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Pregnancy tailors endotoxin-induced monocyte and neutrophil responses in the maternal circulation - PubMed

Pregnancy tailors endotoxin-induced monocyte and neutrophil responses in the maternal circulation

Marcelo Farias-Jofre et al. Inflamm Res. 2022 Jun.

Abstract

Objective: To comprehensively characterize monocyte and neutrophil responses to E. coli and its product [lipopolysaccharide (LPS) or endotoxin] in vitro during pregnancy.

Material or subjects: Peripheral blood was collected from pregnant women during the third trimester (n = 20) and from non-pregnant women (n = 20).

Methods: The number, phagocytic activity, and reactive oxygen species (ROS) production of peripheral monocytes and neutrophils were investigated using flow cytometry. The phenotypes of peripheral monocytes and neutrophils after acute or chronic LPS stimulation were also determined using flow cytometry. Cytokine profiles were quantified for LPS-stimulated peripheral blood mononuclear cells (PBMCs) and a whole blood TruCulture® system using a multiplex immunoassay.

Results: Increased number, phagocytic activity, and ROS production capacity of monocytes and neutrophils were found in pregnant compared to non-pregnant women. Additionally, specific subsets of pro-inflammatory monocytes (IL-6+CD14+ or MIP-1α+CD14+ cells) and neutrophils (IL-1β+CD15+ or MIP-1β+CD15+ cells) were increased in pregnant women in response to acute LPS stimulation. Moreover, distinct subsets of intermediate-activated monocytes expressing CD142, IL-6, and IL-1RA were increased in pregnant women upon chronic LPS stimulation. Last, pregnant women displayed a different cytokine profile than non-pregnant women in LPS-stimulated PBMCs and in whole blood.

Conclusions: Pregnancy tailors the immune responses of circulating monocytes and neutrophils to endotoxin, a Gram-negative bacterial product.

Keywords: Chorioamnionitis; Cytokine; Funisitis; Infection; Inflammation; Phagocytosis.

© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Fig. 1
Fig. 1

Phagocytic activity and ROS production of monocytes and neutrophils. a Peripheral blood was obtained from pregnant (n = 20) and non-pregnant (n = 20) women to determine the phagocytic activity and ROS production of monocytes and neutrophils by flow cytometry. b Number of monocytes (CD14+ cells) per μL in the peripheral blood of pregnant (red triangles) and non-pregnant (blue triangles) women. c Number of neutrophils (CD15+ cells) per μL in the peripheral blood of pregnant (red triangles) and non-pregnant (blue triangles) women. d (Top) Flow cytometry gating strategy used to identify monocytes and neutrophils in peripheral blood samples. (Middle) Representative histograms depicting the increase in phagocytic monocytes and neutrophils (green histograms) upon incubation with fluorescently labeled E. coli. (Bottom) The frequencies of phagocytic monocytes and neutrophils in pregnant and non-pregnant peripheral blood samples. e (Top) Flow cytometry gating strategy used to identify monocytes and neutrophils in peripheral blood samples. (Middle) Representative histograms depicting basal ROS production by monocytes and neutrophils from pregnant (red histograms) and non-pregnant (blue histograms) women. (Bottom) Comparison of ROS production, represented as mean fluorescence intensity (MFI), by monocytes and neutrophils between pregnant and non-pregnant women. ROS: reactive oxygen species. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2
Fig. 2

Response of monocytes and neutrophils to acute LPS stimulation. a Peripheral blood samples were obtained from pregnant (n = 18) and non-pregnant (n = 17) women to isolate leukocytes for acute (4 h) in vitro stimulation with lipopolysaccharide (LPS). Following stimulation, surface marker and cytokine/chemokine expression by monocytes (CD14+ cells) and neutrophils (CD15+ cells) was determined using flow cytometry. b Heatmap representation of the relative expression of immune markers by monocytes from pregnant and non-pregnant women following 4 h LPS stimulation. c Heatmap representation of the relative expression of immune markers by neutrophils from pregnant and non-pregnant women following 4 h LPS stimulation. d Heatmap representation of the relative expression of cytokines and chemokines by monocytes from pregnant and non-pregnant women following 4 h LPS stimulation. e Heatmap representation of the relative expression of cytokines and chemokines by neutrophils from pregnant and non-pregnant women following 4 h LPS stimulation. f Frequencies of CD14+ cells expressing IL-6 in pregnant (red symbols) and non-pregnant (blue symbols) women following 4 h LPS stimulation (circles) or control (triangles). g Frequencies of CD14+ cells expressing MIP-1α in pregnant and non-pregnant women following 4 h LPS stimulation (circles) or control (triangles). h Frequencies of CD15+ cells expressing IL-1β in pregnant and non-pregnant women following 4 h LPS stimulation (circles) or control (triangles). i Frequencies of CD15+ cells expressing MIP-1β in pregnant and non-pregnant women following 4 h LPS stimulation (circles) or control (triangles). *p < 0.05; **p < 0.01; ***p < 0.001. ( +) Stimulated; (−) control

