Objective
Ureaplasma colonization in the setting of polymicrobial flora is common in women with chorioamnionitis, and is a risk factor for preterm delivery and neonatal morbidity. We hypothesized that Ureaplasma colonization of amniotic fluid would modulate chorioamnionitis induced by Escherichia coli lipopolysaccharide (LPS).
Study Design
Sheep received intraamniotic (IA) injections of media (control) or live Ureaplasma either 7 or 70 days before delivery. Another group received IA LPS 2 days before delivery. To test for interactions, U parvum –exposed animals were challenged with IA LPS, and delivered 2 days later. All animals were delivered preterm at 125 ± 1 day of gestation.
Results
Both IA Ureaplasma and LPS induced leukocyte infiltration of chorioamnion. LPS greatly increased the expression of proinflammatory cytokines and myeloperoxidase in leukocytes, while Ureaplasma alone caused modest responses. Interestingly, 7-day but not 70-day Ureaplasma exposure significantly down-regulated LPS-induced proinflammatory cytokines and myeloperoxidase expression in the chorioamnion.
Conclusion
Acute (7-day) U parvum exposure can suppress LPS-induced chorioamnionitis.
Premature birth is a major national health concern as it contributes disproportionately to neonatal morbidity and mortality, accounting for 12.3% of live births and >60% of neonatal deaths in the United States. Both clinical and experimental studies strongly demonstrate a causal link between intrauterine infection and preterm labor. The most common organisms isolated from the amniotic fluid of women with chorioamnionitis are the Ureaplasma species. Furthermore, among the very early preterm infants with chorioamnionitis caused by the Ureaplasma species, about 60% also had coinfection with other organisms. Exposure of preterm infants to Ureaplasma is associated with increased risk for adverse pulmonary, gastrointestinal, and neurological outcomes.
Despite the reports demonstrating adverse outcomes, Ureaplasma infection often is silent clinically. Ureaplasma species have been isolated in amniotic fluid samples from genetic amniocenteses done at 15-21 weeks’ gestation. Although the rates of preterm deliveries were higher in women with second-trimester Ureaplasma species in the amniotic fluid, the majority of the women delivered at term gestation. The reasons why some pregnancies with chronic Ureaplasma infection result in preterm delivery while others deliver at term are unknown. There is minimal information on modulation of innate immune system of the maternal or fetal host by the Ureaplasma species.
We reported that intraamniotic (IA) injection of U parvum in sheep induced a chronic colonization with poor host clearance of the organism from the amniotic fluid. Interestingly, chronic but not acute infection of amniotic fluid with Ureaplasma suppressed Escherichia coli endotoxin (lipopolysaccharide [LPS])-induced inflammation in the fetal lung, indicating innate immune tolerance. Further, we previously demonstrated that multiple exposures to IA LPS also induced endotoxin tolerance in the preterm fetus. We therefore hypothesized that chronic but not acute exposure to Ureaplasma would down-regulate LPS-induced chorioamnionitis. We report inflammatory responses in the chorioamnion to IA exposures to acute and chronic Ureaplasma , to LPS, and the interactions between Ureaplasma and LPS exposures.
Materials and Methods
Animals and IA injections
All animals were studied in Western Australia with approval from the animal ethics/care and use committees of the Cincinnati Children’s Hospital Medical Center (Cincinnati, OH) and the University of Western Australia. The fetal pulmonary data from these animals were previously reported. Time-mated Merino ewes with singleton fetuses were randomly assigned to 6 groups of 5-10 animals ( Figure 1 ). All injections were given by the IA route under ultrasound guidance, and after verification of proper placement in the amniotic compartment by fluid electrolyte analysis of aspirated samples. Sheep received an IA injection of: (1) U parvum serovar 3 (2 × 10 7 colony-forming units [CFU]) or 2 mL of media as a control 70 days or 7 days prior to delivery; or (2) E coli LPS (O55:B5; Sigma-Aldrich, St. Louis, MO) 10 mg in 2 mL of saline or saline only as a control 2 days prior to delivery. To evaluate the interactions, sheep were given IA injections of U parvum 7 days or 70 days prior to delivery plus IA LPS 2 days before delivery. Animals were delivered by cesarean section at 125 ± 1 day of gestational age (term ewe gestation = 150 days). Fetuses were given lethal intravascular doses of pentobarbital for euthanasia. At the time of delivery, rolls of fetal chorioamnion membranes were snap-frozen for RNA analysis, and a roll was fixed in 10% buffered formalin (pH 7.4) for histology. A fresh sample of amniotic fluid was used for pH measurement. Amniotic fluid was also snap-frozen for measurement of cytokine proteins. Ureaplasma species within sheep chorioamnion were confirmed by culture of homogenized tissue in 10B broth medium using previously reported protocols.
