The Role of Immune Factors in Shaping Fetal Neurodevelopment.

Fetal neurodevelopment in utero is profoundly shaped by both systemic maternal immunity and local processes at the maternal-fetal interface. Immune pathways are a critical participant in the normal physiology of pregnancy and perturbations of maternal immunity due to infections during this period have been increasingly linked to a diverse array of poor neurological outcomes, including diseases that manifest much later in postnatal life. While experimental models of maternal immune activation (MIA) have provided groundbreaking characterizations of the maternal pathways underlying pathogenesis, less commonly examined are the immune factors that serve pathogen-independent developmental functions in the embryo and fetus. In this review, we explore what is known about the in vivo role of immune factors in fetal neurodevelopment during normal pregnancy and provide an overview of how MIA perturbs the proper orchestration of this sequence of events. Finally, we discuss how the dysregulation of immune factors may contribute to the manifestation of a variety of neurological disorders.

Maternal gut bacteria drive intestinal inflammation in offspring with neurodevelopmental disorders by altering the chromatin landscape of CD4+ T cells.

Children with autism spectrum disorders often display dysregulated immune responses and related gastrointestinal symptoms. However, the underlying mechanisms leading to the development of both phenotypes have not been elucidated. Here, we show that mouse offspring exhibiting autism-like phenotypes due to prenatal exposure to maternal inflammation were more susceptible to developing intestinal inflammation following challenges later in life. In contrast to its prenatal role in neurodevelopmental phenotypes, interleukin-17A (IL-17A) generated immune-primed phenotypes in offspring through changes in the maternal gut microbiota that led to postnatal alterations in the chromatin landscape of naive CD4+ T cells. The transfer of stool samples from pregnant mice with enhanced IL-17A responses into germ-free dams produced immune-primed phenotypes in offspring. Our study provides mechanistic insights into why children exposed to heightened inflammation in the womb might have an increased risk of developing inflammatory diseases in addition to neurodevelopmental disorders.

Prenatal interleukin 6 elevation increases glutamatergic synapse density and disrupts hippocampal connectivity in offspring.

Early prenatal inflammatory conditions are thought to be a risk factor for different neurodevelopmental disorders. Maternal interleukin-6 (IL-6) elevation during pregnancy causes abnormal behavior in offspring, but whether these defects result from altered synaptic developmental trajectories remains unclear. Here we showed that transient IL-6 elevation via injection into pregnant mice or developing embryos enhanced glutamatergic synapses and led to overall brain hyperconnectivity in offspring into adulthood. IL-6 activated synaptogenesis gene programs in glutamatergic neurons and required the transcription factor STAT3 and expression of the RGS4 gene. The STAT3-RGS4 pathway was also activated in neonatal brains during poly(I:C)-induced maternal immune activation, which mimics viral infection during pregnancy. These findings indicate that IL-6 elevation at early developmental stages is sufficient to exert a long-lasting effect on glutamatergic synaptogenesis and brain connectivity, providing a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders.

Immune synaptopathies: how maternal immune activation impacts synaptic function during development.

In the last two decades, the term synaptopathy has been largely used to underline the concept that impairments of synaptic structure and function are the major determinant of brain disorders, including neurodevelopmental disorders. This notion emerged from the progress made in understanding the genetic architecture of neurodevelopmental disorders, which highlighted the convergence of genetic risk factors onto molecular pathways specifically localized at the synapse. However, the multifactorial origin of these disorders also indicated the key contribution of environmental factors. It is well recognized that inflammation is a risk factor for neurodevelopmental disorders, and several immune molecules critically contribute to synaptic dysfunction. In the present review, we highlight this concept, which we define by the term "immune-synaptopathy," and we discuss recent evidence suggesting a bi-directional link between the genetic architecture of individuals and maternal activation of the immune system in modulating brain developmental trajectories in health and disease.

Fetal brain response to maternal inflammation requires microglia.

