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BOR - Papers in Press, published online ahead of print January 10, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.056143
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BIOLOGY OF REPRODUCTION 76, 738–748 (2007)
DOI: 10.1095/biolreprod.106.056143
© 2007 by the Society for the Study of Reproduction, Inc.

Interleukin 10 Regulates Inflammatory Cytokine Synthesis to Protect Against Lipopolysaccharide-Induced Abortion and Fetal Growth Restriction in Mice1

Sarah A. Robertson 2, Alison S. Care , and Rebecca J. Skinner 

Research Centre for Reproductive Health and Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide, South Australia 5005, Australia

ABSTRACT

Interleukin 10 (IL10) is a potent immune-regulating cytokine and inhibitor of inflammatory cytokine synthesis. To evaluate the anti-inflammatory role of IL10 in pregnancy, the response of genetically IL10-deficient mice to low-dose lipopolysaccharide (LPS)-induced abortion was examined. When IL10-null mutant C57Bl/6 (Il10–/–) and control (Il10+/+) mice were administered low-dose LPS on Day 9.5 of gestation, IL10 deficiency predisposed to fetal loss accompanied by growth restriction in remaining viable fetuses, with an approximately 10-fold reduction in the threshold dose for 100% abortion. After LPS administration, inflammatory cytokines tumor necrosis factor-alpha (TNFA) and IL6 were markedly increased in serum, uterine, and conceptus tissues in Il10–/– mice compared with Il10+/+ mice, with elevated local synthesis of Tnfa and Il6 mRNAs in the gestational tissues. IL1A and IL12p40 were similarly elevated in serum and gestational tissues, whereas interferon gamma (IFNG) and soluble TNFRII content were unchanged in the absence of IL10. Recombinant IL10 rescued the increased susceptibility to LPS-induced fetal loss in Il10–/– mice but did not improve outcomes in Il10+/+ mice. IL10 genotype also influenced the responsiveness of mice to a TNFA antagonist, etanercept. Fetal loss in Il10–/– mice was partly alleviated by moderate or high doses of etanercept, whereas Il10+/+ mice were refractory to high-dose etanercept, consistent with attenuation by IL10 status of TNFA bioavailability after etanercept treatment. These data show that IL10 modulates resistance to inflammatory stimuli by downregulating expression of proinflammatory cytokines TNFA, IL6, IL1A, and IL12, acting to protect against inflammation-induced pathology in the implantation site.

cytokines, immunology, placenta, pregnancy, uterus

INTRODUCTION

The maternal immune response is a key determinant of the success or failure of pregnancy, and disruption of the necessary adaptations to accommodate the conceptus is implicated in unexplained recurrent miscarriage in women [1]. The maternal immune response is principally regulated by an array of cytokines acting in complex networks to orchestrate the changes in leukocyte populations required to protect the conceptus from fetal immune rejection [2, 3] and to facilitate the tissue remodeling processes necessary for adequate placental development [4]. Inflammatory processes induced by host defense to infection or by immune disorders independent of infection are a major challenge to successful pregnancy [1, 5] and are linked to fetal growth restriction [6]. Inflammatory mediators, notably tumor necrosis factor-{alpha} (TNFA), can act in gestational tissues to damage placental blood supply and function [7] and cause fetal injury [8], leading ultimately to placental and fetal demise.

Studies in mice show that inflammatory stress operates primarily via synergistic induction of TNFA and interferon-{gamma} (IFNG) synthesis in macrophages and uterine natural killer (uNK) cells, respectively, which target uterine endothelial cells to elicit vascular injury and placental ischemia [9, 10]. Elevated TNFA and IFNG expression occurs spontaneously in the immune-mediated, abortion-prone CBA/J x DBA/2J mouse model [11], and also contributes to miscarriage due to insufficient luteal progesterone support [12], environmental stress [13], and diabetes [14, 15], highlighting the central role of proinflammatory cytokines in the effector pathways mediating fetal loss, regardless of the eliciting cause. Administration of TNFA or IFNG [16] or their activating cytokine interleukin 12 (IL12) is sufficient to induce abortion in mice [17], whereas inhibition of TNFA or IL1 is protective [18].

The cytokine IL10 has pivotal roles in modulating immune and inflammatory processes [19]. IL10 is a central regulator of the inflammatory response, acting to limit inflammation-induced tissue pathology by terminating monocyte and macrophage synthesis of TNFA and an array of other proinflammatory cytokines and chemokines [19]. Experiments in rodent models and humans implicate IL10 in controlling inflammatory processes in pregnancy. IL10 is expressed abundantly in the decidual and placental tissues in mice [20–22] and humans [23, 24]. Elevated placental and amniotic IL10 synthesis occurs in pathologies of pregnancy, including in utero growth restriction [25] and preeclampsia [26]. Decidual T lymphocytes from women with unexplained recurrent miscarriage are less frequently IL10 positive than clones from normal pregnancies [27].

