Androgens Upregulate Pathogen-Induced Placental Innate Immune Response

Abstract

Group B Streptococcus (GBS) is a leading cause of placental infection, termed chorioamnionitis. Chorioamnionitis is associated with an increased risk of neurobehavioral impairments, such as autism spectrum disorders, which are more prominent in males than in female offspring. In a pre-clinical model of chorioamnionitis, a greater inflammatory response was observed in placenta associated with male rather than female fetuses, correlating with the severity of subsequent neurobehavioral impairments. The reason for this sex difference is not understood. Our hypothesis is that androgens upregulate the placental innate immune response in male fetuses. Lewis dams were injected daily from gestational day (G) 18 to 21 with corn oil (vehicle) or an androgen receptor antagonist (flutamide). On G 19, dams were injected with saline (control) or GBS. Maternal, fetal sera and placentas were collected for protein assays and in situ analyses. Our results showed that while flutamide alone had no effect, a decrease in placental concentration of pro-inflammatory cytokines and infiltration of polymorphonuclear cells was observed in flutamide/infected compared to vehicle/infected groups. These results show that androgens upregulate the placental innate immune response and thus may contribute to the skewed sex ratio towards males observed in several developmental impairments resulting from perinatal infection/inflammation.

Keywords: androgens; autism spectrum disorder; cerebral palsy; chorioamnionitis; cytokines; group B Streptococcus; maternal immune activation; neonatal infection; neurodevelopmental disorders; placenta.

Figure 1

Flutamide acts as an antiandrogen by reducing AGD in pups. (a) The AGD of pups exposed in utero (G18–21) to flutamide or vehicle. (b) Number of live pups/litter. Analyses were done by one-way ANOVA with Tukey’s multiple comparison. Bars represent mean ± SEM. Number (n) of pups in vehicle/control: n = 6, vehicle/GBS: n = 5, flutamide/control: n = 5, flutamide/GBS: n = 5. One male was used per litter for analysis. *** p < 0.001, **** p < 0.0001.

Figure 2

Monitoring of maternal weight and pro-inflammatory response in the blood circulation. Mean maternal weight gain from G18–22 (a). Mean concentrations of IL-1β (b) and IL-6 (c) in maternal sera. Analyses were done by one-way ANOVA with Tukey’s multiple comparison (maternal weight gain, IL-1β) or Kruskal-Wallis with Dunn’s multiple comparison test when data was not normally distributed (IL-6). Bars represent mean ± SEM. Number (n) of dams in vehicle/control: n = 6, vehicle/GBS: n = 5, flutamide/control: n = 5, flutamide/GBS: n = 5. * p < 0.05.

Figure 3

In combination with flutamide, GBS-induced chorioamnionitis is associated with decreased levels of placental proinflammatory cytokines IL-1β, IL-6 and TNF-α. Mean placental concentrations of (a) IL-1β, (b) IL-6, (c) TNF-α detected by ELISA post-GBS inoculation. Analyses were done by one-way ANOVA with Dunnett’s multiple comparison (to vehicle/GBS). Bars represent mean ± SEM. Number (n) of placentas in vehicle/control: n = 6, vehicle/GBS: n = 5, flutamide/control: n = 5, flutamide/GBS: n = 5. One male placenta was used per litter. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 4

Flutamide reduced the infiltration of PMN cells in GBS infected placental compartments. (a) Coronal section of placental compartments showing the maternal (decidua, DC), maternofetal zone (junctional zone, JZ), and fetal (labyrinth, LB) compartments. (b) PMN infiltrations (circular in shape, stained dark purple/brown staining, black arrowheads) in DC, JZ and LB compartments of the placenta.

Figure 5

Mean density of PMNs detected in GBS-infected placental compartments. (a) decidua (DC) (b) junctional zone (JZ), and (c) fetal labyrinth (LB). Statistical analyses were done by one-way ANOVA with Dunnett’s multiple comparison (to vehicle/GBS). Bars represent mean ± SEM. Number (n) of pups in vehicle/control: n = 4–5, vehicle/GBS: n = 3–6, flutamide/control: n = 5–6, flutamide/GBS: n = 6. One male was used per litter for analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Timeline of injections and C-section of dams from G18 to G21.