Cellular immune responses in amniotic fluid of women with preterm clinical chorioamnionitis

Abstract

Objective: Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Some preterm births are associated with clinical chorioamnionitis; yet, this condition has been poorly investigated. Herein, we characterized the amniotic fluid cellular immune responses in women with preterm clinical chorioamnionitis.

Methods and subjects: Amniotic fluid samples were obtained from women with preterm clinical chorioamnionitis and a positive or negative microbiological culture (n = 17). The cellular composition of amniotic fluid was evaluated using fluorescence microscopy, scanning and transmission electron microscopy, and flow cytometry. Women without preterm clinical chorioamnionitis were also examined (n = 10).

Results: Amniotic fluid from women with preterm clinical chorioamnionitis and a positive culture had: (1) abundant neutrophils associated with viable and non-viable bacteria, (2) neutrophils performing phagocytosis, (3) neutrophils forming NETs, (4) increased numbers of neutrophils, monocytes/macrophages, and CD4+ T cells, and (5) high expression of IL-1β by neutrophils and monocytes/macrophages. Amniotic fluid from women with preterm clinical chorioamnionitis and proven infection tended to have fewer monocytes/macrophages and CD4+ T cells compared to those without chorioamnionitis.

Conclusion: We provide the first morphologic and phenotypic characterization of the cellular immune responses in the amniotic cavity of women with preterm clinical chorioamnionitis, a condition associated with adverse neonatal outcomes.

Keywords: Acute chorioamnionitis; Immune Cells; Immunology; Macrophages; Monocytes; Neutrophils; T Cells.

Fig. 1. Detection of live and dead bacteria in amniotic fluid.

Representative bacterial live/dead staining of amniotic fluid from patients with preterm clinical chorioamnionitis and (A) a negative amniotic fluid bacterial culture, with corresponding enlarged image (B), or (C) a positive amniotic fluid bacterial culture, with corresponding enlarged image (D). Green = SYTO 9 stain, red = propidium iodide stain. Magnification = 400X

Fig. 2. Electron microscopy of amniotic fluid neutrophils.

Representative scanning electron microscopy images of amniotic fluid neutrophils from (A) a woman with a negative amniotic fluid microbial culture and (B) a woman with a positive amniotic fluid microbial culture. Representative transmission electron microscopy images of amniotic fluid neutrophils from (C) a woman with a negative amniotic fluid microbial culture and (D) a woman with a positive amniotic fluid microbial culture. Magnifications: (A) Top row: 4000X (left), 2940X (right); bottom row: 10000X. (B) 7000X. (C) 12000X (left), 15000X (right). (D) 7000X

Fig. 3. Flow cytometric analysis of leukocyte populations and innate immune cells in amniotic fluid.

(A) Representative flow cytometry gating strategies showing leukocyte populations in amniotic fluid from women with preterm clinical chorioamnionitis. Immune cells were initially gated within the viability gate and CD45+ gate followed by lineage gating for neutrophils (CD45+CD15+CD14- cells), monocytes/macrophages (CD45+CD14+CD15- cells), T cells (CD45+CD3+CD15-CD14-CD19- cells) and B cells (CD45+CD19+CD15-CD14-CD3- cells). T cells were subsequently gated for CD4+ T cells (CD3+CD4+CD8- cells) and CD8+ T cells (CD3+CD8+CD4- cells). Numbers of (B) total leukocytes (CD45+ cells/mL), (C) neutrophils (CD15+ cells/mL), and (D) monocytes/macrophages (CD14+ cells/mL) in amniotic fluid from women with preterm clinical chorioamnionitis who had either a negative or positive amniotic fluid culture. N = 8–9 per group. Midlines = median, boxes = interquartile ranges, and whiskers = minimum/maximum ranges

Fig. 4. Flow cytometric analysis of adaptive immune cells in amniotic fluid.

Numbers of (A) total T cells (cells/mL), (B) CD4+ T cells (cells/mL), (C) CD8+ T cells (cells/mL), and (D) B cells (cells/mL) in amniotic fluid from women with preterm clinical chorioamnionitis who had either a negative or positive amniotic fluid culture. Lymphocyte populations were gated as shown in Figure 3A. N = 8–9 per group. Midlines = median, boxes = interquartile ranges, and whiskers = minimum/maximum ranges

Fig. 5. Flow cytometric analysis of cytokine expression by innate immune cells in amniotic fluid.

(A) Representative gating strategy for determining the mean fluorescence intensity of IL-1β, IL-8, TNFα, IL-1α, MIP-1α, IL-6, and MIP-1β expressed by amniotic fluid neutrophils and monocytes/macrophages. Mean fluorescence intensity of IL-1β, IL-8, TNFα, IL-1α, MIP-1α, IL-6, and MIP-1β expressed by (B) neutrophils and (C) monocytes/macrophage in amniotic fluid from women with preterm clinical chorioamnionitis who had either a negative amniotic fluid culture (blue bar plots) or a positive amniotic fluid culture (red bar plots). (N=6–7).

Fig. 6. Flow cytometric analysis of amniotic fluid from women with or without preterm clinical chorioamnionitis.

Numbers of (A) total leukocytes (cells/mL), (B) neutrophils (cells/mL), (C) monocytes/macrophages (cells/mL), (D) B cells (cells/mL), (E) total T cells (cells/mL), (F) CD4+ T cells (cells/mL), and (G) CD8+ T cells (cells/mL) in amniotic fluid from women with and without preterm clinical chorioamnionitis who had a positive microbiological culture. Leukocyte populations were gated as shown in Figure 3A. N = 9–10 per group. Midlines = median, boxes = interquartile ranges, and whiskers = minimum/maximum ranges