In utero infection and maternal inflammation can adversely impact fetal brain development. Maternal systemic illness, even in the absence of direct fetal brain infection, is associated with an increased risk of neuropsychiatric disorders in affected offspring. The cell types mediating the fetal brain response to maternal inflammation are largely unknown, hindering the development of novel treatment strategies. Here, we show that microglia, the resident phagocytes of the brain, highly express receptors for relevant pathogens and cytokines throughout embryonic development. Using a rodent maternal immune activation (MIA) model in which polyinosinic:polycytidylic acid is injected into pregnant mice, we demonstrate long-lasting transcriptional changes in fetal microglia that persist into postnatal life. We find that MIA induces widespread gene expression changes in neuronal and non-neuronal cells; importantly, these responses are abolished by selective genetic deletion of microglia, indicating that microglia are required for the transcriptional response of other cortical cell types to MIA. These findings demonstrate that microglia play a crucial durable role in the fetal response to maternal inflammation, and should be explored as potential therapeutic cell targets.

At the crux of maternal immune activation: Viruses, microglia, microbes, and IL-17A.

Inflammation during prenatal development can be detrimental to neurodevelopmental processes, increasing the risk of neuropsychiatric disorders. Prenatal exposure to maternal viral infection during pregnancy is a leading environmental risk factor for manifestation of these disorders. Preclinical animal models of maternal immune activation (MIA), established to investigate this link, have revealed common immune and microbial signaling pathways that link mother and fetus and set the tone for prenatal neurodevelopment. In particular, maternal intestinal T helper 17 cells, educated by endogenous microbes, appear to be key drivers of effector IL-17A signals capable of reaching the fetal brain and causing neuropathologies. Fetal microglial cells are particularly sensitive to maternally derived inflammatory and microbial signals, and they shift their functional phenotype in response to MIA. Resulting cortical malformations and miswired interneuron circuits cause aberrant offspring behaviors that recapitulate core symptoms of human neurodevelopmental disorders. Still, the popular use of "sterile" immunostimulants to initiate MIA has limited translation to the clinic, as these stimulants fail to capture biologically relevant innate and adaptive inflammatory sequelae induced by live pathogen infection. Thus, there is a need for more translatable MIA models, with a focus on relevant pathogens like seasonal influenza viruses.

Profiling metabolome of mouse embryonic cerebrospinal fluid following maternal immune activation.

The embryonic cerebrospinal fluid (eCSF) plays an essential role in the development of the central nervous system (CNS), influencing processes from neurogenesis to lifelong cognitive functions. An important process affecting eCSF composition is inflammation. Inflammation during development can be studied using the maternal immune activation (MIA) mouse model, which displays altered cytokine eCSF composition and mimics neurodevelopmental disorders including autism spectrum disorder (ASD). The limited nature of eCSF as a biosample restricts its research and has hindered our understanding of the eCSF's role in brain pathologies. Specifically, investigation of the small molecule composition of the eCSF is lacking, leaving this aspect of eCSF composition under-studied. We report here the eCSF metabolome as a resource for investigating developmental neuropathologies from a metabolic perspective. Our reference metabolome includes comprehensive MS1 and MS2 datasets and evaluates two mouse strains (CD-1 and C57Bl/6) and two developmental time points (E12.5 and E14.5). We illustrate the reference metabolome's utility by using untargeted metabolomics to identify eCSF-specific compositional changes following MIA. We uncover MIA-relevant metabolic pathways as differentially abundant in eCSF and validate changes in glucocorticoid and kynurenine pathways through targeted metabolomics. Our resource can guide future studies into the causes of MIA neuropathology and the impact of eCSF composition on brain development.

Prenatal inflammation shapes microglial immune response into adulthood.

Hayes and collaborators recently unraveled that maternal immune activation in mice led to a long-lasting decrease in microglial immune reactivity. Thus, microglia exhibited a reduced immune response to a second proinflammatory stressor in adulthood. This altered microglial response impacted both astrocytic reactivity and neuronal circuitry.

Prenatal Infection by Respiratory Viruses Is Associated with Immunoinflammatory Responses in the Fetus.

