Effect of chorioamnionitis on regulatory T cells in moderate/late preterm neonates

Abstract

Regulatory T-cells (Treg) have a protective role for the control of immune activation and tissue damage. The effects of chorioamnionitis (chorio) on Treg in moderate/late preterm newborns are not known. We hypothesized that infants exposed to chorio would have decreased Treg frequency and/or function. We isolated mononuclear cells from adult peripheral blood and cord blood from term and moderate/late preterm infants who were classified for severity of chorio exposure. Mononuclear cells were analyzed by flow cytometry for Treg frequency and phenotype. Treg suppression of activation of conventional T-cells (Tcon) was also quantified. Treg frequencies were similar in all groups of neonates, but lower than that found in adults. Newborn Treg had a naïve phenotype, with decreased levels of CD45RO, HLA-DR, CD39 and TIGIT compared to adult Treg and chorio did not affect the phenotype. Treg from preterm newborns exposed to severe chorio had higher expression of Ki67 compared to the other groups. Treg from preterm newborns were less suppressive than Treg from adults or term, and the level of suppression was reduced with severe chorio. Relative to term, Treg frequency and phenotype were not affected by prematurity and chorio but their functionality was decreased. Lower Treg activity may contribute to inflammation in newborns that is often associated with chorioamnionitis.

Keywords: Fetal inflammation; Full term newborn; Prematurity; Regulatory T cell; Suppression.

Fig. 1

Plasma levels of pro-inflammatory cytokines in newborns. Concentrations of (A) IL-8, (B) IL-6 and (C) MCP-1 were measured in samples from full term neonates (n = 6), preterm neonates without chorio (n = 10), preterm exposed to mild chorio (n= 6) and preterm exposed to severe chorio (n = 10). Bars show the median of the group. Groups were compared using Mann–Whitney U tests.

Fig. 2

Frequency of Treg in peripheral blood from adults and cord blood from newborns. (A) Flow cytometry data from one representative experiment show the Treg characterization in a newborn sample. Three different ways of defining Treg were analyzed in the CD3⁺CD4⁺ gate: CD4⁺FOXP3⁺CD127^Low/− (upper panel), CD25⁺CD127^Low/− (middle panel) and CD4⁺FOXP3⁺ (lower panel). (B) Treg frequency (CD4⁺FOXP3⁺) from adult PBMC and CBMC. Bars show the median of the group. Groups were compared using Mann–Whitney U tests.

Fig. 3

Frequency of Treg and Tcon subsets from adults and newborns in cell cycle. (A) Percentage of Treg expressing Ki67 within the Treg population (CD4⁺FOXP3⁺). (B) Flow cytometry data from one representative experiment show the expression of Ki67 on CD4⁺FOXP3⁺ T cells in term and chorio severe newborns. (C) Positive correlation between plasma IL-6 levels and percentage of Ki67⁺ Treg in newborns. (D) Percentage of Tcon expressing Ki67 within the Tcon population (CD4⁺FOXP3⁻). (E) No correlation between IL-6 levels and percentage of CD4⁺FOXP3⁻Ki67⁺ in newborns. Spearman correlation test.

Fig. 4

Phenotype of Treg in peripheral blood from adults and severe chorio newborns. Flow cytometry data from one representative experiment show the expression of CD45RO, HLA-DR, CD39, TIGIT, PD-1 and CTLA-4 by Treg (CD4⁺FOXP3⁺).

Fig. 5

Treg suppression of Tcon proliferation and cytokine production. (A) Allogeneic Tcon proliferation (CFSE^Low) and TNF-α production were analyzed in Tcon alone (upper panels) and cocultured with Treg (lower panels) (data from one representative term donor). Values represent the percentage of activated cells (% CFSE^Low and % TNF⁺). (B) Representative figure represent the activity Treg suppression of one donor from term, preterm and severe chorio. Values represent the percentage of activated cells (% CFSE^Low). (C) Treg suppression of Tcon proliferation, mean % ± SEM suppression are shown. (D) Treg suppression of TNF-α production. % suppression was calculated in relation to Tcon alone (100%−[response of Tcon in coculture/response of Tcon alone]). Full term (n = 4), preterm without chorio (n = 8), preterm exposed to mild chorio (n = 3) and preterm exposed to severe chorio (n = 3).