Chronic Fetal Exposure to Ureaplasma parvum Suppresses Innate Immune Responses in Sheep

Preterm births account for 12% of all deliveries in the United States and are the most important determinant of neonatal mortality and morbidity (1). Chorioamnionitis or inflammation in the fetal membranes is associated with ∼60% of deliveries before 30 wk of gestation (2). The most common organisms isolated from the amniotic fluid of women with chorioamnionitis are the Ureaplasma species (3). Furthermore, among the preterm infants with chorioamnionitis caused by ureaplasma species, ∼60% also had coinfection with other organisms (3). In addition, ureaplasmas were isolated from amniotic fluid from first or second trimester or from washed semen in assisted reproduction (4–6). Therefore, preterm infants have frequent and often prolonged exposure to Ureaplasmas. Ureaplasma colonization of the upper genital tract can induce preterm labor in nonhuman primates and is associated with preterm labor in humans (5, 7). Exposure of preterm infants to Ureaplasma is associated with increased risk for adverse pulmonary, gastrointestinal, and neurologic outcomes (7–10).

The two species of ureaplasmas, which colonize humans, are Ureaplasma urealyticum (serovars 2, 4, 5, 7, and 13) and Ureaplasma parvum (serovars 1, 3, 6, and 14) (8). Of these, U. parvum is the most common species isolated from preterm neonates and upper genital tracts of women (11, 12). The ureaplasmas are unusual bacteria in that they have a plasma membrane but lack a peptidoglycan cell wall, use urea as the sole source of energy, and are dependent on the host for other metabolic functions (13).

We have reported acute and chronic exposure to ureaplasma species in fetal sheep (14, 15). Intra-amniotic (IA) injection of U. parvum in sheep induces a robust colonization of fetal chorioamnion and lung with poor bacterial clearance (14). Exposure to U. parvum in sheep induces a mild fetal inflammatory response, increases pulmonary surfactant pools, and results in mild transient developmental abnormalities in the lung (16, 17). In contrast, IA injection of LPS induces a robust fetal inflammation in the sheep (18). Interestingly, repeated exposures in vivo to IA LPS induce endotoxin tolerance and cross-tolerance to other Toll-like agonists (19, 20). Ureaplasma species signal via TLR1/2/6 and can increase LPS mediated inflammation in vitro (21, 22). Although coinfection of Ureaplasma spp with other organisms is common in chorioamnionitis (3), the interactions between ureaplasma and LPS in the fetus have not been explored.

On the basis of the in vitro observation that ureaplasma increase LPS-mediated inflammation (21, 22), we hypothesized that exposure to ureaplasma would increase LPS induced fetal inflammation. Pregnant ewes were given an IA injection of U. parvum serovar 3 to induce acute or chronic chorioamnionitis. We subsequently challenged fetal sheep with an IA injection of LPS 2 d prior to preterm delivery at 82% of term gestation. Fetal inflammation was assessed after a single exposure to U. parvum or LPS or after combined exposures.

The animals were studied in Western Australia with approval from the animal care and use committees of the Cincinnati Children’s Hospital (Cincinnati, OH) and the University of Western Australia. Time-mated Merino ewes with singleton fetuses were randomly assigned to study groups of five to seven animals (Table I). The pregnant sheep were given ultrasound-guided IA injections of 1) U. parvum serovar 3 (U. parvum), 2 × 10⁷ CFU or 2 ml media (control) into the amniotic fluid 70 or 7 d prior to delivery, 2) E. coli LPS (O55:B5; Sigma-Aldrich, St. Louis, MO) 10 mg in 2 ml saline 2 d prior to delivery, or saline only (control). To evaluate immune modulatory effects of U. parvum on LPS, separate groups of animals received IA LPS 2 d before delivery after exposure to IA U. parvum 70 or 7 d before delivery. All fetal injections were given with ultrasound guidance and with electrolyte analysis to confirm injection into amniotic fluid (23). All animals were delivered at 124 ± 1 d gestational age, umbilical cord blood was collected for plasma and for circulating leukocytes and fetuses were given lethal intravascular doses of pentobarbital for euthanasia. At autopsy tissue from the right lower lobe of the lung and liver were snap frozen for RNA extraction. For bronchoalveolar lavage fluid (BALF), the left lung was inflated with normal saline to total lung capacity followed by withdrawal, and the procedure was repeated three times (23). BALF were pooled and used for cell counts and protein measurements (24). BALF cell counts were expressed as total cells recovered from the lavage normalized to body weight. Lung compliance was measured from the deflation limb of an air pressure–volume curve with the chest open (24). The right upper lobe of the lung was inflation fixed with 10% buffered formalin at 30 cm H₂O pressure for morphology.

