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date: 20 May 2019

Inflammatory Signals and Sexual Differentiation of the Brain

Summary and Keywords

Sex differences in the brain are established by the differential gonadal steroid hormonal milieu experienced by developing male and female fetuses and newborns. Androgen production by the testis starts in males prior to birth resulting in a brief developmental window during which the brain is exposed to high levels of steroid. Androgens and aromatized estrogens program the developing brain toward masculinized physiology and behavior that is then manifest in adulthood. In rodents, the perinatal programming of sex-specific adult mating behavior provides a model system for exploring the mechanistic origins of brain sex differences.

Microglia are resident in the brain and provide innate immunity. Previously considered restricted to response to injury, these cells are now thought to be major contributors to the sculpting of developing neural circuits. This role extends to being an important component of the sexual differentiation process and has opened the door for exploration into myriad other aspects of neuroimmunity and inflammation as possible determinants of sex differences. In humans, males are at greater risk for more frequent and/or more severe neuropsychiatric and neurological disorders of development, many of which include prenatal inflammation as an additional risk factor. Emerging clinical and preclinical evidence suggests male brains experience a higher inflammatory tone early in development, and this may have its origins in the maternal immune system. Collectively, these disparate observations coalesce into a coherent picture in which brain development diverges in males versus females due to a combination of gonadal steroid action and neuroinflammation, and the latter increases the risk to males of developmental disorders.

Keywords: hypothalamus, preoptic area, estrogens, androgens, microglia, prostaglandins, mast cells

Introduction

What strange bedfellows these make. Inflammation, a complex biological response induced by illness or injury, and sexual differentiation of the brain, a complex biological response induced by gonadal steroids early in development. The two processes seem miles if not worlds apart. Yet the evidence that they are intricately linked keeps accumulating. Evidence comes from many quarters. First, there are empirical facts based on hypothesis-driven research in animal models. Second, there is epidemiological observation of higher male bias toward diseases and disorders associated with inflammation early in development. Last, there is clinical data supporting the concept of greater neuroinflammation in developing males. Each source and its findings are reviewed in turn, after first providing the foundations for inflammation and sexual differentiation.

Inflammatory Signals and Immune Cells

Inflammation is never considered a good thing. It is something that happens when a person is sick or injured. It can be systemic, involving the whole body, or it can be local, limited to the end of that stubbed toe. It can be chronic, lasting months, years, or even decades, or it can be acute and over within hours or days. Despite the fact that inflammation is integral to the healing process, it is inevitably associated with pain and discomfort in the body. But the brain does not feel pain per se, making the detection of inflammation far more difficult. There are many inflammatory signals, but they all initially originate from the resident immune cells at the site of the insult. In the brain, this means the microglia that are tiled throughout the nervous system.

Microglia are not related to the more familiar astrocytes or oligodendrocytes, which have common origins with neurons. Instead they are derived from myeloid precursors located in the embryonic yolk sac and from which they migrate into the brain. This occurs exceedingly early in development, around embryonic day 9 in the rodent and late in the first trimester in the human (Ginhoux, Lim, Hoeffel, Low, & Huber, 2013). Microglia both respond to and produce a wide range of signaling molecules, many of which are considered proinflammatory but also many that are anti-inflammatory. This mixed ability of microglia to be both protective and damaging has spurred an interest in understanding the circumstances and signaling pathways by which such divergent phenotypes are achieved. Likewise, there is a desire to categorize them by phenotype, which has led to classifications as either M1, a proinflammatory microglia, versus M2, an anti-inflammatory microglia (Mills, Kincaid, Alt, Heilman, & Hill, 2000). This appealing simplification has been challenged, however (Ransohoff, 2016), and many now use a 1 to 5 scale based on a combination of morphology and lysosomal enzyme expression to rate microglia as being “activated” versus “ramified” (Hong, Dissing-Olesen, & Stevens, 2016). The term “activated” is indicative of greater production of inflammatory signaling molecules such as prostaglandins and cytokines, while “ramified” is used to donate the surveillance of neighboring cells engaged in by these microglia. Neither function is exclusive to either form but rather a bias along a continuum of states. An additional change comes from our ability to visualize living microglia in brain slices by engineering them to express eGFP. Prior to this ability microglia were thought to be quiescent cells that sat waiting for an injury to occur before leaping into action to clean up the cellular debris. It is now know they are in fact constantly surveying the surrounding cells, with each microglia attending to a stable of neighboring neurons and astrocytes (Nimmerjahn, Kirchhoff, & Helmchen, 2005). Instead of quiescent, the more ramified microglia are now referred to as “surveying,” to reflect their state of constant vigilance. An important question in the context of sexual differentiation of the brain is whether the number and phenotype of microglia differ between males and females.

