Oxidative Stress: A Potential Therapeutic Target in Placental Malaria

18 Pages·2017·2.36 MB·English

Oxidative Stress: A Potential Therapeutic Target in Placental
Malaria
Demba Sarr, Caitlin A. Cooper, Tara C. Bracken, Omar Martinez-Uribe, Tamas Nagy and Julie M.
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ImmunoHorizons is an open access journal published by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
All rights reserved.
ISSN 2573-7732.
RESEARCH ARTICLE
Infectious Disease
Oxidative Stress: A Potential Therapeutic Target in
Placental Malaria
DembaSarr,* CaitlinA. Cooper,* TaraC. Bracken,* OmarMartinez-Uribe,*Tamas Nagy,† and Julie M.Moore*
*CenterforTropicalandEmergingGlobalDiseasesandDepartmentofInfectiousDiseases,CollegeofVeterinaryMedicine,UniversityofGeorgia,
Athens,GA30602;and†DepartmentofPathology,CollegeofVeterinaryMedicine,UniversityofGeorgia,Athens,GA30602 D
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ABSTRACT fro
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Placentalmalaria,characterizedbysequestrationofPlasmodiumfalciparuminthematernalplacentalbloodspaceandassociated h
cinofnlatmribmuatetotroypdlaamceangtea,lcdoanmtraibgueteansdtodpysofourncbtiriothnoduutrcinogmmesalaanrida;ar2e0n0o,0t0c0ominpfalenttedlyeautnhdsearnstnouoadll.yT.oSpinevceifsictigmaetechaapnoistmensttiahlartolefor ttp://w
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oxidativestress,antioxidantgenesandmarkersforoxidativedamagewereassessedbyquantitativePCRandimmunohistochemistry w
inPlasmodiumchabaudiAS-infectedpregnantmice.Widespreadevidenceoflipidperoxidationwasobservedandwasassociated .im
m
withhigherantioxidantgeneexpressioninconceptusesofinfectedmice.Toassesstheextenttowhichthisoxidativedamagemight u
n
contributetopoorbirthoutcomesandbeamenabletotherapeuticintervention,infectedpregnantmiceweretreatedwithN- oh
o
acetylcysteine,afreeradicalscavenger,ortempol,anintracellularsuperoxidedismutasemimetic.Theresultsshowthatmicetreated riz
withN-acetylcysteineexperiencedmalariainduced–pregnancylossatthesamerateascontrolanimalsandfailedtomitigate on
s
placentaloxidativedamage.Incontrast,tempol-treatedmiceexhibitedsubtleimprovementinembryosurvivalatgestationday12. .o
rg
Althoughlipidperoxidationwasnotconsistentlyreducedintheplacentasofthesemice,itwasinverselyrelatedtoembryoviability. b/
Moreover,reducedIFN-gandCCL2plasmalevelsintreatedmicewereassociatedwithmidgestationalembryoviability.Thus, y g
u
althoughoxidativestressisremarkableinplacentalmalariaanditsmitigationbyantioxidanttherapymayimprovepregnancy e
s
outcomes,theunderlyingmechanisticbasisandpotentialtherapeuticstrategiesrequireadditionalinvestigation.ImmunoHorizons, t o
n
2017,1:29–41. N
o
v
e
INTRODUCTION ventionsforcontrollingmalariaanditseffectsduringpregnancyis m
b
e
recommended(2).Malariainpregnantwomenischaracterizedby r 1
AmcacjoorrdpinugbltiocthheealWthorplrdoHbleemalthinOmrgaannyizactoiounn,trmieaslaorfiathreemwaionrslda. fithlteraatciocnu,manudlaetixocnesosifveP.fibfarlicnipdaerpuomsi–tiionnfeicnttehdemRBatCesr,nalelubklooocdytsepaicne- 7, 20
1
8
Despite the progress in reducing malaria cases and deaths, it is (3)oftheplacenta,asyndromeknownasplacentalmalaria(PM).
estimated that 214 million cases of malaria occurred worldwide PMisassociatedwithlowbirthweightandmaternalanemia(4–7).
in2015,leadingto438,000deaths(1).Inareaswithmoderateto InadditiontothesepathogenicfindingsinPM,arecentstudy
high transmission of Plasmodium falciparum, a package of inter- showed that poor pregnancy outcomes may also be attributed
ReceivedforpublicationFebruary14,2017.AcceptedforpublicationMay18,2017.
Addresscorrespondenceandreprintrequeststo:Dr.JulieM.Moore,CenterforTropicalandEmergingGlobalDiseasesandDepartmentofInfectiousDiseases,
CollegeofVeterinaryMedicine,UniversityofGeorgia,500DWBrooksDrive,Athens,GA30602.E-mailaddress:[email protected]
ThisworkwassupportedbytheNationalInstitutesofHealthGrantsHD46860andAI50240toJ.M.M.
ThecontentissolelytheresponsibilityoftheauthorsanddoesnotnecessarilyrepresenttheofficialviewsoftheEuniceKennedyShriverNationalInstituteofChild
HealthandHumanDevelopment,theNationalInstituteofAllergyandInfectiousDiseases,ortheNationalInstitutesofHealth.
