Fetal responses to lipopolysaccharide-induced chorioamnionitis alter immune and airway responses in...

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BASIC SCIENCE: OBSTETRICS

Fetal responses to lipopolysaccharide-inducedchorioamnionitis alter immune and airwayresponses in 7-week-old sheepAndrea J. X. Lee, MSc; Verena A. C. Lambermont, MSc; J. Jane Pillow, PhD; Graeme R. Polglase, PhD;Ilias Nitsos, PhD; John P. Newnham, MD; Manfred W. Beilharz, PhD; Suhas G. Kallapur, MD;Alan H. Jobe, MD, PhD; Boris W. Kramer, MD, PhD

OBJECTIVE: We hypothesized that fetal innate immune responses to li-popolysaccharide-induced chorioamnionitis would alter postnatal sys-temic immune and airway responsiveness.

STUDY DESIGN: Ewes received intraamniotic injections with saline orlipopolysaccharide at 90, 100, and 110 days of gestation. Immune sta-tus and airway responsiveness were evaluated at term and at 7 weeksof age.

RESULTS: At term, lymphocytes, monocytes, and neutrophils were sig-nificantly increased (respectively, 24-fold, 127-fold, and 31,000-fold)
in lungs and blood monocytes became Toll-like receptor 2 responsive
responses in 7-week-old sheep. Am J Obstet Gynecol 2011;204:364.e17-24.

0002-9378/$36.00 • © 2011 Mosby, Inc. All rights reserved. • doi: 10.1016

after lipopolysaccharide exposures. Furthermore, CD4 and CD4/CD25lymphocytes were increased in thymus and lymph nodes. At 7 weeks,airway reactivity decreased and concentrations of CD8 cytotoxic T lym-phocytes changed in the lungs and thymus relative to controls.

CONCLUSION: Early gestational lipopolysaccharide exposure increasedleukocyte responsiveness at term. Decreased airway reactivity andchanges in lymphocytes at 7 weeks postnatal demonstrate persistenteffects of fetal exposure to LPS.

Key words: airway reactivity, hygiene hypothesis, inflammation,
preterm
Cite this article as: Lee AJX, Lambermont VAC, Pillow JJ, et al. Fetal responses to lipopolysaccharide-induced chorioamnionitis alter immune and airway

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Preterm birth (�37 weeks) is fre-quently associated with chorio-

amnionitis.1 Recently, chorioamnionitisas correlated with a 4-fold increased

isk for developing asthma in pretermnfants after correction for confoundingactors.2-4 This observation is in contrast

to the hygiene hypothesis,5-7 in which in-fections during early childhood are asso-ciated with a decreased risk of subse-quent asthma and allergic disease. Theseconflicting observations suggest that the

From the School of Women’s and Infant’s HealNewham, Kallapur, and Jobe), School of BiomoBeilharz), The University of Western Australia,Lambermont and Dr Kramer), School of OncolUniversity Medical Center, Maastricht, The NetCenter (Drs Kallapur and Jobe), University of C

Received July 9, 2010; revised Sept. 29, 2010;

Reprints not available from the authors.

Supported by AI-069716 from the National InstiAustralia) grant, a Viertel Senior Medical Researc(G.R.P.), and the Women and Infants Research016.096.141 from the Dutch Scientific ResearchOncology (GROW), The Netherlands.

A.J.X.L and V.A.C.L contributed equally.

time of exposure to bacterial productsduring childhood may have different ef-fects when compared with antenatal ex-posures. For example, antenatal expo-sure to bacterial products may increasethe risk of asthma in childhood, whereaspostnatal exposure to bacterial productsmay protect from asthma. The mecha-nisms to explain the different outcomesare still unclear.

