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The Journal of Immunology, 2006, 177: 4888-4896.
Copyright © 2006 by The American Association of Immunologists, Inc.

Essential Role for IL-10 in Resistance to Lipopolysaccharide-Induced Preterm Labor in Mice1

Sarah A. Robertson2, Rebecca J. Skinner and Alison S. Care

Research Centre for Reproductive Health and Department of Obstetrics and Gynecology, University of Adelaide, Adelaide, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-10 is highly expressed in the uterus and placenta and is implicated in controlling inflammation-induced pathologies of pregnancy. To investigate the role of IL-10 in regulating preterm labor, the response of IL-10 null mutant mice to low-dose LPS in late gestation was evaluated. When IL-10 null mutant C57BL/6 (IL-10–/–) and control (IL-10+/+) mice were administered LPS on day 17 of pregnancy, the dose of LPS required to elicit 50% preterm fetal loss was 10-fold lower in IL-10–/– mice than in IL-10+/+ mice. Surviving fetuses in IL-10–/– mice exhibited fetal growth restriction at lower doses of LPS than IL-10+/+ mice. Marked elevation of LPS-induced immunoactive TNF-{alpha} and IL-6 was evident in the serum, uterus, and placenta of IL-10–/– mice, and TNF-{alpha} and IL-6 mRNA expression was elevated in the uterus and placenta, but not the fetus. Serum IL-1{alpha}, IFN-{gamma}, and IL-12p40 were increased and soluble TNFRII was diminished in the absence of IL-10, with these changes also reflected in the gestational tissues. Administration of rIL-10 to IL-10–/– mice attenuated proinflammatory cytokine synthesis and alleviated their increased susceptibility to preterm loss. Exogenous IL-10 also protected IL-10+/+ mice from fetal loss. These data show that IL-10 modulates resistance to inflammatory stimuli by down-regulating proinflammatory cytokines in the uterus and placenta. Abundance of endogenous IL-10 in gestational tissues is therefore identified as a critical determinant of resistance to preterm labor, and IL-10 may provide a useful therapeutic agent in this common condition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection and the accompanying inflammatory processes required for host defense comprise a major challenge to successful pregnancy outcome. In women, bacterial infection in late-gestation pregnancy is the predominant cause of preterm labor, which affects ~5–10% of all pregnancies (1, 2, 3). Evasion of serious risk of both fetal and maternal death in the face of escalating infection is the presumed evolutionary rationale. Ascending intrauterine colonization, originating as bacterial vaginosis and progressing to infection of the choriodecidua, the fetal membranes, amniotic cavity, and ultimately, the fetus, activates an inflammatory cascade that culminates in preterm birth. Related molecular pathways activated by immunological dysfunction or other stressors likely account for preterm delivery in the 70% of cases where no infectious agent can be identified (4, 5). Spontaneous preterm labor is the predominant cause of infant morbidity and mortality and is frequently associated with fetal growth restriction and serious developmental disorders that have lifelong consequences for the child (4, 6).

Maternal disposition to infection-induced preterm delivery is linked with the balance in gestational tissue expression of an array of proinflammatory and anti-inflammatory cytokines (7). The process of normal parturition is associated with elevated gene transcription and accumulation of TNF-{alpha}, IL-6, IL-1, and IL-8 in amniotic fluid and gestational tissues, causing activation of PG synthesis which, in turn, leads to the myometrial contractions that expel the fetus (4, 7). In the event of intrauterine infection, inflammatory cytokine expression occurs prematurely despite the anti-inflammatory, progesterone-dominated environment (5, 7). Similar events in rodents administered LPS in late gestation indicate the validity of mouse (m)3 models to study the pathophysiology of preterm labor (8, 9).

IL-10 has been implicated as a key anti-inflammatory modulator in the cascade of cytokine synthesis initiated by intrauterine infection. In other tissues, IL-10 is identified as a critical counterbalance to proinflammatory cytokine synthesis (10). It acts to terminate the inflammatory response and limit inflammation-induced tissue pathology by deactivating macrophages and silencing their synthesis of TNF-{alpha}, IL-6, IL-1{alpha}, IL-8, and an array of other proinflammatory cytokines and chemokines (11, 12), as well as inducing synthesis of the soluble TNF-{alpha} receptor antagonist, soluble TNFRII (13). IL-10 is expressed abundantly in a gestational stage-dependent manner in the maternal uterine decidua and placental tissues in mice (14, 15, 16, 17) and in humans (18, 19, 20, 21). In vitro studies demonstrate the ability of IL-10 to down-regulate synthesis of TNF-{alpha}, IL-6, and PG in human chorion, decidual, and placental cells (22, 23, 24, 25). The potential importance of this cytokine in limiting adverse inflammatory responses in the gestational tissues is suggested by findings of decreased IL-10 expression in preterm labor (26, 27). In rats, exogenously administered IL-10 can attenuate fetal loss and growth restriction induced by LPS (28) and abrogate preterm birth and fetal growth impairment elicited by in utero infection with Escherichia coli (29). Furthermore, treatment of rats with IL-10 can reduce the extent of severe fetal brain injury often evident after uterine bacterial infection (30).

