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. 2018 May;79(5):e12816.
doi: 10.1111/aji.12816. Epub 2018 Jan 25.

Lipopolysaccharide-induced maternal inflammation induces direct placental injury without alteration in placental blood flow and induces a secondary fetal intestinal injury that persists into adulthood

Erin M Fricke  1 , Timothy G Elgin  1 , Huiyu Gong  1 , Jeff Reese  2 , Katherine N Gibson-Corley  1 , Robert M Weiss  1 , Kathy Zimmerman  1 , Noelle C Bowdler  1 , Karen M Kalantera  3 , David A Mills  3 , Mark A Underwood  3 , Steven J McElroy  1
Affiliations

Lipopolysaccharide-induced maternal inflammation induces direct placental injury without alteration in placental blood flow and induces a secondary fetal intestinal injury that persists into adulthood

Erin M Fricke et al. Am J Reprod Immunol. 2018 May.

Abstract

Problem: Premature birth complicates 10%-12% of deliveries. Infection and inflammation are the most common etiologies and are associated with increased offspring morbidity and mortality. We hypothesize that lipopolysaccharide (LPS)-induced maternal inflammation causes direct placenta injury and subsequent injury to the fetal intestine.

Method of study: Pregnant C57Bl6 mice were injected intraperitoneally on day 15.5 with 100 μg/kg LPS or saline. Maternal serum, amniotic fluid, placental samples, and ileal samples of offspring were obtained assessed for inflammation and/or injury. Maternal placental ultrasounds were performed. Placental DNA was isolated for microbiome analysis.

Results: Maternal injection with LPS caused elevated IL-1β, IL-10, IL-6, KC-GRO, and TNF. Placental tissue showed increased IL-1β, IL-6, and KC-GRO and decreased IL-10, but no changes were observed in amniotic fluid. Placental histology demonstrated LPS-induced increases in mineralization and necrosis, but no difference in placental blood flow. Most placentas had no detectable microbiome. Exposure to maternal LPS induced significant injury to the ilea of the offspring.

Conclusion: Lipopolysaccharide causes a maternal inflammatory response that is mirrored in the placenta. Placental histology demonstrates structural changes; however, placental blood flow is preserved. LPS also induces an indirect intestinal injury in the offspring that lasts beyond the neonatal period.

Keywords: cytokines; lipopolysaccharide; microbiome; mouse; placenta.