Studies in IL10-null mutant (Il10–/–) mice show that this cytokine is not essential for normal pregnancy outcome [28–30]. Indeed, Il10–/– females mated with Il10–/– males have increased implantation sites and more viable fetuses than pregnant wild-type (Il10+/+) mice, even in allogeneic pregnancies sired by major histocompatibility complex-disparate Il10–/– males [30], demonstrating that IL10 is not required for adequate maternal immune tolerance of the fetal-placental tissues. However, IL10 does appear to have a necessary role in protecting pregnancy from the adverse effects of inflammatory challenge. Il10–/– mice are more susceptible to lipopolysaccharide (LPS)-induced pathologies of pregnancy, with elevated incidence of miscarriage [31] and preterm labor [32] at doses of LPS that fail to impact pregnancy in wild-type mice. These studies show that endogenous IL10 is a physiologic determinant of pregnancy resilience to inflammatory stress, as suggested by earlier observations that administration of recombinant IL10 can attenuate fetal loss and growth restriction induced in rats by LPS [33] or infection with Escherichia coli [34]. Similarly, exogenous IL10 reduces fetal resorption in pregnancies of CBA/J x DBA/2 mice, whereas anti-IL10 neutralizing antibodies increase fetal loss [35] and lead to growth impairment after birth [36].

The precise physiologic mechanisms through which IL10 acts to protect pregnancy from LPS-induced pathologies are not well defined. While others have reported a role for IL10 in maintaining appropriate phenotypes of uNK cells [31], it is unclear whether other pathways contributing to abortion and fetal growth restriction are affected in Il10–/– mice. IL10 deficiency is associated with sustained and uncontrolled serum levels of TNFA, IL6, IL12, IL1A, and IFNG after low-dose LPS challenge [32, 37]. The current study therefore sought to examine the role of IL10 in protecting against abortion and fetal growth restriction through regulating LPS-induced expression of proinflammatory cytokines in the implantation site. Using Il10–/– mice we have quantified the effect of low-dose LPS in midgestation on fetal viability and growth and investigated the possibility of uncontrolled inflammatory cytokine synthesis as a mechanism underpinning increased disposition to LPS-induced abortion. In particular, the role of TNFA in mediating the adverse effects of IL10 deficiency in pregnancy was evaluated using the soluble TNFA receptor, etanercept.

MATERIALS AND METHODS

Mice

Il10-null mutant mice were generated by targeted mutation of the Il10 gene in 129/Ola embryonic stem cells, propagated on a C57Bl/6 background (Il10–/–) [28]. Null mutant status was confirmed in Il10–/– mice by PCR of DNA extracted from blood or tail tissue of adult mice. PCR primers diagnostic for the Il10-null mutation and the neomycin insertion cassette were as previously reported [28]. Control C57Bl/6 mice (Il10+/+) 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 12L:12D cycle and were administered food and water ad libitum. Il10–/– 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. Animal usage was in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and all experiments were approved by the University of Adelaide Animal Ethics Committee.

Breeding Experiments

In all breeding studies, 1–3 females (Il10–/– or Il10+/+, 8–12 wk old) were housed with a proven fertile male of the same genotype. The day of vaginal plug detection was designated Day 0.5 of pregnancy, when females were removed from the male and housed in groups of two to five. For dose-response experiments, mated Il10–/– and Il10+/+ females were administered LPS (Salmonella typhimurium; 0.05, 0.25, or 2.5 µg; Sigma, St. Louis, MO) in 200 µl PBS i.p. at 1100 h on Gestation Day (GD) 9.5.

In experiments to evaluate the effect of exogenous IL10 replacement on fetal loss, mice were given LPS in combination with recombinant murine IL10 (rmIL10; Biodesign International, Saco, ME). Mice were administered rmIL10 (2.5 µg in 200 µl PBS + 0.1% BSA) i.p. at 0900 h on GD 9.5 prior to LPS injection at 1100 h on GD 9.5 (2.5 µg for Il10+/+ mice and 0.25 µg for Il10–/– mice). Control treatment groups received carrier (PBS + 0.1% BSA) alone or LPS and carrier alone.

For analysis of fetal loss rates and late-gestation pregnancy parameters, Il10–/– and Il10+/+ females were killed by cervical dislocation at 1000 h–1200 h on GD 17.5. The intact uterus of each female was removed and total, viable, and resorbing implantation sites were counted. Each viable fetus was dissected from the amniotic sac and umbilical cord, and fetuses and placentae were weighed.

In an experiment to evaluate the effects of LPS on cytokine synthesis, pregnant Il10–/– and Il10+/+ females were administered 20 µg LPS on GD 9.5 (i.p. at 1100 h). 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 wt), and blood was recovered by cardiac puncture and allowed to clot for 1 h at room temperature prior to centrifugation at 13 000 x g for 10 min and collection of serum. Gestational tissues, including the uterus (from between implantation sites) and conceptus (placenta and fetus), were dissected, and together with serum were snap frozen in liquid N2, then stored at –80°C prior to processing for cytokine ELISA and RT-PCR analysis. Gestational tissue samples were pooled from two implantation sites per pregnant female.

In an experiment to evaluate the effects of the soluble TNFA receptor, etanercept (Enbrel; Wyeth, Baulkham Hills, Australia) on LPS-induced cytokine synthesis, virgin Il10–/– and Il10+/+ females were administered etanercept (50 µg or 500 µg in 200 µl PBS + 0.1% BSA i.p.) or carrier (PBS + 0.1% BSA) at 0900 h on GD 9.5, prior to administration of LPS at 1100 h on GD 9.5 (2.5 µg for Il10+/+ mice and 0.5 µg for Il10–/– mice). Control treatment groups received either PBS alone or etanercept (50 µg or 500 µg) and PBS. Serum was recovered at autopsy 4 h after administration of LPS or PBS, after recovery and processing of blood as described above.

