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The Journal of Immunology, 2007, 179, 8491 -8499
Copyright © 2007 by The American Association of Immunologists, Inc.
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Pulmonary and Systemic Endotoxin Tolerance in Preterm Fetal Sheep Exposed to Chorioamnionitis1

Suhas G. Kallapur2,*, Alan H. Jobe*, Molly K. Ball*, Ilias Nitsos{dagger}, Timothy J. M. Moss{dagger}, Noah H. Hillman*, John P. Newnham{dagger} and Boris W. Kramer{ddagger},§

* Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, OH 45229; {dagger} School of Women’s and Infants’ Health, University of Western Australia, Perth, Australia; {ddagger} University Hospital, Maastricht, The Netherlands; and § University Children’s Hospital, Würzburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In a model of human chorioamnionitis, fetal sheep exposed to a single injection, but not repeated injections, of intra-amniotic endotoxin develop lung injury responses. We hypothesized that repeated exposure to intra-amniotic endotoxin induces endotoxin tolerance. Fetal sheep were given intra-amniotic injections of saline (control) or Escherichia coli LPS O55:B5 (10 mg) either 2 days (2-day group, single exposure), 7 days (7-day group, single exposure), or 2 plus 7 days (2- and 7-day repeat exposure) before preterm delivery at 124 days gestation (term = 150 days). Endotoxin responses were assessed in vivo in the lung and liver, and in vitro in monocytes from the blood and the lung. Compared with the single 2-day LPS exposure group, the (2 plus 7 days) repeat LPS-exposed lambs had: 1) decreased lung neutrophil and monocyte inducible NO synthase (NOSII) expression, and 2) decreased lung cytokine and liver serum amyloid A3 mRNA expression. In the lung, serum amyloid A3 mRNA expression decreased in the airway epithelial cells but not in the lung inflammatory cells. Unlike the single 7-day LPS exposure group, peripheral blood and lung monocytes from the repeat-LPS group did not increase IL-6 secretion or hydrogen peroxide production in response to in vitro LPS. Compared with controls, TLR4 expression did not change but IL-1R-associated kinase M expression increased in the monocytes from repeat LPS-exposed lambs. These results are consistent with the novel finding of endotoxin tolerance in preterm fetal lungs exposed to intra-amniotic LPS. The findings have implications for preterm infants exposed to chorioamnionitis for both responses to lung injury and postnatal nosocomial infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chorioamnionitis or infection/inflammation of the fetal membranes and amniotic fluid is a pregnancy complication associated with up to 70% of preterm deliveries at <30 wk gestation (1). Chorioamnionitis is frequently thought to be chronic with prolonged fetal exposures to inflammatory products in the amniotic fluid. Although infection in amniotic fluid is common, systemic sepsis soon after birth is detected in only 2% of these preterm infants (2). However, ~50% of infants exposed to chorioamnionitis have a systemic inflammatory response, referred to as fetal inflammatory response syndrome (FIRS)3 (3, 4, 5). Unlike the cytokine storm associated with the adult systemic inflammatory response, FIRS is often a more subtle inflammatory response (4, 6). At birth, most preterm infants with chorioamnionitis and FIRS cannot be distinguished clinically from those not exposed to chorioamnionitis, suggesting an adaptive response. Virtually nothing is known about how the fetal lung and innate immune cells in the lung or in the blood respond or adapt to chronic chorioamnionitis.

We developed a preterm lamb model of chorioamnionitis in which intra-amniotic LPS directly contacts the fetal lung and causes inflammatory cell influx and expression of proinflammatory cytokines in the fetal lung (7, 8, 9). We also reported that structural changes resembling bronchopulmonary dysplasia (fewer alveoli, vascular injury responses) occur in the preterm fetal lung after exposure to a single injection of intra-amniotic LPS (10, 11). However, to our surprise, fetal lambs exposed to repeated injections of intra-amniotic LPS had lung morphology indistinguishable from gestation-matched controls when delivered close to term gestation (12). We therefore hypothesized that repeated exposures to intra-amniotic LPS induced an endotoxin tolerance in inflammatory cells and the fetal lung.