A second class of innate immune cells in the brain are the mast cells. These originate in the bone marrow and disperse throughout the entire body but also make up a resident population in the brain. Mast cells tend to exist at interfaces, such as the skin, mucosal linings of the mouth and nose, and around the eyes (Metcalfe, Baram, & Mekori, 1997). Consistent with that, they are concentrated in the meninges around the brain and usually only found in the neuropil when there has been an injury (Moretti et al., 2016). Mast cells are also part of the healthy brain and associated with reproductive endpoints, including production of the gonadotropin-secreting peptide (GnRH; Khalil, Silverman, & Silver, 2003) and responding to chemosensory cues of the opposite sex that promote mating (Kriegsfeld et al., 2003). More important, mast cells are directly implicated in sexual differentiation as they are found in greater numbers in males in a brain region called the preoptic area (POA) and to release histamine, which then signals to neighboring microglia (Lenz et al., 2018).

Inflammatory Signals and Sexual Differentiation of the BrainClick to view larger

Figure 1. Immune cells and inflammatory mediators in developing brain. Varied roles for multiple immune cells in the developing brain are still being discovered. The microglia are derived from myeloid precursors and are the largest population. They both respond to and release various signaling molecules including the inflammatory prostaglandins and cytokines. Mast cells and leukocytes are much less frequent but can be equally impactful by producing very specific signaling molecules such as histamine, serotonin, or the immunoglobulin, IgM.

Microglia and mast cells are innate immune cells of the brain, and they participate in inflammatory responses, but many inflammatory signaling molecules are made by neurons and astrocytes as well, most notably prostaglandins. Prostaglandins were discovered in the prostate gland, hence the name, but are ubiquitous signaling molecules throughout the body derived from arachidonic acid precursors in the cell membrane. The most common over-the-counter drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), are designed for the purpose of inhibiting prostaglandin production. The most famous NSAID is aspirin, which inhibits the actions of the cyclooxygenase enzymes, COX-1 and COX-2 (Vane, 1971). These critical enzymes convert arachidonic acid into a short-lived precursor that forms the basis of all prostaglandins. One prostaglandin in particular, prostaglandin E2 (PGE2), is central to inflammation for its role in producing a fever by acting on heat-inducing neurons of the POA (Blatteis, Li, Li, Feleder, & Perlik, 2005). But the COX enzymes are found in most cells in the body, and so actions are not limited to one region. However, those actions can be very specific and unrelated to inflammation. This, combined with our increasingly sophisticated views on the roles of microglia, and even mast cells, in the brain are causing us to rethink neuroimmunity and what it means for brain health, particularly during development (Figure 1).

Sexual Differentiation of the Brain

The maturing brain is characterized by epochs of sensitivity during which a particular neural circuit, synaptic pattern, or neurochemical phenotype is established. These include fundamental sensory systems such as vision, hearing, and somatosensory sensation, which require stimulation from the appropriate modality during a critical period. This is called a “critical” period because if the stimulation does not occur, then the system will forever be miswired or otherwise dysfunctional (McCarthy, Herold, & Stockman, 2018). This was demonstrated most clearly in the case of the visual system in the Nobel prize winning work of Hubel and Weisel in which kittens raised in an environment devoid of horizontal lines were incapable of recognizing such lines as adults. In the context of sexual differentiation of the brain, the discovery of a critical period had its origins in reproductive physiology with the observation that treating newborn female rats with testosterone rendered them sterile for life. Scientists at the time reasonably thought the source of the sterility was a direct action on the developing ovary. When this proved not to be the case, they turned their attention to the pituitary, and again were denied, leading them inexorably to the brain, in particular the hypothalamus (Barraclough & Gorski, 1961). What the scientists in the 1960s did not know is that the firing pattern of GnRH neurons is markedly different in males and females, and this determines whether luteinizing hormone is released from the pituitary in a continuous pulsatile fashion or in a cycle of surges, the latter being essential for ovulation (MacLusky & Naftolin, 1981). Newborn females treated with androgens adopt the continuous pulsatile pattern of GnRH firing and thus are not able to ovulate and are therefore sterile. The exposure of androgen must occur prior to 10 days of age; after that developmental time point the pattern is set and largely immutable. In this study the females were “sensitive” to the androgens during a “critical” period that proceeds normally in males in response to their own personally manufactured androgens. The same principle was tested and confirmed for the sexual differentiation of mating behavior, which while not explained by the firing pattern of a single neuronal phenotype nonetheless followed essentially the same rule set (Morris, Jordan, & Breedlove, 2004). This framework is frequently referred to as the activational/organizational hypothesis and was established more than 60 years ago (Phoenix, Goy, Gerall, & Young, 1959).

