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.

Histone deacetylase 3 regulates microglial function through histone deacetylation.

As the primary innate immune cells of the brain, microglia respond to damage and disease through pro-inflammatory release of cytokines and neuroinflammatory molecules. Histone acetylation is an activating transcriptional mark that regulates inflammatory gene expression. Inhibition of histone deacetylase 3 (Hdac3) has been utilized in pre-clinical models of depression, stroke, and spinal cord injury to improve recovery following injury, but the molecular mechanisms underlying Hdac3's regulation of inflammatory gene expression in microglia is not well understood. To address this lack of knowledge, we examined how pharmacological inhibition of Hdac3 in an immortalized microglial cell line (BV2) impacted histone acetylation and gene expression of pro- and anti-inflammatory genes in response to immune challenge with lipopolysaccharide (LPS). Flow cytometry and cleavage under tags & release using nuclease (CUT & RUN) revealed that Hdac3 inhibition increases global and promoter-specific histone acetylation, resulting in the release of gene repression at baseline and enhanced responses to LPS. Hdac3 inhibition enhanced neuroprotective functions of microglia in response to LPS through reduced nitric oxide release and increased phagocytosis. The findings suggest Hdac3 serves as a regulator of microglial inflammation, and that inhibition of Hdac3 facilitates the microglial response to inflammation and its subsequent clearing of debris or damaged cells. Together, this work provides new mechanistic insights into therapeutic applications of Hdac3 inhibition which mediate reduced neuroinflammatory insults through microglial response.

Modelling Microglial Innate Immune Memory In Vitro: Understanding the Role of Aerobic Glycolysis in Innate Immune Memory.

Microglia, the resident macrophages of the central nervous system, play important roles in maintaining brain homeostasis and facilitating the brain's innate immune responses. Following immune challenges microglia also retain immune memories, which can alter responses to secondary inflammatory challenges. Microglia have two main memory states, training and tolerance, which are associated with increased and attenuated expression of inflammatory cytokines, respectively. However, the mechanisms differentiating these two distinct states are not well understood. We investigated mechanisms underlying training versus tolerance memory paradigms in vitro in BV2 cells using B-cell-activating factor (BAFF) or bacterial lipopolysaccharide (LPS) as a priming stimulus followed by LPS as a second stimulus. BAFF followed by LPS showed enhanced responses indicative of priming, whereas LPS followed by LPS as the second stimulus caused reduced responses suggestive of tolerance. The main difference between the BAFF versus the LPS stimulus was the induction of aerobic glycolysis by LPS. Inhibiting aerobic glycolysis during the priming stimulus using sodium oxamate prevented the establishment of the tolerized memory state. In addition, tolerized microglia were unable to induce aerobic glycolysis upon LPS restimulus. Therefore, we conclude that aerobic glycolysis triggered by the first LPS stimulus was a critical step in the induction of innate immune tolerance.

Work hard, play hard: how sexually differentiated microglia work to shape social play and reproductive behavior.

Microglia are brain-resident immune cells that play a critical role in synaptic pruning and circuit fine-tuning during development. In the adult brain, microglia actively survey their local environment and mobilize inflammatory responses to signs of damage or infection. Sex differences in microglial gene expression and function across the lifespan have been identified, which play a key role in shaping brain function and behavior. The levels of sex hormones such as androgens, estrogens, and progesterone vary in an age-dependent and sex-dependent manner. Microglia respond both directly and indirectly to changes in hormone levels, altering transcriptional gene expression, morphology, and function. Of particular interest is the microglial function in brain regions that are highly sexually differentiated in development such as the amygdala as well as the pre-optic and ventromedial hypothalamic regions. With a focus on hormone-sensitive developmental windows, this review compares male and female microglia in the embryonic, developing, and adult brain with a particular interest in the influence of sex hormones on microglial wiring of social, reproductive, and disordered behavior circuits in the brain.

Insights Into the Emerging Role of Baf53b in Autism Spectrum Disorder.

Accurate and precise regulation of gene expression is necessary to ensure proper brain development and plasticity across the lifespan. As an ATP-dependent chromatin-remodeling complex, the BAF (Brg1 Associated Factor) complex can alter histone-DNA interactions, facilitating dynamic changes in gene expression by controlling DNA accessibility to the transcriptional machinery. Mutations in 12 of the potential 29 subunit genes that compose the BAF nucleosome remodeling complex have been identified in several developmental disorders including Autism spectrum disorders (ASD) and intellectual disability. A novel, neuronal version of BAF (nBAF) has emerged as promising candidate in the development of ASD as its expression is tied to neuron differentiation and it's hypothesized to coordinate expression of synaptic genes across brain development. Recently, mutations in BAF53B, one of the neuron specific subunits of the nBAF complex, have been identified in patients with ASD and Developmental and epileptic encephalopathy-76 (DEE76), indicating BAF53B is essential for proper brain development. Recent work in cultured neurons derived from patients with BAF53B mutations suggests links between loss of nBAF function and neuronal dendritic spine formation. Deletion of one or both copies of mouse Baf53b disrupts dendritic spine development, alters actin dynamics and results in fewer synapses in vitro. In the mouse, heterozygous loss of Baf53b severely impacts synaptic plasticity and long-term memory that is reversible with reintroduction of Baf53b or manipulations of the synaptic plasticity machinery. Furthermore, surviving Baf53b-null mice display ASD-related behaviors, including social impairments and repetitive behaviors. This review summarizes the emerging evidence linking deleterious variants of BAF53B identified in human neurodevelopmental disorders to abnormal transcriptional regulation that produces aberrant synapse development and behavior.