Chorioamnionitis-mediated inflam-mation exposes the fetus to proinflam-

Ms Lee, and Drs Pillow, Polglase, Nitsos,lar and Biochemical Sciences (Ms Lee and Drh, Australia; Department of Paediatrics (Msand Developmental Biology, Maastrichtlands; Cincinnati Children’s Hospital Medicalnnati, Cincinnati, Ohio.

epted Nov. 2, 2010.

of Health; an Asthma Foundation (Westernellowship (J.J.P.), a NHFA/NHMRC Fellowshipndation, Western Australia; VENI BWKganization and the Research School for

/j.ajog.2010.11.015

APRIL 2011 American

matory mediators primarily by fetalbreathing and swallowing of amnioticfluid.8-10 The fetal lung responds tomediators such as lipopolysaccharide(LPS), interleukin-1 (IL-1), and liveureaplasma with a local inflammatoryresponse. Previous experiments re-vealed that exposure to chorioamnioni-tis changes the lymphocyte populationin the posterior mediastinal lymph node(PMLN) that drains the lung11,12 andcauses systemic immune modulation ofblood monocytes.13-15 Furthermore, thegut responses to LPS with altered inflam-matory cells in the gut wall and regionallymph nodes.8,16 These acute fetal re-ponses were modulated further with re-eated exposure to induce LPS-tolerancend cross-tolerance to other proinflam-atory Toll-like receptor (TLR) ago-

ists.13-15 However, the relationshipbetween exposure to antenatal inflam-mation and postnatal disease is con-founded by gestational age and neonatallung disease in human studies.

We hypothesized that fetal innateimmune responses to LPS-induced cho-

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temic immune and airway responsive-ness resembling asthma later in life. Totest this hypothesis, we evaluated im-mune status at birth and at 7 weeks afterLPS-mediated chorioamnionitis. Weevaluated leukocytes in the bronchoal-veolar lavage fluid (BALF). Transform-ing growth factor-beta 1 (TGF-�1) andelastin were measured in lung tissue.CD4, CD8, CD4/CD25, and gamma-delta subpopulations of T lymphocyteswere measured in the PMLN and thy-mus. Monocytes isolated from bloodwere tested in vitro for responses to LPSand PamCysK4 (a ligand for TLRs 1and 2). The immunologic measurementswere performed at term and at 7 weeks ofage and airway reactivity to metacholine(MCh) was tested at 7 weeks after inha-lation of house dust mite (HDM).

MATERIALS AND METHODSAntenatal treatmentAll animal procedures were approved bythe Animal Ethics Committee of theUniversity of Western Australia, Austra-lia. Ewes received intraamniotic (IA) in-

FIGURE 1Timing of fetal exposure to LPS

Open arrows indicate gestational age of intraamnwith LPS-induced chorioamnionitis. Operative deanimals (n � 3-5 lambs), whereas the remaininglambs were studied at 7 weeks. Immune status wat 7 weeks.LPS, lipopolysaccharide.

Lee. Fetal effects of chorioamnionitis on 7-week-old sheep. Am

jections with saline (controls) or 10 mg

364.e18 American Journal of Obstetrics & Gyneco

Escherichia coli 055:B5 endotoxin (LPS;Sigma-Aldrich, St. Louis, MO) at 90,100, and 110 days of gestation (Figure 1).At 147 days of gestation, lambs from LPS(n � 5) and control (n � 3) groups were

elivered surgically to assess immuneunction of the fetus at term (term is ap-roximately 147 days). The remainingwes delivered spontaneously and air-ay responsiveness and immune statusf lambs were evaluated at 7 weeks (LPS� 5, Control n � 6). Lambs were killedith intravascular pentobarbital at term

nd at 7 weeks.

