of what is observed in young animals. At this point it is unknown why stroke displays such profound sexual dimorphism at different ages, but an enhanced inflammatory response in the aged female brain may be responsible for some of these differences. As levels of gonadal hormones fluctuate throughout the lifespan, interactions between chromosomal factors (XX and XY), sex steroid levels and aging are likely. Adding another layer of complexity is that sex steroid hormones can act via organizational effects which irreversibly commit tissues to a male or female phenotype or through activational effects that are dependent on the continued presence of the hormone (Arnold, 2009b; Becker et al., 2005). One difficulty with studies examining “sex differences” after stroke is that the organizational effects of sex steroids are not reversible by gonadectomy, and cannot be completely eliminated in cell culture models. Current approaches aimed at studying the genetic role of sex in stroke include the evaluation of epigenetic differences, X-chromosomal dosage, and most recently, the analysis of stroke outcomes in the four-core genotype model (Arnold and Chen, 2009; Siegel et al., 2011; Turtzo et al., 2011). This model, in which the testis-determining gene, Sry, has been deleted from the Y chromosome and inserted in an autosome, eliminates many of the activational and organizational effects of gonadal hormones, while maintaining the sex chromosome complement (XX vs. XY).

Sex, Stroke, and Inflammation

Emerging data suggests that stroke-induced inflammation significantly contributes to neuronal injury and clinical outcome (Dirnagl et al., 2003; Han and Yenari, 2003; Ishikawa et al., 2004; Seymour et al., 2003). Local and systemic inflammatory responses following cerebral ischemia have highlighted a pivotal role for the innate immune system in the brain’s response to injury, including activation of glia and myeloid cells (Iadecola and Anrather, 2011; Stoll et al., 1998). Injury also leads to the activation of several transcription factors involved in the inflammatory response, including nuclear factor κB (NF-κB) (Wang et al., 2007) with the subsequent up-regulation of numerous pro-inflammatory genes, such as tumor necrosis factor-α (TNF- α), monocyte chemotactic protein-1 (MCP-1), and interlukin-6 (IL-6), etc. (Altinoz and Korkmaz, 2004; Wang et al., 2007). A growing body of evidence (Kalaitzidis and Gilmore, 2005; Stein and Yang, 1995) has shown that estrogen interacts with many of these inflammatory pathways, but estrogen’s effects on immune cells after ischemic injury has not been as well studied. This review will focus on recent work examining the effect of estrogen on immune cells that could contribute to sex differences in stroke. Potential hormone-independent effects will be highlighted, although few of these studies have been performed to date.

The role of estrogen as a neuroprotectant in stroke is well established, however, much less is known about estrogen’s contribution to the immune response after ischemic injury despite numerous reports suggesting citing its role as a potent anti-inflammatory agent (Straub, 2007; Suzuki et al., 2009). estrogen likely has actions on both resident immune cells as well as on infiltrating peripheral leukocytes but the contribution of each to estrogen’s neuroprotective effects is unknown. Sexual dimorphism in immunity is evidenced by a more robust cellular and humoral response in females, which may partially explain the female bias in developing autoimmune disease (Lleo et al., 2008; McCombe et al., 2009). Females also show an increased resistance to infection and sepsis (Angele et al., 2006). Importantly, these improved outcomes correlate with female reproductive status, with less damage incurred during high estrogen states, suggesting an important contribution of gonadal hormones to these sex differences (Raju et al., 2008). The anti-inflammatory effects of estrogens on immune cells, endothelium, and in the brain are well documented (Kublickiene and Luksha, 2008; Nilsson, 2007; Pozzi et al., 2006; Straub, 2007; Vegeto et al., 2008; Villablanca et al., 2010). Although recent data suggests involvement of an adaptive and local autoimmune component following stroke, this review will focus primarily on the innate response as it relates to local and systemic activation of myeloid cells and how sex and gonadal hormones may influence this response (Becker, 2009; Vogelgesang and Dressel, 2011).

Experimental Evidence of estrogen mediated Anti-inflammatory Effects after Stroke

To date, few studies have detailed the specific role of estrogen in the immunological response to stroke. An early study by Santizo et al. (2000) found that female OVX mice had higher baseline leukocyte adhesion and more sustained adhesion six hours following stroke compared to ovary-intact controls. Subsequent findings have demonstrated that estrogen exposure decreases NF-κB activity, IkB, iNOS, and TNF expression which is associated with ischemic neuroprotection (Iadecola and Ross, 1997; Liao et al., 2002; Nagayama et al., 1999; Wen et al., 2004). Importantly, male rats treated with estrogen prior to MCAO showed a significant reduction in cortical IL-1β and reduced infarct size compared to non-treated males (Chiappetta et al., 2007), suggesting these effects are independent from genetic sex (XX vs. XY). Estrogen can suppress IL-1β-mediated activation of astrocytes, and the induction of COX-2 in cerebral endothelium (Friedman et al., 1996; Ospina et al., 2004). Interestingly, Koerner et al (2008) showed that estrogen-replacement in OVX mice led to a unique temporal pattern of brain cytokine expression following stroke, with an early increase in TNF, IL-1α, IL-1β, and IL-6 levels followed by a delayed reduction in expression of these pro-inflammatory molecules. This suggests that estrogen has temporal anti-inflammatory effects that may reflect a complex, sequential activation and suppression of the cellular immune response. Combination treatment with estrogen and progesterone (Dang et al., 2011) decreased cortical Iba1 (putative microglia) and CD3 (T- /NK cells) expression, and suppressed IL-6 and chemokine (CCL2, CCL5) expression in male rats, suggesting these anti-inflammatory effects of gonadal hormones are independent of chromosomal sex. However, this regimen was also neuroprotective, and the reduced immune response may simply reflect the decreased damage seen in treated males. While it is intriguing to speculate that similar estrogenic effects on cytokine expression might exist in stroke patients, thus far no sex difference has been observed in peripheral mononuclear cells or serum concentration of cytokines (Czlonkowska et al., 2006).