Quantitative RT-PCR for Cytokine mRNAs

Total cellular RNA was extracted from uterus and conceptus tissues using RNAzol B solution (Tel-Test, Friendswood, TX). Following treatment with RNase-free DNase I (500 IU/ml; 60 min/37°C; Boehringer Mannheim, Mannheim, Germany), first-strand cDNA was reverse transcribed from 1 µg RNA employing a Superscript II RNase H Reverse Transcriptase kit (90 min/43°C; Invitrogen, Carlsbad, CA). The cDNA solution was diluted to 100 µl and stored at –20°C. Primer pairs specific for cytokine cDNA sequences were designed using Primer Express software (Applied Biosystems, Foster City, CA). The PCR amplification employed 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 cDNA. The negative control included in each reaction consisted of H2O substituted for cDNA. PCR amplification was performed in an 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 reaction conditions for each primer pair are listed in Table 1. Reaction products were analyzed by dissociation curve profile and by electrophoresis in 2% agarose gel containing 0.5 µg/ml ethidium bromide and were visualized over an ultraviolet light box. Representative PCR products were purified and then sequenced at the Institute of Medical and Veterinary Science (Adelaide, Australia) using Big Dye version 2 or 3 (Applied Biosystems) to confirm primer specificity. Data was normalised for Actb mRNA expression and expressed in arbitrary mRNA units relative to the mean mRNA content of Il10+/+ conceptus tissue, which was assigned a value of 100.


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  		TABLE 1. PCR primer sequences, product size, and GenBank accession numbers for cytokine RT-PCR.

Cytokine Immunoassay

Frozen uterus and conceptus tissues were thawed on ice and solubilized in ice-cold PBS containing protease inhibitors (Complete Mini, EDTA-free; Roche Diagnostics, Penzberg, Germany) at 5 ml/g tissue using an Ultra-Turrax high-speed homogeniser (Janke and Kunkel, Staufen, Germany). 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 assay. The protein content of tissue homogenates was determined using Bradford reagent (Bio-Rad Protein Assay; Bio-Rad Laboratories Inc., Hercules, CA) 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 (Minneapolis, MN), according to the manufacturer's directions, with Maxisorp 96-well microtiter plates (Nunc A/S, Roskilde, Denmark). Cytokine ELISA kits were as follows: TNFA, Quantikine MTA00; IL6, Duoset DY406; IL1A, DuoSet DY400; IFNG, DuoSet DY485; IL12p40, DuoSet DY2398; and the soluble TNFA receptor II (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 to 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 Software Inc., Point Richmond, CA). The cytokine content of tissue homogenates was normalized to protein content to allow data expression as picograms per milligram of tissue. The thresholds for cytokine detection were 10 pg/ml TNFA, 10 pg/ml IL6, 5 pg/ml IL1A, 10 pg/ml IFNG, 10 pg/ml IL12p40, and 5 pg/ml soluble TNFRII.

TNF Bioassay

TNF bioactivity was assayed in cytotoxicity assays employing the TNF-sensitive cell line L929 as described previously [38]. Duplicate serial dilutions of serum were incubated for 24 h with 50 000 L929 cells in 200 µl RPMI-1640 (Gibco) supplemented with 10% fetal calf serum, glutamine (2 mM), streptomycin (100 U/ml), penicillin (100 U/ml), and containing cycloheximide (5 µg/ml). Cell lysis was measured by methyl violet uptake (0.5% in 20% methanol for 10 min at room temperature). Incorporated dye was dissolved in 50% acetic acid and quantitated by measuring absorbance at 570 nm using a multiwell ELISA reader (Bio-Rad Laboratories). The TNF content of serum samples was determined by comparison with a standard curve prepared from serial dilutions of recombinant murine TNFA (R&D Systems). The threshold for detection of TNF bioactivity was 10 pg/ml. More than 95% of the TNF bioactivity detected in serum recovered from IL10+/+ mice 4 h after administration of 2.5 µg LPS could be neutralized by addition of etanercept (50 µg/ml) to the L929 bioassay. This indicates the low abundance of TNF-like factors other than TNFA in serum from LPS-treated mice and relative specificity of the L929 assay for TNFA under these experimental conditions.

Statistical Analysis

All statistical analysis was carried out using SPSS 9.0 software (SPSS Inc., Chicago, IL). Cytokine content, mRNA abundance, and parameters of pregnancy, including numbers of implantation sites or fetal loss, were compared by parametric tests after confirmation of normal distribution of data by Shapiro-Wilk normality test. One-way ANOVA and posthoc 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 the Mann-Whitney U test was used. Categorical data expressed as proportions were compared by chi-square analysis. Differences between groups were considered significant when the probability was P < 0.05.

RESULTS

Effect of Maternal IL10 Deficiency on LPS-Induced Fetal Loss

To examine the effect of IL10 deficiency on susceptibility to LPS-induced fetal resorption, Il10–/– and Il10+/+ female mice were mated with males of the same genotype and then administered one of three doses of LPS or carrier on GD 9.5. Three doses of LPS (0.05 µg, 0.25 µg, and 2.5 µg) were used after preliminary experiments showed their effectiveness in causing substantial, moderate, or negligible fetal loss in pregnant Il10+/+ mice. When mice were examined on GD 17.5, Il10-null mutant mice were seen to be more severely affected by LPS treatment. This was most evident in groups treated with the 0.25-µg dose of LPS, where there was a significant reduction in the proportion of mated Il10–/– mice carrying viable fetuses at GD 17.5 (9% in Il10–/– and 66% in Il10+/+ mice, P < 0.001; Fig. 1A) and a higher rate of fetal resorption in implantation sites (51% in Il10–/– and 97% in Il10+/+ mice, P = 0.002; Fig. 1B). This was reflected in significantly fewer viable implants per mated Il10–/– female (mean ± SEM: 3.0 ± 0.7 in Il10–/– and 0.1 ± 0.1 in Il10+/+ mice, P = 0.011; Fig. 1C). The viable pregnancy rates and the incidence of fetal resorption in Il10–/– females administered 0.25 µg LPS were comparable to those seen in Il10+/+ females at a 10-fold higher LPS dose. Injection with 2.5 µg LPS was fatal in 2 of 14 Il10–/– mice but none of 18 Il10+/+ mice, concurring with previous reports of increased susceptibility to endotoxic shock in Il10-null mutant mice [37, 39].