Airway reactivityAt 7 weeks lambs were sedated 2 days af-ter HDM exposure (nebulized 1 mg)with diazepam 0.25 mg/kg (Sigma NSW,Australia) and Ketamine 5 mg/kg (Par-nell Laboratories, NSW, Australia). Theywere intubated with a 6.0 mm ID tra-cheal tube (Portex Ltd, UK) and venti-lated (Humming V, Metran, Japan) witha peak inspiratory pressure of 25 cmH2O, an inspiratory time 0.7 second anda rate of 40 breaths per minutes. A con-tinuous infusion of intravenous Propo-

c (IA) injections with LPS or saline. Sheep modely was performed at 147 days gestation for somees (n � 5-6) delivered spontaneously and theirvaluated in postmortem tissues at 147 days and

bstet Gynecol 2011.

fol (0.3-0.6 mg/kg/h, Repose, 0.1 mg/kg/ s

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min; Norbrook Laboratories Ltd., Victo-ria, Australia) and Remifentanil (0.3 mg/kg/h, Ultiva 0.05 �g/kg/min; Glaxo-

mithKline, Victoria, Australia) wasommenced, and following confirma-ion of deep anesthesia, neuromuscularlockade was achieved with vecuronium0.1 mg/kg IV; Essex Pharma, Germany).ambs were stabilized for 10 minutes be-

ore baseline measurements, and theneceived for 1 minute aerosols (1 mL) at

minute intervals of saline, followedy increasing concentrations (0.01%,.03%, 0.1%, 0.3%, 1%; w/v) of metha-holine (Acetyl-�-methylcholine chlo-ide; Sigma). Partitioned respiratorympedance (Zrs) was measured usinghe low-frequency forced oscillationechnique (LFOT) using the FlexiVentModule 5; Scireq, Montreal, Canada).fter airway occlusion at commence-ent of expiration, respiratory system

nput impedance (Zrs) measurementsere obtained using a primewave (17utually prime frequencies from 0.5-

9.75 Hz) over a 6-second apneic inter-al. Measurements were repeated every0 seconds until peak response wasbserved (normally approximately 3inutes).Measurements of pressure and flowere transformed to the frequency do-ain and corrected for the impedance of

he tracheal tube and measurement sys-em. The partitioned airway (airway re-istance � Raw; airway inertance � Iaw)nd tissue mechanical variables (tissueamping – G; tissue elastance � H) wereetermined by fitting the resultant im-edance spectrum to the constant phaseodel.17 Hysteresivity (�) was calcu-

lated as G/H. Changes in airway and tis-sue variables were expressed relative tomeasurements obtained at baseline.

Bronchoalveolar lavageAfter collection of the lungs, thymus,and the PMLN, the left lung was lavaged3 times with 0.9% NaCl.18 The BALFs

ere pooled and centrifuged at 500 rpmor 5 minutes. Differential cell countsere performed on cytospin prepara-

ions after staining with Pappenheim-

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Lung tissueLung tissue from the right lower lobe(RLL) was snap frozen and stored at�80°C. For homogenization, a mix of ly-sis buffer (RIPA buffer, Sigma) and pro-tease inhibitor (Sigma) was added tolung tissue. The lung tissue was homog-enized (PRO Quick Connect Generatorspart no. 02-07095; PRO Scientific Inc.,Oxford, CT) and centrifuged at 12 rcf(11.4 rpm) for 5 minutes at 4°C.

Enzyme-linked immunosorbentassay of TGF-�1

Free, bound, and total TGF-�1 (referredto R&D enzyme-linked immunosorbentassay [ELISA] kit; Minneapolis, MN asactive, latent, and total TGF�1) were

easured with R&D ELISA kits (DuoSetLISA, human TGF/�1, no. DY240;&D Systems). Free TGF-�1 was mea-

sured for the original sample. TotalTGF-�1 was measured after acid activa-tion of 150 �L of the sample with 30 �L 1

HCl for 10 minutes at room tempera-ure and 33 �L 1 M NaOH HEPES was

used to stop the activation.19 BoundGF-�1 was calculated as the differenceetween total TGF-� and free TGF-�1.

Elastin stainingWe measured elastin foci of secondarycrests (concentrated vs nonconcen-trated) and elastin fiber deposition in thevessel walls. Elastin fibers were stainedblack with a Hart’s resorcin fuchsin so-lution,20-22 using paraffin slides that

ere dehydrated and stained at 60°C andounterstained with a tartrazine solu-

TABLE 1Physiology

Antenatal treatment Body weight, kg

At term..........................................................................................................

