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Smoking During Pregnancy

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Smoking During Pregnancy
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  BASIC SCIENCE: OBSTETRICS Smoking during pregnancy influences the maternalimmune response in mice and humans Jelmer R. Prins, MD; Machteld N. Hylkema, PhD; Jan Jaap H. M. Erwich, PhD; Sippie Huitema;Gerjan J. Dekkema; Frank E. Dijkstra; Marijke M. Faas, PhD; Barbro N. Melgert, PhD OBJECTIVE:  Duringpregnancythematernalimmunesystemhastoadaptits response to accommodate the fetus. The objective of this study was toanalyzetheeffectsofsmokingonthematernalimmunesystem. STUDY DESIGN:  First-trimester decidual tissue and peripheral blood ofsmokingandnonsmokingwomenwereanalyzedbyrealtimereversetran-scription–polymerase chain reaction (RT-PCR) and flow cytometry. Amouse model was used to further analyze the effects of smoking. Murinetissue was analyzed by flow cytometry, real-time RT-PCR, andimmunohistochemistry. RESULTS:  Smoking caused lower percentages of viable pups in miceand lower birthweights in humans. Smoking mothers, both mice andhuman,hadmorenaturalkillercellsandinflammatorymacrophageslo-cally, whereas systemically they had lower percentages of regulatory Tcells than nonsmoking controls. CONCLUSION:  Maternal smoke exposure during pregnancy influenceslocal and systemic immune responses in both women and mice. Suchchanges may be involved in adverse pregnancy outcomes in smokingindividuals. Key words:  macrophages, maternal immune system, natural killercells, smoking Cite this article as: Prins JR, Hylkema MN, Erwich JJHM, et al. Smoking during pregnancy influences the maternal immune response in mice and humans. Am JObstet Gynecol 2012;207:76.e1-14. D espite the well-known fact thatsmoking adversely affects preg-nancyoutcome, 1 about30%ofpregnantwomen still smoke in The Netherlands. 2,3 Fetal reactions to cigarette smoke were al-ready described in 1935. 4 After a motherstarted smoking a cigarette, an increase infetal heart rate was found, and it was con-cluded that a toxic agent, probably nico-tine, crossed the placenta to the fetus. 4 Currently, it is clear that smoking duringpregnancy causes lower birth weights,an increased incidence of spontaneousabortions and preterm birth, increasedplacental pathology,andincreasedstillbirthrates. 5-9 Interestingly,ithasalsobeenshownthatsmokingwomenhavealowerincidenceof  pregnancies complicated by preeclamp-sia. 10 The mechanisms responsible for thetoxic and protective effects of smoking onpregnancy and preeclampsia are not yetunderstood.Tobaccosmokecontainsmanysubstancesand may affect the fetus directly, but it may also affect placental development and de-cidualization. 1,11,12 Alternatively, smokingmayachieveitseffectsbymodulationofma-ternalimmunologicalandendocrineparam-eters. During pregnancy the maternal im-munesystemplaysakeyroleinthesuccessof pregnancybyadaptingtoaccommodatethesemiallogeneic fetus. 13 Adaptations of thematernalimmunesystemtakeplacelocallyattheimplantationsiteaswellasintheperiph-eralcirculation.Disturbancesinthisadapta-tiontowardmaternaltolerancehavebeenas-sociated with adverse pregnancy outcomesand other reproductive pathologies. 13,14 In-deed, in nonpregnant women and animals,smoking alters immune responses. 15-19 Therefore, adverse pregnancy outcomes insmoking women could be the result of im-munologicalchangescausedbysmoking.Theexactinfluenceofsmokingonthema-ternal immune system during pregnancy isnotknown.Only1studyinvestigatedtheef-fectsofsmokingoncirculatingmaternalleu-kocytes during pregnancy, showing thatsmoking pregnant women had higher per-centagesofCD3  cells,lowerpercentagesof CD56   cells, and a lower expression of CD54 (activation marker) on monocytes inperipheral blood in early pregnancy com-paredwithnonsmokingpregnantwomen. 20 It was concluded that smoking acts both asan inflammatory and antiinflammatory stimulus on the maternal immune systemduringpregnancy. 