Abbreviationsusedinthisarticle:AUC,areaunderthecurve;Cat,catalase;Ct,thresholdcycle;ED,experimentday;HNE,4-hydroxynonenal;Ho,hemeoxygenase;
IHC,immunohistochemistry;IP,infectedpregnant;NAC,N-acetylcysteine;Nrf2,NFerythroidderived2like-2;PM,placentalmalaria;ROS,reactiveoxygenspecies;Sod,
superoxidedismutase;TPL,tempol;UP,uninfectedpregnant.
Theonlineversionofthisarticlecontainssupplementalmaterial.
ThisarticleisdistributedunderthetermsoftheCCBY-NC4.0Unportedlicense.
Copyright©2017TheAuthors.
https://doi.org/10.4049/immunohorizons.1700002 29
ImmunoHorizonsispublishedbyTheAmericanAssociationofImmunologists,Inc.
30 TARGETINGOXIDATIVESTRESSINPLACENTALMALARIA ImmunoHorizons
to malaria-induced oxidative stress in the placenta (8). In that Male and female C57BL/6J (B6) mice were originally
work,thelevelofcellmembranelipidperoxidation,detectedas purchasedfromtheJacksonLaboratoryandwereusedtogenerate
malondialdehyde,wassignificantlyincreasedintheplacentasof breeding stock and experimental animals in the University of
malaria-infectedandanemicwomencomparedwithnoninfected Georgia Coverdell Vivarium (18). All mice were kept under
andnonanemiccounterparts(8).Moreover,oxidativestresswas specificpathogen-freeconditionsattheCoverdellCenterVivar-
observed in a murine model of PM caused by P. berghei ANKA ium(University of Georgia, Athens, GA), and were devoid of
(9–11),whichyieldsmanyfeaturesofhumanPMbutislimitedby Helicobacter infection, which has a clear negative impact on
parasite virulence and maternal mortality, restricting studies to pregnancyinC57BL/6mice(21).P.chabaudichabaudi,strainAS
latepregnancy. (MR4-741), contributed by D. Walliker, was obtained from the
Oxidative stress is defined as an imbalance between the BEIResourcesRepository,NationalInstituteofAllergyandInfec-
productionofreactiveoxygenspecies(ROS),includingsuperox- tious Diseases, National Institutes of Health. The parasite was
ide,hydrogenperoxide,hydroxylanion,andtheabilityofvarious maintained by routine passage through female A/J mice and
antioxidantmechanismstoscavengetheseradicals.Bycatalyzing cryopreservation. Infection in experimental female mice, aged
the dismutation of superoxide radical into oxygen or hydrogen 8–10wk,wasinitiatedintravenouslywith1000infectedRBCsper
peroxide,superoxidedismutase(SOD)reactswithNOandROSas 20 g of body weight on gestation day 0; this is considered D
an antioxidant and mitigates their negative physiological effects experiment day (ED) 0; thus, ED is directly comparable to ow
(12).ThreeisoformsofSODhavebeenidentified.SOD1hascopper gestation day. Parasitemia was detected beginning on ED6 in nlo
a
andzincascofactorsandislocalizedtointracellularcytoplasmic Giemsa-stainedtailbloodthinsmearsbyexaminingatleast1000 d
e
creoamctpiavretmceenntetsr.aSnOdDh2a,stbheeesnelcoocnadliziseodfotormm,ithoacshmonadnrgiaanoefsaeeirnobiitcs RpaBrCassitinemfoiuarwhaigshm-poonwiteorremdicdraoilsycobpyicflfioewldcsy(t3o,m14e–tr1y8)u.sTinhgerSeyatfotexr-, d from
cells.ExtracellularSOD(SOD3)issynthesizedbyonlyafewcell Orange staining (22). Percent of hematocrit was used as an http
typesincludingvascularsmoothmusclecells,andislocalizedin indicatorofanemia.Weightwasalsorecordedeveryday(except ://w
theextracellularmatrixofvarioustissuesincludingthevascular fromED1toED5)ingrams(18),andpercentofweightlossorgain w
w
ewnazllymanedrpelsapcoennstiabl(e13f)o.rCaRtOalSasceo(nCtarot)l,,afnurotthheerrdmeationxiafinetsioaxidparno-t wasAclallcmuilcaetewdearseprseavciroifiucselyddoenscrEibDe1d0(o3)r.ED12 as indicated (3, .imm
oxidantresponsebydegradinghydrogenperoxideintowaterand 14–18).Plasmaandtissueswerecollectedasdescribedbelow.Mice un
o
oxygen.Toourknowledge,theextenttowhichthesemediatorsof were dissected for evaluation of embryo status and isolation of h
o
anantioxidantresponseareactiveinPMduringearlypregnancy tissuesaspreviouslydescribed(14,15,17,18).Briefly,resorptions riz
o
malariahasnotbeenexplored.Toaddressthisissue,analternative or nonviable embryos were identified by their necrotic and/or n
s
murine model (3, 14–18) in which P. chabaudi chabaudi AS (P. smallersizecomparedwithviablenormalembryosastheyappear .org
chabaudiAS)infectioninearlygestationinducesplacentaldamage inuninfectedpregnant(UP)mice.Miceundergoingactiveabor- b/
y