Rationale: Respiratory viral infections can be transmitted from pregnant women to their offspring, but frequency, mechanisms, and postnatal outcomes remain unclear. Objectives: The aims of this prospective cohort study were to compare the frequencies of transplacental transmission of respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), analyze the concentrations of inflammatory mediators in maternal and fetal blood, and assess clinical consequences. Methods: We recruited pregnant women who developed upper respiratory infections or tested positive for SARS-CoV-2. Maternal and cord blood samples were collected at delivery. Study questionnaires and electronic medical records were used to document demographic and medical information. Measurements and Main Results: From October 2020 to June 2022, droplet digital PCR was used to test blood mononuclear cells from 103 mother-baby dyads. Twice more newborns in our sample were vertically infected with RSV compared with SARS-CoV-2 (25.2% [26 of 103] vs. 11.9% [12 of 101]; P = 0.019). Multiplex ELISA measured significantly increased concentrations of several inflammatory cytokines and chemokines in maternal and cord blood from newborns, with evidence of viral exposure in utero compared with control dyads. Prenatal infection was associated with significantly lower birth weight and postnatal weight growth. Conclusions: Data suggest a higher frequency of vertical transmission for RSV than SARS-CoV-2. Intrauterine exposure is associated with fetal inflammation driven by soluble inflammatory mediators, with expression profiles dependent on the virus type and affecting the rate of viral transmission. Virus-induced inflammation may have pathological consequences already in the first days of life, as shown by its effects on birth weight and postnatal weight growth.

Differential effects of early or late exposure to prenatal maternal immune activation on mouse embryonic neurodevelopment.

Exposure to maternal immune activation (MIA) in utero is a risk factor for neurodevelopmental and psychiatric disorders. MIA-induced deficits in adolescent and adult offspring have been well characterized; however, less is known about the effects of MIA exposure on embryo development. To address this gap, we performed high-resolution ex vivo MRI to investigate the effects of early (gestational day [GD]9) and late (GD17) MIA exposure on embryo (GD18) brain structure. We identify striking neuroanatomical changes in the embryo brain, particularly in the late-exposed offspring. We further examined the putative neuroanatomical underpinnings of MIA timing in the hippocampus using electron microscopy and identified differential effects due to MIA timing. An increase in apoptotic cell density was observed in the GD9-exposed offspring, while an increase in the density of neurons and glia with ultrastructural features reflective of increased neuroinflammation and oxidative stress was observed in GD17-exposed offspring, particularly in females. Overall, our findings integrate imaging techniques across different scales to identify differential impact of MIA timing on the earliest stages of neurodevelopment.

Poly (I:C)-induced maternal immune activation generates impairment of reversal learning performance in offspring.

Maternal immune activation (MIA) induces a variety of behavioral and brain abnormalities in offspring of rodent models, compatible with neurodevelopmental disorders, such as schizophrenia or autism. However, it remains controversial whether MIA impairs reversal learning, a basic expression of cognitive flexibility that seems to be altered in schizophrenia. In the present study, MIA was induced by administration of a single dose of polyriboinosinic-polyribocytidylic acid (Poly (I:C) (5 mg/kg i.p.)) or saline to mouse pregnant dams in gestational day (GD) 9.5. Immune activation was monitored through changes in weight and temperature. The offspring were evaluated when they reached adulthood (8 weeks) using a touchscreen-based system to investigate the effects of Poly (I:C) on discrimination and reversal learning performance. After an initial pre-training, mice were trained to discriminate between two different stimuli, of which only one was rewarded (acquisition phase). When the correct response reached above 80% values for two consecutive days, the images were reversed (reversal phase) to assess the adaptation capacity to a changing environment. Maternal Poly (I:C) treatment did not interfere with the learning process but induced deficits in reversal learning compared to control saline animals. Thus, the accuracy in the reversal phase was lower, and Poly (I:C) animals required more sessions to complete it, suggesting impairments in cognitive flexibility. This study advances the knowledge of how MIA affects behavior, especially cognitive domains that are impaired in schizophrenia. The findings support the validity of the Poly (I:C)-based MIA model as a tool to develop pharmacological treatments targeting cognitive deficits associated with neurodevelopmental disorders.

IL-17A Cytokine-Regulated Glut1 Expression in Placenta Cells.