After exsanguination of the fetus, the lung was chopped thoroughly into fine pieces and incubated in RPMI 1640 medium (25). The lung suspension was then gently passed through a 100-μm mesh filter and the suspension was washed twice with PBS. Cells from the suspension were then layered over discontinuous Percoll gradients (1.085 and 1.046 g/ml) (Amersham Biosciences, Piscataway, NJ) to separate the monocytic cells from the other cells at the interface between the Percoll densities (25). Whole blood diluted 1:1 with saline was layered on a 1.046 g/ml Percoll gradient to recover mononuclear cells. Cells were counted using trypan blue to evaluate viability and then plated in culture dishes using media supplemented with 10% heat-inactivated FCS (Sigma-Aldrich, Castle Hill, NSW, Australia). After incubation at 37°C for 2 h, nonadherent cells were removed, and plates were washed twice with PBS. Monocytes were challenged with media only or LPS (100 ng/ml) for 16 h (19). Cell responsiveness as indicated by IL-6 secretion was measured in the media with a sandwich ELISA (coating Ab – mouse anti-ovine IL-6 (number MAB1004; Chemicon, Billerica, MA) and primary Ab rabbit anti-ovine IL-6 (number AB1839; Chemicon) (19).

Total RNA was isolated from fetal tissues using a modified Chomzynski method (24), and mRNA quantitation was performed using real-time PCR. The mRNA was reverse transcribed to yield a single-strand cDNA (verso cDNA kit; Thermo Scientific, Loughborough, U.K.), which was used as a template with primers and TaqMan probes (Applied Biosystems, Carlsbad, CA) specific to sheep sequences. The values for each cytokine were normalized to the internal 18S rRNA. Data were expressed as fold increases over control values.

Inflammatory cells in BALF were counted using a hemocytometer and the differential counts on cytospins were performed using Diff-Quick staining (Baxter Health Care, Deerfield, IL). For preparation of lung for morphology, formalin was removed from the fixed tissue within 24 h by washing in PBS (pH 7.4), and the tissues were transferred to 70% ethanol and embedded in paraffin. After deparaffinization and rehydration of fixed tissue, Ag retrieval was carried out using citric acid buffer (pH 6) with microwave boiling. Endogenous peroxidase activity was blocked with methyl alcohol/hydrogen peroxide. Nonspecific interactions were inhibited with 2% goat serum for both primary and secondary Ab incubations. Sections were incubated with anti-CD3 Ab (1:100, number A0452; DakoCytomation, Carpinteria, CA), anti-PU.1 (number sc-225; Santa Cruz Biotechnology, Santa Cruz, CA), anti-myeloperoxidase (MPO) Ab (catalog number CMC028, Cell Marque, Rocklin, CA) (1:400), and guinea-pig polyclonal anti–MCP-1 (1:1000) Ab generated at our institution (Seven Hills Bioreagents, Cincinnati, OH). Following incubation with the primary Ab at 4°C overnight, sections were incubated with the appropriate secondary Ab for 30 min at room temperature (1:200). Immunostaining was visualized using a Vectastain avidin/biotin complex peroxidase Elite kit to detect the Ag:Ab complexes (Vector Laboratories). Ag detection was enhanced with nickel diaminobenzidine, followed by incubation with TRIS cobalt to give a black precipitate. Nuclei were counterstained with Nuclear Fast Red for photomicroscopy. Blinded scoring of inflammation in the lung was done by counting MCP-1, PU.1, CD3, or MPO-positive inflammatory cells in 10 comparable nonoverlapping high-power fields of each animal (four to six animals per group).

