Objective
We hypothesized that fetal lipopolysaccharide exposures to the chorioamnion, lung, or gut would induce distinct systemic inflammatory responses.
Study Design
Groups of 5-7 time-mated ewes were used to surgically isolate the fetal respiratory and the gastrointestinal systems from the amniotic compartment. Outcomes were assessed at 124 days gestational age, 2 days and 7 days after lipopolysaccharide (10 mg, Escherichia coli 055:B5) or saline solution infusions into the fetal airways or amniotic fluid.
Results
Lipopolysaccharide induced systemic inflammatory changes in all groups in the blood, lung, liver, and thymic lymphocytes. Changes in lymphocytes in the posterior mediastinal lymph node draining lung and gut, occurred only after direct contact of lipopolysaccharide with the fetal lung or gut.
Conclusion
Fetal systemic inflammatory responses occurred after chorioamnion, lung, or gut exposures to lipopolysaccharide. The organ responses differed based on route of the fetal exposure.
Preterm birth at less than 32 weeks’ gestation is frequently associated with a chronic, often clinically silent chorioamnionitis. The inflammatory exposures result in fetal risks ranging from no apparent effect to sepsis and fetal death. Some preterm newborn infants have a fetal systemic inflammatory response syndrome (FIRS) diagnosed histologically by fetal inflammatory cells in the cord or by elevated interleukin (IL)-6 levels in cord blood. The nature of the fetal response to infection/inflammation remains poorly described and is likely quite variable depending on organism, duration of exposure, and host responses. We have used chorioamnionitis induced with lipopolysaccharide (LPS) in fetal sheep to evaluate the responses of the fetal lung to inflammation. This chorioamnionitis also causes a modest systemic inflammatory response. We demonstrated that direct lung contact with the proinflammatory mediators LPS or IL-1 were required for the lung inflammation, injury, and maturation responses and that chorioamnionitis induced by subchoronic LPS infusion did not induce lung maturation. Because so little is known about how a fetus detects and responds to chorioamnionitis, we have surgically manipulated the fetal sheep to allow us to selectively expose the fetus to LPS via the lung, the chorioamnion, or the chorioamnion and gut. We then asked if the targeted fetal exposures would induce lung and systemic inflammatory responses and alter cell populations that modulate immune responses in the blood, thymus and posterior mediastinal lymph node (PMLN) that drain the lung and gut.
Materials and Methods
Animals
Time-mated ewes with singletons were randomized to surgically isolate the respiratory and the gastrointestinal systems from the amniotic sac ( Table 1 ). Ewes were sedated with intramuscular ketamine (10 mg/kg body weight) and xylazine (0.5 mg/kg; Troy Laboratories Pty Ltd., Smithfield, Australia) and anesthetized with halothane in oxygen. The fetal head was delivered through a uterine incision, and the fetal trachea of each fetus was attached to a 2-L plastic bag (Baxter Healthcare Corp, Deerfield, IL) with a short length of silicone rubber tubing (Silastic; Dow Corning, Midland, MI). A fine catheter was passed through the tracheostomy site, positioned in the distal trachea and connected to an Alzet osmotic pump (Alzet, Inc, Chicago, IL) that delivers 2 mL over 24 hours. The pump contained 1 mg LPS in saline solution ( Escherichia coli LPS, serotype 055:B5; Sigma-Aldrich Chemical Co, St. Louis, MO) or saline solution. A second Alzet pump containing either saline solution or 10-mg LPS was tied to an extremity for the intraamniotic exposures. The LPS dose was chosen based on a study demonstrating that an intratracheal dose of 1-mg LPS causes lung maturation, and a 10-mg intraamniotic dose of LPS causes chorioamnionitis. The inflammatory responses in the lungs were similar after the 2 LPS exposure routes. In some animals, the esophagus was ligated with sutures. We randomly assigned fetuses to exposure to LPS infusion into the amniotic fluid, with or without esophageal ligation and to LPS or saline solution infusion into the trachea. The controls received intraamniotic and intratracheal saline solution. The tracheal conduit to the bag to collect fetal lung fluid separated the lungs from the amniotic fluid in all fetuses. Fetal sheep swallow about half the amniotic fluid daily, and esophageal ligation prevented distal gut exposure to LPS. The surgeries were performed 2 or 7 days before preterm delivery at 124 days’ gestation (term is 150 days). The ewes tolerated the surgery well and were healthy until delivery of the fetuses at 124 days’ gestation. Each pregnant ewe was heavily sedated and given spinal anesthetic for delivery of the fetus. Amniotic fluid samples were collected and the fetus was killed with intravascular pentobarbital. A cord blood sample was obtained for a white blood cell count.
