Environmental Toxicant Exposure Paralyzes Human Placental Macrophage Responses to Microbial Threat

Abstract

Exposure to environmental toxicants (such as dioxins) has been epidemiologically linked to adverse reproductive health outcomes, including placental inflammation and preterm birth. However, the molecular underpinnings that govern these outcomes in gravid reproductive tissues remain largely unclear. Placental macrophages (also known as Hofbauer cells) are crucial innate immune cells that defend the gravid reproductive tract and help promote maternal-fetal tolerance. We hypothesized that exposure to environmental toxicants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could alter placental macrophage responses to inflammatory insults such as infection. To test this, placental macrophages were cultured in the presence or absence of TCDD and then infected with the perinatal pathogen Group B Streptococcus (GBS). Our results indicate that TCDD is lethal to placental macrophages at and above a 5 nM concentration and that sublethal dioxin exposure inhibits phagocytosis and cytokine production. Taken together, these results indicate that TCDD paralyzes placental macrophage responses to bacterial infection.

Keywords: GBS; TCDD; antimicrobial responses; environmental toxicants; placental macrophages.

Figure 1.

Isolation of placental macrophages. De-identified placental tissue is collected from non-laboring patients who delivered healthy, full-term infants by Caesarian section. Villous core tissue was mechanically separated and enzymatically digested. Isolation of CD14⁺ cells was performed via magnetic MACS Cell Separation system. Human placental macrophage cells are isolated in pure culture before being used in assays with TCDD and/or GBS. Image created with BioRender.

Figure 2.

Analysis of human placental macrophage viability by ATPLite assay. Macrophages were cultured in medium alone (0) or supplemented with increasing concentrations (0.005, 0.025, 0.1, 1, 5, 10, and 25 nM) of TCDD for 4 h then viability was assessed by ATPLite assay to quantitate cellular ATP levels. Exposure to 5 nM, 10 nm, or 25 nm TCDD resulted in a statistically significant decrease in cellular ATP (P≤0.05, one-way ANOVA with Dunnet’s post hoc multiple corrections test).

Figure 3.

Evaluation of human placental macrophage phagocytosis and internalization of GBS. A.) To evaluate placental macrophage phagocytosis of fluorescently-labeled GBS cells were cultured in medium alone (Control), 0.025 nM TCDD (TCDD), or 5 μM of the known phagocytosis inhibitor cytochalasin D (Cyto D) as a positive control. After quenching extracellular signal with trypan blue stain, intracellular bacterial fluorescence was measured. TCDD and cytochalasin D inhibited placental macrophage phagocytosis of GBS compared to untreated controls; a result that was statistically significant (*P≤0.05, one-way ANOVA with Tukey’s post hoc multiple comparisons test). B.) To evaluate intracellular bacterial numbers, gentamicin protection and quantitative culture assays were performed (B). Our results indicated that TCDD exposure resulted in a significant reduction in intracellular GBS within placental macrophages (*P≤0.05, Mann-Whitney U analysis. N=3 biological replicates from separate placental samples from different donors, error bars indicate standard error mean).

Figure 4.

Analysis of cytokine and chemokine production by human placental macrophages. Placental macrophages were cultured in medium alone or medium supplemented with TCDD (+TCDD) prior to infection with GBS (+GBS) and uninfected negative controls were also included (UI). Supernatants were collected and analyzed by multiplex assay to quantify the cytokine and chemokines produced. A) Heat map analysis of cytokine production by human placental macrophages. Blue indicates low expression (0 pg/mL), white is medium expression (4,000 pg/mL) and yellow is high expression (8,000+ pg/mL). Values used to calculate heat map were derived from the mean of 3 biological replicates. B) Individual bar graphs of cytokines significantly altered by GBS infection and/or TCDD exposure (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA with Tukey’s post hoc multiple comparisons test), #P<0.05, Student’s t test. Bars indicate mean +/− standard error mean with individual data points from different biological replicates derived from separate placental samples overlaid upon the bars (N=3).

Figure 5.

Conceptual diagram of the effects of TCDD exposure on human placental macrophages. Exposure to high levels of TCDD (at or above 5 nm) results in placental macrophage cell death. Exposure to sublethal doses of TCDD (at 0.025 nM) results in decreased placental macrophage phagocytosis and internalization of GBS, and inhibition of cytokine secretion in response to infection. Image created with BioRender.