Control 5.0 � 1.0..........................................................................................................

LPS 5.5 � 1.0...................................................................................................................

7 weeks..........................................................................................................

Control 16.2 � 1.3..........................................................................................................

LPS 15.5 � 1.5...................................................................................................................

Body weight, organ weights, and number of animals studied acontrol group.LPS, lipopolysaccharide; PMLN, posterior mediastinal lymph n

Lee. Fetal effects of chorioamnionitis on 7-week-old sheep

ion. A magnification of 20� was used to h

ave 3 pictures randomly. Counting oflastin foci was blinded, using ImageJ.41o software (Rasband; National Insti-utes of Health, Bethesda, MD) foruantification.

Flow cytometrySingle cell suspensions of thymus andPMLN were made with a strainer, as-sessed for viability, and counted usinga Neubauer chamber (Hawksley, Eng-land).23 The 106 cells were incubated

ith primary monoclonal antibodiesmAbs: CD4, CD8, CD25, and TcR-1 re-eptor [gamma-delta T lymphocytes];MRD USA) for 30 minutes at 4°C. Cellsere washed and incubated with fluores-

ence-conjugated secondary mAbs for0 minutes at 4°C in the dark (FITCnd R-PE labeled antibodies fromEROTEC, Great Britain). Cells werenalysed on a FACScalibur instrumentBecton Dickinson, Franklin Lakes, NJ)sing CellQuest software (Becton Dick-

nson). CD4/CD25 percentages are ex-ressed relative to the population of cellsxpressing CD4. Other lymphocyte per-entages are expressed relative to the to-al population of thymic lymphocytes.11

Hydrogen peroxide assayfor blood monocytesMonocytes and macrophages were iso-lated with Percoll gradients from cordblood (at term) and peripheral blood (at7 weeks) to perform functional tests.15 In

rief, the isolated cells were cultured inPMI 1640 media supplemented by 10%eat-inactivated fetal calf serum for 6

PMLN, g/kg Thymus, g/kg Splee

.........................................................................................................................

0.13 � 0.01 2.07 � 1.07 1.37 �.........................................................................................................................

0.21 � 0.02 3.59 � 0.43 1.74 �.........................................................................................................................

.........................................................................................................................

0.23 � 0.02 2.52 � 0.19 4.20 �.........................................................................................................................

0.26 � 0.02 2.68 � 0.48 4.62 �.........................................................................................................................

h and at 7 weeks of age. Organ weights are standarized per kilog

J Obstet Gynecol 2011.

ours with LPS (a ligand for TLR 4; 100

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g/mL; E coli O55:B5; Sigma-Aldrich)nd PamCysK4 (a synthetic ligand forLRs 1 and 2; 5 �g/mL; EMC Microcol-

ection, Tuebingen, Germany). Pam-ysK4 signals through the TLR2 path-ay and is involved in maturation of the

mmune system. Control cells were ex-osed to saline (media). After washinghe cells with phosphate buffered salinePBS), the production of hydrogen per-xide by 1 � 106 monocytes was mea-

sured with an assay based on the oxida-tion of ferrous iron (Fe2�) to ferric iron(Fe3�) by hydrogen peroxide undercidic conditions (Bioxytech H2O2-560ssay; OXIS International, Portland,R).13,15

Statistical analysisMicrosoft Excel and GraphPad Prism 5were used for statistics. Data are shownas mean � SEM. Statistical differences

etween LPS and control groups werevaluated with Student t test and the

ann-Whitney test. Values were con-idered significant if P � .05 vs the con-rol (saline) group.

RESULTSBody and organ weights at birthand at 7 weeks of ageThe body weights of LPS-exposed lambswere similar to the control group at termand at 7 weeks (Table 1) indicating sim-ilar growth and development. The organweights (PMLN, thymus, spleen, totallung) were not different for the groups at

/kg Total lung, g/kg Animals, n

..................................................................................................................