Trophoblasts, the principal cellular component of the placenta, play an important role in nutrient and gas exchange. Previous studies have indicated that maternal immune activation (MIA) leads to an elevation in IL-17A cytokine levels in maternal serum, subsequently influencing fetal brain development during pregnancy. In this study, we aimed to elucidate the impact of the IL-17A cytokine on placental function. First, we treated JAR and JEG-3, which is a placenta cell line, with IL-17A in a concentration-dependent or time-dependent manner and observed cell morphology and viability. It was confirmed that treatment with IL-17A or a double-stranded RNA mimic (PolyI:C) had no effect on the morphology or cell viability. IL-17A treatment increased the expression of IL-17R at the mRNA and protein levels, and Poly(I:C) increased the levels of IFNγ and TNFα. Additionally, PPARγ, known as a metabolism regulator, was increased by IL-17A treatment. Also, we observed that the expression of Glut1 and Glut3 was increased by IL-17A treatment. To confirm this, we examined the expression of transporters in the placental tissue of the MIA rodent model, and we observed that mRNA expression of glut1 and glut3 was significantly increased. However, the expression of Gltu1 and Glut3 was observed to be significantly inhibited in the brains of MIA-induced offspring. This study suggests that IL-17A increases signaling through IL-17R in the placenta and fetal brain tissue; however, there is a mechanism for regulating the expression of glucose transporters by increased IL-17A in the placenta.

Inhibition of NKCC1 Ameliorates Anxiety and Autistic Behaviors Induced by Maternal Immune Activation in Mice.

Autism spectrum disorder (ASD) is thought to result from susceptibility genotypes and environmental risk factors. The offspring of women who experience pregnancy infection have an increased risk for autism. Maternal immune activation (MIA) in pregnant animals produces offspring with autistic behaviors, making MIA a useful model for autism. However, how MIA causes autistic behaviors in offspring is not fully understood. Here, we show that NKCC1 is critical for mediating autistic behaviors in MIA offspring. We confirmed that MIA induced by poly(I:C) infection during pregnancy leads to autistic behaviors in offspring. We further demonstrated that MIA offspring showed significant microglia activation, excessive dendritic spines, and narrow postsynaptic density (PSD) in their prefrontal cortex (PFC). Then, we discovered that these abnormalities may be caused by overexpression of NKCC1 in MIA offspring's PFCs. Finally, we ameliorated the autistic behaviors using PFC microinjection of NKCC1 inhibitor bumetanide (BTN) in MIA offspring. Our findings may shed new light on the pathological mechanisms for autism caused by pregnancy infection.

Maternal immune activation exerts long-term effects on activity and sleep in male offspring mice.

Exposure to infectious or non-infectious immune activation during early development is a serious risk factor for long-term behavioural dysfunctions. Mouse models of maternal immune activation (MIA) have increasingly been used to address neuronal and behavioural dysfunctions in response to prenatal infections. One commonly employed MIA model involves administering poly(I:C) (polyriboinosinic-polyribocytdilic acid), a synthetic analogue of double-stranded RNA, during gestation, which robustly induces an acute viral-like inflammatory response. Using electroencephalography (EEG) and infrared (IR) activity recordings, we explored alterations in sleep/wake, circadian and locomotor activity patterns on the adult male offspring of poly(I:C)-treated mothers. Our findings demonstrate that these offspring displayed reduced home cage activity during the (subjective) night under both light/dark or constant darkness conditions. In line with this finding, these mice exhibited an increase in non-rapid eye movement (NREM) sleep duration as well as an increase in sleep spindles density. Following sleep deprivation, poly(I:C)-exposed offspring extended NREM sleep duration and prolonged NREM sleep bouts during the dark phase as compared with non-exposed mice. Additionally, these mice exhibited a significant alteration in NREM sleep EEG spectral power under heightened sleep pressure. Together, our study highlights the lasting effects of infection and/or immune activation during pregnancy on circadian activity and sleep/wake patterns in the offspring.

Maternal immune activation induces neurodevelopmental impairments of adult offspring through alterations in tryptophane-kynurenine pathway in the placenta.

