Exploring Sex-Related Differences in Microglia May Be A Game-Changer in Precision Medicine Part 4

Stress Affects Microglia but Sex-Related Changes Are Influenced by Several Factors

Several studies have set out to determine the impact of different stressors on microglia in male and female animal models, partly driven by the need to understand the greater susceptibility of females to stress-linked psychological disorders. 

The relationship between stress and memory is very close. It should be noted that moderate stress can stimulate our memory ability, but excessive stress can greatly affect our memory.

First, moderate stress can keep people alert and focused. When we are under a certain level of stress, our brains automatically release some chemicals, such as adrenaline and norepinephrine, which can promote brain neurotransmission and regulate attention, thereby improving our memory ability. For example, when we are preparing for an important exam or interview, moderate tension and pressure will prompt us to prepare more focused and conscientiously, thereby achieving better results.

However, excessive stress can cause a person's brain to be in a bad state and have negative effects. Once the stress is too great, the human body will have an emergency response, which will prevent our brains from concentrating and release some harmful chemicals that will affect our nervous system and memory. For example, when we are under stress for a long time, our brains will work in a state of lack of oxygen, which will cause damage to our brain cells, thus affecting our memory and thinking abilities.

Therefore, we should learn to face pressure appropriately and not let it overwhelm us. When encountering stress, there are some ways to relieve stress, such as through physical exercise, breathing methods, meditation, and other methods to relax. Through these adjustments, we can balance our emotions and stress, thereby improving memory. We need to have confidence in ourselves, our abilities, and our ability to withstand certain pressures and challenges, and then create better results and value under moderate pressure.

In short, between stress and memory, moderate stress can stimulate our memory ability, while excessive stress can affect our memory. We need to master some methods of coping with stress so that we can perform better when facing pressure. Let us actively face the challenges and difficulties of life, believe in ourselves, and surpass ourselves. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material that has many unique effects, one of which is to improve memory. The efficacy of Cistanche deserticola comes from the many active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health in a variety of ways.

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One study compared the effect of restraint stress in 10–11- 11-week-old male and female rats and, acute stress increased the proportion of primed microglia in male rats and had the opposite effect in females whereas chronic stress decreased the proportion of primed microglia in females and exerted no effect in males (Bollinger et al., 2016). 

This effect seems to be dependent on variables including age, brain area, and type of stress because others reported that maternal separation decreased glial numbers in the ventral tegmental area, in P0–P14 males, compared with females, rats but no change was observed in substantia nigra (Chocyk et al., 2011). 

Prenatal stress also affected microglia in offspring and, specifically, sensitized the cells to inflammatory stimuli such that LPS increased Iba1 immunoreactivity in 2- to 2-month-old male mice (Diz-Chaves et al., 2013). 

In a separate study, chronic unpredictable mild stress exerted no effect on microglial morphology in 3-month-old male or female rats although administration of dexamethasone to the pregnant dams increased the number and length of microglial processes in the hippocampus of females and the nucleus accumbens of males (Gaspar et al., 2021).

Targeting Inflammation in a Sex-Specific Manner in Stroke Is Important

Ischemic or mechanical injury to the brain triggers many changes including an acute inflammatory response that is typified by microglial activation. A recent population-based study that followed over 9 million adults over about 15 years reported that females had a lower lifetime hazard of stroke than males but this ratio fluctuated with age, with premenopausal women less vulnerable and women in older age more vulnerable; overall, the outcome for females is worse (Roy-O'REILLY and Mccullough, 2014; Vyas et al., 2021). 

A key contributor to the increased vulnerability in older women is the loss of the antioxidant, anti-inflammatory, and neuroprotective effects of estrogens, which additionally modulate mitochondrial function (Torrens-Mas et al., 2020) and boost signaling pathways including activating phosphatidylinositol-3- kinase (PI3K) and nuclear factor erythroid 2–related factor 2 (Nrf2) (Ishii and Warabi, 2019). 

