Introduction
Multiple lines of evidence suggest differences in both innate and adaptive immune responses between males and females [
1,
2]. Relative to men, women clear some bacterial and viral infections more rapidly [
1,
3‐
7], show a stronger immunogenic response to certain vaccinations [
8], and have an increased risk of autoimmune diseases [
2]. In addition, sex differences are found in many diseases associated with dysregulated immune responses [
2]. This includes Alzheimer’s disease (AD), in which approximately twofold more women are being diagnosed with the disease compared to men—a difference which cannot be explained by disparities in life expectancy alone [
9]. Accumulation of reactive microglia around amyloid plaques and tau aggregates are consistent neuropathological features of the disease [
10]. Furthermore, genes associated with AD from genome-wide association studies (GWAS) are enriched in microglia, implicating them causally in disease susceptibility [
11‐
16]. In the rodent brain, sex differences have been observed in microglial distribution [
17,
18], morphology [
17,
19], functional output [
19‐
21], transcriptional profile [
17,
18,
21‐
24], and immune responsiveness [
22,
25‐
29]. For example, microglia from female mice exhibit augmented expression of an APOE-driven network of genes associated with aging, amyloidosis, and tau [
30‐
32]. Yet, whilst peripheral human immune cells isolated from men and women show phenotypic and functional variations [
1,
2], there are only limited studies examining sex differences in human microglia [
33‐
35].
Sex-specific phenotypes may be partially explained by differences in exposure to sex hormones. For example, oestrogen exerts both pro- and anti-inflammatory effects on peripheral immune cells depending on the inflammatory stimulus, dose, and cell type [
36]. Sexually dimorphic immune responses also could arise due to incomplete X-chromosome inactivation in females. Many genes contained on the X chromosome have been linked to immune-related functions [
2,
37], and ~ 23% of X-linked genes retain augmented expression in females [
38]. Given the central role of microglia in disease susceptibility and the sex-specific differences in microglial phenotype and function observed in mice, we hypothesised that the greater prevalence of AD in women may be partially explained by sex differences in microglia. To test this hypothesis, we sought to determine whether microglia isolated from women exhibit a transcriptional signature enriched for processes relevant to AD, in comparison with those from men.
Discussion
Sex differences in prevalence can be observed across the spectrum of diseases associated with dysregulated immune responses [
2]. This includes AD, for which genetic evidence suggests that microglia may play a causal role [
12‐
15]. Here, we sought to determine whether microglia isolated from women with no ante-mortem neurological disease exhibit a transcriptional signature that is biased towards pathways relevant to AD relative to those isolated from men. snRNA-seq data previously generated by our laboratory [
39] revealed that genes associated with an increased risk of AD were significantly enriched in microglial nuclei isolated from brains of women without neurological disease relative to those from men. This finding was replicated in a larger, independent brain microglial snRNA-seq data set [
41]. Further analysis determined that brain microglial gene signatures in women were enriched in gene sets whose expression was associated with the accumulation of pathological human β-amyloid or phosphorylated tau species in transgenic mouse models [
31,
43‐
46]. Genes significantly upregulated in microglia from women were primarily associated with a pro-inflammatory immune response. A consistent pattern was observed in similar analyses of peripheral myeloid cell populations (MCs and MDMs). The relative gene set enrichment in peripheral myeloid cells was not modulated by short-term exposure to oestrogen in vitro. These data are consistent with the hypothesis that the increased risk of AD in women may be partly explained by a sex-specific microglial phenotype which is transcriptionally predisposed to processes relevant to the disease pathology. A notable exception to the consistent results found throughout the data sets analysed here is the lack of enrichment of inflammatory and IR-AD gene sets in the microglial nuclei isolated from individuals with AD. This suggests that the sex differences observed in the non-disease populations are reflective of an increased baseline expression of genes relevant to AD pathology in women, potentially predisposing them towards the disease pathology, i.e., it is a factor contributing to susceptibility. This hypothesis is consistent with loss of this difference in AD patients; whilst susceptibility to the disease may be increased, the expression of the disease once established is not changed.
Previous studies of sex differences in the human peripheral blood transcriptome exhibit inconsistent results [
52,
53], but these data may have been confounded by differences in frequencies of immune cell populations between the sexes [
54]. Those studies assessing transcriptional profiles of isolated monocytes alone reported evidence for sex-specific effects consistent with our findings. For example, the transcriptome of peripheral monocytes isolated from individuals with evidence for chronic-low level inflammation found augmented expression of pro-inflammatory genes in women relative to men [
55]. Moreover, in patients diagnosed with early Parkinson’s disease, inflammatory activation of monocytes was observed in cells from women, but not men [
56]. These studies also provided evidence for enrichment of genes associated with the IFN-response in peripheral monocytes from women, as we report here [
52,
55,
56]. Whilst we observe clear sex differences in the non-diseased monocytes, we did not find a data set that allows us to determine whether these differences were retained in monocytes isolated from individuals with AD.