20 FromtheDepartmentofObstetricsandGynecology(DrsPrinsandErwich),Departmentof PathologyandMedicalBiology(DrHylkema,MsHuitema,MrDekkema,andMrDijkstra),DivisionofMedicalBiology,DepartmentofPathologyandMedicalBiology(DrsFaasandMelgert),UniversityMedicalCenterGroningenandUniversityofGroningen,andDepartmentof Pharmacokinetics,Toxicology,andTargeting(DrMelgert),UniversityofGroningen,TheNetherlands.ReceivedFeb.2,2012;revisedApril3,2012;acceptedApril9,2012. ThisstudywassupportedbytheDutchKidneyFoundation(grant2266,toB.N.M.),JanKornelisdeCockFoundation,andGuideGraduateSchool. Theauthorsreportnoconflictofinterest.Presented,inpart,atthe57thAnnualScientificMeetingoftheSocietyforGynecologicInvestigation,March24-27,2010,Orlando,FL,andatthe11thInternationalCongressof ReproductiveImmunology,August15-19,2010,Cairns,Queensland,Australia.Reprintsnotavailablefromtheauthors. 0002-9378/$36.00 ã © 2012 Mosby, Inc. All rights reserved. ã http://dx.doi.org/10.1016/j.ajog.2012.04.017 Research  www. AJOG .org  JULY 2012  American Journal of Obstetrics &  Gynecology  76.e1  Important players in the adaptation of the maternal immune response are T cells(especiallyregulatoryTcells),naturalkiller(NK) cells, and monocytes and macro-phages. 13,21-24 RegulatoryT(Treg)cellsarea subpopulation of T cells that regulateimmuneresponses.Theyareimportantintheprocessoftolerancetoselfandforeignantigens,andtheythereforeplayaroleinau-toimmune diseases, inflammatory diseases,transplantation, and pregnancy. 13,25,26 NKcells are key players in the regulation of re-modeling of spiral arteries and also have arole in regulation of invasion of tropho-blasts. 27 Functional and numerical changesofNKandTregcellsarebothassociatedwithadversepregnancyoutcomes. 13,23,28 Monocytes and macrophages are alsoinvolved in remodeling of spiral arteriesand placental development. 29,30 It hasbecome clear that there are specific sub-sets of monocytes and macrophages,which are thought to have specific rolesin inflammation, tissue remodeling, andvascularization, respectively, M1 (inflam-matory) and M2 (remodeling/regulatory)macrophages. 24,31,32 Macrophages in theplacentaduringhealthypregnancyappearto have a remodeling/regulatory role andarethusmainlyoftheM2subtype. 24 The aim of this study was to analyzethe effects of smoking on the maternalimmune system during pregnancy lo-cally in the uterus and systemically. Inhumans, smoking effects on the localand systemic maternal immune systemwere investigated in first-trimester de-cidual tissue and peripheral blood of smoking women. In this study, we usedunique decidual material, collected dur-ing routine chorionic villous samplingbetween 10 and 12 weeks of pregnancy,which allowed us to study smoking ef-fects in deciduas of ongoing pregnancieswithknownpregnancyoutcomes.Wefur-thermoreusedamousemodeltocomparethe results with human pregnancy andmore specifically study changes in periph-eralandlocalimmuneresponses. M ATERIALS AND M ETHODS Study design First-trimesterdecidualtissuewithaknownpregnancy outcome of 42 women, 21smoking women, and 21 control womenwas selected from a tissue collection andanalyzed by real-time reverse transcrip-tion–polymerase chain reaction (RT-PCR).In addition, data on peripheral blood T cellsubsets of 6 smoking and 12 nonsmokingpregnant women were selected from a con-trolcohortofapreviouslypublishedstudy. 33 Tomorespecificallystudychangesinperiph-eral blood and local immune responses, weusedamousemodel.Therefore,6pregnantmice were exposed to smoke and 6 controlpregnantmicewereexposedtofreshair.Toanalyzetheeffectsofsmokingonthelocal maternal immune system, decidualtissue was analyzed by immunohisto-chemistry and real-time RT-PCR, anduterus-draining lymph nodes were ana-lyzedbyflowcytometry.Fortheanalysisof the systemic immune response, systemiclymphnodesandspleenswereanalyzedby flowcytometry. Human study Collection of human decidual tissue.  Duringroutine chorionic villus sampling, per-formedvaginallybetween10and12weeksof gestation for maternal age, a previously chromosomal abnormal child, or serumscreening-related risk for Down syn-drome, surplus material, which was notneededforkaryotyping,wasobtained.Im-mediately after sampling, decidual tissuewas mechanically separated from villi un-der a microscope by a qualified, experi-enced laboratory technician to minimizetrophoblastcontamination.Thechorionicvilli appeared as free-floating white struc-tures with fluffy, filiforme branches,whereasdecidualtissuehadamoreamor-phous appearance and lacked distinctbranches. Earlier studies showed that tro-phoblastcellswereseldomfoundindecid-ual samples. 