Like estrogens, progesterone suppresses microglial activation (Yilmaz et al., 2019) and, in middle cerebral artery occlusion (MCAO), attenuates microglial activation and neuronal damage, and improves functional outcomes (Zhu et al., 2019). However, despite the apparent beneficial effects of estrogen and progesterone in experimental models, translation into clinical use has failed to provide a convincing case for either because of lack of efficacy and/or significant side effects (Liu and Yang, 2013; Gibson and Bath, 2016). 

Studies in models of stroke have highlighted sex differences in the context of neuroinflammatory changes. For instance, MCAO induced greater microglial activation as assessed by Iba1 immunoreactivity, and infarct area as revealed by diffusionweighted imaging, in male mice compared with females. 

This was attributed to the fact that microglial gene expression in females is reflective of a neuroprotective/neuroreparative phenotype, compared with a more inflammatory phenotype in males (Villa et al., 2018). Interestingly infarct volume was decreased in male mice that underwent MCAO following transplantation with microglia from female mice compared with transplantation with microglia from male mice (Villa et al., 2018). Other factors may partly explain the sex-related differences in response to ischemic injury, including differences in activation of the pathways that trigger poly ADP ribose polymerase (PARP)–/caspase 3-dependent neuronal cell death as recently reviewed (Spychala et al., 2017). 

Significantly, neuronal cell death appears to post-date microglial activation as revealed in a study of time-related changes following transient forebrain ischemia in rats (Hamby et al., 2007). 

In this study, the PARP inhibitor, PJ34, completely blocked the ischemia-induced microglial activation and markedly decreased neuronal death. More recently, it was shown that post-ischemic delivery of PJ34 attenuated the MCAO-induced increases in (ipsilateral) cortical inducible nitric oxide synthase (iNOS), matrix metallopeptidase 9 (MMP9) and TNFα mRNA in male but not female mice although it decreased markers of microglial activation in both males and females (Chen et al., 2020). Consistently, in P9 mice, PJ34 reduced infarct volume in male mice but not females and this was associated with a decrease in Iba1+ COX2+ cells in male mice, which was interpreted by the authors as inflammatory microglia (Charriaut-Marlangue et al., 2018). 

Minocycline, which also inhibits PARP, was found to be neuroprotective in experimental stroke and also in male mice, but not female mice (Li and Mccullough, 2009). This is significant because some promising clinical data suggest that minocycline may be beneficial as a treatment for acute stroke (Malhotra et al., 2018), but sex-related differences, if any, remain to be explored. Proliferator-activated receptor alpha (PPARα) agonists, which reduce inflammatory cytokine production because they decrease nuclear factor κB (NFκB) activation, also offer potential benefits, and one such agonist, fenofibrate, has been shown to reduce MCAO-induced infarct volume albeit in male mice only (Dotson et al., 2016). 

Additional factors that may confer relative protection after experimental stroke in female mice are the increases in IL-10 secreting CD8+ T cells (Banerjee et al., 2013) and upregulation in IL-4 signaling (Rahimian et al., 2019), which are not seen in male mice. 

Significantly, both IL-10 and IL-4 attenuate modulate microglial activation (Lyons et al., 2007a,b; Laffer et al., 2019). The protective effect of IL-4 is consolidated by the finding that the reduced infarct volumes and neurological deficits, which are observed in females during phases of the estrus cycle when estrogen is high, are absent in IL-4-deficient female mice (Xiong et al., 2015). 

On the other hand, PPARγ agonists, which increase IL-4 in the hippocampus (Loane et al., 2009), appear to reduce the risk of stroke (Liu and Wang, 2019). In short, these findings demonstrate that while controlling inflammation may provide a beneficial therapeutic option in stroke, sex-related differences in treatments must be considered (a) to improve efficacy and (b) to avoid misinterpretation of clinical findings.

A Focus on Sex-Related Inflammation in Traumatic Brain Injury

Men are more likely to suffer traumatic brain injury (TBI) than women, particularly work-related injury and as a result of road traffic accidents, but whether recovery is similar in males and females is unclear with mixed evidence from clinical studies, depending on factors that include age and severity of injury. 

