Maternal Antibody and ASD: Clinical Data and Animal Models

Over the past several decades there has been an increasing interest in the role of environmental factors in the etiology of neuropsychiatric and neurodevelopmental disorders. Epidemiologic studies have shifted from an exclusive focus on the identification of genetic risk alleles for such disorders to recognizing and understanding the contribution of xenobiotic exposures, infections, and the maternal immune system during the prenatal and early post-natal periods. In this review we discuss the growing literature regarding the effects of maternal brain-reactive antibodies on fetal brain development and their contribution to the development of neuropsychiatric and neurodevelopmental disorders. Autoimmune diseases primarily affect women and are more prevalent in mothers of children with neurodevelopmental disorders. For example, mothers of children with Autism Spectrum Disorder (ASD) are significantly more likely to have an autoimmune disease than women of neurotypically developing children. Moreover, they are four to five times more likely to harbor brain-reactive antibodies than unselected women of childbearing age. Many of these women exhibit no apparent clinical consequence of harboring these antibodies, presumably because the antibodies never access brain tissue. Nevertheless, these maternal brain-reactive antibodies can access the fetal brain, and some may be capable of altering brain development when present during pregnancy. Several animal models have provided evidence that in utero exposure to maternal brain-reactive antibodies can permanently alter brain anatomy and cause persistent behavioral or cognitive phenotypes. Although this evidence supports a contribution of maternal brain-reactive antibodies to neurodevelopmental disorders, an interplay between antibodies, genetics, and other environmental factors is likely to determine the specific neurodevelopmental phenotypes and their severity. Additional modulating factors likely also include the microbiome, sex chromosomes, and gonadal hormones. These interactions may help to explain the sex-bias observed in neurodevelopmental disorders. Studies on this topic provide a unique opportunity to learn how to identify and protect at risk pregnancies while also deciphering critical pathways in neurodevelopment.

Over the past several decades there has been an increasing interest in the role of environmental factors in the etiology of neuropsychiatric and neurodevelopmental disorders. Epidemiologic studies have shifted from an exclusive focus on the identification of genetic risk alleles for such disorders to recognizing and understanding the contribution of xenobiotic exposures, infections, and the maternal immune system during the prenatal and early post-natal periods. In this review we discuss the growing literature regarding the effects of maternal brain-reactive antibodies on fetal brain development and their contribution to the development of neuropsychiatric and neurodevelopmental disorders. Autoimmune diseases primarily affect women and are more prevalent in mothers of children with neurodevelopmental disorders. For example, mothers of children with Autism Spectrum Disorder (ASD) are significantly more likely to have an autoimmune disease than women of neurotypically developing children. Moreover, they are four to five times more likely to harbor brain-reactive antibodies than unselected women of childbearing age. Many of these women exhibit no apparent clinical consequence of harboring these antibodies, presumably because the antibodies never access brain tissue. Nevertheless, these maternal brain-reactive antibodies can access the fetal brain, and some may be capable of altering brain development when present during pregnancy. Several animal models have provided evidence that in utero exposure to maternal brain-reactive antibodies can permanently alter brain anatomy and cause persistent behavioral or cognitive phenotypes. Although this evidence supports a contribution of maternal brain-reactive antibodies to neurodevelopmental disorders, an interplay between antibodies, genetics, and other environmental factors is likely to determine the specific neurodevelopmental phenotypes and their severity. Additional modulating factors likely also include the microbiome, sex chromosomes, and gonadal hormones. These interactions may help to explain the sex-bias observed in neurodevelopmental disorders. Studies on this topic provide a unique opportunity to learn how to identify and protect at risk pregnancies while also deciphering critical pathways in neurodevelopment.