34,35 Tissue was stored imme-diatelyat–20°Cuntilfurtherprocessingasdescribedbefore. 34 Patients were informed that surplusmaterial could be used for research. Fol-low-up of these pregnancies was avail-able by a questionnaire returned by thepatient postpartum. From this material,we selected decidual tissue from smokingwomen, and for each case we used a non-smokingcontrolwomanmatchedforma-ternal age, parity, and gestational age attime of sampling. Information aboutsmoking was obtained by medical history taking. For cases and controls, only preg-nancieswithaknownpregnancyoutcomelonger than 30 weeks’ gestation were se-lected.Intotal,decidualtissueof21smok-ing and 21 nonsmoking women was se-lectedforanalysis(Table1). To determine whether smoking had adose-dependent effect, we divided thetotal smoking group into a heavy (  10cigarettes per day) and moderate smok-ing group (  10 cigarettes per day). Thelow percentage of male offspring iswithin normal variation and may havebeen influenced by the relatively highmaternal age because there is evidencethat a higher maternal age is associatedwith a shift from a male to a female ma- jority in newborns. 36,37 PCR on human decidual tissue.  Total ri-bonucleic acid (RNA) was isolated fromdecidual tissue using a RNA isolationTrizolkit(Invitrogen,Carlsbad,CA).Tofurther purify RNA, a Turbo DNA freekit and column chromatography wereusedaccordingtothemanufacturer’sin-structions (respectively, Applied Biosys-tems, FosterCity,CA,andBio-RadLabo-ratories, Hercules, CA). Complementary deoxyribonucleicacid(cDNA)wasreversetranscribed using a Superscript-II reversetranscriptasekit(Invitrogen).As a housekeeping gene (HPRT), weused the forward primer 5 = -GGCAGTA-TAATCCAAAGATGGTCAA-3 = -GTCT-GGCTTATATCCAACACTTCGT-3 =  (In-vitrogen), and probe: 6-FAM 5 = -CAA-GCTTGCTGGTGAAAAGGACCCC-3 = TAMRA(Eurogentic,Herstal,Belgium).Forthe other genes, interleukin (IL) 6, IL10,Tbet21 (Th1 response), Gata 3 (Th2 re-sponse), Ror   t (Th17 response), Foxp3(Tregcell),CD56(NKcell),CD68(panmac-rophagemarker),NOS2(iNOS,M1macro-phage), and CD206 (MRC-1, M2 macro-phage),On-Demandgeneexpressionassayswere used (Applied Biosystems). PCR reac-tions were performed in triplicate in a vol-umeof10  Lconsistingof0.1  LofMilliQwater, 5  L PCR mix (Eurogentic), 1  L of reverse and forward primer each, 0.4   Lprobe,and2.5  LcDNAforthehousekeep-inggene.For the other genes, the total volumeof10  Lcontained2  LofMilliQwater,5  LPCRmix(Eurogentic),0.5  Lassay  Research  Basic Science: Obstetrics  www.AJOG.org  76.e2  American Journal of Obstetrics &  Gynecology  JULY 2012  mix, and 2.5   L cDNA. Runs were per-formed by a 7900HT Fast real-time PCR system (Applied Biosystems), and RNAdata were normalized to HPRT messen-ger RNA (mRNA) expression using 2 -  Ct .After the run, it appeared that HPRTmRNA expression of 4 samples (3 con-trols, 1 case) was undetectable; these caseswere excluded from this study. Undetect-ablecyclethreshold(Ct)valuesofthegeneofinterest(morethan40)wereinterpretedasthemaximumCtvalue(40). Human flow cytometry.  To analyze theinfluence of smoking on the systemicmaternal immune system in humans,dataonT-cellsubsetswereselectedfromof a previously published control preg-nant cohort and reanalyzed for effects of smoking. 33 In short, peripheral bloodsamples were obtained from pregnantwomen well before the onset of labor,and T-cell subsets were analyzed usingfluorescently labeled antibodies againstCD4, CD25, and Foxp3 as described be-fore. 33 In total, 6 smoking pregnantwomen were selected and matched by gestational age, parity, and maternal ageto 2 nonsmoking control pregnantwomen per case, leading to a controlgroupof12.Resultsobtainedbyflowcy-tometry were reanalyzed using Winlistsoftwareandcellsubsetswerecalculated.Lymphocyteswereselectedbasedonfor-ward and side scatter plots. Additionalgates were used based on fluorescenceemission wavelengths of the antibodies,andnegativeisotypecontrolswereusedtosetthegates(SupplementalFigure1). Animal study  Animals.  