Endotoxin tolerance is defined as altered responsiveness to a subsequent endotoxin challenge following a first encounter with endotoxin both in vivo as well as in vitro (13, 14, 15). However, relatively little is known about endotoxin tolerance induced by intratracheal endotoxin exposures, as most experiments of endotoxin tolerance in vivo have used intravascular or i.p. exposures (14). There is almost no information in preterm fetuses or newborns regarding endotoxin tolerance other than our initial study demonstrating reduced endotoxin responsiveness of blood monocytes in vitro after exposure to repeated intra-amniotic endotoxin exposures (16). Preterm fetuses in all animal species studied to date have very few alveolar macrophages (17, 18) and have immature blood monocytes (16, 19). The monocytes, macrophages, and dendritic cells are the key cells implicated in pathogenesis of endotoxin tolerance (16, 20). Therefore, the phenomenon of endotoxin tolerance may be different in the preterm compared with adults. To test for endotoxin tolerance, we evaluated both in vivo and in vitro LPS responsiveness following exposures to a single or two injections of intra-amniotic LPS in preterm fetal sheep.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals, intra-amniotic injections, and tissue processing at delivery

All animals were studied in Western Australia with approval from the animal care and use committees of the Cincinnati Children’s Hospital (Cincinnati, OH), and the Western Australian Department of Agriculture. Time-mated Merino ewes with singleton fetuses were randomly assigned to groups of five to nine animals, to receive either a single injection of 10 mg of LPS (Escherichia coli 055:B5; Sigma-Aldrich), two injections of 10 mg of LPS, or an equivalent volume of saline (control) by intra-amniotic injections for intervals of 2 or 7 days or (2 and 7 days) before caesarean delivery at 124 ± 1 days gestational age. The control fetal lambs given one or two injections of intra-amniotic saline were combined because there were no differences in the measurements between the single vs two injections of saline. Intra-amniotic injections were given under ultrasound guidance with verification of needle placement by electrolyte analysis of the amniotic fluid aspirated immediately before injection (21). To obtain bronchoalveolar lavage fluid (BALF), the left lung was instilled with cold normal saline to total lung capacity followed by withdrawal (22). The lavage was repeated three times, the BALF was pooled and used for cell counts and to measure LPS-binding protein (LBP) by ELISA (HBT Biotechnologies). The right upper lobe of lung was inflation fixed with 10% buffered formalin at 30 cm H2O pressure. Pieces of the liver and the right lower lobe of the lung were snap-frozen for RNA analysis.

Plasma cortisol, endotoxin levels, and blood counts

Cord plasma cortisol concentrations were measured by a radioimmunoassay (RIA kit; MP Biomedicals) (21). Endotoxin levels were measured using a Limulus amebocyte lysate assay kit (Cambrex) (23). Automated total white blood cell counts were performed with correction for nucleated RBC and differential counts were performed by a single observer, blind to the treatment groups.

RNA extraction and RNA quantitation

Total RNA was isolated from lung and liver samples using a modified Chomzynski method. Ten micrograms of total RNA were used for IL-1β, IL-6, IL-8, TNF-{alpha} (7), IL-10 (8), serum amyloid A3 (SAA3) (24), and TLR4 mRNA (25) quantitation using RNase protection analysis as previously described. Briefly, solution hybridization was performed overnight (16 h) using a molar excess of [{alpha}-32P]UTP-labeled riboprobes. The unhybridized ssRNA was digested with RNase A/T1 (BD Pharmingen). RNase was then inactivated and protected RNA was precipitated using the RPA III inactivation buffer (Ambion). The ribosomal protein mRNA L32 was used as an internal control. The protected fragments were resolved on 6% polyacrylamide 8 M/L urea gels, visualized by autoradiography, and quantified on a PhosphorImager using ImageQuant version 1.2 software (Molecular Dynamics).

Inducible NO synthase (iNOS) immunohistochemistry and scoring of lung inflammation

After deparaffinization and rehydration, Ag retrieval was conducted with citric acid buffer (pH 6.0). Endogenous peroxidase activity was blocked with methyl alcohol/hydrogen peroxide. Nonspecific interactions were inhibited with 2% goat serum during both primary and secondary Ab incubation. Sections were incubated with monoclonal anti-iNOS (NOSII) Ab (1/250; BD Transduction Laboratories) at 4°C overnight followed by incubation with goat anti-mouse IgG biotin (Santa Cruz Biotechnology) for 1 h at room temperature. Immunostaining was visualized using a Vectastain ABC peroxidase Elite kit to detect the Ag:Ab complexes (Vector Laboratories). The Ag detection was enhanced with nickel diaminobenzidine, followed by incubation with Tris-cobalt to give a black precipitate. Nuclei were counterstained with Nuclear Fast Red for photomicroscopy. Blind scoring of lung inflammation was done by counting iNOS-positive inflammatory cells in 10 nonoverlapping high-power fields of each animal. Areas of the section with large airways or blood vessels were excluded and inflammatory cells in the lung parenchyma and airspaces were combined. Three animals per group were evaluated.