andpregnancylossinC57BL/6andA/Jmice(3,17,18)wasused. tion,definedasevidenceofbloody,mucoidvaginaldischargeand/ g
u
Becauseapoptosisdoesnotseemtoplayauniversalroleinthis orevidenceofembryosintheopencervixorvaginalcanalwere es
placentaldamage(17),wehypothesizedthat,consistentwiththe recorded(3,14,16,18).Theuteruswasseparatedbycuttingdirectly t on
P.bergheimodelofPM(9,11),oxidativestressparticipatesinthe below the oviduct and above the cervix, and the mesometrium N
o
v
pathogenesisofP.chabaudiASinfectionduringearlypregnancy. wasremoved. e
m
Tothisend,oxidativestresswasmeasuredbyquantitativePCRof b
e
markersofanantioxidantresponseandbyimmunohistochemistry Antioxidant treatment and outcome monitoring r 1
7
of a reactive lipid mediator generated during oxidative stress, Groups of 6–16 female mice were paired 2:1 with a male , 2
0
4-hydroxynonenal(HNE).Additionally,theabilityoftreatmentwith overnightandcheckedthefollowingmorningforvaginalplugs. 1
8
antioxidants(N-acetylcysteine[NAC]andtempol[TPL])toblock Observation of a plug prompted randomization, as numbers
malaria-associated oxidative stress and improve pregnancy out- allowed,toinfectedpregnant(IP)andUPgroups.Eachofthese
comewasassessed.Finally,becauseoxidativestressandelevated randomization events (on a single day) is considered to be a
ROSareassociatedwithinflammationinhumanandexperimental discreetexperiment.Thesegroupswerethenfurtherrandom-
malaria (19, 20), systemic cytokine levels were measured in izedoneitherED2orED6toreceivecontrolorvehicle(PBS
antioxidant-treatedanduntreatedanimals. or H O) or antioxidant therapy. The antioxidants used in
2
this study were NAC (pH 7.4) and TPL (4-hydroxy-2,2,6,6-
tetramethylpiperidin-1-oxyl). Pairingwascontinueduntilthe
MATERIALSANDMETHODS desired number of animals was achieved and is detailed for
eachtreatmentregimenbelow.
Animals, parasites, and infection For NAC treatment, mice were initiated on 14 discreet
AllanimalproceduresreportedinthisstudyfollowedtheGuide experiment start dates, with two to five plugged mice obtained
for the Care and Use of Laboratory Animals. Animal protocols perday.Incaseswherefewerthanfourplugswereobserved,mice
wereapprovedbytheUniversityofGeorgiaInstitutionalAnimal werealternatedtoIPorUPgroupsonlytoallowlaterallocationto
CareandUseCommittee. control(PBS)andtreatedgroupswithineachstartdate–defined
https://doi.org/10.4049/immunohorizons.1700002
ImmunoHorizons TARGETINGOXIDATIVESTRESSINPLACENTALMALARIA 31
experiment. In initial experiments, NAC was administered i.p. manufacturer’s instructions (GBI Mukilteo, WA). Samples
fromED6toED12atthedoseof50mg/kgperday,withcontrol were stained the same day with the same reagents to avoid
micereceivinginjectionsofPBS,andsacrificewasscheduled interassay variability. Briefly, unstained tissue sections were
for ED12; for this goal, eight UP NAC, eight UP PBS, five firstdeparaffinizedandrehydratedthroughxyleneandgraded
IP NAC, and five IP PBS mice were generated. When these alcohol series. Ag retrieval was performed for 30 mn with a
experimentsrevealedthatpregnancylosswasoccurringprior Pascal pressurized heating chamber (Code S2800; Dako,
toED12,anadditional21micefromeightdiscreetexperiment Carpinteria,CA).DetectionofHNEadducts,markersforlipid
startdates(twotofivemiceperexperiment;sixUPNAC,six peroxidation,wasachievedwithapolyclonalrabbitanti-HNE
UP PBS, six IP NAC, and five IP PBS) were generated for (ab46545;Abcam,Cambridge,MA)at1/2000for1hinamoist
sacrificeatED10(withtreatmentreceivedfromED6toED10). chamber at 37°C. HNE is one of the most quantitatively
ED10sacrificedmiceallowedcollectionofconceptustissuefor important products of lipid peroxidation (25), a process by
evaluationoflipidperoxidation(seebelow)andcollectionof which oxidants such as free radicals or nonradical species
plasmaforassessmentofsystemiccytokinelevelsatthetimeof attack lipids, especially polyunsaturated fatty acids, yielding
abortion. lipidperoxylradicals,andhydroperoxides(26).Sectionswere
TPL (at the dose of 40 mg/kg per day, dissolved in counterstained with hematoxylin, dehydrated and mounted D
methanol, and diluted in PBS) was first administered i.p. withFlo-Texx(LernerLaboratories,Pittsburgh,PA).Stained ow
n
starting at ED6; control mice received sterile PBS supple- slideswereviewedandphotographedonaNikonEclipseE400 lo