Figure 01
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  		FIG. 1. The effect of Il10-null mutation on rate of fetal loss in response to LPS. Il10+/+ (open bars) and Il10–/– (shaded bars) B6 mice were mated with males of the same genotype and injected i.p. with LPS (2.5 µg, 0.25 µg, or 0.05 µg) or carrier (PBS) on GD 9.5. Data at autopsy on GD 17.5 are the percentage of mated females pregnant with viable fetuses (A), the percentage of total implantation sites undergoing resorption (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-square analysis, and data in B and C were compared by ANOVA and Sidak t-test (**P < 0.001 and *P < 0.05 compared with PBS group for same genotype; #P < 0.05 compared with Il10+/+ group at same LPS dose).

In addition, IL10 deficiency increased the adverse effect of LPS treatment on fetal growth in surviving fetuses. In Il10+/+ mice, fetal weight was not significantly affected by LPS treatment at any of the three doses. In Il10–/– mice, there was a progressive decline in fetal weight with increasing dose of LPS. Mean fetal weight was reduced by 6% compared with the control group in pregnant mice given 0.05 µg LPS (P = 0.012), and it was reduced by 20% compared with the control group in pregnant mice administered 0.25 µg LPS (P < 0.001; Table 2). There was no consistent effect of LPS treatment on placental weight in either genotype, but higher doses of LPS were associated with a reduction in the fetal:placental weight ratio, an index of placental efficiency. The 0.25-µg LPS dose resulted in a 17% reduction in fetal:placental weight ratio in Il10–/– mice (P = 0.004) and an 8% reduction in Il10+/+ mice (P = 0.006).


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  		TABLE 2. The effect of LPS on fetal and placental weights in viable implantation sites in Il10–/– and Il10+/+ mice.

Consistent with our previous findings in larger groups of Il10-null mutant mice [30], pregnancies in the control group of Il10–/– mice were characterized by a trend to higher fetal weight (P = 0.089) and a higher fetal:placental weight ratio (P < 0.001; Table 2). While viable litter size was numerically greater in the control group of Il10–/– mice, this did not reach statistical significance (Fig. 1).

Effect of Maternal IL10 Deficiency on LPS-Induced Inflammatory Cytokine Synthesis

Previous studies in nonpregnant mice showed that the modulating effects of IL10 after LPS administration operate through inhibition of proinflammatory cytokine synthesis [37, 39]. To evaluate the effect of IL10 deficiency on the cytokine content of gestational tissues, a second group of Il10–/– and Il10+/+ females mated with males of the same genotype were injected with 20 µg LPS i.p. on GD 9.5 and sacrificed 4 h later. Maternal serum and homogenized uterine and conceptus tissues (placenta and fetus combined) were analyzed for 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 IL10-deficient mice, with the capacity to discriminate the potential regulatory effect of IL10 [37, 39]. Serum from Il10-null mutant mice contained very high levels of proinflammatory cytokines, with increases in circulating TNFA (230-fold increase; P = 0.002), IL6 (13.6-fold increase; P = 0.021), IL1A (3.7-fold increase; P = 0.024), and IL12p40 (7.2-fold increase; P = 0.001; Fig. 2, A–D). In addition, Il10–/– mice showed trends to smaller increases in the serum content of IFNG (P = 0.083; Fig. 2E), and the endogenous TNFA antagonist, soluble TNFRII (P = 0.059; Fig. 2F).


Figure 02
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  		FIG. 2. The effect of Il10-null mutation on LPS-induced cytokines in serum. Il10+/+ (n = 12, open bars) and Il10–/– (n = 12, shaded bars) B6 mice were mated with males of the same genotype and injected i.p. with LPS (20 µg) on GD 9.5, then killed 4 h later. Data are mean ± SEM content of immunoactive TNFA (A), IL6 (B), IL1A (C), IL12p40 (D), IFNG (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 Il10–/– mice compared with tissues from Il10+/+ mice. The greatest increase was in TNFA, which was elevated 38-fold and 7.7-fold in the uterus and conceptus tissue of Il10–/– mice (P < 0.01; Fig. 3A). IL6 and IL1A also were increased in the uterus (2.5-fold and 5.1-fold, respectively, P < 0.05), but the conceptus tissue content of these cytokines remained unchanged in IL10-deficient mice (Fig. 3, B and C). Increased IL12p40 content also was evident in the uterus and conceptus tissue of IL10-deficient mice (9.4-fold and 4.2-fold, respectively, both P = 0.025; Fig. 3D). The uterine and conceptus tissue content of IFNG and soluble TNFRII were unchanged by IL10 deficiency (Fig. 3, E and F).