INTRODUCTION
The increasing evidence of an immune mediated pathogenesis for neuropsychiatric and neurodevelopmental disorders has shifted the focus of epidemiologic studies to include the contribution of cytokines and brain-reactive antibodies. The brain was originally thought to be an immune privileged organ due to the presence of the blood brain barrier (BBB), a structure composed of endothelial cells knit together by tight junctions and supported by astrocytic endfeet (1). We now know that even though the BBB isolates the central nervous system (CNS) from factors in the blood, it is a dynamic semipermeable structure. Immune molecules including antibodies can access the CNS during both physiologic and pathologic states. Even though antibodies cannot cross the BBB and access brain tissue in healthy adults, these molecules may cross the BBB during in utero development when the BBB is immature and more permeable (2). Alternatively, antibodies can penetrate the adult brain when there is a BBB breach as occurs during inflammation (3)(4)(5)(6) or at sites of limited BBB protection such as the choroid plexus. Factors affecting BBB integrity include: trauma, ischemia, stress, aging, antibodies, and specific agonists of endothelial cell receptors, such as cytokines, complement, and antibodies themselves (7)(8)(9)(10)(11)(12). Once in the CNS, antibodies can lead to pathology if they recognize antigens expressed in the brain or spinal cord. In this review we discuss how maternal brain-reactive antibodies affect fetal brain development, contributing to the risk of neuropsychiatric and neurodevelopmental disorders. We focus on antibodies implicated in Autism Spectrum Disorder (ASD) and propose a role for the microbiome, sex chromosomes and gonadal hormones in determining the susceptibility to the effects of maternal antibody and the development of neurodevelopmental disorders.
The potential for pathology to arise from brain-reactive antibodies accessing the brain parenchyma depends on multiple factors. Vulnerability to the brain-reactive antibodies requires that the anti-brain antibody be present in the CNS at a time when the antigen is expressed. Furthermore, when a BBB breach is necessary for the antibody to penetrate the brain parenchyma, the nature of the BBB insult will restrict access to specific regions of the brain, determining whether the antibody will encounter its cognate antigen. For instance, lipopolysaccharide (LPS) causes a BBB breach in the hippocampus in mice (5) while epinephrine results in a BBB breach in the amygdala (4). Additionally, genetic variants and differences in protein expression between males and females (48)(49)(50) may also influence brain vulnerability to immune assault.
The symptoms and deficits observed in disorders caused by brain-reactive antibodies are dependent on the antigen recognized and its distribution in the brain. For example, antibodies to ribosomal P protein (anti-P antibodies) present in SLE patients cross-react with a brain antigen termed neuronal surface P antigen (NSPA) (19). While these antibodies bind to several regions of the mouse brain, when injected into the lateral cerebral ventricles they lead to smell alterations (51) and depression-like behavior (52), implicating the piriform cortex, the cingulate cortex, and the hippocampus. Furthermore, upon LPS-induced BBB breach, which enables access to the hippocampus, these antibodies cause memory impairment in mice (15). SLE anti-DNA antibodies that cross-react with Nmethyl-D-aspartate receptors (NMDAR) termed DNRAb cause cognitive impairment (5) and an abnormal stress response (4) in mice, consistent with the function of NMDARs in the hippocampus and the amygdala, respectively. While NMDARs are expressed in both the hippocampus and the amygdala, the regional selectivity of symptoms observed in the mouse models is determined by the agent used to compromise BBB integrity; cognitive impairment is seen only when LPS damages BBB integrity in the hippocampus while the abnormal stress response is seen with exposure to epinephrine and antibody penetration of the amygdala. This example highlights the importance of antigen distribution and the region of BBB breach.

MATERNAL BRAIN-REACTIVE ANTIBODIES AND FETAL BRAIN DEVELOPMENT
Brain-reactive antibodies have the potential to alter brain development in utero (53), resulting in damage that can be persistent (54) and lead to neurodevelopmental and neuropsychiatric disorders in the offspring. It has been proposed that AD-related impaired B cell tolerance in women can lead to the production of these antibodies. Indeed, ADs are more prevalent in mothers of children with neurodevelopmental and neuropsychiatric disorders including ASD ( Table 1). A study by our group showed that mothers of children with ASD who had brain-reactive antibodies were significantly more likely to harbor anti-nuclear antibodies (53%) than those lacking brain-reactive antibodies (anti-nuclear antibody = 13.4%) and unselected women of child-bearing age (anti-nuclear antibody = 15%), suggesting a maternal predisposition to producing auto-antibodies. We also observed a significantly greater prevalence of AD in mothers of children with ASD who were also positive for brain-reactive antibodies [rheumatoid arthritis (RA) = 3.86%; SLE = 2.22%] compared to those lacking brain-reactive antibodies (RA = 1.45%; SLE = 0.16%) (67).