Rather surprisingly, studies in IL-10 null mutant (IL-10–/–) mice indicate that this cytokine is not essential for normal pregnancy outcome, even in allogeneic pregnancies sired by MHC disparate males (31, 32, 33). Indeed, pregnancies in IL-10–/– mice were characterized by consistently larger litter sizes and increased fetal weights (33). However, reproductive experiments in IL-10 null mice have generally been conducted in pathogen-free barrier facilities where the artificially clean environment and use of antibiotics protect mice from environmental endotoxin (31, 32, 33). Mice with a null mutation in the IL-10 gene have substantially reduced tolerance to bacterial cell wall LPS with the lethal dose of LPS for IL-10 null mutant mice being 20-fold lower than that for wild-type mice, due to uncontrolled production of TNF-{alpha} and IFN-{gamma} in the absence of IL-10 (34). Inability to terminate inappropriate inflammation in response to normal enteric Ags contributes to the chronic enterocolitis evident in IL-10 null mutant mice (31, 35). Consistent with predisposition to inflammatory responses in the reproductive tract, exposure to LPS in early pregnancy is associated with increased likelihood of miscarriage in mice deficient in IL-10 (36).

The availability of IL-10 null mutant mice provides the opportunity to examine the precise physiological significance of this cytokine in regulating the inflammatory cascade culminating in preterm labor. Previous studies have shown that exogenous IL-10 can attenuate susceptibility to LPS-induced preterm labor (28, 29), but the necessity for endogenous synthesis of this cytokine in regulating responsiveness to LPS in late gestation has not been evaluated. Furthermore, while the inhibition of TNF-{alpha} synthesis has been proposed as a mode of action for IL-10 administration (28), there has been no comprehensive quantitative analysis of the effects of IL-10 on modifying proinflammatory cytokine expression in gestational tissues in vivo. The purpose of the current study was to define the physiological consequences of genetic IL-10 deficiency after low-dose LPS challenge in late-gestation pregnancy. We show that this cytokine has an essential role in mediating resistance to preterm fetal loss. Uncontrolled synthesis of inflammatory cytokines TNF-{alpha}, IL-6, IL-1{alpha}, IL-12, and IFN-{gamma} are identified as probable mediators of the adverse effects of IL-10 deficiency on pregnancy outcome in mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

IL-10 null mice were generated by targeted mutation of the IL-10 gene in 129/Ola embryonic stem cells, propagated on a C57BL/6 background (IL-10–/–) (31). Null mutant status was confirmed in IL-10–/– mice by PCR of DNA extracted from blood or tail tissue of adult mice. PCR primers diagnostic for the IL-10 mutation and the neomycin insertion cassette were as reported previously (31). Control C57BL/6 mice (IL-10+/+) were obtained from the University of Adelaide Central Animal House. All mice were housed under specific pathogen-free conditions at the University of Adelaide Medical School Animal House on a 12:12 light-dark cycle, and were administered food and water ad libitum. IL-10–/– mice received broad-spectrum antibiotics (Oxymav 100, 100 g/kg oxytetracycline hydrochloride; Mavlab) in autoclaved drinking water twice weekly at a concentration of 2 mg/ml to prevent colitis. IL-10+/+ mice also were given antibiotics for 2 wk before and over the duration of experiments. All experiments were approved by the University of Adelaide Animal Ethics Committee.

Treatment protocols

For all experiments, one to three females (IL-10–/– or IL-10+/+, 8–12 wk old) were housed with a proven fertile male of the same genotype and checked daily for vaginal plugs as evidence of mating. The day of vaginal plug detection was designated day 1 of pregnancy, when females were removed from the male and housed in groups of two to five per cage. For dose-response experiments, mated IL-10–/– and IL-10+/+ females showing overt evidence of pregnancy were administered 0.25, 2.5, or 25 µg of LPS (Salmonella typhimurium; Sigma-Aldrich) in 200 µl of PBS i.p. at 1100 h on gestation day (gd) 17.

In experiments to evaluate the effect of exogenous IL-10 replacement on preterm delivery, mice were given LPS in combination with recombinant murine IL-10 (rIL-10; BioDesign). Mice were administered rIL-10 (2.5 µg in 200 µl of PBS + 0.1% BSA i.p., at 0900 h on gd 17 before LPS injection at 1100 h on gd 17 (2.5 µg for IL-10+/+ mice and 0.5 µg for IL-10–/–). Control treatment groups received carrier alone (PBS plus 0.1% BSA) or LPS and carrier alone.

For analysis of preterm delivery, pregnant IL-10–/– and IL-10+/+ females were killed by cervical dislocation at 1200–1400 h on gd 18. Vaginal bleeding or the presence of intact or partial fetal tissue in the cage was noted as evidence for fetal delivery. The intact uterus of each female was removed, and the total number of implantation sites was counted. Implantation sites were classified as viable (presence of live fetus and placenta), dead (fetus anemic, malformed, or severely growth retarded), or delivered (fresh implantation scar with absent fetus and placenta, or implantation site with placenta but no fetus). The percentage preterm loss was calculated as the sum of the number of delivered and dead fetuses, divided by the total number of implantation sites. When viable fetuses were present, each viable fetus was dissected from the amniotic sac and umbilical cord, and fetuses and placentae were weighed.