18 35.3 � 3.3 3..................................................................................................................

20 31.8 � 2.4 5..................................................................................................................

..................................................................................................................

30 12.9 � 0.4 6..................................................................................................................

50 13.7 � 0.4 5..................................................................................................................

body weight. There were no differences to corresponding

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Airway reactivityMeasurements with methacholine chal-lenge at 7 weeks, including heart rate,peak inspiratory pressure, respiratoryrate, and blood gas values (PaCO2,

aO2) were not different between fetalLPS exposed and control lambs (Table2). Representative impedance spectra areshown in Figure 2. There were no differ-

FIGURE 2Dose response curves to inhaled m

Responses to inhaled methacholine are expresseold lambs that were exposed to intraamniotic injec

, tissue damping, C, tissue elastance, and Deactivity. Airway resistance (Raw; P � .008) anecreased with 1% w/v MCh. Tissue elastance

differences compared with the control group.LPS, lipopolysaccharide; MCh, metacholine.


TABLE 2Ventilatory variables of lambs duri

Treatment PaO2, mm Hg

Saline (7 wk) 96 � 4...................................................................................................................

LPS (7 wk) 98 � 1...................................................................................................................

HR, heart rate; LPS, lipopolysaccharide; PaCO2, arterial carbo

Lee. Fetal effects of chorioamnionitis on 7-week-old sheep


ences in baseline measurements betweenthe groups. Airway resistance (Raw; P �008; Figure 2, A) and tissue damping (G;� .037; Figure 2, B) did not increase at

1% w/v MCh in LPS-exposed lambs,whereas they increased in the saline con-trol animals, indicating decreased airwayreactivity with fetal LPS exposure. LPS-exposed lambs and control lambs did not

acholine

elative to measurements at baseline for 7-weeks of LPS or saline (control). A, Airway resistance,

ysteresivity were measured to test the airwayssue damping (G; P � .037) were significantly; P � .151) and hysteresivity (�) showed no

bstet Gynecol 2011.

methacholine challenge

PaCO2, mm Hg RR, breaths/mi

53 � 4 26.5 � 0.5.........................................................................................................................

51 � 1 25.1 � 0.7.........................................................................................................................

xide tension; PaO2, arterial oxide tension; PIP, peak-inflation pres

J Obstet Gynecol 2011.

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respond to lower MCh dosing. Tissueelastance (H; Figure 2, C) and hysteresiv-ity (�; Figure 2, D) were not different atany MCh concentration between LPS-exposed and control lambs.

Pulmonary inflammationMonocytes, lymphocytes, and neutro-phils were greatly increased in the BALFfrom the LPS-exposed term fetal lambsrelative to the controls 37 days after thelast IA injection (P � .001 vs control,Figure 3, A). However, by 7 weeks therewere no significant differences in thenumber of inflammatory cells in BALFfor the LPS-exposed and control lambs(Figure 3, B).

Hydrogen peroxide responsesof blood monocytesAfter stimulation in vitro with LPS,blood monocytes from control termlambs had no increased response, but af-ter PamCysK4 stimulation a 6-fold in-crease was present (Figure 4, A). At 7weeks, blood monocytes from controllambs responded to LPS and PamCysK4(16-fold and 14-fold increases respec-tively; Figure 4, B). This change ofmonocyte responses between term and 7weeks is explained by the maturation ofthe innate immune system in early post-natal life. LPS-exposed animals had a dif-ferent monocyte response pattern. Atterm a stimulation in vitro with LPS andPamCysK4 increased H2O2 5-fold and9-fold, respectively (Figure 4, A). Thisincrease was still present at 7 weeks:6-fold increase with LPS and 5-fold in-crease with PamCysK4 (Figure 4, B).Comparing term LPS-exposed lambsand control lambs after a specific stimu-lation, an increased monocyte responsewas only present after stimulation withPamCysK4 (Figure 4, A). At 7 weeks, no

PIP, cm H2O HR, bpm

25.8 � 1.9 175 � 12..................................................................................................................