The BBB is immature during fetal development, presenting a uniquely vulnerable temporal window when antibodies can enter the CNS (2) while critical neurodevelopmental events are taking place. The antibodies that access the fetal brain are produced by the mother and are transported by the neonatal Fc receptor (FcRn) across the placenta into fetal circulation starting on week 13 of human gestation (68)(69)(70). Once maternal brain-reactive antibodies access the fetal circulation they may result in pathology if the timing of antigen expression also coincides with the period when the fetal BBB is permeable to IgG. Presence of maternal brain-reactive antibodies during pregnancy alone is, therefore, not enough to result in pathology, perhaps contributing to the fact that some mothers of neurotypically developing (NT) children also have anti-brain antibodies.
When assessing the potential for maternal brain-reactive antibodies to cause neurodevelopmental disorders we must also be mindful that the women harboring these antibodies will not necessarily present with neurological deficits resulting from antibody exposure as their BBB is likely to be intact or the impact of the antibody may be developmentally determined. Moreover, the deficits caused by in utero antibody exposure may be transient or long-lasting, and consequently, not all children born to mothers with brain-reactive antibodies will present postnatally with detectable symptoms. Transient insults may be compensated for by plasticity mechanisms in the brain during gestation or post-natally. Furthermore, it is possible for deficits to be present only while the pathogenic antibody has access to the brain parenchyma, which is limited after birth by the maturation of the BBB (2) and the disappearance of maternal antibodies in the circulation of newborn infants (71). Lastly, symptomatology from the neurodevelopmental effects of maternal brain-reactive antibodies may appear later in life or may become apparent only if other stressors are present (72).

MATERNAL BRAIN-REACTIVE ANTIBODIES AND ASD
ASD are a group of neurodevelopmental conditions characterized by impaired communication and social interactions, repetitive behaviors, and restricted interests or activities (DSM-V) (73). They are four times more likely to be diagnosed in males compared to females. Both prevalence and incidence of ASD are increasing, with a current estimate of 1 in 59 children being affected (74). The etiology of ASD is not completely understood; hundreds of genes have been associated with ASD (75, 76) but these account for just 10-20% of the diagnosed cases (77). Furthermore, twin studies indicate that only 37% of the susceptibility to ASD is due to genetic heritability (78). These data suggest that environmental factors also play an important role in determining the susceptibility to ASD. Maternal brain-reactive antibodies present in utero represent a potential environmental risk factor for ASD. Several groups have identified brain-reactive antibodies in mothers of children with ASD which are either absent or found at lower frequency in mothers of unaffected children ( Table 2). Dalton et al. (79) showed that, when injected into pregnant mice, brain-reactive serum from a mother of a child with autism and a child with severe specific language disorder led to decreased exploration, deficient motor coordination, and altered cerebellar metabolites in the offspring compared to the offspring of mice given sera from mothers of NT children. In a study in which blood samples were collected mid-pregnancy, Croen et al. (80) suggested a direct pathogenic role for the antibodies. A significantly higher prevalence of reactivity to proteins from brain lysates of 39 and 73 kDa was detected in mothers of children with ASD compared to the general population control group. Moreover, this pattern of reactivity was seen in mothers of children with early onset ASD.