In additional experiments to evaluate the effects of LPS and exogenous IL-10 on cytokine synthesis, pregnant IL-10–/– and IL-10+/+ females were administered various doses (0.5, 2.5, or 20 µg, as specified) of LPS on gd 17 (i.p. at 1100 h) with or without prior administration at 0900 h of rIL-10 (2.5 µg) as described above. Four hours after LPS injection, mice were anesthetized with avertin (1 mg/ml tribromoethyl alcohol in tertiary amyl alcohol diluted to 2.5% v/v at a dosage of 15 µl/g body weight), and blood was recovered by cardiac puncture and allowed to clot for 1 h at room temperature before centrifugation at 13,000 x g for 10 min and collection of serum. Gestational tissues, including uterus (from between implantation sites), placenta (with fetal membranes removed), and fetus were dissected and, together with serum, were snap-frozen in liquid N2, then stored at –80°C before processing for cytokine ELISA and RT-PCR analysis. Gestational tissue samples were pooled from two implantation sites per pregnant female.

Quantitative RT-PCR for cytokine mRNAs

Total cellular RNA was extracted from uterus, placenta, and fetus tissue using RNAzol B solution (Tel-Test). Following treatment with RNase-free DNase I (500 IU/ml; 60 min/37°C) (Boehringer Mannheim), first-strand cDNA was reverse transcribed from 1 µg of RNA using a Superscript II RNase H reverse transcriptase kit (90 min/43°C) (Invitrogen Life Technologies). The cDNA solution was diluted to 100 µl and stored at –20°C. Primer pairs specific for published cytokine cDNA sequences were designed using Primer Express software (Applied Biosystems). The PCR amplification used reagents supplied in a 2x SYBR Green PCR Master Mix (Applied Biosystems), and each reaction volume (20 µl total) consisted of 0.5–1 µM 5' and 3' primers and 3 µl of cDNA. The negative control included in each reaction consisted of H2O substituted for cDNA. PCR amplification was performed in a ABI Prism 5700 sequence detection system (Applied Biosystems) according to the manufacturer’s instructions to allow amplicon quantification. In preliminary experiments, serial dilutions of cDNA were analyzed to confirm a linear relationship between cDNA content and quantity of product across the amplification range. PCR primers and optimized PCR conditions for each primer pair are listed in Table I. Reaction products were analyzed by dissociation curve profile and by electrophoresis in 2% agarose gel containing 0.5 µg/ml ethidium bromide and visualized over an ultraviolet light box. Data were normalized for beta-actin mRNA expression and expressed in arbitrary mRNA units relative to the mean mRNA content of IL-10+/+ placental tissue, which was assigned a value of 100.


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  		Table I. PCR primer sequences, product size, and Genbank accession numbers for cytokine RT-PCR

 
Cytokine immunoassay

Frozen uterus, placenta, and fetus tissue was thawed on ice and solubilized in ice-cold PBS containing protease inhibitors (Complete Mini, EDTA-free; Roche Diagnostics) (5 ml/g tissue) using an Ultra-Turrax high-speed homogenizer (Janke and Kunkel). Samples were centrifuged at 13,000 x g for 15 min at 4°C to pellet debris, and supernatants were stored at –80°C until cytokine ELISA. The protein content of tissue homogenates was determined using Bradford’s reagent (Bio-Rad Protein Assay; Bio-Rad) as described in the manufacturer’s instructions.

The cytokine content of tissue homogenates and in serum was determined using mouse cytokine-specific sandwich ELISA kits from R&D Systems, according to the manufacturer’s directions, with MaxiSorp 96-well microtiter plates (Nunc). Cytokine ELISA kits were as follows: TNF-{alpha}; Quantikine MTA00, IL-6; Duoset DY406, IL-1{alpha}; DuoSet DY400, IFN-{gamma}; DuoSet DY485, IL-12p40; DuoSet DY2398, and soluble TNFRII; DuoSet DY426. All samples from a given experiment were measured in the same assay, in duplicate, after dilution in PBS containing 1% BSA (1/10–1/500, as determined in preliminary experiments). The cytokine content of samples was determined by comparison of mean values with standard curves generated for each assay plate using the relevant recombinant cytokine, and the four parameter logistic curve fit function of SigmaPlot software (Systat). The cytokine content of tissue homogenates was normalized to protein content, to allow data expression as pg/mg tissue.

Statistical analysis

All statistical analysis was conducted using SPSS 9.0 software (SPSS). Cytokine content, mRNA abundance, and parameters of pregnancy, including numbers of implantation sites or preterm loss were compared by parametric tests after confirmation of normal distribution of data by Shapiro-Wilk normality test. One-way ANOVA and post hoc Sidak t test were used when more than two treatment groups were compared, and independent samples t test was used to evaluate effect of genotype. When data were not normally distributed, the nonparametric Kruskal-Wallis H test followed by Mann-Whitney U test were used. Categorical data expressed as proportions were compared by {chi}2 analysis. Differences between groups were considered significant when of p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of maternal IL-10 deficiency on LPS-induced preterm loss

To examine the effect of IL-10 deficiency on susceptibility to LPS-induced preterm loss, IL-10–/– and IL-10+/+ females were mated with males of the same genotype and then administered one of three doses of LPS or carrier on gd 17. Three doses of LPS (25, 2.5, and 0.25 µg) doses were used after preliminary experiments showed their effectiveness in causing complete, moderate, or negligible preterm loss in pregnant IL-10+/+ mice during the 24-h period subsequent to LPS administration. Preterm loss on gd 18 manifested as either delivery of the fetus, where a fresh uterine scar was evident (with or without a retained placenta), together with evidence of vaginal bleeding and fetal tissue in the cage bedding (~85% affected fetuses), or as fetal death where the fetus was retained in utero but clearly not viable (~15% affected fetuses).