Estrogen and Inflammation: Timing of Therapy

The protective effects of estrogen are well established in the scientific literature, although not without caveats. Efforts to exploit estrogen for its therapeutic value have come up short in clinical trials, as both the Women estrogen Stroke Trial (WEST) and the Women Health Initiative (WHI) trial, reported a surprising increase in stroke incidence in estrogen-treated woman (Viscoli et al., 2001; Wassertheil-Smoller et al., 2003). The explanation for these findings has been debated extensively in the literature (Lisabeth and Bushnell, 2012; Lobo, 2007)). The timing of HRT initiation and the dose used are emerging as key factors in the vascular response to hormonal therapies. In the Nurses’ Health Study (NHS) HRT initiated early after menopause led to a lower stroke risk than if initiated in older cohorts of women. In addition high doses of estrogen were less protective against vascular disease and promoted stroke risk (Grodstein et al., 2000). The proinflammatory effects are also reflected in the serum levels of C-reactive protein (CRP), a marker of vascular risk (Cushman et al., 1999; Lakoski and Herrington, 2005). CRP was elevated 65% in healthy women (>65 years of age) exposed to 12 weeks of a high dose of micronized E2 (1 mg/d), and remained 92% higher than placebo even 12 weeks after treatment was discontinued (Prestwood et al., 2004). Interestingly, in reproductive-age women, endogenous E2 levels negatively correlate with CRP protein levels across the menstrual cycle (Gaskins et al., 2012; Wander et al., 2008), suggesting an overall anti-inflammatory effect with physiological levels of estrogens. In the Women’s Health Initiative (WHI) and the Women’s Estrogen for Stroke Trial (WEST) participants were well beyond menopause when they received HRT/ERT. It has been hypothesized that ERT should be initiated immediately at menopause in order to achieve its protective effects (Suzuki et al., 2007a), an effect recently modeled in preclinical studies (Liu et al., 2011; Selvamani and Sohrabji, 2010). In young animals estrogen treatment immediately following gonadectomy led to a significant reduction in local and plasma concentrations of pro-inflammatory cytokines (MCP-1, IL-6, TNF, GM-CSF) after stroke (Suzuki et al., 2007a). However, this effect was lost in females which had a 10-week delay between ovariectomy and initiation of estrogen-replacement suggesting that prolonged loss of estrogen makes subsequent treatment ineffective, or perhaps even detrimental.

The inflammatory changes seen throughout the lifespan differ in males and females, and may contribute to the sexually dimorphic responses to stroke (Leon et al., 2011; Liu et al., 2009). Importantly, our lab has recently shown that prolonged periods of hypoestrogenicity prior to estrogen replacement therapy (ERT) lead to a dramatically enhanced inflammatory response in aged female mice. Chronic ERT initiated in late middle age, during the estropause (at 14–15 months), markedly decreased nuclear NF-κB translocation, reduced the expression of pro-inflammatory cytokines, and led to a significant decrease in total infarct volume in animals subjected to middle cerebral artery occlusion (MCAO) at the age of 20 months (Liu et al., 2011). However, acute ERT in aged females (initiated 2 weeks prior to stroke) led to enhanced nuclear NF-κB translocation, a pro-inflammatory milieu and poorer behavioral and histological outcomes after injury. Interestingly, aged male mice receiving estrogen treatment, even acutely, demonstrated a significant reduction in pro-inflammatory markers and infarct size following stroke compared to age-matched females. This suggests that there is a poorly understood interaction between sex, hormones, and aging that leads to a sex-specific vulnerability to the detrimental effects of estrogen in aging (Bao et al., 2006; Grodstein et al., 2001; Hao et al., 2007a; Levine and Hewett, 2003; Sunday et al., 2007).

In addition to the initial pro-inflammatory phase, stroke induces a subsequent systemic immunosuppressive response, leading to splenic atrophy, T-lymphopenia and decreased peripheral cytokines levels (Offner et al., 2009; Urra et al., 2009b). Although this may benefit the brain, immunodepression may account for the increased susceptibility to infection in the days following stroke (Klehmet et al., 2009; Vogelgesang et al., 2008; Woiciechowsky et al., 1999). Sex hormones differentially alter this post-stroke immunosuppressive state. Estrogen treatment reduced stroke-induced peripheral immunosuppression in OVX female mice, and restored splenocyte numbers and proliferative capacity compared to untreated OVX mice (Zhang et al., 2010). Both male and female sex steroids seem to have effects on post-stroke immune status, as dihydrotestosterone (DHT)- reduced splenocyte proliferation and increased the number of splenic regulatory T cells following MCAO in male castrates compared to non-treated castrates (Dziennis et al., 2011). Regulatory T cells are a subset of CD4+ Fox3p+ T cells which release TGF-β and IL-10 and are thought to limit inflammation, an effect associated with splenic atrophy and post-stroke immunosuppression (Levings et al., 2006; Offner and Polanczyk, 2006). Somewhat surprisingly, no changes in CD4+ T cells, CD19+ B cells, or CD45+CD11b+ microglia/macrophage populations were seen between treated and non-treated castrates in ischemic brain. Whether these effects are also estrogen mediated via the aromatization of testosterone to estrogen is not yet known. Further studies are needed in both sexes to clarify the hormones vs. non-hormonal contribution to the post-stroke immune response.

CELLULAR COMPONENTS OF THE IMMUNE RESPONSE: HORMONE AND SEX EFFECTS

Astrocytes

Astrocytes are intimately associated with many unique cell types including neurons, endothelial cells, and microglia (Kimelberg and Nedergaard, 2010). It is becoming increasingly evident that astrocytes are endowed with the transcriptional machinery to perform specific immunological functions in the brain which were originally only attributed to central nervous system (CNS) resident myeloidderived microglia and macrophages (Dong and Benveniste, 2001). Inflammatory mediators released by astrocytes have been shown to activate endothelial cells, effect blood brain barrier permeability, regulate activation of microglia, promote B cell survival and differentiation, recruit leukocytes via chemokine signals, and regulate myelination (Farina et al., 2007). The development of neuroprotective agents which target astrocytes may promote neuronal survival and maintain the integrity of the neurovascular unit (Zhao and Rempe, 2010).

