Preterm infants have deficient monocyte and lymphocyte cytokine responses to group B streptococcus

Abstract

Group B streptococcus (GBS) is an important cause of early- and late-onset sepsis in the newborn. Preterm infants have markedly increased susceptibility and worse outcomes, but their immunological responses to GBS are poorly defined. We compared mononuclear cell and whole-blood cytokine responses to heat-killed GBS (HKGBS) of preterm infants (gestational age [GA], 26 to 33 weeks), term infants, and healthy adults. We investigated the kinetics and cell source of induced cytokines and quantified HKGBS phagocytosis. HKGBS-induced tumor necrosis factor (TNF) and interleukin 6 (IL-6) secretion was significantly impaired in preterm infants compared to that in term infants and adults. These cytokines were predominantly monocytic in origin, and production was intrinsically linked to HKGBS phagocytosis. Very preterm infants (GA, <30 weeks) had fewer cytokine-producing monocytes, but nonopsonic phagocytosis ability was comparable to that for term infants and adults. Exogenous complement supplementation increased phagocytosis in all groups, as well as the proportion of preterm monocytes producing IL-6, but for very preterm infants, responses were still deficient. Similar defective preterm monocyte responses were observed in fresh whole cord blood stimulated with live GBS. Lymphocyte-associated cytokines were significantly deficient for both preterm and term infants compared to levels for adults. These findings indicate that a subset of preterm monocytes do not respond to GBS, a defect compounded by generalized weaker lymphocyte responses in newborns. Together these deficient responses may increase the susceptibility of preterm infants to GBS infection.

FIG. 1.

Neonatal and adult MNC lymphocyte cytokine responses to PHA or GBS. CBMC or PBMC were incubated for 48 h with optimal doses of heat-killed GBS (1 × 10⁸ CFU/ml) or PHA. Data shown are box-whisker plots with outliers (Tukey); n = 8 to 10 for preterm (26- to 33-week GA), 43 for term (<37-week GA), and 37 for adult. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

FIG. 2.

Neonatal and adult MNC innate/inflammatory cytokine responses to PHA or GBS. CBMC or PBMC were incubated for 48 h with optimal doses of heat-killed GBS (1 × 10⁸ CFU/ml) or PHA. Data shown are box-whisker plots with outliers (Tukey); n = 8 to 10 for preterm (26- to 33-week GA), 43 for term (<37-week GA), and 37 for adult. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

FIG. 3.

Cell-specific intracellular cytokine production by adult MNC in response to GBS or PHA. PBMC were stimulated for 0, 6, 24, and 48 h with either PHA or HKGBS (1 × 10⁸ CFU/ml), brefeldin A was added for the last 4 h of stimulation at each time point, and cells were stained for cytokine and cell lineage markers. Data shown are means ± SEM (n = 4 donors).

FIG. 4.

Nonopsonic and opsonic phagocytosis of GBS by neonatal and adult monocytes. CBMC or PBMC were incubated for 1 h with pHrodo dye-labeled HKGBS (1 × 10⁸ CFU/ml) with our without RbC. (A) Left panel, representative forward-scatter (FSC) versus side-scatter (SSC) FACS plot showing inclusion gate for monocytes; right panel, representative histograms showing pHrodo-specific fluorescence in monocytes without bacteria or with GBS plus or minus RbC. (B) Percentage of monocytes positive for phagocytosed GBS (means ± SEM). n = 11 for the <30-week group, 15 for the 31- to 33-week and term groups, and 18 for adults. *, P < 0.0151; **, P < 0.0084; Wilcoxon signed rank test comparing phagocytosis of GBS with and without RbC.

FIG. 5.

Monocyte-specific TNF-α and IL-6 production with respect to GBS uptake. CBMC and PBMC were exposed to 1 × 10⁸ CFU/ml pHrodo-HKGBS (plus or minus RbC) for 1 h and then washed and incubated for a further 4 h in the presence of brefeldin A before staining for intracellular cytokine. (A) Left panel, representative forward-scatter versus side-scatter FACS plot showing inclusion gate for monocytes; right panel, representative FACS plot showing monocyte-specific TNF-α production in association with ingestion of pHrodo-GBS. (B and C) Percentage of HKGBS-positive monocytes producing TNF-α or IL-6 (means ± SEM). n = 10 for the <-30-week group, 13 for the 31- to 33-week and term groups, and 14 for adults. P values (Mann-Whitney test) are as displayed.

FIG. 6.

Fresh cord-blood responses to live GBS. (A and B) Fresh cord/venous blood IL-6 and TNF responses to live GBS determined by Bioplex assay of culture supernatants or by intracellular cytokine staining (of monocytes), respectively. Data shown are means ± SEM; n = 6 for preterm infants (<30-week GA), 8 for term infants, and 9 for adults. *, †, ‡, P < 0.05; **, ††, ‡‡, P < 0.01; ***,†††, ‡‡‡, P < 0.001, comparing responses between preterm and term infants (*), preterm infants and adults (†), or term infants and adults (‡) using two-way ANOVA with Bonferroni's adjustment for multiple comparisons. (C) Phagocytosis of HKGBS by neutrophils or monocytes in freshly isolated cord blood from preterm infants (<30-week GA; n = 4) and term infants (n = 5) or venous blood from adults (n = 7), as determined by flow cytometry using pHrodo-labeled HKGBS (5 × 10⁷ CFU/ml). Data shown are means ± SEM. †, P < 0.05, comparing uptake by preterm cells to that by adults using two-way ANOVA with Bonferroni's adjustment for multiple comparisons.