Fig. 5: Border-associated macrophages but not microglia are upregulated in fetal brains from high-dose IAV dams.

A Representative sagittal sections of the E11.5 fetal brain stained with Iba1 (red) and CD206 (green). Microglia are Iba1⁺CD206^- and BAMs are Iba1⁺CD206⁺. Left scale bar = 1000 µm; right scale bar = 100 µm. There were no changes in B microglia or C BAM count per mm² at E11.5, 2 dpi. While there were no changes in D microglia count, E BAM count was upregulated in E16.5, 7 dpi fetal brains exposed to a high dose of IAV. F Representative coronal sections of the meninges and choroid plexus from each treatment group of Iba1 and CD206 co-stained E16.5 fetal brains. Scale bars = 100 µm. G, H Separation of choroid plexus and meningeal BAMs showed that only G meningeal BAMs were increased at 7 dpi. I Evaluation of E16.5 brain macrophage proliferation via Ki67 and Iba1 co-staining revealed no changes in double-positive cells in the J whole brain, K parenchyma, L meninges, or M choroid plexus. Arrows = representative co-staining; scale bars = 100 µm. IAV = influenza A virus, dpi = days post-inoculation, E = embryonic day, MFI = mean fluorescence intensity, T = telencephalon, D = diencephalon, MS = mesencephalon, MT = metencephalon MY = myencephalon, Con = saline control, X31_mod = IAV-X31 10³ TCID₅₀, X31_hi = IAV-X31 10⁴ TCID₅₀. Groups were compared using one-way ANOVA with Tukey post hoc for multiple comparisons. For data containing residuals with unequal variance, Brown-Forsythe and Welch’s ANOVA with Dunnett T3 post hoc multiple comparisons was used. Data are means ± SEM; *p < 0.05, **p < 0.01; dots represent one representative fetus per litter; n = 9–14 per treatment group. See Supplementary Table S11 for complete statistical analysis of all data collected for this figure (individual mean ± SEM per group, p-values, hypothesis test used, and test statistic).