Maternal immune activation (MIA) is recognized as one of the significant environmental risk factors for neuropsychiatric disorders such as schizophrenia in adult offspring. However, the pathophysiological mechanisms remain unknown. The tryptophan (TRP)-kynurenine (KYN) pathway, influenced by inflammation, may be implicated in the pathophysiology of neuropsychiatric disorders. We investigated whether abnormal behaviors in adult offspring could be induced by MIA through alterations in the TRP-KYN pathway. MIA increased not only IL-6 expression in the placenta but also reactive oxygen species (ROS) levels in both the placenta and fetal brain and disrupted cortical layering in the fetal brain. We observed increased levels of 3-hydroxykynurenine (3-HK), a metabolite with oxidative stress properties, in both the placenta and fetal brain. In the knockout mice of kynurenine 3-monooxygenase (KMO), the enzyme responsible for 3-HK production, MIA failed to induce the abnormal behaviors in adult offspring. Notably, RO-618048, a KMO inhibitor that does not cross the blood-brain barrier (BBB), also blocked MIA-induced abnormal behaviors in adult offspring, reduced not only increased IL-6 expression in the placenta but also ROS levels in both the placenta and fetal brain, and prevented abnormal cortical development in the fetal brain. These findings suggest that MIA-induced abnormal behaviors in adult offspring may result from the increase in 3-HK levels through activation of KMO. Therefore, KMO is an attractive target for the prevention of neuropsychiatric disorders associated with MIA.

Autophagy defects at weaning impair complement-dependent synaptic pruning and induce behavior deficits.

Autophagy is crucial for synaptic plasticity and the architecture of dendritic spines. However, the role of autophagy in schizophrenia (SCZ) and the mechanisms through which it affects synaptic function remain unclear. In this study, we identified 995 single nucleotide polymorphisms (SNPs) across 19 autophagy-related genes that are associated with SCZ. Gene Set Enrichment Analysis (GSEA) of data from the Gene Expression Omnibus public database revealed defective autophagy in patients with SCZ. Using a maternal immune activation (MIA) rat model, we observed that autophagy was downregulated during the weaning period, and early-life activation of autophagy with rapamycin restored abnormal behaviors and electrophysiological deficits in adult rats. Additionally, inhibition of autophagy with 3-Methyladenine (3-MA) during the weaning period resulted in aberrant behaviors, abnormal electrophysiology, increased spine density, and reduced microglia-mediated synaptic pruning. Furthermore, 3-MA treatment significantly decreased the expression and synaptosomal content of complement, impaired the recognition of C3b and CR3, indicating that autophagy deficiency disrupts complement-mediated synaptic pruning. Our findings provide evidence for a significant association between SCZ and defective autophagy, highlighting a previously underappreciated role of autophagy in regulating the synaptic and behavioral deficits induced by MIA.

Impact of maternal immune activation and sex on placental and fetal brain cytokine and gene expression profiles in a preclinical model of neurodevelopmental disorders.

Maternal inflammation during gestation is associated with a later diagnosis of neurodevelopmental disorders including autism spectrum disorder (ASD). However, the specific impact of maternal immune activation (MIA) on placental and fetal brain development remains insufficiently understood. This study aimed to investigate the effects of MIA by analyzing placental and brain tissues obtained from the offspring of pregnant C57BL/6 dams exposed to polyinosinic: polycytidylic acid (poly I: C) on embryonic day 12.5. Cytokine and mRNA content in the placenta and brain tissues were assessed using multiplex cytokine assays and bulk-RNA sequencing on embryonic day 17.5. In the placenta, male MIA offspring exhibited higher levels of GM-CSF, IL-6, TNFα, and LT-α, but there were no differences in female MIA offspring. Furthermore, differentially expressed genes (DEG) in the placental tissues of MIA offspring were found to be enriched in processes related to synaptic vesicles and neuronal development. Placental mRNA from male and female MIA offspring were both enriched in synaptic and neuronal development terms, whereas females were also enriched for terms related to excitatory and inhibitory signaling. In the fetal brain of MIA offspring, increased levels of IL-28B and IL-25 were observed with male MIA offspring and increased levels of LT-α were observed in the female offspring. Notably, we identified few stable MIA fetal brain DEG, with no male specific difference whereas females had DEG related to immune cytokine signaling. Overall, these findings support the hypothesis that MIA contributes to the sex- specific abnormalities observed in ASD, possibly through altered neuron developed from exposure to inflammatory cytokines. Future research should aim to investigate how interactions between the placenta and fetal brain contribute to altered neuronal development in the context of MIA.