Lung and blood monocyte isolation and culture

Following vascular perfusion with HBSS of the right lower lobe to remove blood, the fetal lung was chopped thoroughly into fine pieces and incubated in HBSS with shaking at +37°C for 1 h (26). The lung suspension was then gently filtered through a 100-µm mesh and the suspension was washed twice with PBS. Cells from the suspension were then layered over discontinuous Percoll gradients (1.085 and 1.046 g/ml; Amersham Pharmacia Biotech) to separate the lung monocytic cells from the other cells. Monocytic cells were recovered from the interface between the Percoll densities (26). To obtain blood monocytes, cord blood was diluted with PBS (1/1) and layered onto a Percoll gradient as described (16). Cells were counted using trypan blue to evaluate viability and then plated in culture dishes using medium supplemented with 10% heat-inactivated FCS (Sigma-Aldrich). After incubation at 37°C for 2 h, nonadherent cells were removed and plates were washed twice with PBS. To estimate the number of monocytes, cells were scraped from the culture dishes, and differential cell counts were performed on cytospin preparations stained with DiffQuick (Baxter Healthcare). The adherent cell population was 90 ± 3% monocytes for all treatment and control groups.

Hydrogen peroxide and IL-6 measurements

Monocytes were cultured with or without exposure to LPS for 16 h in culture (100 ng/ml, E. coli, serotype O55:B5; Sigma-Aldrich). Production of hydrogen peroxide was measured with an assay based on the oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+) by hydrogen peroxide under acidic conditions (Bioxytech H2O2-560 assay; Oxis International). Control samples were exposed to saline. Concentrations of IL-6 were determined in the supernatant of LPS (100 ng/ml) exposed monocytes by an ovine-specific ELISA (16).

In situ hybridization

SAA3 mRNA expression was evaluated by in situ hybridization of paraffin embedded sections using a digoxigenin-labeled antisense riboprobe. The plasmid psSAA3 (8C3) was digested with NotI for the antisense probe (T7 polymerase; Promega) and with NcoI for the sense probe (SP6 polymerase; Promega). The riboprobes were generated in an in vitro transcription reaction using digoxigenin-UTP (Roche). For hybridization, the sense and antisense probes were diluted in hybridization buffer to a final concentration of 1 µg/ml (100 µl/section) and incubated at 57°C. After hybridization, sections were washed with a buffer containing 50% formamide for 30 min at 65°C. Sections were then treated with RNase A/T1 to reduce nonspecific binding followed by washes. An anti-digoxigenin Ab conjugated with alkaline phosphatase was applied (1/2000; Roche) overnight at 4°C followed by reacting with an alkaline phosphatase substrate containing nitro blue tetrazolium chloride (Roche). Controls for specificity of riboprobe binding included use of lung tissues obtained from lambs exposed to intra-amniotic saline and the use of homologous (sense) probe.

TLR4, IL-1R-associated kinase (IRAK)-4, and IRAK-M protein analysis

Cell surface TLR4 expression was determined by flow cytometry using a sheep cross-reactive anti-TLR4 Ab (BD Biosciences) and an appropriate fluorescence tagged secondary Ab (26). The abundance of IRAK proteins in the monocytes was determined using immunoblots. Protein content of the monocyte cell lysates was determined by the BCA method, using BSA as the standard and resolved by gel electrophoresis (Novex precast gels; Invitrogen Life Technologies). The proteins from the gel were transferred to polyvinylidene difluoride membrane by electroblotting (Invitrogen Life Technologies). Blots were blocked 1 h in 5% nonfat dry milk in TBS with 0.1% Tween 20. These blots were incubated with the primary Ab overnight at 4°C with either IRAK-4 or IRAK-M Ab (Abcam). The primary Ab was diluted in 5% nonfat dry milk in TBS with 0.1% Tween 20. Blots were incubated for 1 h at room temperature with the appropriate conjugated secondary Ab. After washing, bands were visualized by chemiluminescence (ECL kit; Amersham Pharmacia Biotech) and radiographed. The blot was then stripped and reprobed with a β-actin Ab (Sigma-Aldrich) to normalize for protein loading. The autoradiographs were scanned at high resolution and images were acquired using Adobe Photoshop software. The densitometric quantitation was performed using ImageQuant version 1.2 software (Molecular Dynamics).