FemaleandmaleC57Bl6mice,aged 8-10 weeks, were obtained fromHarlan (Horst, The Netherlands). Allmice were held under specific pathogen-free conditions on a 12:12 light-dark cy-cle and were administered food and wa-ter ad libitum. All animal protocols wereapproved by the local committee onanimal experimentation (University of Groningen, Groningen, The Nether-lands) and were performed under strictgovernmental and international guide-lines on animal experimentation.For experimental purposes, female micewere treated with 1.5 IU pregnant mare’sserum gonadotropin and 1.25 IU humanchorionicgonadotrophintoinducesimul-taneous cycling. To induce pregnancy, 2females were housed with 1 male. Matingwas confirmed by vaginal plug detection.Allfemalemicehadbeenmated,4ofthe6smoking females (pregnancy rate 67%),and 5 of the 6 nonsmoking females (preg-nancyrate83%)endedupbeingpregnant(Table 2). The morning after mating wascalled day 0.5 of gestation. Mated femaleswereseparatedfrommalesandwerekilledat day 11.5 of gestation. At day 11.5paraaortic lymph nodes (uterus-draininglymph nodes), inguinal (systemic) lymphnodes,andspleenswereremoved.Inaddi-tion,implantationsiteswereremovedandfixed in 4% paraformaldehyde, zinc fixa-tive  38 or immediately snap frozen at–80°C for immunohistochemical andreal-timeRT-PCRanalysis. Cigarette smoke exposure.  Cigarette smokewas generated using a TE-10 smoke expo-suresystemofTeagueEnterprisesSmokeExposure System (Woodland, CA). Fe-male mice were exposed to fresh air orsmoke in sessions of 7 hours continu-ouslyperday,from7daysbeforematinguntil the day the animals were killed.Mice were exposed to 5 cigarettes thefirst day, 10 cigarettes the second day, 25 TABLE 2 Pregnancy outcome in smoke-exposed and control mice Variable Nonsmoking (n  6) Smoking (n  6) Pregnancy rate, % 83 67 .............................................................................................................................................................................................................................................. Number of implantations 14.4  2.1 14.0  3.4 .............................................................................................................................................................................................................................................. Number of resorptions 0.60  0.6 3.4  1.5 a ..............................................................................................................................................................................................................................................  Viable pups, % 96 74 b .............................................................................................................................................................................................................................................. Maternal weight, g 21.5  1.4 19.6  0.8 .............................................................................................................................................................................................................................................. Data are mean  SD. a P   .01 in a Student  t   test;  b P   .05. Prins.Smokingandthematernalimmuneresponse.AmJObstetGynecol2012. TABLE 1 Characteristics of pregnant women for the decidual mRNA expression data CharacteristicNonsmoking(n  18 )Smoking(n  20 ) < 10 cigarettesper day (n  11) > 10 cigarettesper day (n  9)  Age, y 37.6  2.1 36.8  4.3 37.5  2.8 35.8  5.7 ................................................................................................................................................................................................................................................................................................................................................................................ Primiparity, % 20.0 25.0 27.3 22.2 ................................................................................................................................................................................................................................................................................................................................................................................ Birthweight, g 