The Role of Parvalbumin Interneurons in Autism Spectrum Disorder.

As an important subtype of GABAergic interneurons, parvalbumin (PV) interneurons play a critical role in regulating cortical circuits and neural networks. Abnormalities in the development or function of PV interneurons have been linked to autism spectrum disorder (ASD), a neurodevelopmental disorder characterized by social and language deficits. In this review, we focus on the abnormalities of PV interneurons in ASD, including quantity and function and discuss the underlying mechanisms of impairments in PV interneurons in the pathology of ASD. Finally, we propose potential therapeutic approaches targeting PV interneurons, such as transplanting MGE progenitor cells and utilizing optogenetic stimulation in the treatment of ASD.

Gut microbiota transfer from the preclinical maternal immune activation model of autism is sufficient to induce sex-specific alterations in immune response and behavioural outcomes.

The gut microbiome plays a vital role in health and disease, including neurodevelopmental disorders like autism spectrum disorder (ASD). ASD affects 4:1 males-to-females, and sex differences are apparent in gut microbiota composition among ASD individuals and in animal models of this condition, such as the maternal immune activation (MIA) mouse model. However, few studies have included sex as a biological variable when assessing the role of gut microbiota in mediating ASD symptoms. Using the MIA model of ASD, we assessed whether gut microbiota contributes to the sex differences in the presentation of ASD-like behaviors. Gut microbiota transplantation from MIA or vehicle/control male and female mice into healthy, otherwise unmanipulated, 4-week-old C57Bl/6 mice was performed for 6 treatments over 12 days. Colonization with male, but not female, MIA microbiota was sufficient to reduce sociability, decrease microbiota diversity and increase neuroinflammation with more pronounced deficits in male recipients. Colonization with both male and female donor microbiota altered juvenile ultrasonic vocalizations and anxiety-like behavior in recipients of both sexes, and there was an accompanied change in the gut microbiota and serum cytokine IL-4 and IL-7 levels of all recipients of MIA gut microbiota. In addition to the increases in gut microbes associated with pathological states, the female donor microbiota profile also had increases in gut microbes with known neural protective effects (e.g., Lactobacillus and Rikenella). These results suggest that gut reactivity to environmental insults, such as in the MIA model, may play a role in shaping the sex disparity in ASD development.

Maternal selenium dietary supplementation alters sociability and reinforcement learning deficits induced by in utero exposure to maternal immune activation in mice.

Maternal immune activation (MIA) during pregnancy increases the risk for the unborn foetus to develop neurodevelopmental conditions such as autism spectrum disorder and schizophrenia later in life. MIA mouse models recapitulate behavioural and biological phenotypes relevant to both conditions, and are valuable models to test novel treatment approaches. Selenium (Se) has potent anti-inflammatory properties suggesting it may be an effective prophylactic treatment against MIA. The aim of this study was to determine if Se supplementation during pregnancy can prevent adverse effects of MIA on offspring brain and behaviour in a mouse model. Selenium was administered via drinking water (1.5 ppm) to pregnant dams from gestational day (GD) 9 to birth, and MIA was induced at GD17 using polyinosinic:polycytidylic acid (poly-I:C, 20 mg/kg via intraperitoneal injection). Foetal placenta and brain cytokine levels were assessed using a Luminex assay and brain elemental nutrients assessed using inductively coupled plasma- mass spectrometry. Adult offspring were behaviourally assessed using a reinforcement learning paradigm, the three-chamber sociability test and the open field test. MIA elevated placental IL-1ß and IL-17, and Se supplementation successfully prevented this elevation. MIA caused an increase in foetal brain calcium, which was prevented by Se supplement. MIA caused in offspring a female-specific reduction in sociability, which was recovered by Se, and a male-specific reduction in social memory, which was not recovered by Se. Exposure to poly-I:C or selenium, but not both, reduced performance in the reinforcement learning task. Computational modelling indicated that this was predominantly due to increased exploratory behaviour, rather than reduced rate of learning the location of the food reward. This study demonstrates that while Se may be beneficial in ameliorating sociability deficits caused by MIA, it may have negative effects in other behavioural domains. Caution in the use of Se supplementation during pregnancy is therefore warranted.