Figure 03
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  		FIG. 3. The effect of Il10-null mutation on LPS-induced cytokines in uterus and conceptus tissue. Il10+/+ (n = 7 to 9, open bars) and Il10–/– (n = 6 to 8, shaded bars) B6 mice were mated with males of the same genotype and injected i.p. with LPS (20 µg) on GD 9.5, then killed 4 h later. Data are mean ± SEM content of immunoactive TNFA (A), IL6 (B), IL1A (C), IL12p40 (D), IFNG (E), and soluble TNFRII (F) in homogenates of uterine and conceptus tissue, expressed as nanograms or picograms of cytokine per milligram of protein. Data were compared by independent samples t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

TNFA was the cytokine in gestational tissues most dramatically affected by IL10 deficiency. It was of interest to investigate whether increased tissue abundance of this cytokine was the result of increased local mRNA expression or, alternatively, due to the increase in circulating cytokine synthesized elsewhere. Tnfa and Il6 mRNAs were measured by real-time quantitative RT-PCR in uterine and conceptus tissues recovered from Il10–/– and Il10+/+ mice 4 h after LPS administration. IL10 deficiency was associated with increased Tnfa mRNA expression in both the uterus (4.8-fold; P = 0.001) and the conceptus (2.6-fold; P = 0.016; Fig. 4A), in keeping with the elevated TNFA protein levels seen in both tissues. Il6 mRNA expression was increased in the uterus (8.1-fold; P = 0.006) but not the conceptus (Fig. 4B), consistent with the finding of elevated IL6 immunoactivity in the uterus but not the conceptus of Il10–/– mice.


Figure 04
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  		FIG. 4. The effect of Il10-null mutation on LPS-induced synthesis of Tnfa mRNA (A) and Il6 mRNA (B) in uterus and conceptus tissue. Il10+/+ (n = 8, open bars) and Il10–/– (n = 8, shaded bars) B6 mice were mated with males of the same genotype and injected i.p. with LPS (20 µg) on GD 9.5, then killed 4 h later. Messenger RNA content was measured by quantitative RT-PCR. All data is expressed in arbitrary mRNA units as a mean ± SEM percent of the mean value of the Il10+/+ conceptus group, calibrated such that the mean value of the Il10+/+ conceptus group = 100. Data were compared by independent samples t-test (*P < 0.05; **P < 0.002).

Effect of IL10 Replacement on LPS-Induced Fetal Loss in IL10-Deficient Mice

Our previous experiments have shown that administration of exogenous rmIL10 normalizes the uncontrolled synthesis of inflammatory cytokines in Il10–/– mice while exerting only subtle effects on cytokine levels in Il10+/+ mice [32]. To evaluate the effect of exogenous IL10 replacement on susceptibility to LPS-induced miscarriage, in a third experiment Il10–/– mice were given rmIL10 (2.5 µg) 2 h prior to administration of 0.25 µg LPS on GD 9.5. This dose of LPS was chosen to achieve a viable pregnancy rate of approximately 10% in Il10–/– mice. The dose of rmIL10 was based on previous reports showing effectiveness in reducing endotoxemia [40] and preventing LPS-induced pregnancy pathologies in mice [31, 33]. Administration of rmIL10 increased the proportion of mated mice with viable pregnancies on GD 17.5 from 12% to 83% (P < 0.001), with an accompanying 2.1-fold reduction in the number of resorbing implantation sites (P < 0.001) and 4.4-fold increase in the viable implantations per litter (P = 0.004; Table 3).


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  		TABLE 3. The effect of exogenous recombinant IL10 on fetal loss in Il10–/– and Il10+/+ mice.

Administration of rmIL10 improved the LPS-mediated reduction in fetal weight by 19%, such that fetuses from rmIL10-treated mice were comparable in weight to those in the control group (Table 4). The mean fetal:placental weight ratio also increased after rmIL10 treatment; however, this did not reach statistical significance.


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  		TABLE 4. The effect of exogenous recombinant IL10 on fetal and placental weights in viable implantation sites in Il10 –/– and Il10 +/+ mice.

It also was of interest to determine whether exogenous IL10 might increase resistance to LPS-induced miscarriage in IL10-replete mice. Il10+/+ mice were administered 2.5 µg rmIL10 prior to administration of 2.5 µg LPS, a dose expected to provide a viable pregnancy rate of approximately 10%. Administration of rmIL10 did not alter the proportion of mated mice with viable pregnancies, and there was no effect on the number of fetal resorptions or average litter size (Table 3). Furthermore, there was no effect of rmIL10 on fetal weight or fetal:placental weight ratio (Table 4).

Effect of TNFA Antagonist Etanercept on LPS-Induced Fetal Loss in IL10-Deficient Mice

TNFA is a key cytokine in mediating fetal loss, and exogenous administration of this cytokine can mimic the effects of LPS in causing abortion [10, 16]. In view of the finding that TNFA synthesis in gestational tissues is substantially increased by IL10 deficiency, it was of interest to investigate the effect of the TNFA antagonist etanercept on attenuating susceptibility to LPS-induced miscarriage. Additional groups of Il10–/– mice in the third experiment were administered 50 µg or 500 µg of etanercept or carrier 2 h prior to administration of 0.25 µg of LPS or PBS on GD 9.5. The lower dose of etanercept was chosen on the basis of previous reports showing effectiveness in neutralizing serum TNFA bioactivity [41] and alleviating TNFA-mediated pathologies in mice [41–43]. Treatment with 50 µg etanercept increased the proportion of mated mice with viable pregnancies on GD 17.5 from 12% to 60% (P = 0.035). There also was a reduction in the proportion of resorbing implantation sites (P = 0.025), and although together this resulted in a 2-fold increase in viable implantation sites, the change failed to reach statistical significance (Table 3). Modest increases in fetal weight and fetal:placental weight ratio were seen after administration of etanercept to pregnant Il10–/– mice, but this failed to reach significance, and fetuses from etanercept-treated mice remained smaller than those in the control group (Table 4).