IL-10 null mutation was associated with more severe effects of LPS treatment. This was most evident in groups treated with the 2.5-µg dose of LPS, with IL-10 deficiency causing a reduction in the proportion of mice carrying viable fetuses at gd 18 from 9 of 14 (64%) in IL-10+/+ mice, to 0 of 11 (0%) in IL-10–/– mice (p < 0.001) (Fig. 1A). When the fate of individual fetuses was evaluated, preterm loss was evident in 75 of 75 (100%) of fetuses carried by IL-10–/– females, compared with only 38% (36 of 95) of fetuses in IL-10+/+ females (p < 0.001) (Fig. 1B). This was reflected in a corresponding reduction in the number of remaining viable implantation sites per female (Fig. 1C). At the lowest dose of LPS (0.25 µg), there was no effect on pregnancy in IL-10+/+ females, but viable pregnancy rates and the incidence of preterm loss in IL-10–/– females were comparable to those seen in IL-10+/+ females at a 10-fold higher LPS dose. A 100% fetal loss was evident in IL-10+/+ mice administered 25 µg of LPS. A 25-µg treatment group was not conduced in IL-10–/– mice after maximum fetal loss was achieved at the lower 2.5-µg LPS dose.


Figure 1
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  		FIGURE 1. The effect of IL-10 null mutation on rate of preterm delivery in response to LPS. IL-10+/+ and IL-10–/– B6 mice were mated with males of the same genotype and injected i.p. with LPS (25 µg (IL-10+/+ only), 2.5 or 0.25 µg) or carrier (PBS) on gd 17. Data are the percentage of mated females pregnant at autopsy on gd 18 (A), the percentage of fetuses per litter delivered preterm, or dead at day 18 (B), and the mean ± SEM number of viable implantation sites per mated mouse (C). Numbers of mated mice and numbers of implantation sites are given in parentheses. Data given in A were compared by {chi}2 analysis, and data in B and C were compared by ANOVA and Sidak t test (*, p < 0.05, compared with PBS group for same genotype; #, p < 0.05, compared with IL-10+/+ group at same LPS dose).

 
Effect of maternal IL-10 deficiency on LPS-induced inflammatory cytokine synthesis

The modulating effects of IL-10 in the inflammatory response to LPS are reported to operate through inhibition of synthesis of proinflammatory cytokines including TNF-{alpha} (34, 37). To evaluate the effect of IL-10 deficiency on the proinflammatory cytokine content of gestational tissues, a second group of IL-10–/– and IL-10+/+ females mated with males of the same genotype were injected with 20 µg of LPS i.p. on gd 17, and serum and tissues were recovered 4 h later for analysis of cytokine content by immunoassay. The 20-µg LPS dose and 4-h time point were chosen to ensure detectable cytokine expression in both wild-type and IL-10-deficient mice, with capacity to discriminate the potential regulatory effect of IL-10 (34). Serum from IL-10 null mutant mice contained very high levels of proinflammatory cytokines, with increases in circulating TNF-{alpha} (330-fold increase; p = 0.002), IL-6 (12.1-fold increase; p = 0.004), IL-1{alpha} (2.5-fold increase; p = 0.05), IL-12p40 (14.3-fold increase; p < 0.001), and IFN-{gamma} (7.1-fold increase; p = 0.05) (Fig. 2, A–E). In addition, there was a 2.8-fold decrease in the serum content of the endogenous TNF-{alpha} antagonist soluble TNFRII in IL-10–/– mice (p = 0.01) (Fig. 2F).


Figure 2
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  		FIGURE 2. The effect of IL-10 null mutation on LPS-induced cytokines in serum. IL-10+/+ (n = 9, {square}) and IL-10–/– (n = 8, Figure 2) B6 mice were mated with males of the same genotype and injected i.p. with LPS (20 µg) on gd 17, then killed 4 h later. Data are mean ± SEM content of immunoactive TNF-{alpha} (A), IL-6 (B), IL-1{alpha} (C), IL-12p40 (D), IFN-{gamma} (E), and soluble TNFRII (F) in serum. Data were compared by independent samples t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

 
Proinflammatory cytokines also were elevated in the gestational tissues from IL-10–/– mice, compared with tissues from IL-10+/+ mice. The greatest increase was in TNF-{alpha}, which was elevated 45-, 13.6-, and 58-fold in the uterus, placenta, and fetus of IL-10–/– mice (p < 0.01, Fig. 3A). Increased IL-6 and IL-12p40 content (>10-fold) also was evident in the uterus and placenta of IL-10–/– mice, but in the fetal tissue, these cytokines were present in considerably lower concentrations and were not affected by genotype (Fig. 3, B and D). Less dramatic increases were seen in the uterine tissue content of IL-1{alpha}, IFN-{gamma}, and soluble TNFRII, and these cytokines were unchanged by IL-10 deficiency in the placenta and fetus (Fig. 3, C, E, and F).