Data analysis

Results are given as mean ± SEM. Comparisons between LPS-treated groups and untreated controls were by ANOVA with Student-Newman-Keuls tests used for post-hoc analyses. Statistical significance was accepted at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Physiologic variables at birth

The control lambs exposed to saline and lambs given single or repeated intra-amniotic LPS injections had similar body weights, plasma cortisol, and cord blood gas values (Table I). LPS was not detected in the cord blood from any of the lambs across different groups (data not shown). The lung/body weight was 15% higher in the intra-amniotic LPS single injection 2-day group compared with controls, likely due to lung inflammation and edema (Table I). The repeated-LPS exposure group and all other groups had similar lung/body weight ratios compared with controls. In response to exposure to a single intra-amniotic LPS injection, peripheral blood neutrophils decreased by 60% at 2 days and increased 3-fold at 7 days (Table II). Blood monocyte and lymphocyte counts were similar in the different groups of fetal lambs. The blood neutrophil counts in the lambs exposed to repeated intra-amniotic LPS (2- plus 7-day group) were similar to the intra-amniotic LPS 7-day group (single injection). There were no fetal deaths.


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  		Table I. Physiological variables of preterm lambs at birth

 

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  		Table II. Differential white blood cell counts (x109/L) in the blood

 
Repeated intra-amniotic LPS inhibits inflammatory cell iNOS expression in the fetal lung

Control lambs had no inflammatory cells in the BALF (Table III). Consistent with our previous results (7) and relative to controls, BALF neutrophils, monocytes, and lymphocytes increased at 2 days and remained increased at 7 days after single or repeated exposure to intra-amniotic LPS. Consistent with the BALF data, very few neutrophils or monocytes were detected in the lung parenchyma of the control lungs (Fig. 1, A and B). Activation of inflammatory cells was assessed by iNOS (NOSII) immunostaining. The iNOS expression in the inflammatory cells was striking at 2 days and had decreased 7 days after a single exposure to intra-amniotic LPS (compare Fig. 1, C and D). A prior exposure to intra-amniotic LPS 7 days before delivery inhibited the induction of iNOS expression after intra-amniotic LPS challenge given 2 days before delivery (intra-amniotic LPS 2- plus 7-day group; compare Fig. 1, C and E).


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  		Table III. Inflammatory cells in the bronchoalveolar lavage

 

Figure 1
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  		FIGURE 1. Repeated intra-amniotic LPS decreases induction of iNOS (NOSII) expression in the lung inflammatory cells. Immunostaining was performed on 5-µm sections of fetal lung. A, Ten high-power fields in each of four animals per group were scored for cells positive for iNOS immunostaining and shown as dot plots with median. B, iNOS expression was not detected in control lung. C, Robust iNOS expression was detected in the inflammatory cells (shown by arrow and magnified in the inset) at 2 days after single-dose LPS exposure (IA LPS 2 days). D, iNOS expression decreased 7 days after single-dose LPS exposure (IA LPS 7 days). E, In contrast to B, iNOS expression was decreased after repeated LPS exposures (IA LPS 2 + 7 days). Scale bar, 50 µm; *, p < 0.05 vs control; t, p < 0.05 vs single dose-LPS 2 days.

 
Repeated intra-amniotic LPS inhibits cytokine mRNA expression in the fetal lung

Consistent with our previous results (7), the mRNAs for proinflammatory cytokines were induced in the fetal lung 2 days after a single intra-amniotic LPS exposure: IL-1β (40-fold), IL-6 (8-fold), IL-8 (20-fold), and TNF-{alpha} (2-fold) (Fig. 2, A–D) with a return to control levels 7 days after exposure to a single injection of intra-amniotic LPS. In contrast, a prior exposure to intra-amniotic LPS 7 days before delivery inhibited the induction of IL-1β, IL-6, and IL-8 mRNA expression after intra-amniotic LPS challenge given 2 days before delivery (intra-amniotic LPS 2- plus 7-day group). The mRNA for the anti-inflammatory cytokine IL-10 did not change in the fetal lung after intra-amniotic LPS exposure (Fig. 2E). Similar to proinflammatory cytokines, TLR4 mRNA expression increased 2.5-fold in the fetal lung at 2 days with a return to control levels by 7 days after exposure to a single injection of intra-amniotic LPS (Fig. 2F). TLR4 mRNA expression in the fetal lung of lambs exposed to repeated intra-amniotic LPS (2- plus 7-day group) was similar to those in the control group. The lack of induction of iNOS and cytokine mRNA expression in response to the repeat-LPS group relative to the single LPS-exposure group is consistent with endotoxin tolerance.


Figure 2
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  		FIGURE 2. Repeated intra-amniotic LPS inhibits lung cytokine mRNA expression. Quantification of: A, IL-1β; B, IL-6; C, IL-8; D, TNF-{alpha}; E, IL-10; and F, TLR4 mRNA from fetal lung normalized to L32 (ribosomal protein mRNA). The mean mRNA signal in control animals was given the value of 1 and levels at each time point were expressed relative to controls. Intra-amniotic LPS (single injection) induced cytokine expression in the lung at 2 days with return to control values 7 days after exposure. Repeated intra-amniotic LPS injections did not induce cytokine mRNA (*, p < 0.05 vs control; t, p < 0.05 vs single LPS 2-day exposure).