TGF-β1 in BALF was measured by acidification of lavage fluid to activate latent TGF-β1 and concentrations were measured using a commercial ELISA kit (number DY240 against human TGF-β1; R&D Systems, Minneapolis, MN). Other cytokines were measured using custom ELISAs (26, 27). The Abs used were as follows: IL-1β (coating Ab: rabbit anti-ovine IL-1β and primary Ab guinea pig anti-ovine IL-1β [Seven Hills Bioreagents]), IL-8 (coating Ab: mouse anti-ovine IL-8 [number MAB10445; Chemicon] and primary Ab rabbit anti-ovine IL-8 [number AB1840; Chemicon]), and MCP-1 (rabbit anti-sheep MCP-1 coating Ab, and primary Ab guinea pig anti-sheep MCP-1 detection Ab [Seven Hills Bioreagents]). The detection Ab in all the assays was an appropriate species-specific HRP-conjugated Ab. Plasma haptoglobin was measured by ELISA (anti-bovine kit; ICL, Newberg, OR).

Results are given as mean ± SEM. Comparisons among three or more groups were performed by analyses of variance with Student-Newman-Keuls tests used for post hoc analyses. Comparison of two groups was done by a nonparametric t test (Mann–Whitney U test) for data not distributed normally and Student t test for normally distributed data. Statistical significance was accepted at p < 0.05.

All animals given an IA injection of U. parvum had positive cultures for U. parvum in the amniotic fluid (Table I). The amniotic fluid U. parvum titer tended to be lower (statistically not significant) after a 7-d exposure compared with 70-d exposure, but there was variability within each group. U. parvum did not cause gross developmental abnormalities and the birth weights were similar between infected and uninfected control fetuses (Table I). Consistent with our previous reports (15), compared with controls, the lung gas volumes increased ∼3-fold after chronic but not acute U.parvum exposure (Table I) indicating lung maturation. The 2-d LPS group had increased lung weight relative to body weight but decreased lung volume relative to lung weight. Low-power lung histology demonstrated that the control and the U. parvum only-exposed animals (both 7 and 70 d) had a similar histology (Fig. 1A–C). In contrast, 2-d LPS exposure recruited inflammatory cells in the airways and lung interstitium (Fig. 1D). The histology in animals exposed to acute U. parvum 7 + 2-d LPS was indistinguishable from the 2-d LPS only (compare Fig. 1D, 1E), whereas chronic U. parvum 70 + 2-d LPS-exposed animals had a lung histology similar to controls (compare Fig. 1F, 1A).

Control fetal lambs had very few neutrophils or monocytes in the pulmonary airspaces (Fig. 2A, 2B). Exposure to IA U. parvum for 7 d caused no significant recruitment of neutrophils, whereas a chronic exposure (70 d) recruited qualitatively higher numbers of neutrophils and monocytes to the airspaces. IA LPS induced a large recruitment of neutrophils and monocytes. Fetal lambs exposed to either LPS alone or U. parvum 7 d + LPS had similar numbers of inflammatory cells in the airspaces. However, animals exposed to U. parvum 70 d + LPS had qualitatively fewer inflammatory cells in the pulmonary airspaces.