A recent meta-analysis of 156 studies reported worse outcomes for women than men when the injury was classified as mild moderate, and better when the injury was classified as moderate-severe, although statistical power influenced this conclusion (Gupte et al., 2019). 

In animal models, the evidence is also mixed and, here the confounding issues include the model used, the outcomes assessed, the age of the animals, and the relative lack of focus on the assessment of changes in females. Despite this, the evidence seems to tilt in favor of a protective role for estrogens in TBI as highlighted in recent reviews (Spani et al., 2018; Gupte et al., 2019); 17β-estradiol attenuates TBI-induced damage to blood vessels and blood-brain barrier function, increase in intracellular calcium and mitochondrial dysfunction (Kovesdi et al., 2020). 

A recent review of sex-related differences in response to injury suggests greater detrimental effects in male mice (Rahimian et al., 2019). This confirms data that demonstrated increased COX2 expression coupled with increased apoptosis (Gunther et al., 2015), and more extensive microglial activation and/or increased microglial numbers in male mice compared with female mice after either a penetrating cortical injury (Acaz-Fonseca et al., 2015) or controlled cortical impact (Doran et al., 2019). In the latter model, a more aggressive neuroinflammatory profile was observed in the cortex, thalamus, and dentate gyrus of male mice during the acute and subacute phases post-injury (Villapol et al., 2017). 

These authors also reported an increase in Iba1+ cells in the brains of male mice 1–7 days after TBI, while the number of ramified microglia was increased in female mice. Sex-related changes in inflammatory cytokines were also observed with earlier and more short-lived changes in IL-1β and TNFα mRNA in the cortex of female mice contrasting with more consistent increased expression of both in the cortex of males (Villapol et al., 2017). 

A greater infiltration of macrophages was observed in male mice following injury (Villapol et al., 2017; Doran et al., 2019) perhaps triggering the changes in microglial activation that have been described in in aged rats and APP/PS1 mice (Barrett et al., 2015a,b).

Factors That May Contribute to Sex-Related Differences in Microglia

Sex Hormones

The cause(s) of the sex-related differences in microglia is far from clear with the role of sex hormones being one obvious factor and this influence has been amply demonstrated. 

Estrogen has profound effects on all immune cells, including microglia; receptor-induced intracellular signaling cascades as well as direct activation of estrogen response elements, which are located on promotors of immune-function genes, both exercise control over the production of immune mediators (Acosta-Martinez, 2020). Thus estradiol eliminated the sex-related difference in phagocytic capacity of microglia in neonatal mice (Nelson et al., 2017) and attenuated the LPS-induced changes in microglia from adult male, but not female, rats (Loram et al., 2012). Specific age-dependent effects have been reported. 

For example, the microglial transcriptome, at least in terms of NFκB-regulated genes, was not markedly altered by ovariectomy or 17β-estradiol treatment in young mice (Villa et al., 2018) but in aged mice ovariectomy upregulated inflammatory molecules including TNFα and IL1β and increased responsiveness to LPS (Benedusi et al., 2012) as recently reviewed (Villa et al., 2016; Lenz and Nelson, 2018; Acosta-Martinez, 2020). 

A recent study reported the sexual dimorphic effects of 17α-estradiol and specifically showed that the age-related Iba1 immunoreactivity in the hippocampus and hypothalamus was reduced by treatment of 25-month-old male but not female mice and that the effect in the hippocampus was inhibited by castration, whereas ovariectomy exerted no effect (Debarba et al., 2021), which is at odds with earlier work that showed estrogens reduced the number of microglia in hippocampus of aged C57BL/6NIA female mice (Lei et al., 2003). Some reports suggest that estrogen treatment improves TBI-induced changes in animal models. 

The presence of membrane-bound estrogen receptors on neurons may directly contribute to the increased neuronal survival observed in estrogen-treated animals following injury (Brotfain et al., 2016) (see Figure 3). However, the effect of estrogens on microglia also plays a role in sparing neurons perhaps by inhibiting activation of NFκB through activation of PI3K (Crespo-Castrillo and Arevalo, 2020). 