 
SAA3 expression in the lung and the liver

SAA3 is a class of acute-phase reactant proteins whose transcription is robustly induced by LPS exposure (27). Control animals had minimal expression of SAA3 mRNA in the lung and the liver (Fig. 3). Compared with controls, preterm lambs exposed to a single injection of intra-amniotic LPS increased SAA3 mRNA by 70-fold in the lung and 30-fold in the liver at 2 days. SAA3 mRNA expression decreased at 7 days after exposure to a single-injection of intra-amniotic LPS. Unlike cytokine mRNA expression, lung SAA3 expression increased after the repeated intra-amniotic LPS exposure (2- plus 7-day group) to values similar to the 2-day group. However, unlike the lung SAA3 expression, induction of the liver SAA3 mRNA was blunted after exposure to repeat intra-amniotic LPS exposure (2- plus 7-day group), indicating a different pattern of LPS responsiveness in the lung and the liver.


Figure 3
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  		FIGURE 3. Lung and liver acute-phase reactant SAA3 mRNA expression. A, Quantification of SAA3 mRNA from fetal lung and, B, liver, normalized to L32 (ribosomal protein mRNA). The mean mRNA signal in control animals was given the value of 1 and levels at each time point were expressed as the increases relative to controls. SAA3 mRNA was induced after repeated intra-amniotic LPS in the fetal lung but not in the fetal liver (*, p < 0.05 vs control; t, p < 0.05 vs single LPS 2-day exposure).

 
Cellular localization of lung SAA3 mRNA expression

We evaluated expression of SAA3 mRNA in the fetal lung using in situ hybridization. SAA3 mRNA expression was not detected in control animals (Fig. 4, A and B) or in lung sections from LPS-treated animals using the sense SAA3 probes (data not shown). Two days after the single injection of intra-amniotic LPS, intense SAA3 mRNA expression was detected in the bronchiolar epithelium (Fig. 4C) and the inflammatory cells (Fig. 4D). Based on the characteristic nuclear morphology, most of the inflammatory cells that expressed SAA3 were identified as neutrophils and the remainder were monocytes (data not shown). SAA3 expression decreased in the bronchiolar epithelium and the inflammatory cells 7 days after a single exposure to intra-amniotic LPS (Fig. 4, E and F). However, after the 2- plus 7-day repeat intra-amniotic LPS exposures, bronchiolar epithelial SAA3 expression was not induced. However, the inflammatory cell SAA3 expression was induced in the repeat LPS-exposed group. This response was distinct from the iNOS expression in these fetal lambs demonstrating selective responsiveness to a second intra-amniotic LPS exposure.


Figure 4
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  		FIGURE 4. Cellular localization of the lung SAA3 mRNA. In situ hybridization was performed with digoxigenin labeled antisense sheep SAA3 riboprobe on lung sections. Control-injected intra-amniotic saline (A and B) did not have detectable SAA3 mRNA expression. Intense SAA3 expression was seen in the bronchiolar epithelium (C) and inflammatory cells (arrow in D and magnified in the inset) 2 days after single-dose LPS. SAA3 mRNA expression decreased in all cell types at 7 days (E and F). Exposure to repeated intra-amniotic LPS (2 + 7 days) had reduced SAA3 expression in the bronchiolar epithelium (G) but similar inflammatory cell SAA3 expression (H) compared with the single LPS 2-day group. Scale bar is 50 µm.

 
Responses of lung and blood monocytes in vitro to LPS

Monocytes from both the lung and the peripheral blood had similar responses to an in vitro LPS challenge. Both control and intra-amniotic single injection 2-day LPS-exposed lung and blood monocytes did not significantly increase IL-6 secretion (Fig. 5, A and C) or H2O2 production (Fig. 5, B and D) in response to an in vitro LPS challenge. This unresponsiveness to LPS suggests immaturity of fetal monocytes. However, 7 days after exposure to intra-amniotic LPS, the monocytes responded to an in vitro LPS-challenge with a 4-fold increase in IL-6 secretion and a 5- to 6-fold increase in H2O2 production compared with monocytes from control lambs, suggesting a functional maturity. In striking contrast, monocytes from the repeat intra-amniotic LPS (2 plus 7 days) exposed lambs failed to increase IL-6 secretion or H2O2 production above baseline in response to in vitro LPS.