Very few lymphocytes were detected in the airspaces of animals from any of the groups (data not shown). We therefore evaluated recruitment of CD3⁺ T lymphocytes in the fetal lung. Control and U. parvum-exposed fetal lambs had a few T lymphocytes in the lung (Fig. 2C, 2E, 2G). Exposure to IA LPS recruited T lymphocytes in the lung, and this recruitment was almost completely blocked by a prior chronic but not acute exposure to U. parvum (compare Fig. 2F, 2H). PU.1 is an ets-domain developmental transcription factor that orchestrates the inflammatory processes in myeloid and nonmyeloid cells (25, 28, 29). A prior chronic but not acute exposure to U. parvum blocked IA LPS induced PU.1 expressing cells in the fetal lung (Fig. 2D, compare Fig. 2J, 2L).

Exposure to chronic U. parvum could decrease LPS responsiveness via secretion of a soluble inhibitor or could induce changes in cell responsiveness to LPS. To evaluate these possibilities, monocytes from both the lung and the peripheral blood were cultured and challenged with LPS in vitro. Consistent with immature LPS responsiveness, fetal monocytes from control lambs had a decreased response to LPS compared with blood monocytes and alveolar macrophages from adult ewes to an in vitro LPS challenge (increase in IL-6 secretion: 2-fold versus 16-fold for blood monocytes and 2- versus 9-fold for lung monocytes; p < 0.05) (Fig. 3A, 3B). Exposure to U. parvum alone did not increase responsiveness to LPS in vitro. However, monocytes from fetal lambs exposed to IA LPS increased IL-6 secretion in response to LPS challenge in vitro, indicating a maturational effect. Interestingly, monocytes from IA LPS + U. parvum (7 d) further increased IL-6 secretion in response to an in vitro LPS challenge compared with the 2-d LPS group and similar to levels from adult cells. In striking contrast, monocytes from the IA LPS + U. parvum 70-d group had a blunted response to LPS challenge in vitro.

We previously demonstrated that the fetal lung is a target of inflammation after exposure to IA administration of proinflammatory agonists (24). We therefore measured expression of proinflammatory cytokine, chemokine, acute phase reactant and anti-inflammatory cytokine mRNAs in the fetal lung (Fig. 4). Relative to controls, either acute (7-d exposure) or chronic (70-d exposure) to U. parvum caused an inconsistent or modest increase in expression of IL-1β (4-fold), IL-6 (2-fold), IL-8 (7-fold), serum amyloid A3 (17-fold), and IL-1 receptor antagonist (IL-1ra) (4-fold) expression in the lung. In contrast, exposure to IA LPS for 2 d greatly increased expression of both the proinflammatory cytokines/chemokines (IL-1β, IL-6, IL-8, MCP-1 [CCL2], serum amyloid A3, CXCL9, and CXCL10), and the anti-inflammatory cytokines IL-1RA and IL-10 in the lung. Prior exposure to U. parvum for 7 d did not change responses to LPS. In sharp contrast, chronic exposure to U. parvum significantly downregulated LPS responses to near control levels. To further confirm these findings, we measured IL-1β and IL-8 protein in the BALF. Chronic exposure to U. parvum alone modestly increased secretion of IL-8 in the lung (Fig. 5B). Neither acute nor chronic exposure to U. parvum induced pulmonary IL-1β secretion (Fig. 5A). Consistent with the mRNA responses, IA LPS greatly increased IL-1β and IL-8 secretion (Fig. 5). Similar to the mRNA effects, chronic but not acute prior exposure to U. parvum decreased LPS-mediated induction of IL-1β and IL-8 secretion.

MCP-1 and MPO are expressed by activated neutrophils and monocytes in fetal lambs (27, 30). IA LPS induced expression of MCP-1 predominantly in the inflammatory cells with some expression in the pulmonary epithelial cells (Fig. 6). Consistent with the cytokine expression, U. parvum (either acute or chronic exposure) did not increase MCP-1 expressing cells in the lung. Prior acute exposure to U. parvum did not modify LPS effects, but strikingly, chronic exposure to U. parvum significantly reduced LPS-induced increases in MCP-1 expressing cells. Similar to MCP-1, U. parvum also did not increase MPO expressing cells in the lung (Fig. 7). Prior chronic but not acute exposure to U. parvum decreased LPS induced increasing in MPO⁺ cells in the lung.