Figure 3
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  		FIGURE 3. The effect of IL-10 null mutation on LPS-induced cytokines in uterus, placenta, and fetus. IL-10+/+ (n = 9, {square}) and IL-10–/– (n = 8, Figure 3) B6 mice were mated with males of the same genotype and injected i.p. with LPS (20 µg) on gd 17, then killed 4 h later. Data are mean ± SEM content of immunoactive TNF-{alpha} (A), IL-6 (B), IL-1{alpha} (C), IL-12p40 (D), IFN-{gamma} (E), and soluble TNFRII (F) in homogenates of uterine, placental, and fetal tissue, expressed as nanograms or picograms of cytokine/mg protein. Data were compared by independent samples t test (*, p < 0.05; **, p < 0.01).

 
The most dramatic differences in gestational tissue cytokine content in IL-10 null mutant mice were seen in TNF-{alpha} and IL-6. It was of interest to investigate whether increased tissue abundance of these cytokines was the result of increased local mRNA expression, or simply a reflection of the increase in circulating cytokine synthesized elsewhere. TNF-{alpha} and IL-6 mRNA were measured by real-time quantitative RT-PCR in uterine, placental, and fetal tissue recovered from IL-10–/– and IL-10+/+ mice 4 h after LPS administration. IL-10 deficiency was associated with increased TNF-{alpha} mRNA expression in the uterus (5.1-fold, p = 0.01) (Fig. 4A) and increased IL-6 mRNA expression in the uterus and placenta (13.1-fold, p = 0.01; and 3.0-fold, p = 0.05, respectively) (Fig. 4B). Expression of both cytokines was several hundred-fold lower in the fetus than in the uterus or placenta and was not affected by IL-10 deficiency (Fig. 4, A and B).


Figure 4
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  		FIGURE 4. The effect of IL-10 null mutation on LPS-induced synthesis of TNF-{alpha} mRNA (A) and IL-6 mRNA (B) in uterus, placenta, and fetus. IL-10+/+ (n = 9, {square}) and IL-10–/– (n = 8, Figure 4) B6 mice were mated with males of the same genotype and injected i.p. with LPS (20 µg) on gd 17, then killed 4 h later. Messenger RNA content was measured by quantitative RT-PCR. All data are expressed in arbitrary mRNA units as a mean ± SEM percentage of the mean value of the IL-10+/+ placenta group, calibrated such that the mean value of the IL-10+/+ placenta group = 100. Data were compared by independent samples t test (*, p < 0.05; **, p < 0.01).

 
Effect of IL-10 replacement on LPS-induced preterm loss in IL-10–/– mice

To evaluate the efficacy of exogenous IL-10 replacement on susceptibility to LPS-induced preterm delivery, in a third experiment, IL-10–/– mice were administered 2.5 µg of rIL-10 or PBS 2 h before administration of 0.5 µg of LPS on gd 17. This dose of LPS was chosen to achieve a fetal loss rate of ~60–80% in IL-10–/– mice. Administration of rmIL-10 reduced the proportion of pregnant mice experiencing fetal loss on gd 18 from 85 to 23% (p < 0.001), with an accompanying reduction in the number of dead/preterm delivered fetuses (Table II). LPS was seen to induce fetal growth restriction in the remaining viable fetuses, with a 9% reduction in fetal weight and 11% reduction in placental weight, compared with viable implantation sites in pregnant IL-10–/– females not given LPS (Table III). Administration of rIL-10 did not alleviate the LPS-mediated reduction in fetal and placental weight, although there was evidence of an 8% increase in the fetal: placental weight ratio, a surrogate measure of placental transport function.


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  		Table II. Effect of exogenous rIL-10 on preterm loss in IL-10–/– and IL-10+/+ mice

 

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  		Table III. Effect of exogenous rIL-10 on fetal and placental weights in viable implantation sites in IL-10–/– and IL-10+/+ mice

 
It also was of interest to determine whether exogenous IL-10 might increase resistance to LPS-induced preterm delivery in IL-10 replete mice. IL-10+/+ mice were administered 2.5 µg of rIL-10 before administration of 2.5 µg of LPS, a dose shown to provide a fetal loss rate of ~60%. Administration of rIL-10 reduced the proportion of pregnant mice experiencing fetal loss from 63 to 27% (p < 0.05), with an accompanying reduction in the number of dead/preterm delivered fetuses (Table II). In the remaining viable fetuses, this dose of LPS resulted in a 10% reduction in fetal weight and a 7% reduction in placental weight, compared with viable implantation sites in pregnant IL-10+/+ females not given LPS (Table III). Administration of rIL-10 did not alleviate the LPS-mediated reduction in fetal and placental weight, and did not change the fetal: placental weight ratio (Table III). Indeed administration of exogenous IL-10 caused a further 8% reduction in placental weight, compared with implantation sites in mice receiving 2.5 µg of LPS alone (p = 0.006). A 0.5-µg dose of LPS, the same as that given to induce 50% fetal loss in IL-10–/– mice, did not induce fetal loss or fetal growth restriction in IL-10+/+ mice (Tables II and III).