Figure 5
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  		FIGURE 5. Response of lung and blood monocytes to an in vitro LPS challenge. Monocytes were purified on Percoll gradients and exposed to 100 ng/ml LPS in vitro for 16 h. Response to LPS was quantified in the lung (A and B) and the blood monocytes (C and D). A and C, IL-6 in the culture medium. B and D, Hydrogen peroxide production in the monocytes. The dashed line indicates IL-6 secretion and H2O2 production in monocytes from control fetuses challenged with medium only in vitro. Fetal lung and blood monocyte response to LPS in vitro were minimal in control and single-dose intra-amniotic LPS-exposed lambs at 2 days. Single-dose intra-amniotic LPS-exposed lambs at 7 days had a robust response. In contrast, fetal monocytes obtained from lambs exposed to repeated intra-amniotic LPS had minimal endotoxin responsiveness (*, p < 0.05 vs control; t, p < 0.05 vs single dose-LPS 7 days; {ddagger}, p < 0.05 vs exposure to medium only).

 
Components of TLR4 signaling

To evaluate some elements that may contribute to decreased LPS responsiveness after repeated intra-amniotic LPS exposure, we quantitated expression of several TLR4-signaling molecules. In the fetal liver, TLR4 mRNA was similar in all the groups (Table IV). Lung and blood monocyte cell surface expression of TLR4 was evaluated by flow cytometry. The lung and blood monocyte TLR4 expression was similar in both the control and all the LPS-exposed fetal lambs (Table IV). The LBP levels in BALF from the single LPS-exposure group were similar to the controls (Table IV). Interestingly, there was a 4-fold increase in BALF LBP levels 7 days after single LPS exposure and a 8-fold increase after repeated intra-amniotic LPS exposure (2- plus 7-day group).


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  		Table IV. TLR4 and LBP expression in the lung, liver, and monocytes

 
IRAK-4 is a positive regulator and IRAK-M is a negative regulator of TLR4 signaling (14, 28). Compared with controls, lung monocytes had a 3-fold increase in IRAK-4 protein and an unchanged IRAK-M protein after a 2- or 7-day single-intra-amniotic LPS exposure (Fig. 6, A and B). In fetal lambs exposed to repeated intra-amniotic LPS (2- plus 7-day group), lung monocytes had a 50% decrease in IRAK-4 protein and a 3-fold increase in IRAK-M protein expression as compared with control monocytes. The blood monocyte IRAK-4 and IRAK-M expressions after single or repeat intra-amniotic LPS exposure were similar to the lung monocyte response (Fig. 6, C and D).


Figure 6
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  		FIGURE 6. Exposures to repeated intra-amniotic LPS decreased IRAK-4 and increased IRAK-M expression in lung and blood monocytes. Immunoblots were quantified for IRAK-4 and IRAK-M expression in lysates from monocytes exposed to LPS 100 ng/ml in vitro for 16 h. A, IRAK-4 in lung monocytes. B, IRAK-4 in blood monocytes. C, IRAK-M in lung monocytes. D, IRAK-M in blood monocytes. Protein densitometry (using 50 µg of total protein) was internally normalized to β-actin and the mean normalized protein densitometry value for controls was assigned a value of 1. The densitometry values from treated lambs were expressed relative to controls. Lung and blood monocytes from both the single-dose intra-amniotic LPS-exposure groups had an increased IRAK-4 and a decreased IRAK-M expression compared with controls. In contrast, compared with the single-dose endotoxin group, lambs exposed to repeated intra-amniotic LPS had decreased IRAK-4 and increased IRAK-M expression (*, p < 0.05 vs control; t, p < 0.05 vs single-dose LPS 2 days or single-dose LPS 7 days).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Chorioamnionitis mediated by intra-amniotic LPS induces unique fetal lung and systemic inflammatory responses (7, 8). The normal fetus has intermittent respirations with occasional gasping. Presumably, the intra-amniotic LPS mixes with the fetal lung fluid and comes in contact with the airway epithelium initiating the lung inflammatory response, because surgical separation of the lung from contact with amniotic fluid containing LPS prevents lung inflammation (9). We previously reported that after intra-amniotic injection of 10 mg of LPS injection (dose used in this study), amniotic fluid levels are on the order of 108 endotoxin units/ml (23), but LPS was not detected in the peripheral blood in this study. Taken together, these results indicate that the airway epithelial inflammation is caused by direct LPS exposure, and systemic inflammation may not be mediated by direct LPS exposure. The fetal responses to LPS exposure of the airway epithelium are very different from response to intravascular LPS injection. For example, intra-amniotic LPS injection doses ranging from 4 to 100 mg induce comparable lung inflammatory responses in the fetal sheep (8) and the fetuses do not have acute systemic responses. In contrast, 10 µg of LPS (a 10,000-fold lower dose), given by the intravascular route, is fatal in the fetal sheep (22, 29). Our study is unique in that, similar to human chorioamnionitis, LPS exposure to the fetus is via the epithelia of the airway, the chorioamnion, and the gastrointestinal tract but not the vascular compartment.