Dysregulated gene expression associated with inflammatory and translation pathways in activated monocytes from children with autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex developmental disorder characterized by deficits in social interactions, communication, and stereotypical behaviors. Immune dysfunction is a common co-morbidity seen in ASD, with innate immune activation seen both in the brain and periphery. We previously identified significant differences in peripheral monocyte cytokine responses after stimulation with lipoteichoic acid (LTA) and lipopolysaccharide (LPS), which activate toll-like receptors (TLR)-2 and 4 respectively. However, an unbiased examination of monocyte gene expression in response to these stimulants had not yet been performed. To identify how TLR activation impacts gene expression in ASD monocytes, we isolated peripheral blood monocytes from 26 children diagnosed with autistic disorder (AD) or pervasive developmental disorder-not otherwise specified (PDDNOS) and 22 typically developing (TD) children and cultured them with LTA or LPS for 24 h, then performed RNA sequencing. Activation of both TLR2 and TLR4 induced expression of immune genes, with a subset that were differentially regulated in AD compared to TD samples. In response to LPS, monocytes from AD children showed a unique increase in KEGG pathways and GO terms that include key immune regulator genes. In contrast, monocytes from TD children showed a consistent decrease in expression of genes associated with translation in response to TLR stimulation. This decrease was not observed in AD or PDDNOS monocytes, suggesting a failure to properly downregulate a prolonged immune response in monocytes from children with ASD. As monocytes are involved in early orchestration of the immune response, our findings will help elucidate the mechanisms regulating immune dysfunction in ASD.

MGEnrichment: A web application for microglia gene list enrichment analysis.

Gene expression analysis is becoming increasingly utilized in neuro-immunology research, and there is a growing need for non-programming scientists to be able to analyze their own genomic data. MGEnrichment is a web application developed both to disseminate to the community our curated database of microglia-relevant gene lists, and to allow non-programming scientists to easily conduct statistical enrichment analysis on their gene expression data. Users can upload their own gene IDs to assess the relevance of their expression data against gene lists from other studies. We include example datasets of differentially expressed genes (DEGs) from human postmortem brain samples from Autism Spectrum Disorder (ASD) and matched controls. We demonstrate how MGEnrichment can be used to expand the interpretations of these DEG lists in terms of regulation of microglial gene expression and provide novel insights into how ASD DEGs may be implicated specifically in microglial development, microbiome responses and relationships to other neuropsychiatric disorders. This tool will be particularly useful for those working in microglia, autism spectrum disorders, and neuro-immune activation research. MGEnrichment is available at https://ciernialab.shinyapps.io/MGEnrichmentApp/ and further online documentation and datasets can be found at https://github.com/ciernialab/MGEnrichmentApp. The app is released under the GNU GPLv3 open source license.

Genetic variants drive altered epigenetic regulation of endotoxin response in BTBR macrophages.

The BTBR T+Itpr3tf/J (BTBR) mouse has been used as a complex genetic model of Autism Spectrum Disorders (ASD). While the specific mechanisms underlying BTBR behavioral phenotypes are poorly understood, prior studies have implicated profound differences in innate immune system control of pro-inflammatory cytokines. Innate immune activation and elevated pro-inflammatory cytokines are also detected in blood of children with ASD. In this study, we examined how underlying BTBR genetic variants correspond to strain-specific changes in chromatin accessibility, resulting in a pro-inflammatory response specifically in BTBR bone marrow derived macrophages (BMDM). In response to repeated lipopolysaccharide (LPS) treatments, C57BL/6J (C57) BMDM exhibited intact endotoxin tolerance. In contrast, BTBR BMDM exhibited hyper-responsive expression of genes that were normally tolerized in C57. This failure in formation of endotoxin tolerance in BTBR was mirrored at the level of chromatin accessibility. Using ATAC-seq, we specifically identified promoter and enhancer regions with strain-specific differential chromatin accessibility both at baseline and in response to LPS. Regions with strain-specific differences in chromatin accessibility were significantly enriched for BTBR genetic variants, such that an average of 22% of the differential chromatin regions had at least one variant. Together, these results demonstrate that BTBR genetic variants contribute to altered chromatin responsiveness to endotoxin challenge resulting in hyper-responsive innate immunity in BTBR. These findings provide evidence for an interaction between complex genetic variants and differential epigenetic regulation of innate immune responses.

Chromatin Dynamics and Genetic Variation Combine to Regulate Innate Immune Memory.

Recent work by Ciernia et al. (2020) identified how genetic and epigenetic mechanisms interact to regulate innate immune memory in bone marrow derived macrophages. The authors examined the BTBR strain, a naturally occurring mouse model of Autism Spectrum Disorder (ASD) that captures the complex genetics, behavioral and immune dysregulation found in the human disorder. Immune cell cultures from the BTBR strain compared to the standard C57 showed hyper-responsive immune gene expression that was linked to altered chromatin accessibility at sites with genetic differences between the strains. Together, findings from this work demonstrated that multiple levels of gene regulation likely dictate the formation of innate immune memory and are likely disrupted in immune cells in ASD. Future work will be needed to extend these findings to immune gene regulation in the brain and how changes in immune function are related to abnormal behaviors in brain disorders.