Maternal viral infection causes global alterations in porcine fetal microglia

Abstract

Maternal infections during pregnancy are associated with increased risk of neurodevelopmental disorders, although the precise mechanisms remain to be elucidated. Previously, we established a maternal immune activation (MIA) model using swine, which results in altered social behaviors of piglet offspring. These behavioral abnormalities occurred in the absence of microglia priming. Thus, we examined fetal microglial activity during prenatal development in response to maternal infection with live porcine reproductive and respiratory syndrome virus. Fetuses were obtained by cesarean sections performed 7 and 21 d postinoculation (dpi). MIA fetuses had reduced brain weights at 21 dpi compared to controls. Furthermore, MIA microglia increased expression of major histocompatibility complex class II that was coupled with reduced phagocytic and chemotactic activity compared to controls. High-throughput gene-expression analysis of microglial-enriched genes involved in neurodevelopment, the microglia sensome, and inflammation revealed differential regulation in primary microglia and in whole amygdala tissue. Microglia density was increased in the fetal amygdala at 7 dpi. Our data also reveal widespread sexual dimorphisms in microglial gene expression and demonstrate that the consequences of MIA are sex dependent. Overall, these results indicate that fetal microglia are significantly altered by maternal viral infection, presenting a potential mechanism through which MIA impacts prenatal brain development and function. maternal immune activation | microglia | neurodevelopment | prenatal inflammation | pig Author contributions: A.M.A., M.A.L., and R.W.J. designed research; A.M.A., M.A.L., M.P.C., S.M.M., and B.J.L. performed research; A.M.A., M.P.C., and S.M.M. analyzed data; A.M.A., M.A.L., and R.W.J. interpreted data; and A.M.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission.Published under the PNAS license. NEUROSCIENCEDownloaded by guest on July 10, 2020 3 symbols = P < 0.001, 2 symbols = P < 0.01, and 1 symbol = P < 0.05.Antonson et al.

Lasting and Sex-Dependent Impact of Maternal Immune Activation on Molecular Pathways of the Amygdala

“…The cytokinemediated signaling pathway was also over-represented among the genes under-expressed in MPA relative to CON pigs ( Table 3). Consistent with our results, the expression of 28 genes annotated to immune stimulus had MIA-by-sex interaction effects in the microglia of GD 97 fetuses after GD 76 PRRSV injection (Antonson et al, 2019).…”

“…Several KEGG pathways associated with inflammation and infection, including allograft rejection, Epstein-Barr virus infection, and autoimmune thyroid disease, were enriched, among the genes differentially expressed between MPA and CON pigs ( Table 5 and Supplementary File S1 Table E). This finding is consistent with the significant effect of MIA from GD 76 PRRSV injection on expression of 12 genes in the amygdala of GD 97 fetuses from both sexes (Antonson et al, 2019). At this fetal stage, most of the genes differentially expressed between MPA and CON gilts were annotated to the BP immune response, including genes annotated to the BP cytokine-mediated signaling pathway, and to the toll-like receptor pathway (Antonson et al, 2019).…”

“…Among these categories was the BP anatomical structure formation involved in morphogenesis and angiogenesis ( Table 5). This result is consistent with the under-expression of early growth response 1 (EGR1, a regulator of angiogenic factors) in the GD 97 fetal microglia of MPA relative to CON pigs of both sexes after the GD 76 PRRSV challenge (Antonson et al, 2019). Angiogenesis was also enriched among differentially expressed genes in the frontal cortex of rats that are exposed to valproic acid in utero (Hill et al, 2015) to model ASD (Favre et al, 2013).…”

“…Following anesthetization, pigs were euthanized using an intracardiac injection of sodium pentobarbital (86 mg/kg body weight, Fata Plus, Vortech Pharmaceuticals, Dearborn, MI, United States). Pig brains were extracted, the amygdalae were recognized using the stereotaxic atlas of the pig brain (Felix et al, 1999), dissected out, flash frozen on dry ice, and stored at −80 • C following published protocols (Antonson et al, 2019). RNA was isolated using EZNA isolation kit following the manufacturer's instructions (Omega Biotek, Norcross, GA, United States).…”

“…Research on the lasting effects of MIA in pigs complements the insights offered by rodent models (Antonson et al, 2019). The advantages of studying a pig model stem from the greater homology of humans to pigs, rather than to rodents, when considering organ physiology, size, development and, in particular, brain growth and development processes (Murphy et al, 2014).…”