We previously reported that the half-life of LPS in the amniotic fluid compartment is 29 h (23). Therefore, the 10-mg intra-amniotic LPS injection should have decreased ~18-fold at 5 days, at which time the second 10-mg intra-amniotic injection was given in the repeat LPS-exposure group. Decreased cytokine expression in the lung, and decreased acute-phase response gene SAA3 expression in the liver in response to two injections of intra-amniotic LPS demonstrates altered in vivo pulmonary and systemic endotoxin responsiveness in the preterm fetus. Interestingly, while the liver SAA3 responses were decreased, the lung expression of SAA3 mRNA was not blunted after exposure to repeated intra-amniotic LPS. This result demonstrates that the fetal lung recognized the second LPS exposure and indicates that altered expression of a given gene may differ in different tissues in endotoxin tolerance.

The fetal lung SAA3 expression induced by intra-amniotic LPS was detected in the bronchiolar epithelium and the inflammatory cells recruited to the lung (30). Interestingly, the repeat LPS-exposure group in this study had inflammatory cell expression of SAA3 mRNA but not expression in the airway epithelium. Although the SAA3 mRNA expression in the lung inflammatory cells was easily detected in the repeat-endotoxin exposure group, iNOS expression was decreased demonstrating differential regulation of different gene products during endotoxin tolerance in the fetus as has been reported in adult (31, 32, 33). In addition to altered gene expression, pulmonary edema as indicated by increased lung/body weight ratio was detected after a single but not repeated intra-amniotic LPS exposure, suggesting altered global pulmonary effects. Although altered expression of TNF-{alpha} and IL-10 are important components of endotoxin tolerance in the adult (13, 34), we did not find significant changes in lung TNF-{alpha} or IL-10 mRNA expression in fetal sheep following repeated intra-amniotic LPS exposure. Taken together, the findings of altered responsiveness in the lung epithelium and inflammatory cells after exposure to repeated intra-amniotic LPS appears to be a distinct phenomenon in the preterm fetus.

Consistent with our previous results (16), lung and blood monocytes from control lambs had a minimal response to an in vitro endotoxin challenge suggesting functional immaturity. However, 7 days after a single intra-amniotic LPS exposure, both the lung and the blood monocytes significantly increase IL6 secretion and H2O2 production in response to an in vitro LPS challenge. We also recently reported that lung monocytes precociously mature to macrophages after exposure to intra-amniotic LPS (26). Thus, exposure to intra-amniotic LPS can induce maturation of monocyte function. Therefore, these results underscore the importance of interpreting the results of in vitro LPS challenge in the context of two mutually distinct phenomena in the preterm fetus: maturation or priming vs tolerance after LPS exposure.

The proinflammatory cytokine expression and induction of NOSII in lung inflammatory cells is robustly induced 2 days after an in vivo exposure to LPS with a decrease in response at 7 days. However, the monocytes isolated from the lambs exposed to intra-amniotic LPS exposure at 2 days did not significantly increase IL-6 secretion or H2O2 production in response to an additional in vitro LPS challenge. These apparently discrepant responses are likely due to functional immaturity of the fetal monocytes compared with the adult monocytes (16). Taken together with decreased iNOS expression in lung monocytes, these results demonstrate reduced endotoxin responsiveness in the monocytes after repeated intra-amniotic LPS exposure. Alternatively, intra-amniotic LPS could cause monocytes to be refractory to an in vitro LPS challenge because monocytes from the 2-day intra-amniotic LPS-exposure group also did not respond to an in vitro LPS challenge. The study design was to deliver control and experimental animals at 124 days gestation. Therefore, the 7-day LPS group was exposed to intra-amniotic LPS at 117 days, while the 2-day group was exposed to LPS at 122 days. It is unlikely that the different gestations of LPS exposure influenced lung inflammatory responses, because we have previously demonstrated that 110-day gestation lambs had a similar lung inflammatory response compared with 124-day gestation lambs (30). In the adult animals, endotoxin tolerance denotes a reduced in vitro LPS responsiveness after an in vivo LPS exposure (20). Because our findings differ from those in adult animals, we prefer to characterize our results as endotoxin reprogramming in the preterm fetus exposed to repeated intra-amniotic LPS exposure.