Some of these maternal brain-reactive antibodies have been found to have antigenic specificity for proteins with potential neurodevelopmental roles including: lactate dehydrogenase A and B (LDH-A, LDH-B) (37 kDa band), Y-box bonding protein 1 (YBX1) (39 kDa band), stress-induced phosphoprotein 1 (STIP1) (upper 73 kDa band), collapsing response mediator protein 1 and 2 (CRMP1, CRMP2) (lower 70 kDa band), and guanine deaminase (GDA) (44 kDa band) (83,84). Maternal reactivity to LDH alone or in combination with reactivity to CRMP1/CRMP2/STIP1 has been associated with a greater risk of ASD. Reactivity to LDH, STIP1, and CRMP1 together (the 37 and 73 kDa combined bands) was the most specific pattern for ASD AD in 1st degree relative and increased risk of ASD [OR 6.0] Maternal AD and increased risk of Not identified Low molecular weight bands (∼20-25 kDa) High molecular weight band (larger than 250 kDa) on WB using fetal rat brain proteins as it was detected in mothers of children with ASD but not in mothers of NT children (81,83). Furthermore, presence of this antibody combination was associated with an increased risk of behavioral regression in ASD (81) and impairments in expressive language (82).
Animal studies have shown that exposure to maternal brain-reactive antibodies in utero can permanently alter the brain during development and cause sustained behavioral and cognitive deficits akin to those observed in ASD ( Table 3)  3 | Animal studies demonstrating that in utero exposure to maternal brain-reactive antibodies can permanently alter the brain, leading to behavioral and cognitive deficits.

Target
Animal model Characteristics References intraventricular embryonic injection model to assess the effects of brain-reactive antibodies recognizing LDH/STIP1/CRMP1 (antigens of molecular weights 37 and 73 kDa) on fetal brain development. These antibodies stimulated the proliferation of stem cells in the subventricular zone (SVZ) of the neocortex and ganglionic eminence, increased adult brain size and weight, and enlarged adult cortical neuron volume (96). Additionally, anti-LDH/STIP1/CRMP1 antibodies decreased basal dendritic arborization in layer V neurons of the frontal cortex and reduced the dendritic spine number and density in several brain regions (97). Complementing these studies, mice exposed in utero to maternal brain-reactive antibodies recognizing LDH/STIP1/CRMP1 showed ASD-like characteristics including increased anxiety-like behaviors (93), impaired social interactions (95), longer bouts of spontaneous grooming (95), increased digging (95), and delayed motor and sensory development (93). A limitation of these animal studies is the timing of exposure to the maternal brain-reactive antibodies. Maternal brainreactive antibodies associated with ASD in humans are likely to be present throughout the pregnancy. These studies did not simulate these conditions; they used a single injection into the pregnant mice at mid-gestation or a single intraventricular injection into the embryos. To address this, Jones et al. (98) completed a study in which female mice were immunized prior to pregnancy with antigenic peptides recognized by anti-LDH/STIP1/CRMP1 antibodies. The offspring mice exposed to endogenous maternal anti-LDH/STIP1/CRMP1 antibodies displayed fewer social interactions as juveniles and adults, increased repetitive behaviors/stereotypies assessed through the number and length of grooming bouts, and impaired social communication measured by ultrasonic vocalizations (USVs) neonatally and as adults.

Cerebellar Purkinje cells and brainstem neurons
In other studies identifying pathologic maternal brainreactive antibodies, Singer et al. (88) found that, compared to mothers of NT children, mothers of children with ASD have a significantly higher prevalence of antibodies reactive to a 36 kDa protein present in rat embryonic and human fetal brain. They also noted a trend for a higher prevalence of antibodies recognizing human fetal proteins at 39 kDa in mothers of children with ASD compared to mothers of NT children. Presence of either of these reactivities was significantly associated with behavioral regression in children with ASD. Intraperitoneal administration of the purified maternal ASD-IgG to pregnant mice led to hyperactivity, increased anxiety, and shorter social interactions in the adult offspring relative to the offspring of pregnant mice given IgG from mothers of NT children or saline (99). Preliminary fetal brain studies suggested a role for microglia and IL-12 in the pathological mechanism of the ASD-IgG induced behavioral irregularities (99). Further assessment of the pathological mechanism of these antibodies showed greater cell proliferation in the subventricular and subgranular zones and decreased postnatal day (P)1-born cell density, suggesting reduced survival, in layers 2-4 of the frontal and parietal cortex (100). Due to the similarity in antigen size and the association with behavioral regression, it is possible that the samples from the Braunschweig and Singer studies contain antibodies that recognize the same proteins at 36-39 and 73 kDa, and that these antibodies represent contributors to ASD risk in the general population. Determining the antigenicity of the antibodies identified by Singer et al. (88) will be necessary to resolve this question.
