Maternal Influenza A Virus Infection Restricts Fetal and Placental Growth and Adversely Affects the Fetal Thymic Transcriptome

Abstract

Maternal influenza A viral infections in humans are associated with low birth weight, increased risk of pre-term birth, stillbirth and congenital defects. To examine the effect of maternal influenza virus infection on placental and fetal growth, pregnant C57BL/6 mice were inoculated intranasally with influenza A virus A/CA/07/2009 pandemic H1N1 or phosphate-buffered saline (PBS) at E3.5, E7.5 or E12.5, and the placentae and fetuses collected and weighed at E18.5. Fetal thymuses were pooled from each litter. Placentae were examined histologically, stained by immunohistochemistry (IHC) for CD34 (hematopoietic progenitor cell antigen) and vascular channels quantified. RNA from E7.5 and E12.5 placentae and E7.5 fetal thymuses was subjected to RNA sequencing and pathway analysis. Placental weights were decreased in litters inoculated with influenza at E3.5 and E7.5. Placentae from E7.5 and E12.5 inoculated litters exhibited decreased labyrinth development and the transmembrane protein 150A gene was upregulated in E7.5 placentae. Fetal weights were decreased in litters inoculated at E7.5 and E12.5 compared to controls. RNA sequencing of E7.5 thymuses indicated that 957 genes were downregulated ≥2-fold including Mal, which is associated with Toll-like receptor signaling and T cell differentiation. There were 28 upregulated genes. It is concluded that maternal influenza A virus infection impairs fetal thymic gene expression as well as restricting placental and fetal growth.

Keywords: Mal; fetus; growth; immunity; influenza; thymus.

Figure 1

Maternal influenza reduces fetal (A) and placental (B) weights. Pregnant mice were inoculated intranasally with phosphate-buffered saline (PBS, control) or influenza at E3.5 (3 control dams; 5 influenza dams); E7.5 (8 control dams; 9 influenza dams); or E12.5 (5 control dams; 5 influenza dams). Fetuses and placentae were weighed on E18.5. Data are shown as mean ± SEM (standard error of the mean); n = number of fetuses. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2

Quantification of fetal vasculature in the placental labyrinth of mice either mock-inoculated and influenza virus-inoculated on E7.5 of pregnancy and terminated on E18.5. (A) Average pixel numbers from 18 control placentas (blue) and 14 placentas from influenza virus-inoculated (orange) dams. Ten randomly chosen vascular channels in CD34 immunolabeled specimens from each placenta were measured and averaged. (B) All CD34 positive vascular segments within a 400 µm × 400 µm grid placed over the placental labyrinth were counted for 18 control placentas (blue) and 14 placentas from influenza virus-inoculated dams (orange). **** p < 0.0001.

Figure 3

Immunohistochemical labelling of CD34 in the labyrinth of placenta. (A) Microphotograph of labyrinth in placenta from mock-inoculated control mouse. Fetal vessels are seen as irregular spaces lined by reddish-brown stained endothelial. Immunohistochemical labelling of CD34. Original magnification 200×. (B) Microphotograph of labyrinth in placenta from mouse infected on day 7.5 of pregnancy. Note reduced fetal vasculature as reflected by fewer vascular channels per area and smaller volume of individual vascular segments. Scale bars = 40 µm. Original magnification 200×.

Figure 4

Ingenuity Pathway Analysis (IPA) analysis of genes differentially expressed 1.5-fold or greater in the E7.5 influenza-inoculated fetal thymuses compared to controls collected on day E18.5. (A) Top 10 canonical pathways, total number of genes in each pathway, percent of these genes downregulated and upregulated, and the −log(p-value). (B) Top 10 significant diseases and functions: physiological system: development and function pathways in E7.5 influenza inoculated compared to control fetuses collected on day E18.5. (C) The top 10 analysis-ready molecules based on p value and greatest downregulated and upregulated genes. (D) Disease and Function analysis identified 26 downregulated fetal thymic genes that were associated with respiratory failure in influenza inoculated compared to control fetal thymus. Also see Supplementary Figure S1 describing 1 upregulated and 40 downregulated fetal thymic genes in influenza inoculated thymuses associated with decreased body size and increased risk of neonatal death.

Figure 5

Maternal influenza infection is associated with downregulation of fetal thymic Mal mRNA. On E3.5, E7.5 or E12.5 pregnant mice were inoculated with PBS (control) or influenza. Subsequently, on E18.5 fetal thymuses were examined for gene expression. On E18.5 fetal thymic Mal mRNA was measured by RT-qPCR. Data are expressed as the relative expression (mean ± SEM). (A) E3.5 litters: control (n = 3), influenza (n = 5). (B) E7.5 litters: control (n = 8), influenza (n = 9). (C) E12.5 litters: control (n = 5), influenza (n = 5). * p < 0.05; *** p < 0.001.