| Group/exposure | Fetal exposures | n | Birthweight, kg | LPS in amniotic fluid (EU/mL × 10 3 ) |
|---|---|---|---|---|
| Surgical controls | IA + IT saline solution | 5 | 2.4 ± 0.1 | 0.004 ± 0.001 |
| Lung – 2 d | IA saline solution+ IT LPS | 5 | 2.4 ± 0.1 | 0.06 ± 0.02 |
| Lung – 7 d | IA saline solution + IT LPS | 6 | 2.3 ± 0.1 | 0.03 ± 0.02 |
| Amniotic fluid + gut – 2 d | IA LPS + IT saline solution | 5 | 2.4 ± 0.1 | 4.8 ± 1.2 |
| Amniotic fluid + gut – 7 d | IA LPS + IT saline solution | 3 | 2.3 ± 0.1 | 0.33 ± 0.13 |
| Amniotic fluid – 2 d | IA LPS + IT saline solution + Eso Lig | 5 | 2.5 ± 0.1 | 16.4 ± 3.9 |
| Amniotic fluid – 7 d | IA LPS + IT saline solution + Eso Lig | 5 | 2.4 ± 0.1 | 0.25 ± 0.06 |
Processing of lung
The thorax was opened and the lungs were inflated to 40 cm H 2 O airway pressure to measure a maximal lung volume. An alveolar wash of the left lung with 0.9% NaCl was repeated 3 times. Bronchoalveolar lavage fluid was centrifuged at 500 g for 10 minutes and the cell pellet resuspended in phosphate buffered saline (PBS) solution. Total cells were stained with tryptan blue and counted. Differential cell counts were performed on cytospin preparations after staining with Diff-Quick (Scientific Products, McGaw Park, IN).
Lymphocyte assays
The thymus and PMLN were isolated and weighed, and a cell suspension was made for FACS analysis. Cells were kept on ice and in the dark. After counting of the cells, separate aliquots were incubated with sheep specific monoclonal antibodies against CD4, CD8, CD25, or for gamma delta T lymphocytes. All antibodies, isotype controls, and corresponding secondary antibodies were obtained from VMRD, Inc, Pullman, WA.
Cytokine messenger RNA
Proinflammatory cytokine messenger RNAs (mRNAs) for interleukin IL-1, IL-6, and IL-8 were measured by using RNA extracted from homogenized lung tissue. With ovine-specific riboprobes, the amount of mRNAs were quantified and mRNA for ribosomal protein L32 was the internal control. Results from the control group were standardized to 1 and are expressed as fold increases.
Serum amyloid A as an acute phase reactant
Serum amyloid A (SAA) is an acute phase reactant that is produced in both hepatic and extra hepatic tissues and is a sensitive marker of inflammation. SAA mRNA was quantified by RNAse protection assay with an ovine-specific riboprobe. The SAA riboprobe protected a SAA fragment, which encompassed portions of the 5′ untranslated region and the coding sequence of SAA mRNA, including a conserved region present among all SAA isoforms (1-143 represents amino acids of the translated SAA protein).
LPS quantification
LPS bioactivity was quantified with Limulus amebocyte lysate assays (Biowhitaker, Walkersville, MD) as previously described. In brief, each amniotic fluid sample was diluted with PBS until the sample yielded a value on the linear part of the curve.
LPS tolerance
Monocytes from the lung were isolated as described previously. In brief, after vascular perfusion of the right lower lobe with Hank’s Balanced Salt Solution (HBSS) to remove blood, the lobe was minced thoroughly into fine pieces. After passage through a 100-μm mesh filter, the resulting cell suspension was centrifuged twice to recover the cells. The cells were then layered over a 2-step Percoll (Sigma-Aldrich) gradient (1.085 g/mL and 1.046 g/mL) and centrifuged at 400 g for 40 minutes at 4°C. Monocytic cells were recovered from the interface between the 2 Percoll densities and the cell concentration adjusted to 2.5 × 10 6 cells per milliliter. After incubation at 37°C for 2 hours, nonadherent cells were removed by washing. Monocytes were cultured overnight in the presence of LPS (100 ng/mL). Tumor necrosis factor-alpha (TNF-α) was measured in the supernatant of the cell media with a commercial enzyme-linked immunosorbent assay (Endogen, Rockford, IL).