Fig. 3
Fig. 3

Response of total monocytes and monocyte subsets to chronic LPS stimulation. a Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of pregnant (n = 20) and non-pregnant (n = 20) women and stimulated with LPS for 24 h. Flow cytometry was performed to evaluate the expression of surface markers and cytokines/chemokines by total monocytes and monocyte subsets. b Heatmap representation of the relative expression of immune markers and cytokines/chemokines by total monocytes from pregnant and non-pregnant following 24 h LPS stimulation. c Heatmap representation of the relative expression of immune markers and cytokines/chemokines by classical monocytes (CD14hiCD16− cells) from pregnant and non-pregnant women following 24 h LPS stimulation. d Heatmap representation of the relative expression of immune markers and cytokines/chemokines by non-classical monocytes (CD14loCD16+ cells) from pregnant and non-pregnant women following 24 h LPS stimulation. e Heatmap representation of the relative expression of immune markers and cytokines/chemokines by intermediate monocytes (CD14hiCD16+ cells) from pregnant and non-pregnant women following 24 h LPS stimulation. f Heatmap representation of the relative expression of immune markers and cytokines/chemokines by CD14loCD16− monocytes from pregnant and non-pregnant women following 24 h LPS stimulation. Frequencies of intermediate monocytes expressing g CD142 (tissue factor), h IL-6, and i IL-1RA in pregnant (red symbols) and non-pregnant (blue symbols) women following 24 h LPS stimulation (circles) or control (triangles). *p < 0.05; ***p < 0.001. ( +) Stimulated; (−) control

Fig. 4
Fig. 4

Cytokine release by PBMCs in response to LPS. a Peripheral blood mononuclear cells (PBMCs) were isolated from pregnant (n = 20) and non-pregnant (n = 20) women and stimulated with LPS for 24 h. Cytokine concentrations were then determined in the cell culture supernatants. Concentrations (pg/mL) of b IL-1β, c IL-2, d IL-4, e IL-6, f IL-8, g IL-10, h IL-12p70, i IL-13, j TNF, and k IFNγ in PBMC culture supernatants from pregnant (red symbols) and non-pregnant (blue symbols) women following 24 h LPS stimulation (circles) or control (triangles). *p < 0.05; **p < 0.01; ***p < 0.001. ( +) Stimulated; (−) control

Fig. 5
Fig. 5

Cytokine release by whole-blood samples in response to LPS (TruCulture®). a Peripheral whole blood was obtained from pregnant (n = 20) and non-pregnant (n = 20) women and stimulated using a whole blood culture system (TruCulture®) containing LPS for 48 h. Cytokine concentrations were then determined in the culture supernatants. Concentrations (pg/mL) of b IL-1β, c IL-2, d IL-4, e IL-6, f IL-8, g IL-10, h IL-12p70, i IL-13, j TNF, and k IFNγ in whole blood TruCulture® supernatants from pregnant (red symbols) and non-pregnant (blue symbols) women following 48 h LPS stimulation (circles) or control (triangles). *p < 0.05; **p < 0.01; ***p < 0.001. ( +) Stimulated; (−) control

Fig. 6
Fig. 6

Correlations between cytokine profiles in whole blood and PBMC samples exposed to LPS. a Correlations of IL-1β, IL-8, and IFNγ concentrations between whole blood and PBMC samples of pregnant women exposed to LPS. b Correlations of IL-4, IL-6, and IL-10 concentrations between whole blood and PBMC samples of non-pregnant women exposed to LPS. The Spearman’s correlation coefficient (ρ) and p value are displayed for each graph. A p value < 0.05 was considered significant

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