Consistent with our previous findings in IL-10 null mutant mice (33), pregnancies in the control (PBS) group of IL-10–/– mice were characterized by higher fetal weight (p = 0.012) and a trend, albeit not statistically significant, toward increased viable litter size than in the control (PBS) group of IL-10+/+ mice.

Effect of IL-10 replacement on LPS-induced inflammatory cytokine synthesis

To investigate the ability of exogenous IL-10 replacement to modulate inflammatory cytokine synthesis, IL-10–/– mice were administered 2.5 µg of rIL-10, 2 h before administration of 0.5 µg of LPS on gd 17, and serum was prepared from blood collected 4 h later. Exogenous IL-10 significantly inhibited the LPS-induced production of proinflammatory cytokines, with reductions in circulating TNF-{alpha} (15.6-fold decrease in mean content; p < 0.001), IL-6 (8.3-fold; p < 0.001), IL-1{alpha} (12.1-fold; p = 0.007), and IL-12p40 (31.1-fold; p < 0.001) (Fig. 5, A–D) were seen. There was no change in the serum content of soluble TNFRII (Fig. 5E).


Figure 5
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  		FIGURE 5. The effect of exogenous IL-10 administration on LPS-induced cytokines in serum in IL-10–/– and IL-10+/+ mice. IL-10–/– (n = 6–8 per group, Figure 5, A–E) and IL-10+/+ (n = 8 per group, {square}, F–J) B6 mice were mated with males of the same genotype and were administered rIL-10 (2.5 µg in 200 µl of PBS + 0.1% BSA i.p., at 0900 h on gd 17) or carrier (PBS + 0.1% BSA), before administration of LPS at 1100 h on gd 17 (2.5 µg for IL-10+/+ mice and 0.5 µg for IL-10–/– mice). The PBS control treatment groups received carrier alone. Data are mean ± SEM serum content of immunoactive TNF-{alpha} (A and F), IL-6 (B and G), IL-1{alpha} (C and H), IL-12p40 (D and I) and soluble TNFRII (E and J) in serum. Data were compared by independent samples t test (*, p < 0.05; **, p < 0.01, compared with LPS group for same genotype).

 
Similar effects, albeit to a less striking extent, were seen in IL-10+/+ mice administered 2.5 µg of rIL-10 2 h before administration of 2.5 µg of LPS. Exogenous IL-10 significantly inhibited the serum content of TNF-{alpha} (3.0-fold decrease in mean content; p < 0.001), IL-6 (1.9-fold; p = 0.001), and IL-12p40 (2.6-fold; p = 0.012) (Fig. 5, F, G, and I). There was no change in the serum content of IL-1{alpha} or soluble TNFRII (Fig. 5, H and J).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The cascade of events that override the normal processes of gestation to result in preterm birth, particularly the causal sequence involving inflammatory and anti-inflammatory cytokines, remains ill-defined. Previous studies have shown the efficacy of administering IL-10 in averting preterm labor induced by low-dose LPS or bacterial infection (28, 29) but have not investigated the physiological role of IL-10 in the balance of endogenous regulators governing induction of labor. We have shown that genetic deficiency in IL-10 is associated with a higher susceptibility to preterm loss, with a 10-fold-lower LPS dose required to elicit fetal death and delivery, as well as growth restriction in surviving fetuses, compared with IL-10 replete mice. Administration of rIL-10 before LPS protected IL-10 null mutant females from the adverse effects of LPS on pregnancy outcome. These findings indicate that endogenous IL-10 synthesis determines susceptibility to LPS-induced fetal loss and suggest that variation in IL-10 activity is an important factor in the amount of endotoxin that constitutes a threshold dose during an infectious challenge in pregnancy.

The mechanisms underpinning the adverse effects on pregnancy of IL-10 deficiency were explored and clearly involved uncontrolled proinflammatory cytokine synthesis in gestational tissues. Administration of LPS to pregnant IL-10 null mutant mice resulted in substantially elevated serum levels of TNF-{alpha}, IL-1{alpha}, IL-6, IL-12p40, and IFN-{gamma}, and reduced levels of soluble TNFRII, compared with wild-type mice at 4 h after LPS induction. Administration of IL-10 before LPS dramatically reduced serum levels of TNF-{alpha}, IL-6, IL-1{alpha}, and IL-12p40 in IL-10 null mice. The serum cytokine profiles reported in the current study are consistent in quality and scale with previous experiments in nonpregnant IL-10 null mutant mice (34). In nonpregnant wild-type mice, increases in circulating proinflammatory cytokines are evident within 1–3 h of endotoxin challenge, followed by decline to baseline levels within 6 h. In contrast, absence of IL-10 results in elevated and sustained synthesis of TNF-{alpha}, IL-6, IL-12, IL-1, and IFN-{gamma}, with peak serum levels attaining values several-fold higher than in IL-10 replete mice and remaining very high for ≥6 h after LPS induction (34). Uncontrolled proinflammatory cytokine synthesis is similarly high and linked with increased mortality in mice rendered IL-10 deficient using neutralizing Abs (38), whereas administration of IL-10 can protect mice from otherwise lethal doses of LPS (37, 39).

