Chorioamnionitis Precipitates Perinatal Alterations of Heme-Oxygenase-1 (HO-1) Homeostasis in the Developing Rat Brain

Abstract

Chorioamnionitis (CHORIO), placental insufficiency, and preterm birth are well-known antecedents of perinatal brain injury (PBI). Heme-oxygenase-1 (HO-1) is an important inducible enzyme in oxidative and inflammatory conditions. In the brain, HO-1 and the iron regulatory receptor, transferrin receptor-1 (TfR1), are known to be involved in iron homeostasis, oxidative stress, and cellular adaptive mechanisms. However, the role of HO pathway in the pathophysiology of PBI has not been previously studied. In this study, we set out to define the ontogeny of the HO pathway in the brain and determine if CHORIO changed its normal developmental regulation. We also aimed to determine the role of HO-1/TfR1 in CHORIO-induced neuroinflammation and peripheral inflammation in a clinically relevant rat model of PBI. We show that HO-1, HO-2, and TfR1 expression are developmentally regulated in the brain during the perinatal period. CHORIO elevates HO-1 and TfR1 mRNA expression in utero and in the early postnatal period and results in sustained increase in HO-1/TfR1 ratios in the brain. This is associated with neuroinflammatory and peripheral immune phenotype supported by a significant increase in brain mononuclear cells and peripheral blood double negative T cells suggesting a role of HO-1/TfR1 pathway dysregulation in CHORIO-induced neuroinflammation.

Keywords: HO-1; TfR1; neural-immune; neurodevelopment; neuroinflammation; perinatal brain injury; peripheral immune activation.

Figure 1

Placenta gene expressions for HO-1, HO-2, and TfR1 24 h after CHORIO, at E19, 24 h after CHORIO, at E19, no significant differences in placental gene expressions of HO-1 ((A) n = 4 for CHORIO, n = 3 for sham), HO-2 ((B) n = 4 for CHORIO, n = 3 for sham) or TfR1 ((C) n = 3 for CHORIO, n = 3 for sham) was detected compared to sham. p > 0.05, data normally distributed, Welch’s t-test, 2-tailed, mean ± SEM.

Figure 2

Ontogeny of HO-1, HO-2, and TfR1 in Sham Brains. Normal developmental regulation of HO-1 ((A) n = 3, 5, 10, 7, P2 vs. E18.5, * p = 0.0394), HO-2 ((B), n = 6, 8, 7, 7, P7 vs. E18.5, *** p = 0.0006, P7 vs. E19, *** p = 0.0009), and TfR1 ((C), n = 7, 8, 6, 7, P2 vs. E18.5, * p = 0.0169, P7 vs. E18.5, **** p < 0.0001, P7 vs. E19, * p = 0.0275) is displayed at E18.5, E19, P2, and P7 respective postnatal ages. p significant when <0.05, Kruskal–Wallis test, mean ± SEM.

Figure 3

Ontogeny of HO-1, HO-2, and TfR1 in CHORIO. Effect of CHORIO on the developmental regulation of HO-1 ((A), n = 8, 4, 3, 7, E18.5 vs. P7, ** p = 0.0028), HO-2 ((B), n = 6, 3, 5, 7, P7 vs. E18.5, * p = 0.0011) and TfR1 ((C), n = 7, 6, 6, 7, P7 vs. E18.5, ** p = 0.0022, P7 vs. P2, ** p = 0.005) is displayed at E18.5, E19, P2, and P7 respective postnatal ages. p significant when <0.05, Kruskal–Wallis test, mean ± SEM.

Figure 4

CHORIO alters HO-1, HO-2, and TfR1 expressions in brain compared to Sham. At E18.5, CHORIO upregulated HO-1, ** p = 0.0061 (A), HO-2, * p = 0.0152 (B), and TfR1, *** p = 0.0006 (C) in brains compared with sham. At E19 and P2, CHORIO upregulated HO-1, * p = 0.0317 and * 0.0490, respectively (A) in brains compared with sham. At P7, CHORIO downregulated constitutional HO-2 expression, * p = 0.0175 (B) significantly compared with sham. p significant when < 0.05, Kruskal–Wallis test, mean ± SEM.

Figure 5

CHORIO-induced increase in HO-1/HO-2 and HO-1/TfR1 ratios in developing brain. Acute alteration of HO-1/HO-2 ratios in CHORIO brains compared with sham 6 h after initial insult (A). Initial acute alteration of HO-1/TfR1 ratios in CHORIO brains is sustained until P2, 7-days post initial insult in CHORIO brains compared with sham (B). Arrows indicate increased HO-1/HO-2 at E18.5 and HO-1/TfR1 at E18.5, E19 and P2 when compared with Sham.

Figure 6

CHORIO-induced increase in brain mononuclear cells at P7. Single cells, live cells and CD45⁺ CD11b/c positive mononuclear cell population were identified by sequential gating. CD45 FMO and CD11b/c FMO controls were utilized to identify gating boundaries as shown in (A). No change in total CD45⁺ was detected between CHORIO and sham brains (p > 0.05, not normally distributed, Mann–Whitney) (B). Significant increase in the percentage of CD45 + CD11b/c⁺ mononuclear cells in CHORIO brains at P7 compared with sham (** p = 0.0098, normally distributed, Welch’s t-test, mean ± SEM), (C).

Figure 7

CHORIO induced increase in peripheral blood CD45⁺CD3⁺CD4⁻CD8⁻ T cells at P21. Representative gating strategy for PBMC analysis from a CHORIO pup at P21 (A). FMO controls were utilized for all fluorophores in the panel. CHORIO resulted in a significant increase in the percentage of CD45⁺CD3⁺CD4⁻CD8⁻ T cells from P21 pups compared with sham (* p = 0.0275, normally distributed, Welch’s t-test, mean ± SEM), (B).