3388.6  591.6 3339.4  600.2 3612.0  574.8 3165.0  548.5 a ................................................................................................................................................................................................................................................................................................................................................................................ Sex of child (% male) 28.6 35.0 36.4 33.3 ................................................................................................................................................................................................................................................................................................................................................................................ Duration of pregnancy, d 273.1  18.1 270.9  31.1 268.6  41.8 273.6  14.3 ................................................................................................................................................................................................................................................................................................................................................................................ Smoking average, (cigarettes per day) 0 9.2  6.3 b 4.8  2.6 b 15.0  4.3 b ................................................................................................................................................................................................................................................................................................................................................................................ Data are mean  SD. a P   .001 compared with controls when analyzing birthweight percentiles for gestational age;  b P   .001 compared with controls. Prins.Smokingandthematernalimmuneresponse.AmJObstetGynecol2012.  www.AJOG.org   Basic Science: Obstetrics  Research JULY 2012  American Journal of Obstetrics &  Gynecology  76.e3  cigarettes the third day, 50 cigarettes thefourth, and 60 cigarettes on the fifth day and thereafter. Total particulate mattercounts were at least 100. Kentucky 2R4Fresearch-reference filtered cigarettes(The Tobacco Research Institute, Uni-versity of Kentucky, Lexington, KY)were used.  Mouse decidual real-time RT-PCR.  Frozenimplantation sites were sectioned trans-verselyat4  mandstainedwithhematox- ylin. Frozen tissue of 1 control mouse wasnotavailable.Decidualandplacentaltissuewas identified, selected, and cut from theslides using laser dissection microscopy (Leica,Rijswijk,TheNetherlands).Decid-ual and placental tissue was collected sep-arately. Total RNA was isolated from de-cidualandplacentaltissueusinganmRNAeasy kit (QIAGEN, Valencia, CA) accord-ing to the manufacturer’s instructions.cDNA was reverse transcribed using aSuperscript-II reverse transcriptase kit(Invitrogen).Forthehousekeepinggene(B2M)andfor the other genes (IL6, IL10, Tbet21[Th1 response], Gata 3 [Th2 response],Ror   t [Th17 response], Foxp3 [Tregcells],Nk1.1[NKcells],CD68[panmac-rophage marker], NOS2 [M1- macro-phages; iNOS], and CD206 [M2-macro-phages; MRC-1]), On-Demand geneexpression assays were used (AppliedBiosystems). PCRs were performed intriplicateinavolumeof10  Lconsistingof 2   L of MilliQ water, 5   L PCR mix (Eurogentic), 0.5   L assay mix, and 2.5  L cDNA. Runs were performed by a7900HT Fast real-time PCR system (Ap-plied Biosystems), and mRNA data werenormalized to B2M mRNA expressionusing 2 -  Ct . Undetectable Ct values(greater than 40) were interpreted as themaximum Ct value (40).  Mouse flow cytometry.  