Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery

Abstract

Objective: The purpose of this study was to determine in a mouse model whether uterine natural killer (uNK) cell cytotoxic activation induces infection/inflammation-associated preterm labor and delivery.

Study design: Wild type or interleukin (IL)-10(-/-) mice were injected intraperitoneally with lipopolysaccharide on gestational day 14. Mice were either killed for collection of uteroplacental tissue, spleen, and serum or allowed to deliver. Uteroplacental tissue was used for histology and characterization of uNK cells.

Results: Low-dose lipopolysaccharide treatment triggered preterm labor and delivery in IL-10(-/-), but not wild type mice, in a manner independent of progesterone levels. Preterm labor and delivery in IL-10(-/-) mice was associated with an increased number and placental infiltration of cytotoxic uNK cells and placental cell death. Depletion of NK cells or tumor necrosis factor (TNF)-alpha neutralization in these mice restored term delivery. Furthermore, TNF-alpha neutralization prevented uNK cell infiltration and placental cell apoptosis.

Conclusion: The uNK cell-TNF-alpha-IL-10 axis plays an important role in the genesis of infection/inflammation-induced preterm labor/delivery.

Figures

Figure 1. Cytotoxic activation of uNK cells by LPS

Impact of lipopolysaccharide (LPS) on uterine (Ut) natural killer (NK) cell number and cytotoxic activation. LPS (0.5 μg/mouse) or saline was injected intraperitoneally on gestational day (gd) 14 in wild type (n=6) or interleukin (IL)-10−/− (n=8) mice. A, Ut mononuclear cells (UMC) were isolated on gd 16 from LPS- and saline-treated wild type and IL-10−/− mice as described in “Materials and Methods” section. Proportions of Ut and splenic (Sp) NK cells were analyzed. Percentage of NK1.1+CD3− cells is gated as part of CD45+ mononuclear cells and indicated in upper left quadrant of each graph. B, Isolated UMC on gd 16 were analyzed for NK cell cytotoxicity using YAC-1 target cells as described in “Materials and Methods” section. Uterine horns from 3 mice each of wild type and IL-10−/− mice were processed for isolation of mononuclear cells for experiments in A and B. E:T, effector: target cell ratio. Murphy. Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery. Am J Obstet Gynecol 2009.

Figure 2. LPS-induced migration of uNK cells

Uterine natural killer (uNK) cell localization in wild type and interleukin (IL)-10−/− mice. Wild type and IL-10−/− mice were injected intraperitoneally with saline or lipopolysaccharide (LPS) (0.5 μg/mouse) on gestation day (gd) 14 and killed on gd 16. Histologic sections of uteroplacental tissue were analyzed with periodic acid-Schiff-hematoxylin staining to visualize NK cells. In both saline-injected B, wild type and A, IL-10−/− mice, uNK cells remain localized to mesometrial decidual regions (M1 and M2, arrowheads) and are not found within placenta (P). In contrast, LPS-injected C, IL-10−/− mice exhibited profound infiltration of uNK cells into P zone (P1 and P2, arrowheads), which was not seen in P and D, wild type mice. Scale bar, 150 μm; insets, scale bar 15 μm. All analyses were carried out with tissues from at least 3 different experiments. Murphy. Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery. Am J Obstet Gynecol 2009.

Figure 3. Depletion of uNK cells

Depletion of uterine natural killer (uNK) cells in lipopolysaccharide (LPS)-treated interleukin (IL)-10−/− mice. A, IL-10−/− mice were treated with either anti-asialo GM1 (AGM1) or anti-NK1.1 antibodies intraperitoneally on gestational day (Gd) 8, 11, and 14 or appropriate Isotype control antibodies. These mice then received 0.5 μg LPS on gd 14. Uterine mononuclear cells (UMC) were isolated and analyzed by flow cytometry. Both anti-AGM1 and anti-NK1.1 treatments successfully depleted uNK cells. B, Consistent with successful uNK cell depletion, NK cell cytotoxicity was greatly diminished in UMC preparations from anti-AGM1 and anti-NK1.1 treated mice. E:T, effector: target cell ratio. Murphy. Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery. Am J Obstet Gynecol 2009.

Figure 4. Impact of TNFα neutralization

Evidence for tumor necrosis factor (TNF)-α-mediated uterine natural killer (NK) cell cytotoxic activation in lipopolysaccharide (LPS)-treated interleukin (IL)-10−/− mice. A, IL-10−/− mice were treated with 0.5 μg LPS or saline vehicle on gestational day (gd) 14 and uterine mononuclear cells (UMC) were isolated at gd 16. These cells were then stained for CD3, NK1.1, and either TNFα or interferon (IFN)-γ. Shown are events gated on NK1.1+CD3− population. In response to LPS, TNFα, and IFN-γ production was examined by intracellular staining and fluorescence-activated cell sorting analysis. B, IL-10−/− mice were treated with 0.5 μg LPS on gd 14 and UMC were then isolated at gd 16 and subjected to cytotoxicity assay using Yac-1 cell targets. C, Yac-1 cells were cultured in the presence of indicated concentrations of TNFα and analyzed for cell death. E:T, effector: target cell ratio. Murphy. Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery. Am J Obstet Gynecol 2009.

Figure 5. TNFα production by uNK cells

Neutralization of tumor necrosis factor (TNF)-α blocks uterine natural killer (uNK) cell infiltration into placenta (P) and appearance of TUNEL-positive regions in interleukin (IL)-10−/− mice. A, IL-10−/− mice were treated with 0.5 μg lipopolysaccharide (LPS) or saline vehicle on gestational day (gd) 14. Animals that received LPS also were treated with either anti-TNFα antibody or irrelevant IgG on gd 13 and 15. Mice were then killed and utero-P tissue was analyzed for uNK cell migration by periodic acid-Schiff (PAS) staining and cell death via TUNEL histology (without haematoxylin staining). uNK cells were not observed in P of mice receiving saline vehicle alone. Extensive P uNK cell (arrowhead) infiltration was observed in animals treated with LPS and control IgG. This uNK cell infiltration was associated with extensive P cell apoptosis as demonstrated by TUNEL-positive region (arrow). B, IL-10−/− mice were treated with anti-TNFα antibody on gd 13 and 15 along with 0.5 μg LPS on gd 14. Mice were then killed and utero-P tissue was analyzed for uNk cell migration by PAS/haematoxylin histology. Anti-TNFα antibody treatment prevented LPS-induced uNK cell invasion into P. uNK cells remained localized to mesometrial decidua (M) in these mice. Scale bar, 150 μm; insets, scale bar 15 μm. All analyses were carried out with tissues from at least 3 different experiments. Murphy. Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery. Am J Obstet Gyencol 2009.

Figure 6. Serum progesterone levels in mice

Lipopolysaccharide (LPS)-induced preterm birth is not due to loss of P4 levels in treated mice. Circulating levels of P4 were estimated on gestational day 16 in wild type (WT) and interleukin (IL)-10−/− mice (n=3) treated with either saline of LPS with or without anti-asialo GM1 (AGM1) or anti-natural killer (NK)1.1. No treatment groups showed any statistically significant change in levels of P4 as compared with saline treated controls. Murphy. Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery. Am J Obstet Gynecol 2009.