Pro-inflammatory cytokine-stimulated first trimester decidual cells enhance macrophage-induced apoptosis of extravillous trophoblasts

Abstract

Objective: As human blastocyst-derived extravillous trophoblasts (EVTs) invade the early decidua, they are positioned to interact with immune cells and resident decidual cells, and remodel spiral arteries into high capacity vessels that increase blood flow to the developing fetal-placental unit. Shallow EVT invasion elicits incomplete vascular transformation and reduces uteroplacental blood flow that presages adverse pregnancy outcomes. Excess macrophages in the decidua induce EVT apoptosis via tumor necrosis factor-alpha (TNF-α) secretion. Our previous observation that pro-inflammatory cytokines enhance neutrophil and macrophage activator granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in first trimester decidual cells is now extended to include: (1) the specific macrophage activator M-CSF; (2) macrophage activation and subsequent enhancement of EVT apoptosis by both GM-CSF and M-CSF.

Study design: Quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay assessed M-CSF expression in first trimester decidual cells incubated with interleukin-1 beta (IL-1β) or TNF-α. Peripheral monocyte-derived macrophages pre-incubated with conditioned media from decidual cell cultures were co-cultured with a first trimester EVT cell line, HTR-8/SVneo cells. Macrophage activation was examined and EVT apoptosis evaluated by DNA fragmentation, caspase activation and cell membrane asymmetry.

Results: IL-1β or TNF-α significantly enhanced M-CSF expression in first trimester decidual cells. The conditioned media from these cultures activates macrophages, which promote caspase 3/7-dependent EVT apoptosis with antibodies against GM-CSF or M-CSF blocking this effect.

Conclusions: Pro-inflammatory cytokines increases synthesis of M-CSF in first trimester decidual cells. Both GM-CSF and M-CSF activate macrophages, which initiate caspase-dependent EVT apoptosis.

Fig. 1

IL-1β and TNF-α increase M-CSF mRNA and protein expression in first trimester decidual cells. Leukocyte-free first trimester decidual cells were primed with estradiol (E) + medroxyprogesterone acetate (M), then incubated with steroids alone (EM) or with steroids plus IL-1β (EMI) or TNF-α (EMT) for 6 h (for qRT-PCR) or 24 h (for ELISA). The results are normalized to β-actin and total cell protein for (A) qRT-PCR and (B) ELISA for M-CSF mRNA and protein, respectively. The results are reported as the mean ± SEM. (n = 3–6; *P < 0.05, **P < 0.01, ***P < 0.005).

Fig. 2

First trimester decidual cell-derived GM-CSF and M-CSF mediate the activation of macrophages. Flow cytometric analysis of the expression of activation markers, (A) HLA-DR, (B) CD16, (C) CD18, (D) CD86, on Mϕs cultured with CM from 1st trimester decidual cells treated with or without IL-1β (EMI) or TNF-α (EMT) in the presence or absence of anti-human GM-CSF (α-GM) or anti-human M-CSF (α-M) neutralizing antibody for 48 h. The results are reported as the mean ± SEM. (n = 7–8; *P < 0.05, **P < 0.01, ***P < 0.005).

Fig. 3

DNA fragmentation of HTR-8 cells. PI staining for DNA content in PKH26 (Red)-labeled HTR cells co-cultured with Mϕs in CM from 1st trimester decidual cells treated with or without IL-1β (EMI) or TNF-α (EMT) for 24 h. Arrows indicate the subdiploid peaks of DNA histogram. (A) Representative DNA histograms; (B) Percentage of cells in subdiploid peak; (C) TUNEL assay. The results are reported as the mean ± SEM. (n = 4; *P < 0.05, ***P < 0.005).

Fig. 4

Caspase 3/7 expression in HTR-8 cells. (A) Quantitative caspase Glo-3/7 colorimetric assay; Western blotting analysis of (B) caspase 3 and (C) caspase 7 expression in HTR-8 cells (H) co-cultured with Mϕs in CM from 1st trimester decidual cells treated with or without IL-1β (EMI) or TNF-α (EMT) for 24 h. Caspase signaling intensity was normalized to the abundance of the housekeeping protein, hsp90, in Western blotting. The results are reported as mean ± SEM. (n = 3; *P < 0.05, **P < 0.01).

Fig. 5

Apoptosis of HTR-8 cells. (A) Apoptosis in HTR-8 cells (H) co-cultured with Mϕs under the influence of CM from 1st trimester decidual cells treated with or without IL-1β (EMI) or TNF-α (EMT) in the presence or absence of anti-human GM-CSF (α-GM) or anti-human M-CSF (α-M) neutralizing antibody for 24 h was examined by 7-AAD/annexin V co-staining. (B) & (C) The total percentage of HTR-8 cells undergoing apoptosis was calculated as the sum of cells in early apoptosis with 7-AAD⁻/annexin V⁺ (right lower quadrant) and late apoptosis are 7-AAD⁺/annexin V⁺ (right upper quadrant). The results are reported as mean ± SEM. (n = 4; *P < 0.05, **P < 0.01).