Mating behavior in mammals is largely controlled by the same nodes in the networks regulating social behavior, such as recognition and interaction with conspecifics, including play, nurturing, and aggression. These circuits further intersect with those of reward, presumably because being social, either by having sex or playing with a friend, is rewarding. The neurocircuitry of mating also intersects with those controlling fear as mating should not be engaged in when there is a predator baring down. The same goes for feeding: the hunger drive needs to be repressed when one is seeking to mate. The same circuit exists in males and females, but in rodents the behavioral output of males versus females is notably different. Male mating behavior is characterized by mounting and attempting to intromit the penis to a female whose own behavior consists of adopting a receptive posture called lordosis because it involves an inverse curvature of the spine. There are critical nodes in the neural circuit of mating that regulate these two different responses, and the divergence is established during the same perinatal sensitive period when the profile of an luteinizing hormone (LH) surge versus a LH pulse is determined. Moreover, the same hormones are involved, meaning that the production of androgens by the fetal and neonatal male testis organizes the neural circuit to be conducive to mounting and intromitting while the absence of androgens results in a neural circuit conducive to lordosis (McCarthy, Pickett, VanRyzin, & Kight, 2015). This early life programming happens at a time when the animal and its brain are remarkably immature (eyes closed, no fur, cannot walk), and the behavioral impact is not evident until after puberty. This raises the question regarding the cellular events that are happening in the neonatal brain and how they persist until the animal becomes a reproductively mature adult.

Microglia Are Purveyors of Sex Differences in Synaptic Patterning

The POA is an essential node in the neural circuit of mating and is critical to the process of masculinization, defined here as an adult animal that shows male mating behavior. Thus, not surprisingly, this region shows pronounced neuroanatomical sex differences. The sexually dimorphic nucleus (SDN) of the POA was the first large neuroanatomical difference reported in the mammalian brain, with “large” being relative since it is a small collection of Nissl dense cells, but large in both the magnitude of the difference between males and females and the impact of its discovery on the field (Gorski, Harlan, Jacobson, Shryne, & Southam, 1980). But there is also a profound sex difference in synaptic patterning in this region, with males having twice the number of excitatory synapses located on spines per unit dendrite as females (Amateau & McCarthy, 2004). When considering the origins of a sex difference in synapses, it seems obvious to suspect a dominant role for neurotransmitters. But this was not the case. Instead, it was discovered that PGE2 is the dominant signaling molecule establishing a higher density of dendritic spine synapses in males (Amateau & McCarthy, 2004). PGE2 is synthesized by the COX-1 and COX-2 enzymes, and these are upregulated by estradiol aromatized from endogenous testosterone within the developing POA. The higher PGE2 produced in males results in the activation of the cAMP-linked EP2 and EP4 receptors, activating protein kinase A located in the necks of dendritic spines, leading to phosphorylation of the glutamate ionotropic receptor AMPA type subunit receptors and, in ways not entirely understood, to an increase in spine synapses (Lenz, Wright, Martin, & McCarthy, 2011; Wright & McCarthy, 2009). The synthesis of PGE2 by microglia is promoted by histamine release from mast cells in the POA, which are more numerous and more active in males (Lenz et al., 2018).