Sex differences have also previously been reported in the human microglial transcriptome. Using computational cell mapping techniques to predict microglial gene expression from bulk transcriptomic data, Bonham et al. [
33] inferred dysregulation of microglial genes in AD patients in the cerebellum and temporal cortex, and these perturbations were observed to a greater extent in women compared to men. Moreover, using snRNA-seq Mathys et al. [
57] identified a subcluster of AD-associated microglia enriched in the brains of women relative to men. However, due to the relatively rarity of microglia in the brain, in this un-enriched population only a low resolution of microglia was be obtained (
n = 1920 microglial nuclei compared to
n = 27,592 in our enriched discovery data set [
39] and
n = 148,606 in the replication data set [
41]). However, despite the large number of nuclei, these data sets were derived from a small number of individuals. Additionally, considering APOE is the main genetic risk factor for AD, it would be of interest to confirm these results in a larger sample size in which the APOE genotype is known. Nevertheless, the results from these studies support the hypothesis that the female bias observed in AD diagnosis may be partially driven by sex differences in microglial phenotypes.
The lack of evidence in our study for an E2-mediated effect on the GWAS and AD related gene sets analysed suggests that the sex differences we observed arise as a consequence of differential regulation of expression of genes on the sex chromosomes. The X chromosome includes the largest number of annotated immune related genes in the human genome [
2], including the X-linked helicase
DDX3X, an upstream regulator of type I interferon [
58]; the toll-like receptors,
TLR7 and
TLR8 [
59]; and
IRAK1,
NEMO,
NKRF, and
NKAP—members of the NF-κB signalling pathway [
59]. Moreover, genes which have been shown to escape X-chromosomal inactivation, including
TLR7 and
IRAK1 [
38], have the potential to functionally influence phenotypic diversity between male and female immune cells. Future research could study this directly by exploring sex-specific enhancer-promoter interactions for the sex chromosomes in microglia.
Although we have made convergent observations using different myeloid cell data sets generated both in house and externally, our study has several limitations. We have based our rationale on an interpretation of the predominant AD associated gene expression enrichment for microglia in the context of cells in the brain yet relied in part on supportive evidence from data concerning peripheral monocytes and macrophages. This genomic evidence could be interpreted more broadly as implicating sex-specific myeloid (as well as yolk sac derived microglia) cells in determining the relatively higher frequency of AD amongst women. Moreover, as the monocyte data set was not designed to study AD, information on their amyloid and tau burden was not available. However, as the median age was 67, it is likely that only around 0.6% of individuals had any form of dementia [
60] which would be too small to account for the transcriptomic differences we report. Moreover, whilst amyloid deposits have been found in the brains of ~ 22% of cognitively normal individuals, no significant sex differences in total amyloid burden were reported [
61,
62] and thus this is unlikely to account for the transcriptional enrichments observed here. Furthermore, due to the difficulties in obtaining post-mortem microglia from younger donors, the age-independence of the sex differences in myeloid cells transcriptome was based on the analysis of peripheral populations. Berchtold et al., has previously identified sexually dimorphic changes in gene expression across the brain as a consequence of aging [
63]. Thus, we cannot rule out that there may be an interaction between the effects we describe and the age of the donors.
Relevant in vivo differences in monocytes across the menstrual cycle may have been lost as a consequence of the 7-day in vitro MDM culture and our study of the effects of E2 treatment used only short treatment periods. Moreover, due to practical considerations only a small number of male and female iPSC lines were used. Nevertheless, our conclusion that the observed expression enrichments are independent of sex hormones is supported by the fact that these enrichments were detected in both monocytes and post-mortem microglia from post-menopausal women. It is important to note, however, that our determination of a post-menopausal state was based on age alone and thus the donors may not all have been at the same menopausal stage.
Finally, all of our work is based on transcriptomic evidence because of its broad coverage and the confidence with which it can be related to genetic association study data. Similar interrogations of expressed proteins are needed as comparable cell-specific untargeted proteomic data of sufficient size becomes available.
In conclusion, we present transcriptomic data from multiple human-derived myeloid populations supporting the hypothesis that the increased risk of AD in women may be partly explained by a sex-specific microglial/myeloid phenotype which is biased towards processes involved in the disease pathology. Our identification of molecular evidence for sex-specific susceptibility effects for AD suggests that future research into mechanisms for sex-associated gene regulation may provide novel targets for modification of disease susceptibility.
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