The present study used the XY* mouse model to examine the effect of sex chromosomes on stroke sensitivity, and several important findings were revealed. Firstly, the second X chromosome (but not the Y chromosome) contributes to sex differences seen in stroke. This effect is independent of the acute activational effect of gonadal hormones as the same sex differences were seen in the absence of gonadal hormones in a cohort of surgically gonadectomized mice. Secondly, sex differences in the inflammatory response to stroke are also impacted by the chromosome compliment. Significantly higher post-stroke inflammation was seen in mice with two vs. one X chromosome. Similar patterns were seen in the activation of microglia, the infiltration of peripheral immune cells in the ischemic brains, and the level of circulating cytokines. Thirdly, two X chromosome genes named Kdm5c and Kdm6a are more highly expressed in microglia having two vs. one X chromosome both before and after stroke. Based on our knowledge, this is the first study that specifically investigated the contribution of the second X chromosome to stroke sensitivity and post-stroke inflammation. This has further extended our understanding of the biology and etiology of sex differences in stroke.
Our previous studies have recapitulated the “aged female sensitive” stroke phenotype seen in stroke patients. Aged female mice have larger infarcts and worse neurobehavioral deficits compared to age matched males after MCAO [5]. The conclusion was further confirmed in another study using aged FCG mice that also showed worse stroke outcomes in XX vs. XY mice with two vs. one copy of X chromosome [8]. However, these prior studies could not differentiate the effects of the X chromosome from that of the Y chromosome, as all mice were genetically XX vs. XY. The present study replicates and extends our previous finding in FCG mice by using XY* mice model. This model is designed to specifically examine the effect of an X or Y chromosome on stroke sensitivity by comparing XX vs. XO and XY vs. XXY (X effect), or XX vs. XXY and XO vs. XY (Y effect). We primarily used aged XY* mice as stroke mainly affects the elderly, and this also allowed us to minimize the effect of circulating hormones, as gonadal hormone levels were equivalently low in aged XY* mice of all four genotypes. However, hormonal activational effects [32] at puberty may still impact stroke outcomes. Therefore, we included a cohort of mice that were gonadectomized at weaning age to eliminate the activational effects of gonadal hormones. Similar sex differences as those seen in aged mice remained in GDX mice, i.e. XX and XXY mice had worse outcomes compared to XO and XY respectively (Fig. 2G-I), suggesting the detrimental X chromosomal effect on stroke outcomes is independent of the activational effects of gonadal hormones. We cannot exclude organizational effects of hormonal steroid that occur before or after birth and are permanent [35]. Nevertheless, the consistency of the data in both our aged and GDX young cohorts strongly indicates a deleterious effect of X chromosomal dosage to stroke sensitivity. Some reports suggest that the Y chromosome plays a pivotal role in various diseases [36–40]. However, the present study suggests that Y chromosome does not contribute to stroke sensitivity. Nevertheless, the contribution of Y chromosome to the stroke sensitivity is not conclusive, and warrants further investigation.
Microglia are the brain resident immune cells and are one of the first immune cells activated after brain injury [41]. Microglial activation after stroke has been increasingly recognized as a key element in initiating and perpetuating post-stroke inflammation [42–44]. Our data suggested that both pro- (TNFα, IL-1β) and anti-inflammatory (IL-10) activation in microglia is also regulated by the second X chromosome (Fig. 3), and this effect extends to effects on peripheral immune cell infiltration (Fig. 4) and circulating cytokine levels. The immune responses to stroke are both causative and resultant to the ischemic injury [45], and lead to secondary neuronal death after stroke [46]. Studies by our laboratory and that of other groups have consistently revealed sex differences in the immune responses to stroke [13, 27, 47–50]. The present study has further determined that X chromosomal effects could be the driving force for sex differences in post-stroke inflammation. The immune system has long been known to develop in a sexually dimorphic manner that results in a sex bias in infectious diseases, autoimmune and inflammatory diseases and cancer [51]. Our data suggest that there is a mechanistic link between X chromosomal genes and sex differences seen in the inflammatory response to stroke.
The XX vs. XY differences in post-stroke inflammation and outcomes in aged mice could be explained by inherent differences in XX and XY cells. Our interest focuses on a group of genes that escape the X chromosome inactivation (XCI). The process of XCI reduces expression of genes on one of the two X chromosomes in each XX cell, bringing the dose of expression in XX cells to within the range of XY cells [52, 53]. However, some X genes escape inactivation, and are expressed by both X chromosomes, so that expression is constitutively higher in XX than XY cells [54]. We have previously found two X escapee genes, Kdm5c and Kdm6a, have higher expression in aged female vs. male microglia. KDM5C and KDM6A are histone demethylases of H3K4me3 and H3K27me3 respectively [55, 56]. Demethylation of H3K4me3 and H3K27me3 by KDM5C and KDM6A to their me1 forms confer repressive and activational effect on gene expression respectively [57, 58]. Since Kdm5c and Kdm6a escape XCI, the demethylation levels of the two histones are different between males and females, which might lead to sex specific, epigenetic modification of inflammatory genes. Our previous study suggested that two important interferon regulatory factors (IRF), IRF4 and IRF5, are targets of these KDMs [13], and IRF4/IRF5 are the key determinants of microglial anti- and pro-inflammatory activation respectively [21, 22, 59, 60]. The present study confirmed higher expression of these two KDMs in microglia having two vs. one X chromosome (Fig. 6), although our primary focus was on the determination of X vs. Y chromosomal effects on stroke sensitivity and post-stroke inflammation. Ongoing studies in the lab are using conditional knock out mouse model in which Kdm5c or Kdm6a is specifically deleted in microglia, to investigate the molecular mechanistic link between the KDMs and IRFs.
In summary, the present study employed the XY* mouse model and investigated the role of sex chromosomes in ischemic stroke outcomes and inflammation. We revealed that the number of X chromosomes determines the stroke sensitivity in aged mice and these effects are not attributable to circulating hormone levels. Microglial activation and the inflammatory responses to stroke are also impacted by the second X chromosome, which might be a driving source for the sex differences seen in aged stroke. The Y chromosome seems to play a minimal role in mediating stroke sensitivity, and X escapee genes likely initiate a downstream mechanistic cascade that leads to the sex differences in stroke.