a
mentedasappropriatewithdiluent(methanol).Overall,eight microscope with a Nikon DS-L1-5M imaging camera at 3200 d
e
experiment start days were used with the number of mice finalmagnificationtomonitorstainingandassessresults.Scoring d fro
varyingfromthreetosixperexperiment,ultimatelyyielding5 for HNE in murine spleen and placental tissues was done by m
UsaPcrTifiPcLe,d7oUnPEDP1B2S., 7 IP TPL, and 11 IP PBS mice that were eoxnaam0in–i4nsgc1a0lep.oHwigehr-fireelsdosluotfitohneismtaaigneesdfsolriddeaatandpraessseignntaintigonaswcoerree http://w
Insubsequentexperiments,TPLwasdeliveredadlibitum takenwithanOlympusBX41microscopecoupledwithanOlympus w
in drinking water at 40 mg/kg per day (23, 24) with daily DigitalcameraDP25,savedasTIFFfiles,andcroppedandresized w
.im
changes,andwasinitiatedateitherED2orED6throughED10 usingAdobePhotoshopCS6. m
orED12.Thisconcentrationwascalculatedtobecomparable un
o
onaper-daybasistotheconcentrationdeliveredbyinjection. Antioxidant gene expression by quantitative PCR h
o
TPLconsumptionwasconfirmedbytrackingthevolumeofliquid Total RNA from mouse conceptuses (ED9, 10, and 11) was riz
o
consumed over the entire cage of mice and average per-mouse extractedusingTrizolReagent(Ambion,CarlsbadCA),DNase- n
s
.o
consumptioncalculated.FortheED6TPLtreatmentregimen,nine treated (Ambion), reverse-transcribed with High Capacity rg
experimentstartdateswereincluded,withdailyinclusionranging cDNAReverseTranscriptionKit(AppliedBiosystems,Foster b/
y
fromthreetosixmice.Totalgroupsizeswere4UPTPL,6UPH O, City, CA). Genes of interest (Table I) were quantified with g
2 u
s15acIrPifiTcPedL,oanndED6I12P.HFo2OrtfhoeraTtPoLtadlorifn3k1inmgicree.gAimlleonftshtaersteemdiocnewEDer2e, PUo.Kw.e)rwSyitbhrGthreeenC1M0a0s0teTrMouicxh(ATphpeliremdaBlioCsyysctleemrs(,CWFXar9ri6ngRtoenal, est on
20experimentstartdates(numbersofmicerangingfromthreeto Time Systems; Bio-Rad, Hercules CA). Each sample was N
o
v
six)wereperformedandyielded45mice(6UPTPL,5UPH2O,12IP assayed in duplicate for all targets (antioxidant genes) and em
TPL,10IPH O)forsacrificeonED10and40mice(5UPTPL,12UP thehousekeeping(18SRNA)gene.Averagethresholdcycle(Ct) b
2 e
H2O,10IPTPL,6IPH2O;nineexperimentstartdates)forsacrifice values were normalized to the average of 18S RNA Ct values r 17
onED12. andrelativeexpressionwascalculatedforeachtargetgeneby , 2
ThemeanvolumeofTPLsolutiondrunkpermouseperday the DDCt method (27). Details of mouse-specific primer sets 01
8
wasrespectively4.9360.29and4.4860.09inUPTPLandIP (sequence forward and reverse) as well as the size of the
TPL,whereasthemeanvolumeofwaterdrunkwas5.266 0.18 amplification products and gene name are summarized in
and4.2860.09inUPH OandIPH O.VariationinwaterandTPL TableI.
2 2
uptakewasnotsignificantbetweentreatmentgroups.
ELISA for TNF, IL-6, IFN, and CCL2
Placental histology and immunohistochemistry PlasmawascollectedfromIPandUPmicebycardiacpuncture
At sacrifice (ED10 and ED12), conceptuses and spleens were at sacrifice on ED10. Cytokines and chemokine levels were
immediately harvested from TPL- and vehicle-treated mice assessed by ELISA using reference standard curves with
and fixed in 10% neutral formalin for 12 h. The fixed tissues knownamountsofrecombinantcytokines(TNF,IL-6,andIFN)
wereembeddedinparaffinandsectionedat7mm.H&E-stained andchemokine(CCL2)followingthemanufacturer’sinstructions
organ sections were used by a pathologist to assess malaria- (BDandR&Dasappropriate).FormeasurementofCCL2inmouse
associated histopathology and the effects of TPL treatment. conceptuses,totalproteinwasisolatedaspreviouslydescribed(17).
Unstained7mmtissuesections(3)wereusedforimmunohis- TotalproteinswerequantifiedbyCBQCAProteinQuantification
tochemistry(IHC)accordingtopreviouslyreportedmethods Kitanddilutedinassaydiluentattheconcentrationof1mg/mlfor
(17) with a Polink-HRP kit for rabbit Abs following the useinELISA.
https://doi.org/10.4049/immunohorizons.1700002
32 TARGETINGOXIDATIVESTRESSINPLACENTALMALARIA ImmunoHorizons
Statistical analysis
Unless otherwise noted, the Graph Pad Prism Statistical
Software (Version 5.03; GraphPad Software, La Jolla, CA)
was used for quantitative data analysis. Sample sizes were in
linewithpreviousliterature(cumulative5–12micepergroup).