26.8 � 2.3 161 � 7..................................................................................................................

; RR, respiratory rate.

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after PamCysK4 or LPS in vitro stimula-tion in LPS-exposed lambs when com-pared with control lambs (Figure 4, B).

TGF-�1 in lung tissueAt term large amounts of bound TGF-�1

were detectable with only a small con-centration of free TGF-�1, in lung tissue

omogenate of the control group (Fig-re 5, A). In LPS-exposed fetuses, boundGF-�1 in lung homogenate was de-reased relative to controls at term (P �002). However, there was no increase inree TGF-�1, which is the biologic activeomponent of TGF- �1 that can impair

lung development and cause inflamma-tion and airway remodeling. At 7 weeksof age, free TGF-�1 concentration wasincreased in the lung of LPS-exposedlambs (P � .048, Figure 5, B).

ElastinAt term, control and LPS-exposed lambshad a normal amount of elastin stainingas concentrated elastin foci, with no dif-ferences between groups (Figure 6, A)and no differences in the elastin deposi-tion in the blood vessel walls (Figure 6, Band C). At 7 weeks, about 80% of theelastin was nonconcentrated in both an-imal groups, indicating lungs alveo-larization and maturation (Figure 6,D).There was less elastin deposition inthe blood vessel walls of the LPS-exposedlungs at 7 weeks of age than in controls(Figure 6, E and F).

Lymphocytes in thethymus and PMLNLymphocyte populations in the thymusand PMLN had an increased percentage ofCD4 positive lymphocytes after LPS expo-sure at term (Figure 7, A). CD8 positiveand gamma-delta positive lymphocyteswere not different. In contrast, at 7 weeks(Figure 7, B), percentages of CD4 positivewere not different in the LPS-exposedlambs compared with the control group.However, in the thymus, there was a lowerpercentage of CD8 positive lymphocytesand in the PMLN CD8 positive lympho-cytes were increased. There was no effect ofLPS exposure on gamma-delta positivelymphocytes in thymus and PMLN at 7weeks of age. Fetal LPS-induced inflam-mation was associated with an increase in
CD4/CD25 double positive lymphocytes
(Figure 7, C) in both thymus and PMLN atterm. After 7 weeks, no differences weremeasured in the thymus. However, thepercentage of CD4/CD25 double positivewas decreased in PMLN after LPSexposure.

COMMENTIn this study, we examined the effects offetal chorioamnionitis induced by 3 IA

FIGURE 3Leukocytes in BALF from lambs at

Leukocytes in BALF from A, lambs at term and Bneutrophils were increased (P � .001 vs controsignificant differences were evident at 7 weeks,present at term.BALF, bronchoalveolar lavage fluid; LPS, lipopolysaccharide.


FIGURE 4Hydrogen peroxide production by b

Hydrogen peroxide production by blood monocyydrogen peroxide production was significantly inweeks of age, this response was reversed, sug

LPS, lipopolysaccharide.

* vs media; � vs control group; (o) LPS vs PamCysK4.


APRIL 2011 American

injections of LPS from 90 to 110 days’gestation on sheep at term and at 7 weeksof age. In previous experiments was re-ported profound changes in the pulmo-nary immune system after fetal exposureto LPS with an increase of immune cellsand subsequent maturation of systemicmonocytes into PU.1 expressing macro-phages.24,25 In the current study, we alsonoticed significant increases in pulmo-

m and at 7 weeks of age

t 7 weeks of age. Monocytes, lymphocytes, andthe LPS-exposed group at term. However, no

cating resolution of the pulmonary inflammation

bstet Gynecol 2011.

d monocytes

A, at term and B, at 7 weeks of age. At term,ased after stimulation of cells with PamCysK4. Atsting maturation of the immune system.