Data analysis
Results are given as means ± standard error of mean. Comparisons between the groups were performed by analysis of variance (ANOVA) with Student-Newman test as post hoc analysis. Comparisons between groups were with 2-tailed t tests. Significance was accepted at P < .05.
Results
Animals and LPS in amniotic fluid
The treatment groups and birthweights of the animals are given in Table 1 . All animals survived the surgery and were apparently healthy at delivery with no metabolic acidosis (data not shown). LPS in amniotic fluid was detected in high levels 2 days after the intraamniotic LPS and was low after intratracheal LPS and at 7 days.
Lung effects
Lung maturation increased with the intratracheal exposure to LPS as indicated by large increases in lung gas volumes and in the amount of saturated phosphatidylcholine recovered in the bronchoalveolar lavages (BAL) ( Table 2 ). There was no indication of lung maturation in the other groups. Similarly, inflammatory cells expressed as the sum of neutrophils and monocytes/macrophages increased in the BAL at 2 and 7 days only in the group exposed to intratracheal LPS. Proinflammatory cytokine mRNA for IL-1β, IL-6, and IL-8 was increased at 2 days only with intratracheal LPS. These results demonstrate that lung inflammation/maturation requires contact of the proinflammatory agonist with the fetal lung. Chorioamnionitis alone does not cause fetal lung inflammation or maturation.
| Measurements | Surgical controls (IA + IT saline solution) | Lung (IA saline solution + IT LPS) | Amniotic fluid + gut (IA LPS + IT saline solution) | Amniotic fluid (IA LPS + IT saline solution + Eso Lig) |
|---|---|---|---|---|
| Lung gas volume at 7 d (mL/kg at 40 cm H 2 O pressure) | 5.7 ± 0.6 | 28.8 ± 3.8 a | 7.9 ± 1.5 | 8.7 ± 1.3 |
| Saturated phosphatidylcholine at 7 d (μmol/kg) | 0.06 ± 0.02 | 2.4 ± 0.8 a | 0.10 ± 0.05 | 0.31 ± 0.10 |
| Inflammatory cells in BAL – 2 d (×10 6 /kg) | 6.6 ± 4.5 | 129 ± 41 a | 4.0 ± 0.6 | 5.6 ± 2.7 |
| Inflammatory cells in BAL – 7 d (×10 6 /kg) | 6.6 ± 4.5 | 141 ± 27 a | 6.1 ± 0.1 | 19.6 ± 5.5 |
| Cytokine mRNA in lung at 2 d (fold increase over control) | ||||
| IL-1β | 1.0 ± 0.1 | 5.6 ± 2.0 a | 1.3 ± 0.1 | 1.0 ± 0.1 |
| IL-6 | 1.0 ± 0.1 | 2.3 ± 0.5 a | 1.1 ± 0.1 | 0.9 ± 0.1 |
| IL-8 | 1.0 ± 0.1 | 11.3 ± 3.5 a | 1.3 ± 0.2 | 1.2 ± 0.2 |
Systemic inflammation
Neutrophil and monocyte numbers in cord blood 2 days after exposure were similar to the control group ( Figure 1 ). However, neutrophils increased with LPS exposure in all 3 LPS groups 7 days after exposure. Therefore, fetal neutrophils can be induced by LPS exposure to the lung or amniotic fluid. Only an amniotic plus gut exposure increased cord blood monocytes at 7 days, suggesting that gut exposure contributes to the increase in fetal blood monocytes.
The mRNA for acute phase reactant gene SAA increased in the fetal liver at 2 days after all LPS exposures but not in the control group that underwent the same surgical procedures ( Figure 2 ). SAA mRNA levels had returned to control levels by 7 days (data not shown). This finding indicates a similar liver inflammatory response after contact of fetal gut, lung, or membranes with LPS. Therefore, systemic acute phase and inflammatory responses can be induced by the exposure of the fetal lung or chorioamnion by LPS.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