Animal studies of the pathogenic role of ASD-IgG have generally used IgG that was pooled from several mothers of children with ASD or endogenous polyclonal antibody following an immunization protocol. Thus, it has not been possible to identify the antibodies that are pathogenic from those that are not. Furthermore, specific ASD-like characteristics in the animal studies may result from exposure to distinct monoclonal antibodies. There is also likely to be a different proportion of potentially pathogenic antibodies in the pooled sample compared to the composition present in each of the mothers. As a result, the effects of antibodies at low concentration might be obscured by those at high concentration or they may not be detectable because the threshold concentration of antibody necessary to produce pathology may not have been reached. Moreover, the studies discussed above do not definitively identify the targeted antigen as it remains possible that the critical antibodies bind not only to the identified antigens (most of which are intracellular) but also cross-react with a neuronal membrane antigen. We addressed this concern by developing a protocol to generate monoclonal brain-reactive antibodies from mothers of children with ASD and a brain-reactive serology.
One of the monoclonal antibodies that we generated recognizes the extracellular domain of Caspr2, a protein encoded by the gene Contactin Associated Protein-Like 2 (CNTNAP2). Caspr2 is a cell-adhesion molecule expressed in the spines, dendrites, axons, and soma of neurons (103,104). Both rare and common variants of CNTNAP2 have been linked to an increased risk of ASD or ASD-related endophenotypes including language delay and developmental language disorders (105)(106)(107)(108)(109)(110)(111)(112)(113)(114)(115). Furthermore, CNTNAP2 deficient mice exhibit ASDlike phenotypes including increased repetitive behaviors, and impaired communication and social interactions (116). The CNTNAP2 deficient mice also suffer from seizures, show neuronal migration abnormalities and have ectopic neurons in the corpus callosum (116), similar to cortical dysplasia-focal epilepsy (CDFE) syndrome, a syndromic form of ASD associated with mutant CNTNAP2 (114).
Given the link between mutations in CNTNAP2 and ASD in human pedigrees and the presence of ASD-like phenotypes in CNTNAP2 deficient mice, we asked whether exposure to monoclonal anti-Caspr2 antibody (C6) in utero leads to ASDlike characteristics in mice (91). Indeed, in utero C6 exposure led to a thinner cortical plate and fewer proliferating cells in the ventricular zone, and to a reduction in the number of neurons in the entorhinal cortex and in the number of GABAergic neurons in the hippocampus of adults. We also observed decreased dendritic arborization and a reduced spine density in CA1 pyramidal neurons in adult mice exposed to C6 in utero when compared to the controls. Finally, these mice showed ASD-like behavioral abnormalities such as stereotypic behaviors, impaired flexible learning, and impaired social interactions. Interestingly, effects of C6 were only detected in male mice.

MATERNAL BRAIN-REACTIVE ANTIBODIES AND SEX-BIAS
Neuropsychiatric conditions often display a bias for one sex over the other. Neuropsychiatric conditions diagnosed earlier in life are more frequently diagnosed in males while those manifesting during puberty or later in life show a female preponderance. For example, there is a male bias in ASD, ADHD, dyslexia, Tourette Syndrome, and learning disabilities [reviewed by (117)]. Conversely, anorexia nervosa and internalizing disorders such as depression (118) and anxiety are more prevalent in females (119). Neuropsychiatric conditions that are diagnosed earlier in life have been proposed to have a neurodevelopmental origin (119). Given the unique conditions during pregnancy that allow for the in utero environment including maternal antibodies and cytokine levels to influence development, it is reasonable that early-onset neuropsychiatric disorders are more frequent in children of mothers with AD ( Table 1).