The COX enzymes are expressed by a wide range of cell types, leaving unanswered the question of where the PGE2 is being generated. The astrocytes of the POA are also sexually dimorphic and are more stellate and complex in males than in females (Amateau & McCarthy, 2002), making them one contender as the source of prostaglandin. Intriguingly, astrocytes also respond to PGE2 with glutamate release (Bezzi et al., 1998), suggesting they provide stimulation of the glutamate AMPA receptors found on the heads of dendritic spines in the POA. Even more intriguingly, microglia both respond to and produce prostaglandins, and the morphology of these cells also diverges in male versus female POA. More specifically, in males the microglia take on a more “ameboid” like shape, which is consistent with an activated state and the production of prostaglandins (Lenz, Nugent, Haliyur, & McCarthy, 2013). In females the microglia of the POA are predominantly surveying, but they change their shape to ameboid in response to treatment with a masculinizing dose of steroid. If female pups are treated with steroid and a drug called minocycline, which calms microglia, the process of masculinization is blocked as evident by no increase in dendritic spine synapses during developing and no expression of male mating behavior in adulthood (Lenz et al., 2013). The critical role of microglia during the developmental critical period is confirmed by temporarily depleting them in the neonate and assessing the effect on adult behaviors. Males in which the microglia were depleted as neonates exhibit a complete absence of mating behavior as adults (VanRyzin, Yu, Perez-Pouchoulen, & McCarthy, 2016). They are not feminized; instead, they are asexual in nature. Thus these innate immune cells, both microglia and mast cells, are essential to the process of normal masculinization of mating behavior, a process that occurs only during a restricted developmental critical window.

The role of microglia in inducing synapse formation in the POA is contrasted by the synaptic pruning conducted during development of other brain regions, most notably the lateral geniculate of the visual system (Schafer et al., 2012). Here microglia eliminate synapses by engulfing them, presumably via phagocytosis, through a process that also involves innate immunity by signaling through the complement system. Development of the visual system is a classic example of a critical period, and research suggests that the well-known phenomenon of activity-dependent synaptic pruning is in fact the purview of microglia (Schafer, Lehrman, & Stevens, 2013). There have been no sex differences in microglia-mediated synaptic pruning reported to date, but the potential for such an effect certainly seems high.

Enduring Effects of Early Life Programming of Sex Differences by Steroid Hormones

The original conceptualization of the organizational/activational hypothesis of brain sexual differentiation was framed in the context of our understanding of the brain in the mid-1950s. There was no thought of synaptic “plasticity,” no potential for rewiring connections, and certainly no potential for ongoing cell proliferation, including neurogenesis. And so when considering the question of how early organizing effects of steroids endured in the brain, it was considered self-evident that this was because the brain is “hard-wired.” But in the era of modern neuroscience it is known this is not true, as the birth of new cells, including neurons, and formation and elimination of synapses are both possible and prevalent in the mature brain. Moreover, the fact that the brain is in an extremely immature state at the time of sexual differentiation should have made it obvious that some sort of imprinting occurs in order to program responses of neurons and other cells in the postpubertal adult brain. The best, and perhaps only, way that such imprinting can be achieved is via epigenetic modifications that both direct and maintain gene expression profiles that are conducive to the physiology and behavior of one sex versus the other. Epigenetics refers to processes that occur “above the genome,” meaning modifications of the DNA or surrounding histones that do not change the genetic code but instead impact whether a gene is expressed or not. While our understanding of sex differences in epigenetic modifications remains modest, there is strong evidence that it is in fact occurring (McCarthy et al., 2009; Murray, Hien, de Vries, & Forger, 2009; Shen et al., 2015), and indeed the closing of the critical period for sexual differentiation may be a consequence of the loss of the epigenetic modifying ability of steroids as brain maturation progresses (Nugent et al., 2015). Genome-wide bisulfate sequencing of DNA from the POA of neonatal males and females revealed that females have a more highly methylated genome than males (Nugent et al., 2015). In general, methylation of cytosine nucleotides found proximal to guanines is restrictive to gene expression. Consistent with this, when males and females were treated with a drug known to demethylate the DNA, many more genes were upregulated in females than males, suggesting that more genes were being repressed in females. RNA sequencing revealed that many of the genes that were expressed following demethylation were related to the immune system and inflammatory signaling molecules. Moreover, treating newborn females with a drug that demethylates the DNA was sufficient to masculinize their behavior in adulthood, suggesting that a gene expression profile had been de-repressed that then programmed and maintained a masculine profile (McCarthy, Nugent, & Lenz, 2017).