Changes in percent starting weight, hematocrit, and para-
sitemiabetweengroupswereassessedbycomparisonofarea
underthecurve(AUC).AUCwascalculatedinGraphPadPrism
for weight using the ratio of starting weight (weight at day x
dividedbyweightatday0).AUCforhematocritandparasitemia
were calculated using the raw data for hematocrit and para-
sitemia, respectively. Nonparametric Mann–Whitney U test
comparisons and Kruskal–Wallis test with Dunn multiple com-
parison test wereappliedwhendatawere notnormally distrib-
uted for pairwise and multiple group comparisons, respectively. D
o
Otherwise,Studentttestandone-wayANOVAwithBonferroni w
n
multiplecomparisontestwereused.Atwo-tailedFisherexact lo
a
testwasusedtocompareembryoviabilitybetweenantioxidant- de
d
strtaetaitsetdicavlelyrssuigsncifioncatrnotl. mice. A p value ,0.05 was considered FIGURE1.Malaria-inducedantioxidantresponseandlipidperoxidation from
inP.chabaudiAS–infectedC57BL/6(B6)conceptuses. h
RESULTS q(Au)aTnrtaifniesdcrbipytqlueavnetlsitaotfivmePoCusReuNsirnfg2,foHrow,aSrodda1n,dSoredv2e,rsSeopdr3im,earnsdeCqautewnceeres ttp://w
w
showninTableI.ExpressioninIPmiceisrelativetothemeanexpression w
Antioxidant gene expression is elevated in P. chabaudi valueinUPmiceandnormalizedtomouse18SRNAvalues.Columnsand .imm
AS–exposed conceptuses bars represent mean 6 SEM of the relative quantities (fold-changes) for un
OurmousemodelofPMhasconsistentlyfoundthatmiceinfected eachgenefromatleastthreeconceptusespermousewithaminimumof oh
o
withP.chabaudiASatED0aborttheirembryosatmidgestation fivemicepergroup.RepresentativeimagesofUP(B)andIP(C)placenta riz
withnoviableembryosremainingonED12(3,16–18).Toverifythe stained for lipid adducts using an anti-HNE Ab. Arrows indicate stained on
s
trophoblast; arrowheads depict HNE staining in parasite-infected and .o
extent to which embryos and their placentas are subjected to rg
oxidative stress in this model, expression of genes involved in uninfectedRBCsinmaternalbloodspaceandwithinerythrophagocytosing b/
driving the transcriptional response to oxidative stress, NF trophoblast.(D)SummaryscoresforHNEstaining.*p,0.05. y g
u
erythroid derived 2 like-2 (Nrf2), and in detoxification of ROS, es
superoxidedismutase1(Sod1),Sod2,Sod3,andCat aswellasheme t on
oxygenase (Ho), mRNA were assessed by quantitative PCR. On ofPMinthismodeland,inaddition,thattherapeuticinterventions N
o
ED9,1dbeforeabortionstartsinIPB6mice(3,16–18),Nrf2,Sod1, targetingoxygenradicalswillimprovepregnancyoutcome.Tothis ve
m
Sod2,andCatincreased4-to5-foldrelativetoUPconceptuses, end,theefficacyofNACwasfirsttested.NACinteractswithROS b
e
whereas Sod3 was upregulated with the fold increase reaching suchassuperoxideandhydrogenperoxide(28–33),anddecreases r 1
7
almost20(Fig.1A).Sod2remainedelevatedthroughED10;Sod3 thegenerationoffreeradicalsbyincreasingglutathionesynthesis , 2
0
wasstronglyupregulatedthroughED11,peakingatanearlya30- (34, 35). Treatment was started on ED6 and ended on ED12, 1
8
fold increase on ED10 (Fig. 1A). Ho was modestly increased encompassingthetimeofdetectableparasitemiatopregnancy
roughly2-foldduringthetestedperiod(Fig.1A). lossinuntreatedP.chabaudiAS–infectedpregnantB6mice(3,
14, 16–18). Treatment efficacy was assessed by two outcome
Mouse PM is associated with lipid peroxidation measures:embryoviabilityanduterusweightatED12.Contrary
ToverifythatoxidativestressoccursinP.chabaudiAS–infected toexpectations,i.p.-deliveredNAC(36)didnotimprovepregnancy
mouseplacentas,insitugenerationofHNEatED10wasassessed. outcome(SupplementalFig.1,TablesI,II).Infact,50%ofNAC-
Asshowninrepresentativeimages(Fig.1B,1C),infectedplacentas treatedIPmiceabortedearlier(ED8)thanPBS-treatedIPmice,
showedsignificant(p=0.0137;Fig.1C,1D)lipidperoxidationby indicatingapotentialtoxiceffect.IPuteriweresignificantlylighter
IHCrelativetouninfectedplacentas(Fig.1B,1D). that UP uteri (Supplemental Fig. 1G) and all embryos were
nonviable at ED12 regardless of treatment (Table II). By AUC
AntioxidantNACdoesnotrescuepoorpregnancyoutcomes analysis, percent weight change (Supplemental Fig. 1B), percent
in B6 mice hematocrit (Supplemental Fig. 1D), and percent parasitemia
Basedontheobservationsofincreasedantioxidanttranscriptional (SupplementalFig.1F)werenotsignificantlydifferentbetween
responseandlipidperoxidationininfectedconceptuses(Fig.1),we NAC- and PBS-treated mice. These results indicate that, at the
hypothesizedthatoxidativestresscontributestothepathogenesis doseusedinthisstudy,NACfailedtoimprovepregnancyoutcome
https://doi.org/10.4049/immunohorizons.1700002
ImmunoHorizons TARGETINGOXIDATIVESTRESSINPLACENTALMALARIA 33
TABLEI.Listofmouse-specificforward(PF)andreverse(PR)primers suggeststhat,unlikeNAC,TPLhasamodestprotectiveeffectfor
usedinrealtimePCRfortheamplificationofpro-oxidantsand pregnancyoutcomesinP.chabaudiAS–infectedpregnantB6mice.