Next, we asked whether U. parvum modulated IA LPS induced systemic fetal inflammation. We measured the expression of acute phase response genes Serum amyloid A3 and CRP in the fetal liver (Fig. 8A, 8B) and haptoglobin levels in the plasma (Fig. 8C). The expression of serum amyoid A3, CRP and plasma haptoglobin were low in control lambs and those exposed to U. parvum alone (Fig. 8). Exposure to IA LPS increased expression of the acute phase reactants. Prior acute or chronic exposure to U. parvum had no effect on LPS induction of acute phase reactants.

To evaluate some elements that may contribute to decreased LPS responsiveness after prior chronic U. parvum exposure, we measured TGF-β1 expression in the lung because of its known anti-inflammatory properties (31). Control lambs and those exposed to acute U.parvum had low levels of TGF-β1 expression in the airways (Fig. 9A). Chronic U. parvum, LPS, or acute U. parvum + LPS exposure equivalently increased TGF-β1 secretion. Interestingly, the group chronic U. parvum + LPS had the highest levels of BALF TGF-β1. Next, we quantified expression of several TLR4 signaling molecules. IL-1 receptor associated kinases (IRAKs) 1 and 4 are positive regulators, whereas IRAK-M is a negative regulator of TLR4 signaling (32, 33). TLR4 and IRAK-M mRNA expression increased 2.5- to 3-fold in the fetal lung exposed to IA LPS or IA LPS + U. parvum (7 d) (Fig. 9B, 9E), whereas the expression of IRAK-1 and IRAK-4 mRNA were similar to controls (Fig. 9C, 9D). Compared with controls, chronic exposure to U. parvum or U. parvum + LPS did not change expression of TLR4, IRAK-1, IRAK-4, or IRAK-M mRNAs in the fetal lung.

We report a profound decrease in responsiveness to LPS after a chronic exposure to U. parvum. Expression of all the measured genes with the sole exception of TGF-β1 did not increase in the lung in response to LPS in fetal lambs chronically exposed to U. parvum. Consistent with previous reports (21), we observed augmentation of LPS responses in vitro in monocytes from the acute U. parvum + LPS group compared with the LPS group or controls. However, LPS responsiveness was blunted in monocytes from the chronic U. parvum + LPS group. Endotoxin tolerance is now understood to be a complex reprogramming of inflammatory and noninflammatory cells to repeated exposures to bacterial products (34). Although a large body of literature on endotoxin tolerance is derived from in vitro studies, historical studies have reported endotoxin tolerance in patients recovering from typhoid fever (35). Also, leukocytes from patients with sepsis display endotoxin tolerance (36). Our findings differ from these observations in several respects: Similar to human chorioamnionitis, IA injection of U. parvum causes local colonization of the organism in the organs contacting the amniotic fluid (e.g., lung, chorioamnion, gut, and the skin) but infrequently causes systemic bacteremia (10, 14). Second, we found a near global decreased capacity to respond to LPS in the fetal lung after a chronic U. parvum exposure rather than a complex reprogramming of inflammation. Third, the downregulated LPS responsiveness was demonstrated 70 d after exposure but not after a 7-d exposure. Our study is unique in that, the host is a preterm fetus with developmental immaturity of the immune system.