Others have suggested that there is no beneficial effect of estrogen in male or female mice following TBI (Bruce-Keller et al., 2007). Progesterone receptors are also expressed on neurons and glia, and in animal models of brain injury, a protective role for progesterone in TBI has also been reported (Webster et al., 2015; Spani et al., 2018; Gupte et al., 2019) but clinical studies have failed to identify an overall beneficial effect of progesterone (Skolnick et al., 2014; Chase, 2015; Brotfain et al., 2016) and a meta-analysis of seven randomized controlled trials determined that progesterone treatment exerted no significant effect on outcomes of acute TBI in patients (Lin et al., 2015). 

Similarly, despite the benefits in animal models, the argument favoring the use of estrogens in clinical settings is unconvincing (Kovesdi et al., 2020). If a case is to be made for further clinical study, a focus on potential sex-specific benefits of hormone treatment would be warranted (Khaksari et al., 2018). There is little argument that loss of estrogen at menopause is linked with increased adiposity, insulin resistance, and the associated inflammation, all of which represent disease risks and confer a greater risk of AD. 

Despite the obvious possibility that hormone replacement therapy may reduce risk, clinical trials have provided little encouragement for such a therapeutic approach. However, a recent metanalysis reported that there was an overall beneficial effect in the 16 studies that were assessed (Song et al., 2020) although there are confounding issues that remain to be addressed one of which is to establish whether there is a critical window for hormone therapy in AD, as has been suggested (Song et al., 2020; Wu et al., 2020). 

A focus on analysis of change in microglia during the estrus cycle would deepen our understanding of the impact of hormonal control but there is a paucity of information linking the estrus cycle with microglial status. However, it has been reported that stress-induced IL-1β expression in the paraventricular nucleus, which may reflect microglia status, is attenuated during metestrus (Arakawa et al., 2014). 

Additionally, a transcriptomic analysis of changes in the medial prefrontal cortex across the estrus cycle identified a vast array of differences during the cycle that were markedly more profound than differences between samples from males and females. 

The differences reflect changes in genes that code for proteins that support multiple cell functions from metabolism to cell signaling. Although the authors discussed the changes in the context of neuronal function, many including cell signaling, cell adhesion, and membrane function may potentially also reflect changes in microglia (Duclot and Kabbaj, 2015).

Other Factors

Chromosomal makeup contributes significantly to sex-related differences given that genes on X and Y chromosomes encode for proteins with different immune functions. This is well illustrated by the finding that LPS-induced inflammatory cytokine production from monocytes obtained from male subjects is greater than from females and also from individuals with Klinefelter syndrome, who are phenotypically male but carry an extra X chromosome (Klein and Flanagan, 2016; Lefevre et al., 2019). The development of recent models has triggered an increase in understanding the contribution of chromosomes to sex-related differences in some diseases. 

For example. EAE induced by proteolipid protein was more severe in SJL-castrated male mice that were XX Sry compared with XY−Sry and also in ovariectomized female XX compared with XY− mice (Smith-Bouvier et al., 2008). The authors reported that the relative protection was associated with higher production of Th2 cytokines in XY mice. Furthermore, adoptive transfer of lymph node cells from ovariectomized female XX into WT females induced more severe disease than cells from XY− mice and this was associated with evidence of enhanced inflammation as suggested by an increase in CD45+ cells in the thoracic spinal cord. 

A similar approach was used to evaluate the contribution of chromosomes to mortality in a model of AD, given that the longevity of women with AD is greater than men. It was reported that the addition of an X chromosome conferred resilience. Specifically, XY-APP mice died earlier than XXAPP mice of either gonadal phenotype and cognitive testing revealed that the performance of XY-APP mice was poorer (Davis et al., 2020). 