The effects of estrogen on astrocytes were first observed over 30 years ago and we direct the reader to excellent reviews by Garcia-Segura and colleagues (Azcoitia et al., 2010). Following stroke, there is an increase in estrogen receptor (ER)-α expression in astrocytes, a protective mechanism induced by the injured brain (Blurton-Jones and Tuszynski, 2001). Cortical ER-α gene expression increases rapidly after stroke as a result of a demethylation of the ER promoter in females, allowing for transcription of estrogen-responsive genes (Dubal et al., 2006; Westberry et al., 2010; Wilson and Westberry, 2009). At this point it is not known if this occurs in neurons, astrocytes, resident immune cells or possibly even infiltrating immune cells, but this does appear to be sexually dimorphic, as the increase in ER-α expression following MCAO was seen only in females (Wilson et al., 2011). In a recent study (Spence et al., 2011) utilizing the Cre-loxP system for ER-α, the neuroprotective actions of ERsignaling were found to be primarily mediated by astrocytes and not neurons in experimental allergic encephomyelitis (EAE) but similar studies have not yet been performed in stroke. In contrast to in vivo data suggesting a beneficial effect of astrocytic ER-signaling, in vitro studies are less consistent. For instance, primary rat astrocyte cultures demonstrated a decrease in ER-α expression after OGD (Al-Bader et al., 2011), and others have implicated ER-β as the receptor mediating the beneficial effects of estrogen after lipopolysaccharide (LPS)-induced injury (Saijo et al., 2011). Interestingly, in mice, females have as many as ~20% more hippocampal astrocytes compared to males, an effect that becomes more pronounced with aging (Mouton et al., 2002). Whether this is secondary to the loss of estrogen with gonadal senescence or due to a true “sex difference” is not yet known. OVX mice were not examined in this study, and even with removal of activational effects of steroids, early organizational effects remain. Estrogen does appear to have a suppressive effect on glial activation and proliferation after stroke (see Figure 1). Real-time imaging of GFAP-luciferase expression demonstrated lower GFAP expression during stages of the estrous cycle with the highest estrogen levels (Cordeau et al., 2008), an effect independent of infarct size.

Figure 1. The potential estrogenic effects on glial cells in the ischemic brain.

The anti-inflammatory effects of estrogen in the brain are primarily mediated by glial cells. Estrogen promotes a resting phenotype in microglia, as evidenced by increased ramified morphology and decreased MHCII expression. These actions serve to limit microglial activation, proliferation, and migration toward the injured site. Similar changes occur in astrocytes, ultimately leading to decreased glial scar formation. Estrogen-mediated decrease in aquaporin expression prevents osmotic imbalance and may lower risk of edema. Pro-inflammatory cytokines and chemoattractant signals normally released by activated astrocytes and microglia are suppressed in the presence of estrogen, thereby reducing extravasation of infiltrating immune cells. As well, estrogen enhances astrocytic expression of glutamate transporters which facilitates glutamate uptake, mitigating injury due to neuronal excitoxicity. In addition, astrocytes increase synthesis of endogenous steroid production after injury, and secrete growth factors like VEGF, which promote angiogenesis. Together, these actions have a profound impact on curbing neuronal injury due to ischemia by conferring protection via local immunosuppression. The green arrow represents enhancing or promoting action, and the red arrow represents inhibitory action.

Abbreviations: E2 (17 beta-estradiol), MHCII (major histocompatibility complex class II), VEGF (vascular endothelial growth factor)

Although ovarian-derived estrogens lead to neuroprotection in many animal models of brain injury (McCullough and Hurn, 2003), estrogens can also be synthesized by the local conversion of testosterone by aromatase in cells outside the gonads. Brain aromatase expression increases following MCAO in OVX stroke-prone hypertensive rats, consistent with other studies citing increased aromatase activity following CNS injury (Carswell et al., 2005; Garcia-Segura et al., 1999). Increasing local estrogen concentrations by enhancing glial aromatase expression may represent an intrinsic neuronal survival mechanism. Aromatase knockout female mice had significantly more damage after induced stroke compared to wild-type OVX littermates, suggesting that local, extra-gonadal production of estrogen contributes to neuroprotection (McCullough et al., 2003; Saldanha et al., 2009). Work by Liu et al. (2007a) also provided evidence of a sexual dimorphic response to aromatase-mediated neuroprotection. Female-derived astrocytes were resistant to OGD and H2O2-induced cell death compared to astrocytes derived from males, secondary to a female-specific increase in aromatization (Liu et al., 2008a). However, it is unclear if this is secondary to early developmental effects of estrogen (or testosterone) exposure, leading to a life-long increase in aromatase signaling in females, or is secondary to non-hormonal chromosomal effects. Sex steroid binding globulin maintains steroid levels in the low range after birth (Kanova and Bicikova, 2011), but ongoing work in our lab suggests major developmental effects of pre- and early postnatal sex steroid exposure on aromatase expression which can modify the response to stroke in adulthood.

Microglia

Microglia are CNS-resident macrophage-like cells derived from primitive myeloid precursors early in development. The role of microglia in ischemic injury has been extensively studied (see Iadecola and Anrather (2011), Yenari et al. (2010), and Jin et al. (2010)). Evidence suggests that sex differences exist in both absolute microglia number and their responsiveness to injury. Estrogens inhibit microglial proliferation in vitro (Ganter et al., 1992). On average, female mice have 20–40% more Mac-1+ microglia in the (C57Bl/6J) hippocampus than age-matched males (Mouton et al., 2002). Aged females had ~20% more microglia than young females, implicating sex hormones as a causal factor. Follow up studies revealed that estrogen replacement in OVX female mice suppressed this age-induced increase in microglia numbers (Gyenes et al., 2010; Lei et al., 2003a). Estrogen-replacement decreases microglial activation in several other neurodegenerative models (see Table 1). In addition to inhibiting proliferation, estrogen also promotes a resting phenotype, as evidenced by restoration to a ramified morphology and decreased MHCII expression (Barreto et al., 2007; Prat et al., 2011). In our studies, ovariectomy increased microglial numbers, an effect evident in both sham and stroke mice, suggesting that estrogen has a restraining effect on microglial activation (Figure 2). Hippocampal ER-β expression in microglia was selectively upregulated after ischemia in monkey, however, the contribution of ER subtype activation on microglia, at least as it relates to neuroprotection, is still a matter of debate (Baker et al., 2004; Saijo et al., 2011). ER-α knockout mice have spontaneous microglial activation, but subsequent studies have shown larger infarcts in mice with selective loss of neuronal (CamKII-Cre) ER-α compared to myeloid (LysM-Cre) ER-α knockout mice after permanent MCAO (Elzer et al., 2010). This suggests that the myeloid-specific loss of ER-α is not critical, at least to ischemic neuroprotection.

Figure 2. Microglial activation after stroke.

Coronal section of mouse brain at low (10×) and high (20×) power demonstrating increased Iba staining (a marker for microglia) in the penumbral region (indicated by a box in the schematic section) in ovariectomized females both at baseline (sham) and after stroke. DAPI was utilized as a nuclear counter stain.