There are many questions begging to be asked as to precisely how the epigenome contributes to adult sex differences in brain and behavior, but a central one is which cells carry the epigenetic memory? Potential candidates are (a) neurons, and they would seem to be a leading one given their central importance; (b) astrocytes, which are as long-lived as neurons and intimate partners with them; and (c) microglia, the brain’s innate immune system. The innate immune system is not generally considered to be the source of immunological memory; that is a job left to the acquired immune system, as the name suggests. But the phenomenon of “priming” has prompted immunologist to rethink this dichotomy. Priming refers to when an innate immune cell is exposed to a pathogen or toxin and upon re-exposure shows an enhanced response, such as higher cytokine production than witnessed following the first exposure (Netea et al., 2016). In the periphery, the priming response is mediated by permissive epigenetic modifications of latent enhancers in the promoters of proinflammatory genes, resulting in greater expression upon second exposure. Microglia are innate immune cells and are known to be “activated ” following exposure to pathogens or toxins, leading to the suggesting that this is also a case of priming (Haley, Brough, Quintin, & Allan, 2017). The priming response can last for weeks to months, meaning the majority of the lifetime of a mouse. This raises the interesting question of whether a similar phenomenon might happen in response to steroids in which they “prime” microglia in males to have a more robust production of prostaglandins and other inflammatory signaling molecules into adulthood. The transcriptome of microglia isolated from adult male and female mice is notably different, and this difference is maintained when female-derived microglia are transplanted into male brains, where they are neuroprotective in a model of ischemic stroke (Villa et al., 2018). At least a portion of the sex-specific transcriptome appears to be established developmentally during the process of sexual differentiation, but a role for genetic sex and adult steroid hormonal programming cannot be ruled out.

Sex Differences in Microglia in Cognitive Brain Regions

The hippocampus is an essential brain region for spatial learning and memory, as well as the central origin of negative feedback within the hypothalamic-pituitary-adrenal stress axis. A major challenge in neuroscience is to associate variation in neuroanatomical features with behavioral output. Naturally occurring variation in behavior of males and females provides a valuable hook by which to achieve this goal. Sex differences in spatial learning strategies and in stress responding have long been recognized but are confounded by complex influences such as context, past experience, and current physiology (McCarthy, 2016). This is particularly true in the adult due to both the acute impact of sex differences in circulating steroids and the enduring influence of developmental events. But early development provides an opportunity to observe sex differences as they are established and to largely eliminate, or at least control for, the influences of environment and experience.

The sex difference in the size of the SDN of the POA is due to differential cell death, not cell proliferation. The opposite is true in the hippocampus as it develops, with an almost twofold higher rate of cell proliferation in the neonatal male hippocampus and dentate gyrus compared to female littermates (Bowers, Waddell, & McCarthy, 2010). This does not double the size of the hippocampus, however, but instead likely contributes to a sex difference in the rate of maturation of the synaptic profile of hippocampal pyramidal neurons (Weinhard et al., 2017). Females have a greater density of dendritic spine synapses across the first two weeks of life, but this is equalized by the time of sexual maturation. Intriguingly, females also exhibit more robust microglial development during the same period, suggesting, but not proving, that synapse formation and microglial maturation are functionally linked (Weinhard et al., 2017). Female microglia are also more phagocytic during this same period (Nelson, Warden, & Lenz, 2017). Whether this contributes to the higher rates of cell proliferation seen in males, be eliminating newborn cells in females, is not yet known but would be consistent with the ability of microglia to control cell number in the developing cortex (Brown & Neher, 2012; Cunningham, Martinez-Cerdeno, & Noctor, 2013).

Immune Responses to Developmental Insult Vary in Males and Females

In addition to being active participants in the sexual differentiation process, immune cells of the brain can also alter the response to extraneous insults or injuries. A differential response to the same developmental challenge could occur for two principle reasons. The first is that the maturational state might not be the same for males and females of equal ages, and this may vary by brain region, as has been shown in humans (Lenroot et al., 2007), or it might involve specific cell populations as has been suggested for microglia (Hanamsagar et al., 2017). The rate at which microglia migrate into and populate a particular brain region also varies by sex (Schwarz, Sholar, & Bilbo, 2012). Thus, when an insult occurs, the substrate it is impacting is not the same in males and females and therefore has different effects. Alternatively, there could be intrinsic differences in immune cells of males and females (other than maturational state), as has been demonstrated in the highly divergent transcriptomes of mast cells from male and female mice (Mackey et al., 2016; Villa et al., 2018).