Because Ureaplasma spp are common colonizers of the lower genital tract, there is some debate about the pathogenicity of the organism in pregnancy. However, several studies have unequivocally demonstrated the association of female upper genital ureaplasma colonization with preterm deliveries, fetal inflammation and adverse neonatal outcomes (8, 9, 13). Furthermore, Ureaplasma species can induce inflammatory cells to produce proinflammatory cytokines via TLR2/6 in vitro (21, 22). Consistent with its low virulence, we observed a persistent colonization but low-grade lung inflammation both at 7 and 70 d after IA injection of U. parvum. Lung inflammation after IA Ureaplasma exposure is likely via direct colonization, because we previously reported a 2-log order higher Ureaplasma titer in the fetal lung fluid compared with the amniotic fluid (14). Ureaplasmas are known to produce biofilms, which could trap inflammatory products thereby mechanically block inflammatory signaling (37). However, this is an unlikely explanation for the profound hyporesponsiveness to LPS in our experiments, because LPS responses were preserved when U. parvum exposure was for 7 d. Furthermore, both lung and blood monocytes from fetal lambs exposed to U. parvum for 70 but not 7 d demonstrated decreased responsiveness to LPS in vitro. Whether these responses are dependent on the degree of prematurity is not known. Also, the precise mechanism(s) by which a chronic exposure to U. parvum can decrease LPS responses remain to be identified.

Exposure to chronic but not acute U. parvum infection decreased both the influx of pulmonary inflammatory cells and expression of activation markers induced by IA LPS. Therefore, a question is whether the decreased expression of activation markers reflected decreased inflammatory cells in the fetal lung. The inflammatory cell composition in the lung 2 d after IA LPS is ∼60% neutrophils and 35% monocytes with few lymphocytes. Compared with effects of LPS alone, a prior exposure to chronic U. parvum reduced the neutrophil influx by ∼30% and monocytes by 60% (these reductions were not statistically significant). In contrast, the reductions in expression of cytokines, PU.1⁺, MCP-1⁺, and MPO⁺ cells were much larger, and the monocytes from fetal lambs with chronic U. parvum + LPS were poorly responsive to an in vitro LPS challenge. Taken together, these findings suggest that chronic exposure to U. parvum decreased activation and possibly recruitment of leukocytes to the fetal lung in response to LPS.

Endotoxin tolerance causes a complex reprogramming of inflammatory responses (34). Proinflammatory cytokine expression is downregulated, whereas there is no change or even an increase in the expression of anti-inflammatory genes, antimicrobial genes, and genes mediating phagocytosis (34). Indeed, microarray analysis of tolerant versus nontolerant monocytes demonstrate a host of genes that are downregulated, whereas other genes are not downregulated (38). The net result of endotoxin tolerance appears to prevent host organ injury while maintaining antimicrobial functions. However, almost all the information regarding endotoxin tolerance is derived from gene expression in functionally mature monocytes or macrophages. Because endotoxin tolerance is an adaptive host response, the characteristics of endotoxin tolerance might vary depending on the inflammatory context. We previously reported that preterm sheep fetuses exposed to repeated doses of intraamniotic LPS demonstrate endotoxin tolerance and cross-tolerance to other Toll-like agonists (19, 20). The genes reported not to be downregulated after endotoxin tolerance include IL-10, IL-1ra, TGF-β1, serum amyloid, and others (38). Although IL-10 and IL-1ra were also downregulated in the chronic U. parvum + LPS animals in our study, TGF-β1 increased relative to controls. Another class of genes not downregulated during endotoxin tolerance is the TRIF-mediated IFN-inducible genes (39). Furthermore, IFN signaling negatively regulates TLR4 signaling and can abrogate endotoxin tolerance in blood monocytes (40, 41). In the present experiment however, the pattern of expression of the IFN-inducible genes CXCL9 (monokine induced by IFN-γ) and CXCL10 (IFN-γ–inducible protein 10) mRNA expression in the lung after chronic U. parvum + LPS exposure was similar to the other proinflammatory cytokines. These results suggest that chronic exposure to U. parvum induced downregulation of LPS responses were more global in nature than previously reported in endotoxin tolerance.