Monocytes

Monocytes undergo rapid mobilization in response to inflammatory signals released at sites of tissue injury. Monocytes do not proliferate, thus turnover is constitutive, and required for proper renewal of macrophage and dendritic cell populations. Monocytes exhibit a high degree of heterogeneity, based on cell surface molecule expression and functionality. In fact, recent reports describe a subpopulation of ‘inflammatory monocytes’ which contribute to disease (Prinz et al., 2011). While monocytes have been shown to infiltrate the ischemic brain following stroke, their presence may be both detrimental and beneficial to the progression of the sterile inflammatory response (Garcia et al., 1994; Urra et al., 2009a). The number of circulating monocytes in patients following stroke is correlated with the risk of post-stroke infection (Chamorro et al., 2006).

In women, monocyte levels are higher both during ovulation (Maoz et al., 1985), and during gestation as compared to postpartum (Pitkin and Witte, 1979). Physiological estrogen levels have a significant impact on myelopoeisis (Barak et al., 1986; Wang et al., 2006). Evidence also points to a role for estrogen in regulating monocyte migration behavior (Miyagi et al., 1992; Okada et al., 1997) and estrogen treatment has been shown to reduce CXCR2 (interleukin 8 receptor beta), a chemokine receptor important for both monocyte and neutrophil migration (Lei et al., 2003b). Interestingly, the estrogen effect on monocyte adhesion may be temporal, in that it has been observed that estrogen is less effective at reducing monocyte adhesion when endothelial cells are exposed to cytokines (TNF, IL-1β) chronically (Mikkola and St Clair, 2002). Taken together, the ability of estrogen to reduce the migratory and adhesive capacity of monocytes may serve to limit entry and further amplification of the stroke-induced inflammatory response, but a specific role for monocytes in mediating ischemic sexual dimorphism remains to be determined.

Macrophages

In addition to microglia, distinct populations of hematopoietic-born macrophages are resident in the CNS. These include choroid plexus, meningeal, and perivascular macrophages. Because microglia and macrophages share many overlapping features, the contribution of brain macrophages to stroke has been difficult to assess. It is now understood that microglia populate the brain during a finite period in development, while CNS macrophages continually renew via hematopoeisis in the bone marrow. However, the ability to accurately discriminate between local proliferation of microglia and infiltrating monocyte-derived macrophages following CNS injury is still an unresolved issue (Guillemin and Brew, 2004). Thus, current knowledge of brain macrophage function is limited to- and deduced from- studies on peripheral macrophages and cell lines.

In a recent study by Scotland et al. (2011), compartmental sex differences in immune cell compositions and phenotypes in rodents was seen, with higher numbers of resident leukocytes in the peritoneal and pleural cavities in females compared to males. These resident female macrophage populations exhibited greater Toll-like receptor (TLR) expression, phagocytic activity, and NADPH oxidase activity compared to male macrophages, while cytokine production was attenuated, allowing for a dampened (and more controlled) inflammatory cell recruitment and leukocyte influx in response to sepsis. Although this more efficient response may be responsible for the female survival advantage seen in severe sepsis, it is not yet known if these differences are seen in sterile injury models such as stroke. Interestingly no difference in TLR expression was seen in female blood vessels, suggesting sex-specific up-regulation of TLR expression is restricted to female leukocytes. OVX reversed some of the differences, enhancing basal chemokine function to levels seen in males, and equalizing macrophage phenotype, function, and numbers. However, OVX had no significant impact on T-lymphocyte populations, which appeared to be regulated independently of activational levels of gonadal steroids. Sex differences in T cell function, although described for autoimmune diseases, are less well documented in stroke models but are an area of active investigation (Lakhan et al., 2009; Rubtsov et al., 2010).

Neutrophils

In response to infection or injury, neutrophils migrate to sites of inflammation where they function to contain the spread of injury though phagocytic removal of dead cells and release of inflammatory signals which amplify the response and serve to recruit more leukocytes. Historically, the large influx of neutrophils into the brain during a sterile inflammatory event has been viewed as detrimental (McColl et al., 2007). This view has been shaped by observations that neutrophil accumulation is associated with poor outcome after injury (Akopov et al., 1996; Price et al., 2004). However, neutrophil activation is an integral component of the host immune response, thus some studies suggest that neutrophilia may be a beneficial host response following stroke (Beray-Berthat et al., 2003; Hao et al., 2007b; Hayward et al., 1996). Evidence for sex differences in neutrophils came from early studies which described a positive correlation between estrogen levels and neutrophil counts in the blood (Bain and England, 1975; Pitkin and Witte, 1979). Interestingly, several studies have shown that women have comparably higher myeloperoxidase activity in neutrophils than men, which correlated with the menstrual cycle (Jansson, 1991; Kabutomori et al., 1999). Physiological levels of estrogen (10nM) enhanced neutrophil degranulation and promoted release of myeloperoxidase, elastase, and superoxide (Chiang et al., 2004). Interestingly, estrogen levels were positively correlated with ER expression levels, but differed between sexes; as estrogen upregulated both ER-α and ER-β in women, but only ER-α in men (see Table 2). The dynamics of neutrophil infiltration into the female brain and the result of activation following stroke has been yet to be determined.

Table 2.