At this stage we do not yet know why the immune response to insult varies in developing males and females, but we know that it happens. Diesel exhaust particles are a toxicant that induces inflammation in the fetal brain if exposed in utero but mostly, if not exclusively, in males, resulting in impaired cortical development (Bolton et al., 2017). Microglia in the developing male cortex exposed to diesel exhaust appear more activated with higher expression levels of TLR4 receptors, a hallmark of inflammation. This is just one example of what surely are going to be many as more researchers incorporate the impact of sex and the role of microglia in developmental injury models.

Peripheral Immune System Also Contributes to Brain Development

Inflammatory Signals and Sexual Differentiation of the BrainClick to view larger

Figure 2. Microglia migration and maturation. Microglia migrate into the brain early in development and begin proliferating and tiling throughout the brain, followed by a period of maturation. As a result, they are poised to modulate many of the process known to be critical to establishment of sex differences in the brain including astrocyte and neurogenesis, synaptogenesis and synaptic pruning, myelination, and apoptosis. Microglia can modulate these diverse functions via secretion of signaling molecules or by direct interaction including phagocytosis of synapses and cells. Many of these processes are not equal in males and females depending on brain region and precise timing. A future goal is to understand how such diverse developmental changes are coordinated.

Our understanding of how the immune system impacts the developing brain is in its earliest stages, with surprising new findings appearing almost daily. As is evident from this discussion, the majority of emphasis to date has been on microglia, an emphasis appropriately placed given the early migration into the brain of these essential cells (Figure 2). Because they take up permanent residence prior to the closing of the blood-brain barrier, microglia are considered the innate immune system of the brain. More recently another class of immune cells, the peripheral leukocyte B-1a cell, has been implicated in promoting myelination by increasing the proliferation of oligodendrocytes in the developing corpus callosum (Tanabe & Yamashita, 2018). The B-cells are attracted to the brain from the blood by secretion of chemoattractant cytokine from the choroid plexus and meninges of the lateral ventricles. Secretion of the immunoglobulin M (IgM) by the B-cells stimulates neuronal stem cells to differentiate into oligodendrocytes. Depletion of B-1a cells impairs appropriate myelination, a process that has also been associated with hypoxic/ischemic brain damage following birth complications and in developmental neuropsychiatric disorders such as autism spectrum disorder (ASD) and schizophrenia, all of which are more frequent and more severe in males (Aleman, Kahn, & Selten, 2003; Hill & Fitch, 2012; Werling, 2016). Similarly, the appearance of maternal antibodies in the serum of newborn infants has been associated with ASD and schizophrenia (Kowal, Athanassiou, Chen, & Diamond, 2015). Much about this particular process remains to be learned; nonetheless, it demonstrates the potential for sensitive windows of development during which the immune and nervous system are engaged in a critical conversation directing how the brain is constructed.

Males Suffer More From Neurodevelopmental Disorders Associated With Inflammation

ASD and schizophrenia are considered highly heritable but in a complex polygenic manner. In both cases the number of genes identified from genome wide association studies number in the hundreds, with each mutation explaining only a small proportion of the cases. Attention deficit and associated hyperactivity disorders also have a genetic component, albeit much less well understood, as do major depressive disorder, bipolar disorder, and substance abuse. There is both shared and specific gene regulation across all these disorders (Gandal et al., 2018), attesting to the importance of extraneous influences such as environment and experience. One of the most powerful of these extraneous influences is the presence of inflammation during gestation. This can occur for a variety of reasons but the most prevalent is maternal flu and bacterial infections, such as acute cystitis. The risk of schizophrenia and ASD both increase markedly following early life inflammation, and this is now well modeled in the rodent via maternal immune activation (Patterson, 2009). Of central interest is to marry the understanding of genetics with the understanding of immune responding but what has been largely left out of the equation is the importance of sex. Males are significantly more likely to be diagnosed with ASD—as much as 4.5 times more (Lai, Lombardo, Auyeung, Chakrabarti, & Baron-Cohen, 2015). They also have an earlier onset of schizophrenia diagnosis with more severe symptoms (Abel, Drake, & Goldstein, 2010). ASD is usually diagnosed prior to three years of age and so is clearly developmental in origin, but schizophrenia and increasingly attention deficit hyperactivity disorder are also considered developmental disorders, despite diagnosis sometimes not occurring until the second or even third decade of life (Lewis & Levitt, 2002; Shaw et al., 2007). Taken together, there is an association between processes associated with very early development, immune activation, genetics, and maleness. Yet none of the high confidence genes associated with any of the disorders are found on the Y chromosome, nor do they appear to be strongly regulated by gonadal steroids. The strongest convergence is between inflammation and maleness, with animal studies suggesting that males have inherently more neuroinflammation than females even in an unperturbed healthy state. But is this true in humans?

