Poster U16 - Maternal influenza infection alters offspring’s epigenomic and transcriptomic dynamics in the mouse frontal cortex

Introduction: Maternal infection during pregnancy has been identified as one of the environmental insults that could potentially affect the common developmental trajectory of the brain, thereby increasing the risk of several neurodevelopmental psychiatric such as schizophrenia and autism spectrum disorder (ASD). Rodent models of prenatal environmental insults have established the concept that maternal immune activation during pregnancy, rather than a specific pathogen, induces neurodevelopmental changes in mouse offspring. Previous studies have shown that maternal infection can lead to impairments in synaptic function and transmission through immune alterations during brain development. Specifically, maternal infection activation affects neurotransmitter systems, including the GABAergic, glutamatergic, dopaminergic, and cholinergic systems. Furthermore, microRNA or histone modifications regulate the expression of genes encoding proteins involved in these signaling systems, demonstrating the potential association between maternal immune activation and epigenetic regulation.
In our study, we combined MOWChIP-seq and SMART-seq2 techniques to profile H3K27ac and H3K4me3 histone modifications, as well as transcriptomes in adult mice born in influenza virus-infected mothers and controls. We focused on the effects of maternal influenza viral infection on the epigenetic and transcriptomic landscapes in the frontal cortex. By examining cell-type-specific features, we aimed to enhance our understanding of the molecular mechanisms underlying the association between increased risk of neurodevelopmental disorders and maternal viral exposure. Additionally, we performed cross-fostering experiments to evaluate the effects of maternal behavior after delivery in the adult offspring.

Materials and

Methods: Experiments began with pregnant CD-1 dams. On Gestational Day 9.5, mice received intranasal (i.n.) infections with 5  103 pfu of influenza A/WSN/33 (H1N1) virus, or mock PBS. On the day of birth, half of the offspring born to influenza virus-infected mothers and control dams were removed from the original mother and then placed with a surrogate rearing mother. Male mice offspring aged ten weeks were then sacrificed, and their frontal cortices (bregma 1.9 to 1.40 mm) were promptly frozen. Using an anti-NeuN antibody to sort nuclei, we isolated NeuN-positive (NeuN+) nuclei (approximately 60,000 nuclei) for ChIP-seq (~10,000 nuclei per assay) and RNA-seq (~6,000 per assay). ChIP-seq and RNA-seq libraries were prepared following established protocols (MOWChIP-seq and SMART-seq2, respectively). Differential analysis was conducted for both ChIP-seq and RNA-seq data, and weighted gene co-expression network analysis was also performed for RNA-seq data. Integrating H3K27ac histone modification data with RNA-seq data, we employed the Taiji pipeline to identify crucial transcription factors and regulated genes. LDSC was used to evaluate the LD score regression between samples and the established GWAS traits.

Results, Conclusions, and Discussions: We obtained 347 genome-wide profiles from 60 male mice offspring, comprising 118 for H3K4me3, 115 for H3K27ac, and 114 for RNA. Our ChIP-seq and RNA-seq libraries generated have minimal background noise and surpass ENCODE database standards for quality control. Utilizing various data analysis methods such as weighted gene co-expression network analysis (WGCNA), differential analysis, key transcription factors and regulate gene analysis, and GWAS linkage disequilibrium score analysis, we investigated the prenatal and postnatal effects of MIA on mice offspring.
In the comparison group for prenatal effects, a significant number of differential peaks were observed compared to the negative control group. KEGG terms such as the GABAergic synapse pathway, cAMP signaling pathway, Glutamatergic synapse pathway, and neurodegenerative disease pathway consistently emerged in WGCNA and differential analysis, which confirm the prenatal effect of MIA on mice offspring.
In addition, offspring of infected mothers undergoing cross-fostering showed distinct differences in both differential and WGCNA analysis compared to adult mice born to influenza virus-infected mothers without cross fostering, suggesting role of prenatal environmental insults as a primer that exacerbates adverse conditions like cross fostering for psychiatric disorders.
In conclusion, our study provides insights into the prenatal and postnatal effects of maternal viral infection on mice offspring, emphasizing the critical link between maternal health and offspring neurodevelopment.

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