The anti-inflammatory effects of estrogen on myeloid cells

Monocytes
Author	Model	Drug/Treatment/mutation	Downstream Effect
(Kanda and Watanabe, 2002, 2003)	In vitro - Human	E2	↑NGF, VEGF
(Stefano et al., 1999)	In vitro - Human	E2	↑NO release
(Pioli et al., 2007)	In vitro - Human	E2 + LPS	↓CXCL8
(Kramer et al., 2004)	In vitro - Human	E2	↓CD16, TNF, IL-1 β, IL-6
(Nathan et al., 1999; Pervin et al., 1998)	In vivo - Rabbit	E2 + hypercholesteremia	↓VCAM, MCP-1
(Lei et al., 2003b)	In vivo - Rat	E2 + hypercholesteremia	↓CXCR2
Macrophages
Author	Model	Drug/Treatment/mutation	Downstream Effect
(Ghisletti et al., 2005)	In vitro - Mouse	E2 + LPS	↓NF-κB translocation
(Adamski et al., 2004)	In vitro - Mouse	E2 + IFN-γ	↓MHCII
(Huang et al., 2008)	In vitro - Mouse	E2 + H2O2	↓TNF, IL-1 β, MIP-2, MCP-1
(Murphy et al., 2010)	In vitro - Human	E2 + LPS	↑KappaB-Ras2, miR-125b ↓TNF, let-71 miRNA
(Jawaheer et al., 2010; Suzuki et al., 2007c)	In vivo – Mouse, Rat	E2 + trauma-hemorrhage	↓IL-6, TNF, MIP-1a, MIP-2, TLR4
(Zheng et al., 2006)	In vivo - Mouse	E2 + hypoxia	↓MyD88, Src, IL-6
(Scotland et al., 2011)	In vivo - Mouse	Physiological E2	↑TLR2/3/4, MYd88, CCL2, CX3CR1, CXCL1, CXCL12, CCR1, CCR2, CXCR4, NAPDH oxidase
Atherosclerosis
(Uzui et al., 2011)	In vitro - Human	E2 + Ox-LDL	↓MMP-9
(Chiba et al., 2011)	In vivo - Mouse	E2 + apoE(−/−) hypercholesteremia	↑neutral cholesteryl ester hydrolase activity
(Rayner et al., 2009)	In vivo - Mouse	E2 + apoE(−/−)/(HSP27o/e)	↑HSP27 ↓Ox-LDL uptake
Dendritic Cells
Author	Model	Drug/Treatment/mutation	Downstream Effect
(Bengtsson et al., 2004)	In vitro - Human	E2 + LPS	↑IL -6, IL-8, MCP-1, osteoprotegerin, stimulative capacity, migratory activity
(Yang et al., 2006a; Yang et al., 2005)	In vitro - Mouse	E2	↑MHCII, IL-6, IL-10, CD40, CD54, viability, stimulative capacity ↓NF-kBp65, endocytosis
(Polanczyk et al., 2006)	In vitro/In vivo - Mouse	E2 + EAE	↑PD-1, Treg activity ↓ T-cell proliferation
(Papenfuss et al., 2011)	In vitro/In vivo - Mouse	Estriol + EAE, LPS	↑Th2 response, CD80, CD86, PD-L1/2, B7-H3/4, IL-10, TGF-β ↓IL-6, IL-12, IL-23, T-cell proliferation
(Kawasaki et al., 2008)	In vitro/In vivo - Mouse	E2 + trauma-hemmorhage, LPS	↑MHCII, CD83, Cd40, TNF, IL-6, IL-12p40, antigen-presenting capacity ↓Apoptosis
Neutrophils
Author	Model	Drug/Treatment/mutation	Downstream Effect
(Geraldes et al., 2006)	In vitro - Pig	E2 + IFNγ	↓CD40, CD40L, adhesion
(Chiang et al., 2004)	In vitro - Human	E2 + fMLP	↑Myeloperoxidase, elastase, superoxide, LDL oxidation
(Miller et al., 2004)	In vitro/In vivo - Rat	E2 + Vascular Injury	↓Chemotaxis, IL-1, IL-6, CINC-2α
(Doucet et al., 2010)	In vivo - Rat	E2 + trauma-hemorrhage	↓Respiratory burst activity
(Yu et al., 2006)	In vivo - Rat	E2 + trauma-hemorrhage	↓Myeloperoxidase, CINC-1, CINC-3, ICAM-1
(Garcia-Duran et al., 1999)	In vivo - Human	E2	↑nNOS ↓CD18
(Kabutomori et al., 1999)	In vivo - Human	Physiological E2	↑Myeloperoxidase activity
(Nadkarni et al., 2011)	In vivo - Human	E2	↑Annexin A1 ↓Adhesion
Platelets
Author	Model	Drug/Treatment/mutation	Downstream Effect
(Bracamonte et al., 2002)	In vivo - Pig	Physiological E2	↓PDGF-BB, TGF-β1, TGF-β2
(Jayachandran et al., 2003)	In vivo - Pig	Physiological E2	↓Aggregation, ATP secretion, MMP-2 ↑MMP-14
(Jayachandran et al., 2005b)	In vivo - Pig	E2	↑Tissue factor ↓CD40, CD40L
(Selles et al., 2001)	In vitro - Rat	E2	↑NO ↓Aggregation
(Leng et al., 2004)	In vitro - Mouse	ADP, collagen-related peptide, agonist-induced fibrinogen (sex effect)	↑Female platelet aggregation

Dendritic cells

Known for their remarkable capacity to stimulate memory and naïve T-cells, dendritic cells (DC) are considered to be the only ‘professional’ antigen presenting cell. DC’s are active players in the innate response and participate in phagocytosis, and cytokine and chemokine release. Much consideration has been given to DCs in recent years, for their unique role in bridging the innate and adaptive responses in diseases like atherosclerosis, multiple sclerosis, and more recently, stroke. Evidence that DCs may have a role in ischemic brain injury was first put forth by Reichmann et al. (2002) and Kostulas et al. (2002), whom both showed an increase in DC recruitment to the rodent brain after injury. Later work by Felger et al. (2010) indicates that a local population of DCs exhibits high MHCII and CD80 expression at 72hrs post-reperfusion, a time-point associated with maximal lymphocyte infiltration. These studies parallel the work of many others showing increased accumulation of DCs in the brain in diverse models of neuroinflammation (Newman et al., 2005; Pashenkov et al., 2001; Pashenkov and Link, 2002; Serafini et al., 2000; Suter et al., 2003). Taken together, these findings suggest that the number of DCs in the brain following injury positively correlate with the degree of injury. Estrogen-treated EAE mice had improved outcome and reduced levels of brain DCs, and decreased expression of pro-inflammatory cytokines in DCs in the spleen. Several recent studies (see Table 2) have confirmed the effects of estrogen on DC’s.

Platelets

In the initial response to vascular injury, platelets bind to collagen and extracellular matrix proteins and become activated, signaling the release of inflammatory mediators, attracting additional platelets, and inducing thrombus formation. Adherence of platelets to leukocytes induces a proinflammatory response potentiating thrombus formation and increases the risk of embolization (Cerletti et al., 2011). Antiplatelet therapy has become a mainstay in both primary and secondary stroke prevention, and interestingly, there has been some recent clinical evidence that women may benefit more from therapy than men, at least for the prevention of stroke (Ridker et al., 2005).