A 10-fold higher dose of etanercept was used in an effort to achieve greater efficacy in protecting Il10–/– mice from LPS. Treatment with 500 µg etanercept yielded results similar to 50 µg in terms of pregnancy outcome and fetal growth parameters in Il10–/– mice (Tables 3 and 4). Etanercept administered at either the 50-µg or 500-µg dose in the absence of LPS did not adversely affect pregnancy outcome (Table 3) or fetal growth parameters (Table 4) in Il10–/– mice.

To determine whether etanercept exerted a similar protective effect on LPS-induced miscarriage in IL10-replete mice, Il10+/+ mice were given 50 µg or 500 µg etanercept prior to administration of 2.5 µg LPS. Similar to effects in Il10–/– mice, treatment with 50 µg etanercept increased the proportion of mated mice with viable pregnancies from 21% to 78% (P < 0.001), substantially reduced the number of fetal resorptions (P < 0.001), and increased the average litter size (P < 0.001; Table 3). There was no effect of 50 µg etanercept on fetal weight or fetal:placental weight ratio in Il10+/+ pregnancies (Table 4). However in contrast to Il10–/– mice, the 10-fold higher dose of etanercept failed to protect mice from the adverse effects of LPS, and there was a trend to fewer viable pregnancies with this treatment (P = 0.068; Table 3). Treatment with 50 µg etanercept in the absence of LPS did not adversely affect pregnancy outcome (Table 3) or fetal growth parameters (Table 4). However, treatment with 500 µg etanercept in the absence of LPS caused a 9% reduction in fetal weight compared with the PBS control (P < 0.001; Table 4).

Etanercept is a dimeric form of the soluble TNFA receptor and has been reported to have both TNFA antagonist and agonist (carrier) effects, depending on the relative concentrations of etanercept and TNFA [41]. A high etanercept:TNFA ratio can act to increase synthesis and/or protect breakdown of plasma TNFA [41]. The relationship between etanercept-bound and free TNFA can be explored by evaluating the relative abundance of TNFA immunoactivity and bioactivity, since TNFA bound to etanercept can be detected by immunoassay but is biologically inactive. To investigate whether the interaction between Il10 status and responsiveness to high-dose etanercept might be associated with differential effects on TNFA bioavailability, virgin Il10+/+ and Il10–/– mice were given 500 µg etanercept 2 h prior to administration of LPS (2.5 µg for Il10+/+ mice and 0.5 µg for Il10–/– mice), and serum was recovered 4 h later and analyzed for bioactive TNF and immunoactive TNFA content. In Il10–/– mice, high-dose etanercept tended to reduce serum TNF bioactivity (63% reduction; P = 0.076), despite a substantial increase in serum TNFA immunoactivity (12.5-fold increase; P < 0.001). In serum of Il10+/+ mice, high-dose etanercept increased both TNF bioactivity (10.6-fold increase; P = 0.014) and TNFA immunoactivity (14.7-fold increase; P < 0.001; Table 5). In addition, this dose combination of LPS and etanercept attenuated levels of other serum cytokines in both IL10-deficient and IL10-replete mice. In Il10+/+ and Il10–/– mice, respectively, serum IL6 content was reduced by 63% (P < 0.001) and 47% (P < 0.001), and serum IL12p40 content was reduced by 47% (P = 0.042) and 38% (P = 0.007). Serum IL1A content was reduced by 70% in Il10–/– mice (P = 0.046) but was unchanged in Il10+/+ mice, whereas serum TNFRII levels were unchanged in both genotypes, and serum IFNG was undetectable in this experiment (Table 5).


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  		TABLE 5. The effect of etanercept administration on LPS-induced TNFA immunoactivity and TNF bioactivity in serum from Il10–/– and Il10+/+ mice.

DISCUSSION

The cross-regulatory balance between cytokines in the placental-decidual interface is a major determinant of the viability and the developmental potential of a fetus, and perturbations in the endometrial cytokine balance are associated with miscarriage in women [1] and mice [16]. Inflammatory cytokine excess causes activation of macrophages and uNK cells, which elicit vascular damage that in turn compromises placental blood supply and function [10]. This impairs fetal growth, and when the inflammatory insult is severe or sustained, fetal death ensues due to placental ischemia. Previous studies have demonstrated the efficacy of administering IL10 in averting fetal growth restriction and fetal loss, and have shown that increased synthesis of IL10 occurs in the placenta and uterus as a natural response to endotoxin challenge [33]. However, the physiologic significance of endogenous IL10 in modulating inflammatory cytokine synthesis in utero has not been defined. In this study we show that genetic deficiency in IL10 is associated with a higher susceptibility to abortion and fetal growth restriction in response to an inflammatory insult compared with IL10-replete mice. These adverse effects in IL10-deficient mice are preceded by uncontrolled inflammatory cytokine synthesis, with marked elevation in TNFA, IL1A, IL6, and IL12p40 and a trend to increased IFNG compared with wild-type mice. Treatments that inhibit inflammatory cytokine synthesis, including recombinant IL10 or the TNFA antagonist etanercept, protected Il10–/– females from the consequences of LPS challenge. These findings confirm that endogenous IL10 synthesis is a pivotal determinant of susceptibility or resilience to LPS-induced fetal pathologies due to the central role of IL10 in suppressing inflammatory cytokine synthesis in the implantation site.