antioxidantgenesaswellashousekeeping18SrRNAandsizeoftheir
amplificationproductinthisstudy
TPL delivered via drinking water partially improves
GeneName ForwardandReversePrimers Size pregnancy outcomes in P. chabaudi AS–infected mice
PF:59-CCAGAGCGAAAGCATTTGCCAAGA-39
GiventhepromiseofTPLtreatmentbyinjection,wenexttested
18SRNA PR:59-TCGGCATCGTTTATGGTCGGAACT-39 106
whethertheoutcomecouldimprovewithconstantadministration
PF:59-CTACTCCCAGGTTGCCCACA-39
Nrf2 PR:59-CGACTCATGGTCATCTACAAATGG-39 103 via drinking water at the same dose (40 mg/kg per day) as
PF:59-GATGAAGAGAGGCATGTTGGA-39 previously described in models for fetal growth restriction and
Sod1 PR:59-CCATTGTACGGCCAATGATGGAATG-39 123 preeclampsia(23,24).OneregimenstartedonED6(Supplemental
PF:59-CACAAGCACAGCCTCCCAG-39
Fig.2)andthe secondon ED2(toallowlongerexposureto the
Sod2 PR:59-CGCGTTAATGTGTGGCTCC-39 63
drug; Fig. 3). In both cases, ED12 was initially chosen as the
PF:59-TTCTTGTTCTACGGCTTGCTAC-39
Sod3 PR:59-CTCCATCCAGATCTCCAGCACT-39 174 endpoint.WeightgainwassuppressedincontrolIPmicerelative
PF:59-AAATGCTTCAGGGCCGCCTT-39 toUPmiceasexpected(Fig.3A,3B)(3,14,16–18).Withtreatment
Cat PR:59-GTAGGGACAGTTCACAGGTA-39 93 startingonED2,weightchangeamongthedamswasnotdifferent D
PF:59-GGTGATGGCTTCCTTGTACC-39 o
between the TPL-treated groups, apparently due to reduced w
Ho PR:59-AGTGAGGCCCATACCAGAAG-39 155 n
weightgaininUPmice(Fig.3A,3B).Asobservedwithinjected lo
a
TPL,treatmentdidnotimproveanemia(Fig.3C,3D),noralterthe de
d
in P. chabaudi AS–infected mice. Due to the observed toxicity developmentofparasitemia(Fig.3E,3F).Likewise,uterusweight fro
of NAC and its failure to mitigate lipid peroxidation in spleen was reduced with infection regardless of treatment (Fig. 3G). m
(SupplementalFig.2L)andplacenta(SupplementalFig.2H,2J), BecauseuterusweightincontrolUPmicewascomparabletothat http
thistherapeuticoptionwasabandoned. intreatedUPmice(Fig.3G),reducedweightgaininthelatter(Fig. ://w
3A)wasnotduetoembryotoxicity;infact,theproportionofviable w
w
TouPtLcodmeleisveinrePd.i.cph.aibnacuredmi eAnSt–ailnlyfeicmtepdromviecsepregnancy eIIm)bwraysoseqinuiTvPalLe-nttr.eNatoetdab(9ly3,%u)terreulastwiveeitgohUtiPnctorenatrteodlsI(P94m%ic;Teawbales .imm
GiventhefailureofNACtoalleviatepoorpregnancyoutcomesin notdifferentfromthatoftreatedUPmice(Fig.3G),likelyowingto un
o
P.chabaudiAS–infectedpregnantmice,we nextturnedto TPL, improvedoutcome(increaseduterusweight)in2outof10IPmice. ho
anitroxidecompoundthathasbeenstudiedextensivelyinanimal Improvedoutcomeisalsoreflectedinthenumberofembryosper riz
o
modelsofincreasedROSandoxidativestress(37).Itmetabolizes uterus(Fig.3H),whichdifferedacrossthegroups(Kruskal–Wallis ns
superoxide anions via a reducible nitroxide group (38) to form test, p = 0.0218; pairwise post hoctests did not yield significant .org
hydrogen peroxide, accounting for its superoxide dismutase results). Indeed, the proportion of viable embryos in IP TPL- b/
y
mimetic action (37, 38). An advantage of using TPL is that it is treated mice was significantly higher than in the untreated IP g
u
cell permeable, specifically targeting superoxide anions that are cohort (Table II). Consistent with this, the number of viable es
generatedintracellularly(39).IntraperitoneallydeliveredTPLdid embryos and uterus weight were positively correlated in the t on
N
notimpacttheoutcomeofinfectionintermsofweightchange(Fig. treatedIPmice(Spearmanr=0.86;p=0.0045;Supplemental o
2A,2B),anemia(Fig.2C,2D),orcourseofparasitemia(Fig.2E,2F). Fig.3). ve
m
Althoughuterusweightwasreducedwithinfectionregardlessof Finally,similarimprovedpregnancyoutcomeswereobserved b
e
treatment(Fig.2G),thenumberofembryosperuterus(Fig.2H) with oral TPL treatment initiated at ED6 (Supplemental Fig. 4, r 1
7
was equivalent in all groups (p . 0.05). Importantly, IP TPL- TableII). Collectively, these results indicate that TPL delivered , 2
treated mice showed a significantly higher proportion of viable orally ad libitum has a limited but significant positive effect on 01
8
embryos relative to untreated IP mice (Table II). This result embryosurvivalinIPmice.