Immunohistochemistry and scoring of inflammation
Inflammatory cells in the chorioamnion were quantified by immunohistochemistry for markers of inflammatory cell activation using 5-μm paraffin sections from formalin-fixed chorioamnion. The sections were deparaffinized and rehydrated, and antigen retrieval was carried out using citric acid buffer, pH 6.0, with boiling in a microwave. Endogenous peroxidase activity was blocked with methyl alcohol/hydrogen peroxide. Nonspecific binding was inhibited with 2% goat serum during both primary and secondary antibody incubation. Sections were incubated overnight at 4°C with anti-myeloperoxidase (MPO) antibody (1:400) (catalog no. CMC028; Cell Marque, Rocklin, CA), or guinea-pig polyclonal anti-monocyte chemoattractant protein (MCP)-1 antibody (1:1000) (Seven Hills Bioreagents, Cincinnati, OH). After washing, sections were incubated with the appropriate secondary antibody for 30 minutes at room temperature (1:200). Immunostaining was visualized using a Vectastain ABC peroxidase Elite kit (Vector Laboratories, Burlingame, CA) and enhanced with nickel-diaminobenzidine, followed by incubation with trisaminomethane-cobalt to give a black precipitate. Nuclei were counterstained with Nuclear Fast Red for photomicroscopy. Scoring of MPO- and MCP-1-positive cells in 10 comparable nonoverlapping high-power fields for each animal was done by a single investigator blinded to study groups. For qualitative scoring of chorioamnionitis, the sections were stained with hematoxylin and eosin, and staged and graded for degree of inflammation in a blinded manner using a modified Redline grading system. Scores denoting the magnitude and tissue distribution of neutrophil infiltration within the chorioamnion were reported as follows: 0 = no chorioamnionitis; 1 = stage 1, grade 1 chorioamnionitis; 2 = stage 1, grade 2 chorioamnionitis; 3 = stage 2, grade 2 chorioamnionitis; and 4 = stage 3, grade 2 chorioamnionitis. In this scoring system, the stage of chorioamnionitis denotes the tissue plane of inflammation and the grade denotes the severity of inflammation. Stage 1 chorioamnionitis refers to inflammation restricted to the choriodecidua, stage 2 is inflammation in the choriodecidua + amnion, and stage 3 denotes necrosis of the amniotic epithelium.
Cytokine messenger RNA quantitation
Total RNA was isolated from the frozen chorioamnion samples. The quantitation of messenger RNA (mRNA) was performed by real-time polymerase chain reaction after reverse transcription of the mRNA yielding a single-strand complementary DNA (cDNA) (Verso cDNA kit; Thermo Scientific, Waltham, MA). cDNA was then used as a template with primers and TaqMan probes (Applied Biosystems, Carlsbad, CA) specific to sheep sequences for interleukin (IL)-1β, IL-6, IL-8, IL1-rA, MCP-1, and serum amyloid A3 (SAA3). The values for each cytokine were normalized to the internal 18S ribosomal RNA value. Final expression data are represented as fold increase over the control value.