E2 reduces both platelet aggregation and aggregate size (Haque et al., 2001; Nakano et al., 1998; Selles et al., 2001). Platelet aggregation and secretion of ATP, PDGF, and MMP-2 are increased in OVX pigs, an effect which is mitigated by estrogen replacement (Jayachandran et al., 2005a). Yet, the findings of a potentially anti-thrombotic role for HRT are challenged by clinical data showing that both exposure to oral contraceptives in younger patients (DeLoughery, 2011) and HRT in post-menopausal women (Norris et al., 2002) increases thrombotic risk. Age, the overall health of the endothelium, and the route of hormone administration (as transdermal ERT is much less thrombogenic) are probably key factors in determining the overall effect of E2 on platelet and fibrinolytic function (see Figure 3) (Canonico et al., 2007; Scarabin et al., 1997). Several conflicting studies have reported enhanced reactivity in female compared to male-derived platelets, indicating a pro-aggregating role of sex or hormonal exposure (Jayachandran et al., 2005b; Johnson et al., 1975; Kurrelmeyer et al., 2003; Leng et al., 2004; Moro et al., 2005). To date only one study has directly examined the effects of E2 selectively on platelet activation following experimental stroke. OVX rats had increased platelet reactivity as evidenced by increased P-selectin expression, which was abrogated by estrogen-treatment in an eNOS-independent manner (Littleton-Kearney et al., 2005). However, because inflammation is also prothrombotic, the MCAO model of arterial occlusion by ligation does not accurately mimic the atherogenic/thrombogenic setting in humans, and thus, future studies examining the effects of estrogen on platelet responses in spontaneous or embolic models of stroke may prove more insightful.

Figure 3. The atheroprotective effects of estrogen.

Primary and secondary stroke prevention centers around vascular dysfunction associated with atherosclerosis and blood clotting, respectively. Macrophages are key players in atherogenesis, the development of arterial plaques. Estrogen acts to suppress pro-inflammatory cytokine production, and enhance removal of dead cells from the atherosclerotic tissue. Decreased uptake of modified lipoproteins prevents foam cell formation. Estrogen also prevents lipid buildup by increasing cholesterol ester metabolism and enhancing efflux transport of cholesterol out of the cell. Excessive blood clotting in response to vascular injury following stroke can impede reperfusion of blood flow back to ischemic tissue. Platelets are major contributors in the formation of blood clots. Estrogen has been shown to increase platelet- and endothelial derived NO levels, which plays an important role in reducing adhesion, aggregation, and recruitment. Decreased platelet-leukocyte aggregation due to estrogen exposure prevents lesion progression, plaque rupture, thrombus formation, and the risk of embolization. In sum, these changes prevent clotting and re-establish blood flow back to the ischemic site. The green arrow represents enhancing or promoting action.

Abbreviations: Ox-LDL (oxidized low-density lipoprotein), NO (nitric oxide), ATP (adenosine triphosphate)

In addition, many of the pro-thrombotic effects of hormone therapy are related to changes in circulating plasma levels of several endogenous anticoagulants (antithrombin, protein S, and plasminogen activator inhibitor) which decrease in response to estrogen, and are regulated by the menstrual cycle (Mendelsohn and Karas, 1999), rather than direct effects on platelets. As most strokes occur in post-menopausal women, understanding the effect of loss of E2 with menopause on both platelet function and coagulation factors is critical. Sex differences are present in the response to thrombolytic as well as antiplatelet therapy (Meyer et al., 2011). In a pooled analysis of thrombolytic trials (Hill et al., 2006; Kent et al., 2005), women had a greater margin of benefit compared to men when treated for stroke with thrombolytic drugs compared with placebo. Using noninvasive vascular imaging to assess vessel patency within 72 hours of IV tissue plasminogen activator (tPA) treatment, recanalization (TIMI score 1–3) was more likely in women after thrombolysis (Savitz et al., 2005). Aging and female sex have significant procoagulant effects (Roeloffzen et al.). For example, plasminogen activator inhibitor (PAI)-1 (a procoagulant) increases post-menopause (Koh et al., 1997) and higher plasma levels of PAI-1 are associated with thrombolysis failure (Kim et al., 2005). Future studies are urgently needed in this area as use of reperfusion therapies increases.

The potential influence of genetic sex on immunity and the immune response to stroke

Sex differences in X-linked and Y-linked gene expression have been widely documented (Xu et al., 2002; Xu and Disteche, 2006) and sex differences in X-chromosome gene expression in the blood leukocytes of ischemic stroke patients has been recently reported (Stamova et al., 2011). Female-specific expression of X-chromosome genes involved in natural killer cell signaling, TNFR1 signaling and axon guidance, transforming growth factor-signaling, and IL17 signaling (tissue inhibitor of matrix metalloproteinase-1) were seen. Male-specific changes in expression involved genes associated with development, cell-trafficking, and cellular movement, suggestive of a more robust inflammatory response in men.

Evidence for the potential role of genetics underlying sex differences in the neuroinflammatory response may be found in studies which observe an escape from X-chromosomal inactivation by certain X-linked genes that lack a Y counterpart (Libert et al., 2010). This phenomenon not only increases allelic diversity in females which could alter the immune response, but the presence of two functional copies increases gene dosage relative to males. X-chromosome genes which have been shown to escape inactivation and may be relevant to the stroke response include tissue inhibitor of metalloproteinase 1 (TIMP1), NF-kB activating protein (NKAP), and IL-1R-associated kinase (IRAK1) (Carrel and Willard, 2005).

The contribution of X chromosome dosage (XX vs. XO) to experimental stroke outcome was recently evaluated in our laboratory. Female XX or XO gonadally intact, OVX, and ovariectomized females supplemented with estrogen were subjected to temporary MCAO (Turtzo et al., 2011). Infarct sizes were equivalent between ovariectomized XX and XO mice, between intact XX and XO mice, and between estrogen-supplemented ovariectomized XX and XO mice. Estrogen was neuroprotective in both XX and XO mice. While this investigation showed no overall effect of X chromosome dosage on stroke, as with all negative studies, potential for type 1 error exists. Individual X-linked genes may be more important in humans, as up to 20% of X-linked genes escape X inactivation in humans (Carrel and Willard, 1999). In contrast, a much smaller percentage of X-inactivation escapees exist in mouse (Disteche et al., 2002; Tsuchiya and Willard, 2000). In addition, homologs of some genes present on the human X chromosome are located on autosomes in mouse (Carrel and Willard, 1999; Disteche et al., 2002; Tsuchiya and Willard, 2000). For these genes, escape from X inactivation may be crucial for dosage in humans, but irrelevant in mouse. In addition, which genes escape from X-inactivation can vary in a tissue-specific and age-dependent manner (Lopes et al., 2010). As this study only examined young mice, and X-linked contributions to the inflammatory response are probably more relevant in the aging brain, future studies are needed. Whether a stressor, such as cerebral ischemia, can trigger expression of X-inactivation escapees in humans or in mice has yet to be investigated. Sex differences could also be secondary to the male-limited expression of genes in the non-recombining region (NRY) of the Y chromosome (Sekido and Lovell-Badge, 2009; Skaletsky et al., 2003; Teuscher et al., 2006) or in the Sry gene itself.