A sex-bias has indeed been described in multiple studies of the effects of maternal brain-reactive antibodies on development and behavior. For example, we observed a significant male bias for all the fetal brain developmental and adult behavioral effects of the C6 anti-Caspr2 antibody (91). Females exposed to C6 in utero were not affected while males developed disrupted brain anatomy and ASD-relevant behaviors. The animal studies of maternal anti-LDH/STIP1/CRMP antibodies have also noted a male-bias in ASD-like traits including impaired communication and social interactions (93,98). Lastly, Wang et al. (102) observed a significantly higher rate of fetal death after embryonic day (E) 15 in female offspring exposed to SLE DNRAb compared to males. While females had a greater fetal death risk, males in this model were born with cognitive impairment (101). This difference between the sexes suggests that sex-dependent factors play a role in determining not only the impairments resulting from maternal brain-reactive antibody exposure but also the severity of the outcomes.
Sex chromosomes and gonadal hormones may influence the susceptibility to maternal brain-reactive antibodies. Sex chromosome genes contribute to sexual dimorphisms, including sex-specific patterns of brain development and function, independently from gonadal hormone influences (50,120). For example, sex chromosomes have been implicated in the density of vasopressin fibers (121), the number of tyrosine hydroxylase expressing neurons (120,122), social interactions (123,124), aggression (125)(126)(127), and anxiety (128). Genes found in "sex specific" regions in the X and the Y chromosomes may account for some of these sexual dimorphisms. These genes are highly expressed in the brain (48,50) and show expression, spatial, and temporal differences between sexes (50). Furthermore, sex differences may be in part explained by gene dosage as some genes on the X chromosome escape inactivation (129)(130)(131), and X chromosome imprinting can affect gene expression in the brain (132,133). Indeed, X chromosome imprinting has been associated with social impairment in Turner syndrome (134). Higher expression of these sex chromosome genes may be protective if they encode the antigens recognized by the maternal brain-reactive antibodies when binding of the antibody leads to protein internalization and partial loss of function but is only pathogenic if protein expression falls below a certain threshold. Conversely, if antibody interaction with its cognate antigen induces cell signaling cascade activation or apoptosis, then the sex with higher expression would be at an increased risk for developing the antibody-induced phenotypes. Furthermore, temporal differences in gene expression between sexes may be important as antibody exposure must coincide with this period in order to cause pathology. Moreover, the effects of sex chromosome genes on the susceptibility to maternal brainreactive antibodies may be indirect if the proteins encoded modulate the expression or activity of the proteins recognized by the antibodies. For instance, the DNRAb mediated female fetal loss may be in part due to sex chromosome genes acting as regulating factors and contributing to the higher expression of the NMDAR subunit NR2A in the female brainstem by E17 (102).
Sex hormones also influence brain development, aiding normal maturation of the fetal brain or altering normal development. Estrogen has neurotrophic and neuroprotective functions including modulation of neuronal apoptosis, migration, and spinogenesis and neurite growth (135)(136)(137)(138)(139)(140). Estrogen administration during fetal development leads to masculinization of mouse neural pathways and behaviors (141,142). Human fetal testosterone has been linked to narrow interests (143) and greater impairments in social skills and empathy in offspring (143)(144)(145). Additionally, treatment with estrogen reversed or mitigated some of the ASD-relevant behavioral phenotypes in animal models of ASD, importantly, CNTNAP2 mutant zebrafish (146) and Reeler heterozygous mice (147). As estrogen treatment showed a decrease in phenotypic behavior, these data suggest that estrogen may have a protective role in ASD and could therefore account for the lower prevalence in females. Overall, gonadal hormones modulate processes in brain development and maturation that could lead to compensation for or exacerbation of the pathologic effects of maternal brain-reactive antibodies. The specific effects of individual gonadal hormones are likely to depend on the neural pathways affected by the antibodies.