Macrophages, monocytes, and dendritic cells are the cell types implicated in the pathogenesis of endotoxin tolerance in the adult (13, 20). Alveolar macrophages are either absent or numerically greatly reduced in preterm fetuses compared with adult animals (17, 18) and very little information is available regarding the dendritic cells in the fetal lung. The initial response of the fetal lung to endotoxin is presumed to be an epithelial response because heat shock protein 70 and SAA3 expression increased in the airway epithelium within 5 h after exposure to intra-amniotic LPS (30, 35). Whether lung dendritic cells, which also come in contact with the airway, also initially respond to intra-amniotic LPS is not known. The availability of species-reacting Abs in the sheep is limited compared with rodents or humans, restricting our ability to evaluate whether the lung monocyte preparation contained dendritic cells. Because the lung monocytic cells in culture had a similar response to an in vitro LPS challenge as the blood monocytes, the monocytes appear to be an important component of the endotoxin reprogramming in the fetus exposed to repeated intra-amniotic LPS injections. Whether the altered airway epithelial signaling is required for the induction of reprogramming in the monocytes and neutrophils in the fetal lung remains to be studied.

We also demonstrated endotoxin reprogramming in the fetal liver. Whether the liver response is secondary to the lung responses to intra-amniotic LPS is not known. The altered responsiveness to LPS in other fetal organ systems was not studied. Nevertheless, the findings suggest complex interactions between the fetal lung, circulating leukocytes, and the fetal liver when LPS is delivered by the intra-amniotic route.

Both intracellular negative regulators and extracellular soluble factors have been implicated in the mechanism of endotoxin tolerance. Extracellular/humoral factors that potentially mediate endotoxin signaling include steroid hormones (36), heat shock protein 70 (37), and IL-10 (38). The plasma cortisol levels and lung expression of IL-10 mRNA did not change significantly in these fetal lambs, suggesting other mechanisms of endotoxin tolerance in these animals. LBP levels have been reported to either not change or increase during endotoxin tolerance (14). The LBP levels in the BALF increased, however, the downstream positive regulator IRAK-4 levels decreased and the negative regulator IRAK-M expression increased in lung and endotoxin tolerant blood monocytes, suggesting a postreceptor mechanism of endotoxin reprogramming. A growing list of intracellular mediators including MyD88 short (39), IRAK-M (28), single immunoglobulin IL-1 protein (40), suppressor of cytokine signaling-1 (41), and others have been proposed as mediators of endotoxin tolerance. The precise molecular pathways to endotoxin reprogramming in the fetuses remain to be identified.

There are several clinical implications from these results. Chronic or repeated exposure to Gram-negative bacterial chorioamnionitis may induce endotoxin tolerance which may limit inflammation induced lung and systemic injury responses. Indeed, we have reported that preterm lamb fetuses exposed to a single injection of intra-amniotic LPS had lung remodeling similar to bronchopulmonary dysplasia, but exposures to repeated intra-amniotic LPS did not result in progressive lung remodeling (10, 11, 12). In contrast, endotoxin tolerance may inhibit host responses to invading pathogens. Endotoxin tolerance increases the risk of infections and mortality and thus is maladaptive in the intensive care setting (42). Approximately 25% of very low birth weight preterm neonates develop postnatal nosocomial sepsis (43). However, it is not known whether exposure to chorioamnionitis can increase the susceptibility of preterm infant to nosocomial sepsis.

In summary, the experiments demonstrate a novel finding that repeated exposures to intra-amniotic LPS induce lung and systemic endotoxin reprogramming, demonstrated by altered in vivo and in vitro LPS responsiveness in a preterm fetus.


   Acknowledgments
 
We thank Amy Whitescarver, Daniele Herbst, and Silvia Seidenspinner for their expert technical assistance.


   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 work was supported by National Institutes of Health K08 HL 70711 (to S.G.K.), AI-069716 and HL-65397 (to A.H.J.), Grant A-27 of the Interdisciplinary Center for Clinical Research, University of Wurzburg/Germany (to B.W.K.), and CDA-303261 from the National Health and Medical Research Council, Australia (to T.J.M.M.). Back

2 Address correspondence and reprint requests to Dr. Suhas G. Kallapur, Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address: suhas.kallapur{at}cchmc.org Back

3 Abbreviations used in this paper: FIRS, fetal inflammatory response syndrome; BALF, bronchoalveolar fluid; LBP, LPS-binding protein; SAA3, serum amyloid A3; iNOS, inducible NO synthase; IRAK, IL-1R-associated kinase. Back

Received for publication July 27, 2007. Accepted for publication October 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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