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364.e22 American Journal of Obstetrics & Gynecology APRIL 2011

nary immune cell populations, neutro-phils in particular at term. This is re-markable as the fetal LPS exposure was37 days before the increased neutrophilswere detected. Neutrophils were not in-creased at 7 weeks of age. Monocytes andlymphocytes numbers that were elevatedat term remained elevated at 7 weeks ofage in lambs exposed as fetuses to LPS.These changes in leukocyte populationsmay indicate an accelerated develop-ment of the pulmonary immune systemafter LPS-induction.

At the systemic level, IA LPS also in-duced an inflammatory response.18 Previ-

usly, our group showed that the func-ional response of blood monocytes fromreterm LPS-exposed lambs were similaro the blood monocytes of adult sheep.13,14

A functional assay in the current studydemonstrated increased H2O2 productionafter PamCysK4 (a synthetic ligand forTLRs 1 and 2) stimulation of blood mono-cytes at term in fetal LPS-exposed animalscompared with controls. This result dem-onstrated that blood monocytes at termwere competent to respond to TLR2, be-cause of the fetal LPS exposure. At 7 weeks,controls and LPS-exposed lambs were nowsimilarly able to respond to PamCysK4stimulation.

Changes in lymphocyte populationsof the thymus and the PMLN of LPS-exposed preterm/fetal sheep were dem-onstrated in previous experiments.11

Furthermore, LPS-induced chorioam-nionitis resulted in a reduced size of thethymus.23 In the current study, the per-centage of cytotoxic lymphocytes de-creased in the thymus after fetal LPS ex-posure, but increased in the PMLN ofLPS-exposed lambs. This could be ex-plained by the migration of immune ef-fector cells to the site of inflammation.In addition, the percentage of CD4�

CD25� T lymphocytes were significantlydecreased in the PMLN suggesting lesssuppression of effector cells by regula-tory T lymphocytes (Tregs). In the liter-ature, it was proposed that a decrease inTregs may play a role in the dysregu-lation of airway inflammation inasthma.6,26,27 Normally, Tregs can sup-press established airway inflammationand airway hyperresponsiveness. Schaub

FIGURE 5TGF-�1 was measured in homogenates of lung tissue from lamb

TGF-�1 was measured in homogenates of lung tissue from lambs A, at term and B, after 7 weeks ofge. At term, the concentration of total and bound TGF-�1 were significantly decreased after LPS

exposure, but free TGF-�1 was unchanged. At 7 weeks a significant increase of free TGF-�1 wasetected in the LPS group, but no differences in bound or total TGF-�1 were found.

LPS, lipopolysaccharide; n.s., not significant; TGF-�1, transforming growth factor-beta 1.

ee. Fetal effects of chorioamnionitis on 7-week-old sheep. Am J Obstet Gynecol 2011.

FIGURE 6Elastin in lung tissue from lambs

Elastin (stained in black ) in lung tissue from lambs A, C, at term and B, D, at 7 weeks of age. A, B,, Elastin foci were distinguished between concentrated (black arrow ) and nonconcentrated (red

arrow ) foci. C, D, F, Elastin scoring in the vessel wall was performed on a scale from 0-3. At 7 weeks,ess elastic fibers were present in the lungs after exposure to LPS. No differences in elastic foci wereoticed at term and at 7 weeks of age.

LPS, lipopolysaccharide.

Lee. Fetal effects of chorioamnionitis on 7-week-old sheep. Am J Obstet Gynecol 2011.

et al28 demonstrated that cord blood

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from offspring of atopic mothers hadfewer innate-induced Tregs, indicating apotential mechanism by which intra-uterine immune modulation can occurand may influence further atopic diseasedevelopment. It also points to the intra-uterine environment as a critical im-mune modulator. Interestingly, Szépfa-lusi et al29 demonstrated that allergenscould be transferred across the placenta,supporting the theory that fetal T lym-phocytes are exposed during gestation tomaternally derived allergens and that theuterus may not be completely sterile.However, the effect of antenatal andpostnatal exposure to allergens may havereinforcing effects because exposure toallergens early in life enhanced the devel-opment of airway hyperresponsivenessand impairment of lung function atschool age.30