The adverse effects on pregnancy outcome of low-dose LPS in Il10-null mutant mice can be directly attributed to uncontrolled inflammatory cytokine synthesis. Each of the cytokines shown to be upregulated in Il10–/– mice is implicated directly or indirectly in the sequence of events culminating in placental pathology and fetal demise. TNFA and IFNG are both potent abortifacient cytokines [10, 16], and TNFA is implicated as a major mediator of fetal growth restriction elicited by LPS treatment [44, 45]. IL12 also potentiates pregnancy loss in the abortion-prone CBA/J x DBA/2 mouse model [17].

The contribution of IL6 to the cytokine cascade underpinning abortion is less clear. IL6 is upregulated in implantation sites in CBA/J x DBA/2 mice [46]. However, IL6 administration to CBA/2 x DBA/J mice does not cause fetal loss and, in contrast, appears to support pregnancy maintenance [47]. This is consistent with preliminary observations of an elevated abortion rate in IL6-null mutant mice (Robertson, unpublished observations) and can be explained by an anti-inflammatory role for IL6 in inhibiting synthesis of TNFA and other inflammatory cytokines [48]. The soluble TNFA receptor antagonist, soluble TNFRII, also has an anti-inflammatory role, acting to sequester and reduce bioavailability of TNFA and thus to limit the toxic effects of this cytokine [41, 49].

The serum cytokine profiles induced by LPS in the current study are generally similar in quality and scale to those reported in previous experiments in nonpregnant Il10-null mutant mice [37] and in Il10-null mutant mice given LPS late in gestation to induce preterm labor [32]. In nonpregnant wild-type mice, increases in circulating proinflammatory cytokines are evident within 1–3 h of LPS challenge, followed by decline to baseline levels within 6 h. In contrast, absence of IL10 results in elevated and sustained synthesis of TNFA, IL6, IL12, IL1, and IFNG, with peak serum levels attaining values several-fold higher than in IL10-replete mice and remaining very high for 6 h or more after LPS induction [37]. Uncontrolled proinflammatory cytokine synthesis also is linked with increased mortality in mice rendered IL10 deficient using neutralizing antibodies [50], whereas administration of IL10 can protect mice from otherwise lethal doses of LPS by suppressing the toxic effects of circulating TNFA [40, 51]. The trend to increase in soluble TNFA receptor antagonist TNFRII we observed in the absence of IL10 is consistent with TNFA-regulated induction of this natural antagonist [49] but contrasts with the decrease we observed in Il10-null mutant mice given LPS late in gestation [32].

As well as the serum increase, similar changes in TNFA, IL6, IL12, and IL1A occurred in the uterus and conceptus tissues of Il10-null mutant mice, whereas there was no detectable change in the IFNG or TNFRII content of conceptus or uterus tissue. Quantitative RT-PCR experiments to examine whether this was the consequence of local mRNA transcription or blood-borne cytokine showed clearly that under the regulatory control of IL10, both TNFA and IL6 are differentially synthesized in uterine and conceptus tissues. Tnfa mRNA synthesis was elevated in the absence of IL10 in both compartments, whereas Il6 mRNA production was considerably higher in the uterus than in the conceptus, and IL10 regulation was not evident in the conceptus. Increased Tnfa and Il6 mRNA transcription in the uterus and conceptus of Il10-null mutant mice therefore is likely to substantially account for the increase in gestational tissue content. This result is comparable to data obtained in late-gestation uterus and placental tissues, where IL10-regulated differences in the content of TNFA and IL6 protein result from modulation of local mRNA synthesis [32]. Interestingly the elevated TNFA synthesis occurs in gestational tissues despite the concurrent local increase in the TNFA-modulating cytokine IL6 [48], showing the dominant suppressive role of IL10 over that of IL6.

Activated maternal macrophages positioned at the interface between the uterine and placental tissues are implicated as the major source of inflammatory cytokines, particularly TNFA and IL6 [9, 46, 52]. This response is amplified by LPS-induced uNK cell synthesis of IFNG [53]. These cytokines may have direct apoptotic effects in trophoblast cells, and in addition cause trophoblast cell death via induction of macrophage nitric oxide [52]. IL12 potentially contributes to this pathway by activating cytotoxic activity and amplifying IFNG synthesis in uterine NK cells. However, experiments in IFNG-unresponsive mice indicate that the effects of inflammatory cytokines are exerted largely in maternal decidual tissue as opposed to the placenta, and implicate a pathway involving TNFA and IFNG-induced ischemic damage in maternal endothelial cells [10]. In the absence of IL10, elevated and sustained synthesis of TNFA and IL12 would result in uncontrolled activation of macrophages and uNK cells, leading to greater tissue damage than in wild-type mice.

IL10 is reported to execute its anti-inflammatory effect principally by signaling termination of macrophage synthesis of TNFA, IL1, IL6, and nitric oxide [19] using transcriptional and posttranscriptional mechanisms involving induction of the suppressor of cytokine signaling 3 [54] secondary to STAT3 activation [55]. Direct inhibitory effects of IL10 on NK cell IFNG synthesis would be augmented indirectly through suppression of macrophage-derived IL12 and TNFA [56]. Thus, it seems clear that the protective effects of IL10 in the implantation site would be exerted through inhibitory effects on both macrophages and uNK cells, with the net consequence of dampening the synergistic activation that is mediated by TNFA, IL12, and IFNG cross talk between these two cell populations. The current observations therefore are consistent with a previous report of activated uNK cells mediating fetal loss in Il10–/– mice administered low-dose LPS [31], and they expand upon our understanding of the anti-inflammatory role of IL10 in the implantation site by showing its pivotal regulatory role in the cytokine network regulating not only uNK cells but also macrophages, endothelial cells, and trophoblast cells.