Both intracellular negative regulators and extracellular soluble factors have been implicated in the mechanism of endotoxin tolerance. Extracellular/humoral factors that potentially mediate endotoxin signaling include steroid hormones (42), heat shock protein 70 (43), and IL-10 (44). The plasma cortisol levels (data not shown) and lung expression of IL-10 mRNA did not increase in these fetal lambs. However, TGF-β1 expression in the airways was higher in the lambs with downregulated LPS responses compared with controls. These data are consistent with the known anti-inflammatory properties of TGF-β1 (31). We previously reported increased expression of TGF-β1 and the downstream mediator phospho-SMAD2 after exposure to IA LPS (45). Both TGF-β1 and phospho-SMAD2 are detected in multiple lung cell types in the epithelium and the interstitium of fetal lung. The expression of mRNAs for the receptor TLR4 or the downstream transcription factors IRAK-1, IRAK-4, or IRAK-M in the lung did not change. Similarly, a growing list of intracellular mediators including MyD88s (46), IRAK-M (33), single Ig IL-1 receptor related (47), suppressor of cytokine signaling-1 (48), and others have been proposed as mediators of endotoxin tolerance. We previously reported that TLR4 mRNA is expressed ubiquitously in most of the lung cell types in the preterm fetus (49). In the absence of cell-type expression data for downstream mediators of TLR signaling, a caveat in the interpretation of changes in expression is that whole-lung homogenates used for quantification of mRNAs may dilute the changes in the relatively nonabundant recruited inflammatory cells.

Regulatory T cells expressing FOXP3 are abundant in the fetal circulation and downregulate inflammatory responses (50). However, in our study, acute or chronic exposure to U. parvum did not change FOXP3⁺ cells in the mediastinal lymph node or the lung (data not shown). Another mechanism for endotoxin tolerance is chromatin remodeling, changes in histone acetylation, and methylation induced gene silencing (38, 40). Although the precise molecular pathways of downregulated endotoxin responses in chorioamnionitis remain to be determined, increased TGF-β1 expression was demonstrated in the lungs of fetal lambs with downregulated LPS responses.

In contrast to sepsis, exposure of the fetus to bacterial components in chorioamnionitis is via the epithelia of the airway, the chorioamnion, and the gastrointestinal tract but not the vascular compartment. The resulting systemic inflammatory response is a mild increase in cytokines and acute phase reactants rather than the cytokine storm associated with sepsis in the adult (51, 52). Liver expression of acute-phase reactant genes CRP and serum amyloid A3 and plasma haptoglobin increased after IA LPS as expected, but the expression was not downregulated in lambs exposed to chronic U. parvum + LPS. These findings suggest that liver, the major source of acute-phase reactants, was not subject to downregulated LPS responses after exposure to chronic U. parvum. However, the blood monocytes from these chronic U. parvum + LPS animals were poorly responsive to LPS. Taken together, these findings suggest that LPS responsiveness in the fetus differed in different organs.

There are several clinical implications of our findings. Although Ureaplasma spp are the organisms most commonly associated with chorioamnionitis, ∼60% of cases with chorioamnionitis have coinfection with ureaplasma and other microorganisms (3). Our findings suggest the possibility of decreased fetal inflammation in response to Gram-negative organisms with concomitant ureaplasma exposure. In contrast, analogous to adults with sepsis (36), diminished innate immune responses could cause adverse fetal outcomes. Similarly, postnatal nosocomial sepsis occurs in ∼25% of very low birth weight preterm neonates (53). Our data suggest the possibility that ureaplasma chorioamnionitis could diminish innate immune responses and thereby increase the susceptibility of preterm infant to nosocomial sepsis. In summary, the experiments demonstrate a novel finding that a chronic exposure to intraamniotic U. parvum decreases both in vivo and in vitro lung endotoxin responsiveness in a preterm fetus.

We thank Prof. Jane Pillow for helping with animal use, Dr. Kathy Heel and Tracey Lee-Pullen (Center for Microscopy Characterization and Analysis, University of Western Australia, Perth, WA, Australia) for consultation on flow cytometry, and Amy Whitescarver, Manuel Alvarez, Jr., Avedis Kazanjian, Relana Nowacki, Richard Dalton, Joe Derwort, Carryn McLean, Jennifer Henderson, Andrea Lee, Shaofu Li, and Masatoshi Saito for expert technical assistance.

The authors have no financial conflicts of interest.