Amniotic fluid protein analysis
Cytokine protein quantification was performed using custom sandwich enzyme-linked immunosorbent assay. The following antibody sets were used: (1) IL-1β (coating antibody rabbit anti-ovine IL-1β and primary antibody guinea-pig anti-ovine IL-1β [Seven Hills Bioreagents]); and (2) IL-6 (coating antibody mouse anti-ovine IL-6 [Chemicon no. MAB1004; Millipore, Billerica, MA] and primary antibody rabbit anti-ovine IL-6 [Chemicon no. AB1839]). The detection antibody in all assays was an appropriate species-specific horse radish peroxidase-conjugated antibody. The dynamic range of detection was 0.2–12 ng/mL for IL-1β and IL-6. The correlation coefficient was 0.94-0.99 for all assays.
Data analysis
Results are reported as mean ± SEM. All comparisons were specified a priori. Comparisons among ≥3 groups were performed by Kruskal-Wallis nonparametric analyses of variance. Comparison of 2 groups was done by a nonparametric t test (Mann-Whitney U test) for data not normally distributed (GraphPad Prism, version 5.04 for Windows; Prism, La Jolla, CA). The control group was compared vs 7-day U parvum , 70-day U parvum , and 2-day LPS. To specifically examine the effect of U parvum infection on LPS response, 2-day LPS was compared vs 7-day U parvum + 2-day LPS, and 70-day U parvum + 2-day LPS. Statistical significance was accepted at P < .05.
Results
IA infection with U parvum did not cause any gross fetal developmental abnormalities or mortality. Consistent with Ureaplasma utilizing urea as a nutritional source with liberation of ammonia, the amniotic fluid pH values differed significantly among groups ( P = .01) by analyses of variance ( Figure 2 ). By post hoc analysis, the only group with a higher pH compared to control was the 70-day U parvum group. The 70-day U parvum + 2-day LPS group ( Figure 2 ) had only 2 animals and hence statistical difference could not be established.
Histological chorioamnionitis after IA injections of LPS and U parvum
To evaluate the degree of histologic chorioamnionitis among groups, the infiltration of neutrophils in the chorioamnion were scored qualitatively ( Figure 3 ). Relative to the control, all treatment groups had significantly increased recruitment of neutrophils and higher scores for chorioamnionitis ( P = .004), but there were no differences between the LPS and U parvum groups.
To further evaluate the activation of inflammatory cells in the chorioamnion, MPO in the inflammatory cells was quantified ( Figure 4 ). Relative to controls, MPO-positive cells significantly increased in all groups except in those sheep with acute U parvum + LPS exposure. Compared to LPS exposure alone, MPO-positive cells significantly decreased in the 7-day U parvum + LPS group, but not after chronic U parvum exposure (70 days) and LPS challenge.
Acute exposure to U parvum down-regulated LPS-induced cytokine expression in the chorioamnion/amniotic fluid
We previously demonstrated that the chorioamnion is a target of inflammation after exposure to IA administration of proinflammatory agonists. We therefore measured expression of proinflammatory cytokine, chemokine, acute-phase reactant, and antiinflammatory cytokine mRNAs in the chorioamnion ( Figure 5 ). Relative to controls, acute (7-day) exposure did not increase cytokine mRNA expression while chronic (70-day) exposure to U parvum induced increases in mRNA expression for IL-1β (35 fold), IL-6 (34 fold), IL-8 (31 fold), SAA3 (300 fold), and IL-1rA (5 fold). The responses in 70-day U parvum + 2-day LPS were similar to 70-day U parvum alone ( P = .6-.8 for different cytokines for 70-day U parvum vs 70-day U parvum + 2-day LPS). In contrast, exposure to IA LPS for 2 days greatly increased expression of both the proinflammatory cytokines/chemokines: IL-1β (148 fold), IL-6 (30 fold), IL-8 (246 fold), MCP-1 (336 fold), SAA3 (800 fold), and the antiinflammatory cytokine IL-1rA (14 fold). Prior exposure to U parvum for 70 days did not consistently change responses to LPS (MCP-1 mRNA decreased compared to LPS alone, while IL-1β, IL-6, IL-8, SAA3, and IL-1rA did not). In sharp contrast, acute exposure to U parvum (7 days) significantly and consistently down-regulated LPS responses to near control levels. The cytokine expression in the 7-day U parvum + 2-day LPS group was similar to 7-day U parvum alone with the exception of IL-8 ( P = .04) and IL-6 ( P = .07).
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