Erythropoietin prevents LPS-induced preterm birth and increases offspring survival

Abstract

Problem: Preterm delivery is the leading cause of neonatal mortality and contributes to delayed physical and cognitive development in children. At present, there is no efficient therapy to prevent preterm labor. A large body of evidence suggests that infections might play a significant and potentially preventable cause of premature birth. This work assessed the effects of erythropoietin (EPO) in a murine model of inflammation-associated preterm delivery, which mimics central features of preterm infections in humans.

Method of study: BALB/c mice were injected i.p. with 20 000 IU/kg EPO or normal saline twice on gestational day (GD) 15, with a 3 hours time interval between injections. An hour after the first EPO or normal saline injection, all mice received two injections of 50 μg/kg LPS, also given 3 hours apart.

Results: EPO significantly prevented preterm labor and increased offspring survival in an LPS induced preterm delivery model. EPO prevented LPS-induced leukocyte infiltration into the placenta. Moreover, EPO inhibited the expression of pro-inflammatory cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α) in maternal serum and amniotic fluid. EPO also prevented LPS-induced increase in placental prostaglandin (PG)E2 and uterine inducible nitric oxide synthase (iNOS) production, while decreasing nuclear factor kappa-B (NF-κβ) activity in the myometrium. EPO also increased the gene expression of placental programmed cell death ligand 1 (PD-L1) in LPS-treated mice.

Conclusions: Our results suggest that EPO could be a potential novel therapeutic strategy to tackle infection-related preterm labor.

Keywords: PDL1; erythropoietin; inflammation; offspring survival; preterm birth; prostaglandins.

FIGURE 1

The percentage of mice without delivering after LPS injection. In LPS groups, the pregnant mice were i.p. injected with 0.2 mL normal saline twice on GD15, with a 3 hours time interval between injections. One hour after the first normal saline injection, mice then received two injections of 50 μg/kg LPS, also given 3 hours apart. In EPO/LPS groups, the pregnant mice were i.p. injected with 20 000 IU/kg rhEPO twice on GD15, with a 3 hours time interval between injections. One hour after the first rhEPO injection, mice then received two injections of 50 μg/kg LPS, also given 3 hours apart. All mice were observed for preterm delivery every hour after the first LPS or vehicle solution injection

FIGURE 2

Effect of EPO on LPS‐induced upregulation of PGE2 in placenta. Placentae were collected. PGE2 levels were assessed at 12 hours after the first LPS or vehicle solution injection on GD15.The level of PGE2 was determined by immunohistochemistry. Representative photomicrographs of placental histological specimens from mice treated with normol saline + normol saline (A as control), normal saline + EPO (B), normal saline + LPS (C), and EPO + LPS (D) are shown. PGE2 was observed in placental trophoblast cells (black arrowheads). (C) showed the upregulation of PGE2 in placenta and PGE2 expressed in placental trophoblast cells. The expression of PGE2 in placenta is almost invisible in (A) (B) and (D). Original magnification, ×100 or 400. Scale bars 100 µm for original magnification, ×100. Scale bars 25 µm for original magnification, ×400

FIGURE 3

Effect of EPO on LPS‐induced leukocyte infiltration in placenta. Placentae were collected. Total density of leukocytes in placenta was assessed at 12 hours after the first LPS or vehicle solution injection on GD15. Total density of leukocytes in placenta was determined by hematoxylin and eosin. Representative photomicrographs of placental histological specimens from mice treated with normol saline + normol saline (A as control), normal saline + EPO (B), normal saline + LPS (C), and EPO + LPS (D) are shown. (C) shows prominent leukocyte infiltration in placenta (black arrows). In (A), (B) and (D), the infiltration of leukocytes is barely visible. Original magnification, ×100 or 400. Scale bars 100 µm for original magnification, ×100. Scale bars 20 µm for original magnification, ×400

FIGURE 4

Effect of EPO on LPS‐induced release of IL‐1β, IL‐6, and TNF‐α in maternal serum. Maternal serum was collected. IL‐1β (A), IL‐6 (B), and TNF‐α (C) levels were assessed at 12 hours after the first LPS or vehicle solution injection on GD15. The levels of IL‐1β, IL‐6, and TNF‐α were determined by enzyme‐linked immunosorbent assays. Values are presented as mean ± SD. N = 12. ****P < .0001 vs the control. ***P < .001, ****P < .0001 vs LPS group

FIGURE 5

Effects of EPO on LPS‐induced release of IL‐1β, IL‐6, and TNF‐α in amniotic fluid. Maternal amniotic fluid was collected. IL‐1β (A), IL‐6 (B), and TNF‐α (C) levels were assessed at 12 hours after the first LPS or vehicle solution injection on GD15. The levels of IL‐1β, IL‐6, and TNF‐α were determined by enzyme‐linked immunosorbent assay. Values are presented as mean ± SD. N = 12. ****P < .0001 vs the control. ***P < .001, ****P < .0001 vs LPS group

FIGURE 6

Effect of EPO and LPS on uterine phosphorylated‐NF‐κB‐65 and iNOS protein levels. Uterine strips were collected, phosphorylated‐NF‐κB‐65 and iNOS protein levels were assessed at 12 hours after the first LPS or vehicle solution injection. The levels of phosphorylated‐NF‐κB‐65 and iNOS were determined by Western blots. The levels of phosphorylated‐NF‐κB‐65 (A) and iNOS (B) were determined. Values are presented as mean ± SD. N = 5. * P < .01 vs the control. *P < .05, **P < .01 vs LPS group

FIGURE 7

Effects of EPO on the expression of placental PD‐L1 after LPS injection. Placentae were collected. PD‐L1 protein levels were assessed at 12 hours after the first LPS or vehicle solution injection on GD15. The level of PGE2 was determined by immunohistochemistry. Representative photomicrographs of placental histological specimens from mice treated with normol saline + normol saline (A as control), normal saline + EPO alone (B), normal saline + LPS (C), and EPO + LPS (D) are shown. (D) showed the upregulation of PD‐L1 in placenta and PD‐L1 expressed in placental trophoblast cells, including giant trophoblasts (black arrowheads). The expression of PD‐L1 in placenta is almost invisible in (A), (B) and (D). Original magnification, ×100 or 400. Scale bars 100 µm for original magnification, ×100. Scale bars 25 µm for original magnification, ×400