In 2004, Bourdeau and colleagues identified 12,515 ERE (estrogen-responsive element) sites in the human genome, 11,810 in the mouse genome, and 660 gene-proximal EREs that are conserved between mouse and human (Bourdeau et al., 2004). In addition to estrogen-responsive autosomal genes, many sex chromosomal genes are also regulated by E2. Indeed, the X chromosome contains a high number of ERE sequences, although few have been validated to date. A list of ERE-containing genes on the X chromosome is provided in Table 3. The clinical significance of estrogen-responsive X-chromosomal genes is poorly understood, however, post-menopausal loss of estrogen could potentially alter gene expression, provided one allele is X-inactivated (or an X-escapee) and the other is constitutively regulated by estrogen. In such a scenario, it is plausible that X-chromosomal gene dosage could become severely disturbed following menopause. In a comprehensive analysis of male and female gene expression differences between sexes, Yang and company (2006b) screened over 300 mice and revealed more than 10,000 genes with sex-biased expression in four different somatic tissues. Approximately 13% of actively transcribed genes in the brain showed a sexual dimorphism, with 355 genes more highly expressed in females and 257 genes more highly expressed in males. Given the heterogeneity of gene expression in specific brain regions, these numbers likely underestimate the degree of sexual dimorphism in the brain (Arnold, 2004; Yang et al., 2006b). In addition to tissue-specific enrichment of sexually dimorphic genes on autosomes, the authors reported significant enrichment on the X-chromosome, which contained more female-biased genes. While the organizational and activational effects of endogenous sex hormones likely contributed to these differences, the epigenetic regulation of autosomal gene expression by sex chromosomes has also been widely reported, as evidenced by differences in chromatin and gene dosage in X and Y chromosomes (see review by(Wijchers and Festenstein, 2011).

Table 3.

Inflammation-associated ERE-containing X-chromosomal genes and X-encoded MicroRNAs.

Author	X-linked genes containing ERE sites
(Bourdeau et al., 2004)	tlr8
	tnfsf5
	cxcr3
	cldn2
	c6
	IL-2rg
	IL-13ra
	TLR-7
	TIMP-1
	Factor VIII
	Factor IX
	IL-1Rap12
	IKBKG
	IRAK1
Author	X-linked MicroRNAs
(Pinheiro et al., 2011)	miR-18b
	miR-98
	miR-106
	miR-223

Sex Differences in MicroRNA Expression and Implications for Stroke

X-linked differences in the expression of X-encoded miRNAs may also alter regulation of the immune response and provide a basis for sex-linked disease (Pinheiro et al., 2011). The reader is referred to an excellent recent review for details on the miRNA’s located on the X chromosome that have been implicated in immunity and cancer (Pinheiro et al., 2011). Analysis of the genomic distribution of miRNAs found higher densities of miRNAs positioned on the X chromosome (Guo et al., 2009) and remarkably, to date, no miRNAs have been identified on the Y chromosome. Many of these X-linked microRNAs escape meiotic sex chromosome inactivation (Song et al., 2009). Although evidence is still scant, specific X-located miRNAs have been implicated in multiple sclerosis and in the regulation of both the innate immune response and monocytopoiesis (Otaegui et al., 2009; Pinheiro et al., 2011). Recent work in our lab has also provided evidence for miRNA in the etiology of sex differences in stroke. In males cell death is triggered by the mitochondrial release of apoptosis-inducing factor, a mechanism that is caspase-independent. In contrast, females are exquisitely sensitive to cell death induced by caspase activation (Siegel et al., 2010). As X-linked inhibitor of apoptosis (XIAP) is the primary endogenous inhibitor of caspases, we explored its regulation in the response to injury in females. Stroke induced a significant decrease in XIAP mRNA in females, whereas no changes were seen in the male brain. However, XIAP protein levels decreased in both sexes after injury. This was secondary to expression of miR-23a, a microRNA that directly bound the 3' UTR of XIAP. This sex-specific miRNA regulation was independent of activational levels of E2 (Siegel et al., 2011), and may explain the sensitivity of females to caspase-induced cell death. Furthermore, miR-23 is among other miRNAs found to be differentially regulated in the blood of ischemic stroke patients (Kulshreshtha et al., 2008; Tan et al., 2009). More recently, Selvamani and colleagues (2012) describe the neuroprotective effects of anti-Let7f treatment which regulates microglial expression of insulin-like growth factor 1 (IGF-1), a protein known to possess both neurotrophic and angiogenic properties. Interestingly anti-Let7f only reduced injury in gonadally intact females, with no effects in males or ovariectomized females, suggesting that the actions of miRNAs are influenced by local steroid levels. Rapidly evolving clusters of miRNA have been documented on the X chromosome in primates (Zhang et al., 2007) which are preferentially expressed in testis (Bentwich et al., 2005). However, the functional significance of X-linked miRNA expansion is unknown.

To specifically dissociate the organizational effects from sex chromosome effects, several investigators have capitalized on the recently developed “four core genotype” (FCG) mouse model (Arnold, 2009a; Arnold and Chen, 2009). Several studies utilizing the FCG mice have successfully demonstrated the importance of sex chromosome complement to male sexual behaviors, pain responses, addiction behavior and neuroinflammation (Bonthuis et al., 2012; Gioiosa et al., 2008; Smith-Bouvier et al., 2008). Recent work has found that mean arterial pressure was greater in XX mice compared with XY mice in the GDX state, suggesting that sex chromosome effects encoded within the XX sex chromosome complement contribute to hypertension in post-menopausal women, the major modifiable risk factor for stroke (Caeiro et al., 2011; Ji et al., 2010). Stroke studies on these mice should help us dissect out the contribution of genetics and hormones to ischemic stroke sensitivity.