TABLEII.Comparativepercentofembryoviabilityinantioxidant-treatedmiceversusvehicle-treatedmice
UninfectedPregnant(UP) InfectedPregnant(IP)
Drug Route StartedED Treated Vehicle pValue Treated Vehicle pValuea
Viable/Total(%;n) Viable/Total(%;n) Viable/Total(%;n) Viable/total(%;n)
NAC Injection 6 41/43(95;5) 44/46(96;5) NS 0/43(0;6) 0/47(0;6) NS
TPL Injection 6 39/43(90;5) 42/44(95;5) NS 7/65(10;7) 0/55(0;6) 0.01
TPL Drinking 2 40/42(95;5) 80/85(94;10) NS 15/84(18;10) 0/44(0;6) 0.002
TPL Drinking 6 38/41(93;4) 62/67(92;6) NS 12/69(17;12) 0/25(0;4) 0.01
aThepvalueswereobtainedwithatwo-tailedFisherexactprobabilitytestwhencomparingUP-treatedversusvehicle-treatedandIP-treatedversusIP-vehicle,
respectively.BoldpvaluesindicatesignificancebetweenIP-treatedandIP-vehicle.
%,proportionofviableembryos;ED,experimentdayorgestationday,referringtothedaywhentreatmentwasstarted;TPL,tempol;viable/total,thenumberofviable
embryosdividedbythetotalnumberofembryosonED12.
https://doi.org/10.4049/immunohorizons.1700002
34 TARGETINGOXIDATIVESTRESSINPLACENTALMALARIA ImmunoHorizons
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FIGURE2.CourseofP.chabaudiASinfectionandpregnancyoutcomeinmicereceivingi.p.-deliveredTPLfromED6toED12. 18
UnlikeUPmiceTPL-treatedornot(UPPBS),percentweightchange(A)andAUCforweightchange(B)showevidenceofIPmicelosingweight.
Percenthematocrit(C)andAUCforhematocrit(D)alsodepictanemiainIPmicewhereasUPmicehavestablehematocrit.Percentparasitemia(E)
and AUC for parasitemia (F) are comparable in TPL-treated versus control mice. Pregnancy outcome as indicated by uterus weight (G) shows
incrementalimprovementinpregnancyoutcomeinIPTPL-treatedversuscontrolmice.(H)Numberofembryosperuterus.Datashownrepresent
cumulativedatafor30micefromeightexperiments.IP-PBS,opencircle;IP-TPL,opendiamond;UP-PBS,closedcircle;UP-TPL,closeddiamond.
*p,0.05,**p,0.01,***p,0.001.
Chronic TPL exposure does not significantly mitigate Lipid peroxidation was evident in placentas from some
placental lipid peroxidation TPL-treated (Fig. 4D, 4H), and all control mice (Fig. 4B,
To assessthe extent to which TPL treatmentimprovesembryo 4F).Interestingly,therewassignificantvariabilityinHNE
viability by reducing placental oxidative stress, placental lipid staining among IP placentas, with some specimens having
peroxidationatED10inmicereceivingoralTPLfromED2was stainingscorescomparabletothosefromUPdams(Fig.4N).
comparedwithcontrolmice. Thissuggestsvariableefficacyforsuperoxidescavengingby
https://doi.org/10.4049/immunohorizons.1700002
ImmunoHorizons TARGETINGOXIDATIVESTRESSINPLACENTALMALARIA 35
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FIGURE 3. Course of P. chabaudi AS infection and pregnancy outcome in ad libitum orally delivered TPL treatment from ED2 to ED12. 8
Percentweightchange(A)withAUC(B)areindicated.Percenthematocrit(C)andAUC(D)depictanemiainIPmice,whereasUPmicehavestable
hematocrit.Percentparasitemia(E)andAUC(F)arecomparableinmiceorallydeliveredTPLversusmiceunderdrinkingwater.Uterusweight(G)
showsrelativeimprovementofpregnancyoutcomeinmicereceivingtreatmentversusthoseunderdrinkingwater,whereasnumberofembryos
peruterusisthesameregardlessofinfectionortreatmentstatus(H).Thedatafromthisexperimentcumulativelyrepresent31mice(5UP-TPL,10
UP-H O,10IP-TPL,and6IP-H O)generatedwith10experimentstartdays.IP-H O,opencircle;IP-TPL,opendiamond;UP-H O,closedcircle;
2 2 2 2
UP-TPL,closeddiamond.Numberofembryosperuterusvariesacrossthegroups(Kruskal–Wallistest,p=0.0218).Pairwiseposthoctestsarenot
significant.*p,0.05,**p,0.01,***p,0.001.ns,notstatisticallysignificant.