Immediately afterthe animals were killed, paraaortic lymphnodes (uterus-draining lymph nodes), in-guinal (systemic) lymph nodes, andspleenswereremovedandthoroughlydis-persedintosinglecellsuspensionsinfluo-rescence-activatedcellsorting(FACS)buf-fer (consisting of PBS with 2% fetal calf serum).RedbloodcellsincellsuspensionswerelysedusingNH 4 Clsolution.Forflowcytometricanalysis,1millioncells were stained according to standardmethods. In short, cells were incubatedfor 5 minutes with 10% normal mouseserum to block a-specific binding. Cellsfrom the lymph nodes were then incu-bated with a cocktail of fluorescently la-beled antibodies for 30 minutes, includ-ing the following: Pacific Blue-labeledanti-CD3 (17A2; BioLegend, San Diego,CA), PerCP-labeled anti-CD4 (RM4-5;BD Pharmingen, San Diego, CA), Alexa700-labeled anti-CD8 (53-6.7, Bioleg-end), allophycocyanin (APC)-labeledanti-CD25 (3C7; BD Pharmingen), andPE-Cy7-labeled anti-NK1.1 (PK 136;BioLegend). After surface staining, in-tracellular staining was performed ac-cording to the Foxp3-staining kit in-structions (eBioscience, San Diego, CA)usingfluoresceinisothiocyanate(FITC)-labeledanti-Foxp3(FJK-16s;eBioscience).Cellsfromthespleenwerenotonlyin-cubatedwiththeT-cellantibodycocktaildescribed in the previous text but werealso incubated with a monocyte anti-body cocktail: PerCP-labeled anti-CD4(RM4-5; BD Pharmingen), APC-labeledanti-CD11b (M1/70; BD Pharmingen),phycoerythrin (PE)-labeled anti-CD43(1B11; Biolegend), PE-Cy7-labeled anti-Gr-1(RB6-8C5;Biolegend),PacificBlue-la-beled anti-F4/80 (BM8; Biolegend), andFITC-labeled anti-MHC-II (2G9; BDPharmingen).Cells were analyzed on a LSRII (Beck-ton Dickinson, Lincoln Park, NJ) flow cytometer. Results were analyzed withFacsDivasoftware(BecktonDickinson).Lymphocyteswereselectedbasedonfor-ward and side scatter plots and subse-quently staining for CD3, CD4, CD25,and Foxp3. To identify cells positive forthe labeled antibodies, additional gateswere set based on fluorescence emissionwavelengths of the used antibodies andnegative controls (Supplemental Figure1). For monocytes all viable cells weregated for CD11b and F4/80. CD11b-hiand F4/80-intermediate cells were con-sideredmonocytes,andtheseweregatedfor CD43 and GR1 to distinguish theGR1-hi and GR1-lo subset (Supplemen-tal Figure 2). (Immuno)histochemical analysis mice ute-rine tissue.  Implantation sites were fix edin paraformaldehyde and zinc fixative 38 for 24 hours, transferred into 70% etha-nol,andembeddedinparaffinaccordingto standard methods and sectionedtransversely at 7   m. Slides were dew-axed in xylene and rehydrated. For gen-eral histological examination, sectionswere stained with hematoxylin-eosin.First, myometrial, decidual, and placentalsizes, and ratios of decidual spiral artery vessel to lumen area were measured. 39 TcellsandFoxp3  cells were stained us-ing primary antibodies against CD3(ab16669;Abcam,Cambridge,MA),andFoxp3 (FJK16s; eBioscience). Antigenretrieval was performed by incubatingsections (15 minutes at 400 W) in citratebuffer.Sections were incubated with primary antibodies diluted 1:400 (CD3) or 1:50(Foxp3) overnight at 4°C. Thereaftersections were incubated for 30 minuteswith the appropriate secondary and ter-tiary peroxidated antibodies (CD3), orslideswereincubatedwithabiotinylatedsecondary antibody and were incubatedthereafterwithastreptavidin-peroxidasecomplex (Foxp3). Positive staining waslocalized using diaminobenzidine tetra-  A, HigherCD56mRNAexpressioninsmokingpregnantwomen(  graybars  ,n  20)comparedwithnonsmokingpregnantwomen(  whitebars  ,n  18). B, Whenfurther analyzing dose effects of smoking on mRNA expressions in nonsmokers (  white bars  , n  18), moderate smokers (   10 cigarettes per day, n  11,  gray bars   ), and heavy smokers (   10 cigarettes per day, n  9,  black bars   ), a higher expression of Gata3, CD56, CD68, IL6, and iNOS was found in heavy smokingpregnantwomencomparedwithnonsmokingpregnantwomen. C, TherewasadoseeffectofsmokingonmRNAexpressionsofmacrophagesubsetmarkers,withhigher ratios of M1 macrophage markers in heavy smokers. Data are expressed as mean  SEM, statistical analysis was performed using 1-way analysis ofvariance and Dunnet post hoc testing on log transformed data.  Asterisk   indicates  P   .05.  Double asterisks   indicate  P   .01. Prins.Smokingandthematernalimmuneresponse.AmJObstetGynecol2012. Research  Basic Science: Obstetrics  www.AJOG.org  76.e4  American Journal of Obstetrics &  Gynecology  JULY 2012
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