The soluble TNF-{alpha} receptor antagonist soluble TNFRII has not been examined previously in IL-10 null mutant mice, although a likely physiological role for IL-10 in regulating soluble TNFRII is indicated by in vitro experiments in human moncytes where IL-10 induces mRNA expression of both membrane-associated and soluble TNFRII (40). We found that soluble TNFRII protein levels were increased 2.8-fold in the absence of IL-10, although exogenous IL-10 did not attenuate synthesis in response to LPS. This is consistent with a role for IL-10 in limiting the actions of TNF-{alpha} through coordinately down-regulating TNF-{alpha} synthesis and amplifying production of its natural antagonist.

The uterus and placental tissue of IL-10 null mutant mice showed increases in inflammatory cytokine content comparable in scale to the increases in serum. Quantitative RT-PCR experiments to determine the extent to which this reflected local synthesis vs blood-borne cytokine influx showed clearly that both TNF-{alpha} and IL-6 are synthesized in uterine and placental tissue, although in the case of IL-6, synthesis was considerably higher in the uterus than in the placenta. Increased TNF-{alpha} and IL-6 transcription in the uterus and placenta of IL-10 null mutant mice is, therefore, likely to substantially account for the increase in gestational tissue content. This result confirms and extends a previous report of modulating effects of exogenous IL-10 on uterine TNF-{alpha} content in rats (28). The bioavailability of TNF-{alpha} in the gestational tissues would be further elevated due to reduced serum content of soluble TNFRII in IL-10 deficient mice.

In the case of TNF-{alpha} and to a lesser extent IL-6, there also was substantial elevation in fetal tissue content, compared with wild-type mice. However, mRNA for both cytokines was barely detectable in the developing fetus, suggesting that the elevated cytokine content in IL-10–/– fetal tissues was due to maternal cytokine transferred across the placenta, rather than local synthesis. This implies either that LPS is not transported across the placenta and/or that the fetus has alternative mechanisms that block up-regulation in TNF-{alpha} and IL-6 gene expression. Such mechanisms would be expected to be of benefit in sparing the developing fetus from sublethal effects of inflammatory cytokines, particularly the brain which is at risk of intraventricular hemorrhage and white matter damage elicited by endotoxin-induced cytokines during intrauterine infection (30, 41). Our findings of differential cytokine content and synthesis in different compartments of the gestational tissues is consistent with a previous report on cytokine production after intrauterine infection in wild-type mice, where considerably more IL-1, IL-6, and TNF-{alpha} immunoactivity was detected in the uterus than the placenta, and fetal content was lower still (42).

The adverse effects on pregnancy outcome in IL-10 null mutant mice given low dose LPS are directly attributable to uncontrolled proinflammatory cytokine synthesis. Infusion of exogenous TNF-{alpha} or IL-1 into the amniotic cavity is sufficient to induce PG synthesis and preterm labor in rabbits (43). Similarly, systemic administration of rIL-1 to mice in late gestation induces preterm delivery; however, rIL-6 is not effective (44). Blockade of IL-1 action through administration of IL-1 receptor antagonist can avert LPS-induced preterm delivery (45); however, deletion of responsiveness to both IL-1 and TNF-{alpha} using IL-1R1 and TNFRI double-mutant mice is reported to confer more effective protection than deletion of IL-1 responsiveness alone (9).

Activated macrophages accumulating in the uterine and placental tissue after LPS administration are implicated as the major source of inflammatory cytokines (46). Uterine macrophages respond to LPS with increased expression of proinflammatory cytokines and up-regulated NO synthesis (47, 48). This response is amplified by LPS-induced uterine NK cell synthesis of IFN-{gamma} (47). Cytokine responses in decidual leukocytes are augmented by increased production of a range of inflammatory cytokines, including TNF-{alpha}, IL-1, IL-6, and IL-12 in choriodecidual membranes (49) and placental trophoblast cells (50).

Inflammatory cytokine synthesis results in preterm delivery through cytokine induction of PG synthesis in a variety of cell lineages in the gestational tissues. PG plays a central role in the initiation and progression of labor through their transformation of quiescent uterine tissue into a state of active contractility (4). Studies in human uterine and placental tissues show that inflammatory cytokines, particularly TNF-{alpha} and IL-1, induce synthesis of PGE2 and PGI2 in myometrial cells, amnion cells, and placental trophoblast cells through induction of PG H synthase-2 (4). In vitro experiments demonstrate an inhibitory action of IL-10 in placental trophoblast and chorion PG H synthase-2 synthesis (25), as well as choriodecidual production of IL-8 and TNF-{alpha} (51); however, the effects of IL-10 in fetal membranes depend on the precise tissue site because the adjacent amnion responds to IL-10 with increased PGE2 and IL-8 production (51). LPS-activated macrophages also are a potent source of cyclooxygenase –2 (COX-2), which is the first enzyme in the biosynthetic pathway leading to PG synthesis (52). Studies in IL-10 null mutant mice identify IL-10 as a pivotal regulator of this aspect of macrophage function, with COX-2 mRNA expression and PGE synthesis both dramatically elevated after 12 h exposure to low-dose LPS in spleen cells from IL-10 null mutant mice (53). The effects of IL-10 in blocking COX-2 mRNA synthesis appear to be direct (by regulating COX-2 mRNA transcription and mRNA stability) and indirect, because COX-2 is up-regulated in response to inflammatory cytokines (53, 54).