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Figures

FIGURE 1
FIGURE 1
Schematic representation of experimental time points
FIGURE 2
FIGURE 2
Increased doses of intraperitoneal lipopolysaccharide (LPS) result in higher rates of preterm birth without significant effect on maternal survival. A, Pregnant mice were given increasing doses of LPS to assess effect on maternal and neonatal survival. There was no effect on maternal survival below a dose of 2000 μg/kg (n = 60 pregnant mice). B, Rates of preterm birth were 0%, 15%, 50%, and 100% for doses of 50, 100, 200, and 500 μg/kg, respectively (n = 55 litters). C, The proportion of litters that survived to term and then 7 d of life were 100%, 50%, 25%, and 0% at doses of 50, 100, 200, and 500 μg/kg, respectively. D, To assess maternal response to injection, serum cytokines were obtained 4 h following injection. There were significant elevations in the levels of IL-1β, IL-10, IL-6, KC-GRO, and TNF (P < .0009 for all serum cytokines; Sham n = 5, LPS n = 10 pregnant mice, P < .0001)
FIGURE 3
FIGURE 3
Maternal injection with lipopolysaccharide (LPS) increases inflammatory cytokines in the placenta, but not in the amniotic fluid. A, IL-1B, IL-6, and KC-GRO were significantly elevated, and IL-10 was decreased in placental tissue (P = .0001; n = 8 pregnant dams per group, only 1 placenta was used from each animal). B, There were no significant differences in cytokine levels in the amniotic fluid compared to sham controls (P < .0001; n = 8 pregnant dams per group with roughly 3 amniotic fluid compartments sampled and pooled per mother). C, Placental tissue samples were stained for IL-6 and examined at 6× (total placenta) and 20× (intervillous space). LPS-exposed animals had increased staining compared to sham with a trend toward intracellular staining (n = 6)
FIGURE 4
FIGURE 4
Placental histopathology demonstrates increased levels of mineralization and necrosis in placental tissue exposed to maternal lipopolysaccharide (LPS). A, Mineralization scoring criteria. B, Analysis of placental mineralization demonstrates increased mineralization in tissue exposed to LPS (P = .001; n = 8 pregnant dams in the sham group and 10 pregnant dams in the LPS group, only 1 placenta was used from each animal). C, Necrosis scoring criteria. D, Analysis of placental necrosis demonstrates increased necrosis in tissue exposed to LPS (P = .0004; n = 8 pregnant dams in the sham group and 10 pregnant dams in the LPS group, only 1 placenta was used from each animal). E, Sham example: rare zones of coagulation necrosis (circle). Variable-sized zones of mineral accumulation in vascular spaces (arrows). Inset (40× magnification) highlights an area of mineralization characterized by accumulation of a basophilic granular material. F, LPS-exposed example: extensive, multifocal necrosis (circle) with severe, extensive mineralization (dashed circle). Inset (40× magnification) highlights multifocal, variably sized zones of placental necrosis
FIGURE 5
FIGURE 5
Intraperitoneal injections of lipopolysaccharide (LPS) do not cross the placenta. A, LPS was detected in 1 of 3 maternal serum and in (B) 2 of 3 maternal peritoneal fluid in the LPS-treated dams but not in the 2 sham-treated dams. C, LPS was not detected in either the amniotic fluid (n = 45) or (D) placenta (n = 45). All samples were obtained from 2 separate sham-treated and 3 separate LPS-treated pregnant dams
FIGURE 6
FIGURE 6
Maternal exposure to lipopolysaccharide (LPS) does not alter umbilical or uterine artery blood flow. At 6 (n = 5 Sham pregnant dams, 5 LPS pregnant dams) and 30 h (n = 6 Sham pregnant dams, 8 LPS pregnant dams) following injection, Doppler ultrasounds of the umbilical and uterine artery were performed. A, Maternal heart rate (beats/min) was not significantly different in the LPS-treated group compared to Sham controls. B, Placental thickness (mm) was not significantly different between LPS-treated mothers and Sham controls. C, Fetal heart rate was significantly lower in the LPS-treated group compared to the sham group at 6 h (P = .026), but if the single outlier was removed, this significance is lost. There were no significant differences at 30 h (P = .88). No changes were seen in (D) umbilical artery blood flow (P = .42 [6 h], P = .32 [30 h]), in (E) uterine artery blood flow (P = .42 [6 h], P > .99 [30 h]) following maternal LPS exposure, or in (F) calculated systolic:diastolic ratio. Sample pulse wave Doppler tracings are shown for umbilical (G) and uterine (H) arteries
FIGURE 7
FIGURE 7
Rare bacterial DNA was isolated from placental tissues, but lipopolysaccharide (LPS) did not significantly alter detected bacterial ratios. Placental DNA was isolated from both treatment groups under sterile conditions (n = 9 Sham placentas and 8 LPS placentas, 1 placenta sampled per pregnant dam). A, DNA was only isolated in 3 of 9 of sham samples and 1 of 8 LPS samples. B, The majority of bacteria in both groups were bacteroides and firmicutes with small proportions of tenericutes, actinobacteria, and proteobacteria. Statistical analysis could not be performed based on inadequate sample size
FIGURE 8
FIGURE 8
Exposure to lipopolysaccharide (LPS)-induced maternal inflammation generates intestinal injury in her offspring. Intestinal ileal samples showed significantly higher injury scores in mice who had been exposed to maternal LPS-induced inflammation compared with controls (n = 8 Sham and 8 LPS offspring from at least 3 separate pregnant dams at each time point). Significant differences in injury score were seen at PO (P = .0259), P7 (P = .0134), and P56 (P = .0031). Differences at P14 and P21 were not statistically different. Scores as shown as a percentage of the total (A) and as an average for each age (B)
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