Given the sex-bias of neurodevelopmental and neuropsychiatric disorders in humans, understanding what causes maternal brain-reactive antibodies to affect preferentially one sex over the other will aid in our understanding of the pathological mechanisms of these conditions while also providing information that could lead to the discovery of new treatments. Of note, exposure to gonadal hormones in utero in litter-bearing animals differs from that in humans due to the "intrauterine position phenomenon." Fetuses between two males will be exposed to higher levels of testosterone while fetuses between two females will be exposed to higher levels of estrogen [reviewed by (148)]. Consequently, post-natal sexually dimorphic characteristics including brain anatomy and behavior are influenced by fetal position [reviewed by (148)]. Nonetheless, the importance of gonadal hormones can be addressed in studies in which these are administered to neonates. Alternatively, treatment with gonadal hormone receptor agonists or antagonists, and the use of gonadal hormone receptor knockout mice can not only help us to identify which hormones play a role in the sex-bias that is observed but can also lead to the identification of the specific molecular mechanisms involved. The "Four Core Genotypes" (FCG) mouse model offers the unique opportunity to isolate sex chromosome from gonadal hormone contributions to sex-bias. This mouse strain combines two mutations that allow for gonadal determination to be independent from sex chromosome complements. Specifically, the testes determining gene (Sry) was deleted from the Y chromosome (Y − ) and a Sry transgene (TgSry) was inserted into autosomal chromosome 3, resulting in four genotypes: gonadal females with XX or XY − , and gonadal males with XY − TgSry or with XX TgSry (121,149). This model has been informative in understanding the sex bias in autoimmune diseases such as SLE and multiple sclerosis (150).
The gut microbiota may act in concert with maternal brainreactive antibodies, genetic variants and sex-specific factors to modulate ASD susceptibility pre-and post-natally. In the model of maternal immune activation (MIA), specific maternal gut microbiota are necessary for the development of MIA-associated behavioral phenotypes and neurodevelopmental abnormalities. Segmented filamentous bacteria (SFB) -specific T H 17 cells are stimulated by dendritic cells primed by poly (I:C) to produce high levels of IL-17a (174), a key cytokine for ASD-like phenotype induction in the MIA model (175,176). Furthermore, during fetal development, metabolites produced by the maternal gut microbiota can alter BBB tight junction protein expression thus modifying BBB permeability. Offspring of germ-free mice have a decreased expression of occludin and claudin-5 which contributes to the increased BBB permeability observed from the fetal stage until adulthood (3). This increased BBB permeability makes the offspring more susceptible to the neurological effects of immune molecules including antibodies as these are then more likely to access the brain parenchyma (3). Finally, shifts in offspring microbiota composition can be induced by factors in the in utero environment and contribute to the development of ASD-like behavioral deficits. For example, the offspring of poly (I:C) treated mice have an altered gut microbiota diversity which leads to altered serum metabolites and increased IL-6 and gut permeability (177). Hsiao et al. (177) propose that the increased IL-6 expression alters tight junction protein expression, leading to increased gut permeability, and leaking of harmful metabolites into systemic circulation. Post-natal treatment of the offspring with Bacteroides fragilis reversed some of the ASD-like behavioral phenotypes and improved gut barrier permeability, possibly by restoring IL-6 expression, which in turn leads to a partial correction of tight junction protein expression (177).

CONCLUSIONS
The association between maternal brain-reactive antibodies and the pathogenesis of neurodevelopmental disorders has been well-established by both epidemiologic and animal studies. Maternal autoimmune disease and brain-reactive antibodies have been shown to increase the risk of neurodevelopmental disorders. Moreover, animal studies have shown that in utero exposure to maternal brain-reactive antibodies is sufficient to permanently alter brain anatomy and cause aberrant cognition or behavior mimicking certain neurodevelopmental syndromes. Specific neurodevelopmental disorders and the severity of symptomatology are likely determined by an interplay between genetics and environmental risk factors including maternal brain-reactive antibody, maternal cytokines, gonadal hormones, and the microbiome (Figure 1). As the prevalence of neurodevelopmental disorders has been significantly increasing (178), research on the in utero environment, including maternal brain-reactive antibodies, is of great biomedical importance. Identifying potentially pathogenic antibodies and understanding their mechanisms of fetal brain injury provide an opportunity to detect and protect fetuses at risk.

AUTHOR CONTRIBUTIONS
AG-G contributed to the concept and writing of the manuscript. BD contributed to the concept, writing, and reviewing of the manuscript.