In both IL-10 null and wild-type mice receiving LPS at the threshold dose for preterm loss, some mothers at autopsy contained dead and or severely growth-restricted fetuses that had not been expelled from the uterus. This is consistent with an additional route of LPS-induced inflammatory cytokine action independent of induction of myometrial contractions. Fetal growth impairment and/or demise likely involves several pathways, which culminate in placental deterioration and impaired fetal support. TNF-{alpha} is a key cytokine in this regard, with previous experiments linking this cytokine with fetal loss in mice (55, 56) and growth impairment in humans (57). TNF-{alpha} targets the maternal uteroplacental arteries to activate a prothrombotic cascade mediated by fgl2 prothrombinase (56) and can act directly in the placenta to induce apoptosis in trophoblast cells (58). These pathways both cause damage to the placental structure and transport function, resulting in placental ischemia which, depending on the severity would, in turn, cause fetal growth restriction or fetal death.

Our previous studies show that IL-10 deficiency in utero is linked with accelerated fetal growth in late gestation and larger viable litter sizes (33) and this improved reproductive outcome was confirmed in the current experiments. The explanation for this likely relates to placental structure and function, with IL-10 constraining development of the placental labyrinth compartment where maternal-fetal nutrient exchange occurs, partly through inhibiting vasodilation of the fetal-placental circulation (59). It is possible that an effect on placental blood flow underpins the failure of exogenous IL-10 administration to alleviate the LPS-induced fetal growth restriction seen in the current study. In both IL-10+/+ and to a lesser extent in IL-10–/– mice treated with LPS, placental size was in fact further diminished after administration of rIL-10, suggesting that this cytokine can affect acute effects on placental structure. This effect might be elicited through IL-10 down-regulation of placental production of NO (28), a key regulator of vasodilation and hemodynamics of the fetal and placental vasculature thought to be critical in maintaining nutrient and oxygen delivery to the fetus (60). Although IL-10 can also influence cytotrophoblast differentiation and invasiveness, it seems unlikely that changes in cell composition would account for effects of IL-10 in the placenta late in pregnancy when its morphogenesis is complete (59). The lack of benefit of IL-10 treatment for fetal mass contrasts with previous experiments in rats where LPS-induced fetal growth restriction was alleviated with exogenous IL-10 supplied at similar doses (28, 29). However, consistent with our findings, human clinical studies link elevated placental and amniotic IL-10 synthesis with in utero growth restriction (61).

In conclusion, these experiments show the physiological importance of endogenous IL-10 synthesis in gestational tissues in resistance to infection-induced preterm labor. The protective action of IL-10 is attributed to its anti-inflammatory role in deactivating macrophages and inhibiting their LPS-induced synthesis of inflammatory cytokines, particularly TNF-{alpha}, IL-1, and IL-6, as well as COX-2 and other inflammatory mediators in placental cells and fetal membranes. These findings have direct implications for understanding the pathways underpinning infection- or inflammation-induced preterm labor in women and may inform new therapeutic strategies for preventing or delaying this common pathology of pregnancy. It is clear from in vitro studies that IL-10 has similar regulatory effects in down-regulating TNF-{alpha}, IL-6, and PG in human gestational tissues (22, 23, 24, 25). Variation in physiological levels of IL-10 synthesis, or responsiveness to IL-10 induction through polymorphisms in IL-10 pathway genes (62) potentially confers disposition to preterm labor as suggested by reports of reduced IL-10 expression in tissues of women experiencing preterm labor (26, 27). Exogenous IL-10 or IL-10 homologs or small molecule regulators of the IL-10 pathway (62) hold promise as novel tocolytic agents for preventing preterm birth. A likely advantage is that, unlike existing therapies that target uterine smooth muscle cell contractions or inhibit PG production (4, 7), IL-10 targets events occurring earlier in the causal progression leading to delivery. However, caution is warranted in view of the expected high variation in patient responsiveness and the potential for unintended downstream immune-modulating effects of this cytokine (62). Given the potential effects of IL-10 in restricting placental function and fetal growth in mice (33, 59), the possibility of adverse effects of sustained IL-10 administration on the fetus also would need to be considered.


   Acknowledgments
 
The technical assistance of Leanne Srpek is gratefully acknowledged.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by project and fellowship grants from the National Health and Medical Research Council (Australia). Back

2 Address correspondence and reprint requests to Dr. Sarah A. Robertson, Research Centre for Reproductive Health, University of Adelaide, Adelaide, SA 5005 Australia. E-mail address: sarah.robertson{at}adelaide.edu.au Back

3 Abbreviations used in this paper: m, mouse; gd, gestation day; COX-2, cyclooxygenase-2. Back

Received for publication April 11, 2006. Accepted for publication June 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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