Therapeutic Potential of Hormone Therapy in Ischemic Brain Injury

Despite the promise of early animal studies and observational human data indicating the potential benefits of HRT, the failure to translate in clinical trials in both the primary (WHI) and secondary (HERS; WEST) prevention of stroke has been disappointing (Hulley et al., 1998; Simon et al., 2001; Viscoli et al., 2001; Wassertheil-Smoller et al., 2003). While reports have been mixed, many studies suggest an increased risk of stroke occurrence following HRT or ER treatment (Anderson et al., 2004; Barrett-Connor et al., 2006; Fisher et al., 1998; Hendrix et al., 2006; Tannen et al., 2007; Wassertheil-Smoller et al., 2003). Moreover, a meta-analysis of 28 clinical trials found that hormone replacement therapy was associated with an increased risk of ischemic stroke (not hemorrhagic) and an increased risk of fatal stroke (Bath and Gray, 2005). The importance of these trials is reflected in the national guidelines which note that HRT provides no benefit for stroke risk reduction and should be reserved for the acute treatment of peri-menopausal symptoms (Lisabeth and Bushnell, 2012; Sacco et al., 2006). The timing, duration, dosage, and route of administration (ie., transvaginal, oral, transdermal) are critical parameters in determining a HRT regimen and must individualized to minimize risk (Shoupe, 2011). Postmenopausal or disease-related changes in estrogen receptor expression patterns and genetic variation in ERE binding sites may also influence the actions of estrogen (Higaki et al., 2012; Wang et al., 2012; Yu et al., 2011). The higher incidence of ischemic stroke following HRT, suggests a link between estrogen, inflammation, and thrombogenesis. In fact, evidence from several clinical trials indicates an increased risk of occurrence of venous thrombosis following HRT (Brass, 2004; Canonico et al., 2007). Such risks are offset when estrogen is administered via transdermal delivery as opposed to the more conventional oral route (Olie et al., 2010; Speroff, 2010). In particular, low dose, short duration, transdermal estrogen (with or without progestogen) is not associated with an increased risk of stroke, and may present a safer alternative therapeutic option (Renoux et al., 2010; Renoux and Suissa, 2011). The transdermal approach avoids the first-pass effect in the liver associated with the oral route, which is known to increase hepatic protein synthesis of clotting factors, inflammation markers, and sex hormone binding globulin (SHBG) (Goodman, 2012).

The potential for HRT or other hormone based therapies to be developed for stroke treatment remains unclear as existing studies enrolled patients that were at high risk for stroke. To definitively address the potential context-dependent role for estrogen, we must await the results of the Kronos Early Estrogen Prevention Study (KEEPS), a four-year, controlled, randomized, clinical trial designed to answer whether initiating HT (low dose estrogen and progestin) either as an oral or transdermal formulation in recently menopausal women slows the rate of atherosclerosis as measured by carotid intima-media thickness (IMT) (Harman et al., 2005). Importantly, the vast majority of experimental trials have demonstrated the efficacy of estrogens as neuroprotective agents in acute injury paradigms (induced stroke, oxygen-glucose deprivation etc.). Ongoing trials of hormone-based therapy (both progestins and E2) for acute traumatic brain injury (TBI) may help pave the way for future stroke trials (Stein and Wright, 2010). One caveat is that TBI patients are traditionally much younger than stroke patients. The efficacy and safety of therapy will have to be confirmed in older individuals prior to any potential trial, especially with recent preclinical data demonstrating a pro-inflammatory effect of acute estradiol in aged female animals (Liu et al., 2012). However in that study, “acute” exposure was a two week pre-treatment paradigm that was given to mimic prior HRT exposure before an induced stroke. Studies that give acute doses of estrogen post-stroke have shown clear efficacy with a prolonged therapeutic window (6 hours) in animal models but have yet to be performed in aged animals (Liu et al., 2007b). Pilot trials utilizing acute transdermal estrogen treatment to reduce injury in stroke patients should be considered. This may avoid potential the detrimental changes in coagulation and inflammation seen with chronic oral therapy, and could be tested in both sexes. Rationale trial design with measurement of perfusion status by MRI or CT, and concomitant measurements of serum biomarkers (i.e., CRP) may improve patient selection. Emerging pre-clinical studies demonstrating that estrogen treatment enhances stroke-induced neurogenesis may also be of relevance for the development of future therapeutic trials examining chronic functional recovery after stroke (Li et al., 2011; Suzuki et al., 2007b).

Summary

While the host response to sterile inflammatory events is generally viewed as a beneficial response, overactive or dysregulated signaling can lead to homeostatic imbalances which give rise to chronic inflammation. Given that prolonged inflammation can be detrimental to outcome and that stroke-induced immunosuppression can increase susceptibility to infection, recent efforts have been aimed at targeting the immune system to develop novel neuroprotective therapies. Inflammation is an attractive target for therapy due to its wide therapeutic window. The current FDA-approved drug, tPA, is only effective in treating stroke within four and a half hours after the onset of symptoms (Carpenter et al., 2011; Hacke and Lichy, 2008). Because patients may not recognize symptoms, or arrive to the hospital too late to receive treatment, the need to develop new treatments with wider treatment windows is paramount.

In conclusion, factors beyond the activational effects of hormones contribute to ischemic sexual dimorphism. The contribution of sex chromosome dosage, early prenatal organizational effects of hormones, and epigenetic factors, or the interaction between these in stroke related inflammatory signaling promises to be a complex but rewarding area for future researchers in both the clinic and in the laboratory.

Highlights.

• A literature review on potential sex differences in the immune response to stroke

• The underlying basis of sex differences in stroke: hormones versus genetics

• Discussion of hormone and sex effects of the cellular components of the immune response

Abbreviations

OGD

oxygen-glucose deprivation

NMDA

N-methyl-D-aspartic acid

estrogen

17 beta-Estradiol

OVX

ovariectomy

TNF-α

tumor necrosis factor-α

MCP-1

monocyte chemotactic protein-1

ERT

estrogen replacement therapy

MCAO

middle cerebral artery occlusion

DHT

dihydrotestosterone

CNS

central nervous system

LPS

lipopolysaccharide

EAE

experimental allergic encephomyelitis

ER

estrogen receptor

TLR

Toll-like receptor

DC

dendritic cell

tPA

tissue plasminogen activator

NRY

non-recombining region

XIAP

X-linked inhibitor of apoptosis

FCG

four core genotype

NHS

Nurses’ Health Study

WHI

Women’s Health Initiative

WEST

Women’s Estrogen for Stroke Trial

HERS

Heart and Estrogen/progestin Replacement Study