In our study, we tested the airway re-sponsiveness to metacholine in lambs at7 weeks of age. Airway resistance and tis-sue damping did not increase in LPS-ex-posed lambs at the highest MCh dosage(1.0% MCh) as it did in the controls. In-terestingly, these results of decreased air-way reactivity are in contrast with clini-cal observations of increased asthmaafter chorioamnionitis exposure.2 How-ver, airway reactivity and immune dys-unction of the lungs do play a role in theevelopment of asthma. The currenttudy demonstrated structural changesn the vascular wall of the lungs. Lesslastin was detected in blood vessels butore in lung parenchyma. This findingas surprising because elastin deposi-

ion is typically increased in blood vesselsn ventilated animal models of lung in-ury.31 The low pulmonary blood flow intero and spontaneous breathing afterirth are not comparable to ventilatednimal models to our model. Fur-hermore, decreased concentrations ofound and total TGF-�1 were measured

after chorioamnionitis at term. We spec-ulate that the altered deposition of elas-tin in the vessel wall may be the result ofthe structural changes initiated beforebirth and that this is partially mediatedby TGF-�1. However, these changes donot explain the decreased airway reactiv-ity. In real life, children are exposed to
many allergens, which are additional t
stimulators for airway reactivity and thelater development of asthma. The aller-gen exposure was not controlled in thesefree living lambs to 7 weeks of age. Inter-estingly, Eder et al32 reported that expo-sure to LPS in early life protects againstallergic sensitization, but not againstasthma. This might be an explanation forthe different outcome of airway reactiv-ity in our study. Bischof et al33 demon-trated that age is important for asthmatudies in sheep. Significant effects in theirway responsiveness were measurednly after 5 months of age. Our studies atweeks may have been in animals thatere too young, altered responses to fetal

xposure have not been tested previ-usly. Another variable that may changehe postnatal outcome is the gestationalime of fetal exposure to a proinflamma-

FIGURE 7Percentages of specific T lymphoc

A, At term, the percentages of CD4 lymphocytes(34% and 46%, respectively) of LPS-exposed lagamma-delta lymphocytes were not different. B, Tby 7 weeks (P � .039) in the thymus of the LPSncreased (P � .028) in PMLN. CD4 and gammaat 7 weeks. C, CD4 coexpressing CD25 lymphocy16%, respectively, at term. In the thymus no signHowever, in the PMLN a significant decrease of CLPS-exposed lambs at 7 weeks (P � .02).LPS, lipopolysaccharide; PMLN, posterior mediastinal lymph node

ee. Fetal effects of chorioamnionitis on 7-week-old sheep. Am

ory stimuli.

APRIL 2011 American

In conclusion, our results demon-trate that early fetal exposure to LPSaused residual lung inflammation, buthe pulmonary inflammation had re-olved by 7 weeks. However, minortructural and functional changes, de-reased airway reactivity, and changes inymphocytes were noted at 7 weeks. Thelinical relevance of these results is theemonstration that fetal exposure tohorioamnionitis can modulate postna-al immune status and responses. f

ACKNOWLEDGMENTWe thank Jennifer Collins for helpful commentson the manuscript.

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re increased significantly in thymus and PMLN. After chorioamnionitis, CD8 lymphocytes andpercentage of CD8 lymphocytes were decreasedposed lambs. However, CD8 lymphocytes werelta lymphocytes showed no significant changeswere increased in thymus and PMLN of 12% andant changes were present after 7 weeks of age.lymphocytes coexpressing CD25 was noticed in

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yte

wembshe-ex-detesificD4

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https://www.researchgate.net/publication/12502140_Intrauterine_Infection_and_Preterm_Delivery?el=1_x_8&enrichId=rgreq-4c520448-cabf-436f-8f21-4665199127d6&enrichSource=Y292ZXJQYWdlOzQ5Nzc2NDU2O0FTOjI3MzUzOTI2MTUzMDEzMEAxNDQyMjI4MjUwNTA0




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