Both studies demonstrate the impact of chromosomal makeup in these 2 disease models, the pathology of which includes inflammation, but neither direassessesses details of inflammatory change or changes in microglia. There is a paucity of literature exploring the impact of sex chromosomes on microglia.

There is little doubt that microglia are subject to epigenetic control (Cheray and Joseph, 2018) and indeed epigenetic changes underpin LPS-induced immune memory in microglia in the APP23 model of AD and trigger neuropathology (Wendeln et al., 2018) although sex-related differences were not explored. Multiple sex-related differences indicative of epigenetic changes have been described in the brain; for example, DNA methylation and methylated CpG sites are more pronounced in female brains compared with males, expression of ERa is modulated by promoter modulation and the effect of maternal behavior induces differential epigenetic changes in males and females that may impact on behaviors/pathologies in later life (Ratnu et al., 2017). 

However, epigenetic changes that specifically target microglia in a manner that may explain sex differences remain to be identified. Among other factors that influence microglial phenotype and function are microRNAs (miRNA) which have multiple roles including regulation of immune networks, and miRNAseq analysis in microglia has revealed sex-related differences (Kodama et al., 2020). 

Depleting Dicer in microglia, which markedly reduced miRNA, differentially affected the transcriptome in microglia from male and female mice and led to upregulation of genes reflecting immune activation and inflammation in males. Another factor that may impact microglial activation is the infiltration of immune cells. In this context, it has been shown that infiltration of macrophages into the brain of aged rats and APP/PS1 mice is associated with increased inflammation (Barrett et al., 2015a,b), while macrophages from both models are sensitized to inflammatory stimuli suggesting that they contribute to the inflammation. 

Importantly, flow cytometric analysis has revealed that the number of CD11b+/CD45high macrophages was greater in the brain of a 12-month-old female, compared with male APP/PS1, mice (Unger et al., 2018) suggesting that the more inflammatory microglia observed in older female mice might, at least in part, result from macrophage infiltration. In contrast, in adult mice, infiltration of macrophages following traumatic brain injury was greater in males than females (Doran et al., 2019) paralleling the greater microglial activation and evidence of neuronal damage, at least in the early stages following injury.

CONCLUSION

The focus of this review has been on conditions that are unified by the sex differences in microglial activation and neuroinflammation that characterize and contribute to their pathogenesis. The question therefore arises as to whether sex-specific therapies, perhaps particularly those targeting neuroinflammation and microglial activation, will provide a step toward precision medicine. 

A challenge is to address the under-representation of women in clinical trials which, although improving, persists (Labots et al., 2018; Steinberg et al., 2021). A report of 38 studies conducted on drugs approved by the FDA between 2000 and 2009 highlighted that a gender imbalance occurred in Phase I but not Phase II and III trials (Labots et al., 2018) but an analysis of 2020 clinical trials between 2000 and 2020 revealed that sex bias continues to exist in clinical trials (Steinberg et al., 2021) and a recent study highlighted the important fact that women are less likely to meet pre-screening criteria for inclusion in clinical trials in AD in Spain and education was a key factor (Rosende-Roca et al., 2021). 

Sex-related differences in absorption and excretion, distribution, and metabolism of drugs have been described and more adverse drug reactions have been reported in females compared with males (Whitley and Lindsey, 2009) highlighting sexual dimorphic responses to drugs, notably drugs that affect the CNS. In the context of AD, a recent review and metanalysis reported that only 7 out of the 56 randomized clinical trials in AD included reported sex-stratified results and concluded (Martinkova et al., 2021) and the relative lack of data on sex-related differences in drug efficacy has been noted (Ferretti et al., 2018). 

In light of this, a call has been made for a specific analysis of gender effects on drug treatments in AD (Schwartz and Weintraub, 2021) but this cannot be confined to AD and the principle should apply broadly, but particularly in conditions where clear sex-related differences in disease incidence exist.

AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor to this work and has approved it for publication.

FUNDING

This work was supported by Principal Investigator grants to ML from the Science Foundation Ireland (15/iA/3052 and 11PI/1014).

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