Two pieces of evidence suggest that indeed the human male brain experiences a higher inflammatory tone during early development. Reanalysis of the transcriptome from fetal cortex that disaggregated the data by sex found cohorts of genes associated with astrocyte and microglial activation (i.e., inflammation) to be higher in healthy males versus females (Werling, Parikshak, & Geschwind, 2016). Intriguingly, when the cortical transcriptome of adult males that were considered normative controls was compared to males diagnosed with ASD, the inflammatory profile was still higher in those with ASD. No comparison was made with females as there were too few ASD cases to provide confidence (Werling et al., 2016).

The second piece of evidence comes from a completely different quarter. In attempting to understand the biological origins of sexual partner preference in humans (i.e., heterosexual versus homosexual), Ray Blanchard and colleagues long ago noted that the number of older brothers was a predictor of the relative frequency of male homosexuality, with no influence on female homosexuality. They also noted that with each successive male offspring from the same mother, the birthweight decreased but not so for females. This led them to postulate the maternal immune hypothesis of male homosexuality (Blanchard, 2001), which predicts that maternal antibodies against male fetuses increase with each successive pregnancy, an idea consistent with innate immune priming. They further predicted that the specificity of the immune attack on males had its origins in the Y chromosome. This prediction was recently fulfilled with the observation that women with sons had higher titers of antibodies against a synaptic protein, NLGN4Y, a neuroligand that is made only by the Y chromosome, than did control women or men and that women who had gay sons had the highest antibody titer of all (Bogaert et al., 2018). Whether those antibodies penetrate into the male fetal brain and precisely how they might impact brain development in a manner so highly specific that it alters partner preference is unknown. But the finding is consistent with the concept that the fetal environment is distinctly different for males than it is for females, and at least one component of that environment includes a more aggressive maternal immune response. If the maternal immune activation against male fetuses is broader than a single neuroligand, which seems likely, and if it is a regulated process by variables such as stress, diet, genetics, and so on, it could be a pervasive underlying risk factor that contributes to the broad-based vulnerability of developing males to neuropsychiatric disorders of development as well as the probability of and response to prematurity and birth-associated injuries.

Conclusions and Unanswered Questions

Elucidating the role of the immune system in sexual differentiation of the brain is in its earliest stages with far more unanswered questions than firm conclusions. One thing that is clear is that mechanisms involving one type of immune cell or signaling molecule in a particular region of the brain may not generalize to another region. Put differently, every brain region that exhibits a sex difference in anatomy or physiology achieves that end in its own unique way, which contributes to an overall mosaicism of relative maleness, femaleness, and sameness within any individual brain (McCarthy, 2016). In humans we have the added complexity of past experience, current context, and cultural expectations, which likely enormously influence the brain both as it develops and during adulthood, greatly confounding our ability to separate biological from nonbiological influences (Joel & Fausto-Sterling, 2016). But it is the gender biases in diseases of the nervous system that compel us to try and understand the sources of vulnerability and resilience for the potential benefit of both sexes.

Among the many questions to be asked, an essential one is where the regional specificity comes from. If the maternal immune system is reacting to a male fetus differently than a female fetus, how is it that only a particular brain region seems to be affected? Or is it? Perhaps we should keep looking where the light is and fulfilling our predisposed notions of what we think should be impacted. As neuroscience advances with big data approaches that range from the big picture, as in functional magnetic resonance imaging of the entire brain, to the smallest details, as in single cell transcriptomics, we may find insight into the rules of governance for both regional and cellular specificity. We also need to integrate this information with the impact of particular insults and with genetic predispositions, a daunting challenge given the technical hurdles associated with big data approaches. A recent high-dimensional single cell mapping of the immune cell population in the mouse brain revealed enormous diversity across age and disease state (Mrdjen et al., 2018), with no consideration for the impact of sex, which is likely to be substantial. Thus we also need focused hypothesis-driven experiments that will test specific predictions and advance our understanding by both rejecting and accepting null hypotheses. Translating these findings into the human condition, with all the associated caveats in the realm of sex differences, will be the greatest challenge of all.

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