The current study highlights the central role of endogenous IL10 in preventing fetal growth restriction and protecting against fetal death. IL10 deficiency was linked with reduced growth in viable fetuses after exposure to very low doses of LPS that failed to affect wild-type mice, presumably as the result of effects on placental function that were insufficiently severe to cause fetal death. This implies that IL10 not only determines fetal survival after inflammatory challenge, but also influences the long-term health prospects of surviving fetuses. Growth impairment in utero is linked with altered growth trajectory after birth, disproportionate organ and tissue size, and a range of metabolic disorders that manifest in adult life [57, 58]. In humans, survival of an inflammatory challenge in utero is likely to be one of a range of insults that impact development after birth and disease resilience in adult life [6].

Interestingly, whether IL10 has growth-promoting or growth-limiting effects on the placenta and fetus appears to depend on the presence or absence of inflammatory stress. Our previous studies show that in a clean environment, IL10 deficiency in utero is linked with accelerated fetal growth in late gestation and larger viable litter sizes [30], and similar effects were seen in control groups in the current experiments. The explanation for this likely relates to IL10 constraining development of the placental labyrinth compartment where maternal-fetal nutrient exchange occurs through regulation of development of the fetal-placental circulation [59]. Proinflammatory factors such as IFNG and nitric oxide are required to achieve normal vasodilatation in the placental vasculature [4, 60]. Therefore, in the absence of an inflammatory challenge, lack of IL10 would increase their synthesis and improve placental capacity, whereas surplus IL10 would have the opposite result. An adverse effect of IL10 excess probably underpins the failure of exogenous IL10 administration to alleviate the LPS-induced fetal loss and the exacerbated growth restriction in wild-type mice seen in this study. The lack of benefit of IL10 treatment in normal mice contrasts with previous experiments in rats where LPS-induced fetal growth restriction was alleviated with IL10, although these studies used higher doses of LPS later in pregnancy [33, 34]. Consistent with our findings in this study and previous experiments [32], human clinical data link elevated placental and amniotic IL10 synthesis with in utero growth restriction [25]. These observations might be reconciled by proposing that optimal pregnancy outcome requires an appropriate balance between proinflammatory and anti-inflammatory cytokines, with dynamic adjustment to suit the level of inflammatory challenge. A complex range of factors would impact this balance, including the nature and strength of external and internal inflammatory stimuli and stressors, as well as maternal and fetal genetic determinants of cytokine bioactivity.

Similarly, variable responsiveness to the TNFA antagonist etanercept depending on endogenous IL10 synthesis might be explained by the interaction between etanercept and the prevailing in vivo concentrations of TNFA, endogenous TNFA antagonists such as TNFRII, and inhibitors of TNFA synthesis, including IL10 and IL6. Etanercept can have both TNFA antagonist and agonist (carrier) effects, sequestering cytokine and protecting it from clearance [41]. Difference in TNFA regulatory networks subject to IL10 control is likely to account for to the genotype-specific effects of etanercept. However, TNFA bioactivity does not fully explain the differential effects of high-dose etanercept depending on IL10 status, since similar TNF bioactivity levels were compatible with better reproductive outcomes in Il10–/– mice than in Il10+/+ mice, suggesting that other cytokines attenuated by etanercept, such as IL1A, potentially also contribute. In humans, high-dose etanercept can exert adverse effects in sustaining LPS-induced inflammation, mediated through elevated TNFA activity as well as TNFA-independent pathways, including the stress hormone response [61].

In summary, this study provides evidence for the physiologic importance of endogenous IL10 synthesis in gestational tissues in resistance to LPS-induced abortion and fetal growth restriction. The protective action of IL10 is due to its anti-inflammatory action in deactivating macrophages and inhibiting their synthesis of inflammatory cytokines, particularly TNFA, IL1, IL12, and IL6. These findings have direct implications for understanding the pathways underpinning inflammation-induced miscarriage in women, and may inform new therapeutic strategies for treatment of recurrent spontaneous abortion. Furthermore, the current study implicates the IL10 axis as a potentially important determinant of in utero fetal programming in humans. Variation in physiologic levels of IL10 synthesis, or responsiveness to IL10 induction through polymorphisms in IL10 pathway genes [62], might predispose to miscarriage and fetal growth restriction, as suggested by reports of altered IL10 expression linked with fetal loss and low birth weight [25, 27]. While there is growing experimental support for targeting the IL10 pathway in devising new therapeutic interventions for pregnancy pathologies, 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 effect of IL10 dose and interaction with proinflammatory factors in determining whether fetal growth is enhanced or constrained in mice, the possibility of adverse effects of sustained IL10 administration on the human fetus also would need to be considered. Finally, a better understanding is needed of the interplay between proinflammatory and anti-inflammatory components in the complex regulatory networks in the implantation site before the potential utility in pregnancy of anti-inflammatory drugs such as etanercept can be evaluated in the clinic.

ACKNOWLEDGMENTS

The technical assistance of Ms. Leanne Srpek is gratefully acknowledged.

FOOTNOTES

1Supported by project and fellowship grants from the National Health and Medical Research Council (Australia). Back

Correspondence: 2FAX: 618 8303 4099; e-mail: sarah.robertson{at}adelaide.edu.au

Received: 14 September 2006.

First decision: 9 October 2006.

Accepted: 22 December 2006.

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