TPL. Importantly, embryo viability was negatively corre- placentas, splenic HNE was substantially and universally
lated with the IHC score (Spearman r = 20.82; p = 0.0341; reducedinTPL-treatedanimals(Fig.4K,4L).Thus,whereas
Fig.4O),indicatingthatincasesinwhichTPLtreatmentdid orally administered TPL effectively scavenges ROS and
mediate reduction in placental lipid peroxidation, a pro- protectsthespleenagainstlipidperoxidationinIPmice(Fig.
tectiveeffect(i.e.,enhancedembryoviability)wasachieved. 4M),thistreatmentisinconsistentlyeffectiveattheplacental
In contrast to the lipid peroxidation patterns observed in level(Fig.4N).
https://doi.org/10.4049/immunohorizons.1700002
36 TARGETINGOXIDATIVESTRESSINPLACENTALMALARIA ImmunoHorizons
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FIGURE4.Immunostainingfor4-hydroxynonenal,markeroflipidperoxidation,inED10placentaandspleenfrommiceorallyadministeredTPL n
s
beginningatED2versusmiceunderdrinkingwater(H O). .o
2 rg
The two upper panels show immunostaining in the placental junctional zone [JZ; (A–D)] and labyrinth [LB; (E–H)] and the lower panels show b/
immunostainingin thespleen[SP; (I–L)] ofUP andP. chabaudi AS–IP mice. (M) showsHNE stainingscoresin thespleenand panel (N) shows y g
u
staining in the placenta, whereas (O) depicts the correlation between viable embryos and HNE immunostaining scores in the placenta of TPL- e
s
treatedIPmice.Thedatafromthisexperimentweregeneratedoncewithatleastfivemicepergroup,representing45mice[6UPTPL,5UPH O,12 t o
2 n
IPTPL,10IPH O;10experimentalstartdays(threetosixmiceperexperiment)].*p,0.05,**p,0.01.ns,notstatisticallysignificant. N
2 o
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b
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Impact of chronic TPL treatment on local and systemic 6B).TissueCCL2(Fig.6F)differedacrossthegroups(Kruskal– r 1
7
inflammatory markers Wallis test, p , 0.05), but pairwise post hoc tests did not yield , 2
To assess the impact of TPL treatment on the inflammatory significantresults.NeitherIL-6(Fig.6C)norIL-10(Fig.6D)levels 01
8
response in pregnant mice receiving treatment from ED2, the variedsignificantlyasafunctionoftreatmentoroutcome.
levels of cytokines (TNF, IL-6, and IFN-g) and the chemokine
CCL2wereassessedatED10inplasmaandproteinextractsfrom
DISCUSSION
conceptustissuebyELISA(Fig.5).Irrespectiveoftreatment,the
levels of TNF, IL-10, and CCL2 were universally higher in IP
comparedwithUPmice(Fig.5B,5D,5E);CCL2wasalsoelevated In this study, a potential role for placental oxidative stress in
inconceptuslysatesfromIPmice(Fig.5F).IFN-g(Fig.5A)and compromise ofearly pregnancy in P. chabaudi AS infectionwas
IL-6(Fig.5C)alsovariedacrossthegroups(Kruskal–Wallistest, explored.Placentalantioxidantgeneexpressionandlipidperox-
p , 0.05), but pairwise post hoc tests did not yield significant idationinmouseconceptuseswereexaminedandtheimpactof
results.Whenthelevelsofthesefactorswereassessedasafunction antioxidanttreatmentonmidgestationalpregnancyoutcomesin
ofpregnancyoutcome(abortingornot)amongIPmice,bothIFN- malaria-infected mice was assessed. We present evidence that
g(Fig.6A)andCCL2(Fig.6E)werefoundtoclearlysegregateto supportsthehypothesisthatoxidativestresscontributestopoor
lowlevelsinnonabortingmiceandhighlevelsinabortingmicein pregnancyoutcomesduringPM.
TPL-treatedmice,butnotinthecontrolgroup.Interestingly,TPL Previous studies in murine P. berghei NK65 infection in late
treatmentappearedtosuppressTNFamongabortingmice(Fig. pregnancymalariaandinhumanPMhavesuggestedthatmalaria
https://doi.org/10.4049/immunohorizons.1700002
ImmunoHorizons TARGETINGOXIDATIVESTRESSINPLACENTALMALARIA 37
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FIGURE5.LevelsofcytokinesandchemokinesinTPL-treated(fromED2)andcontrol(H2O)miceatmidgestation(ED10). brg/
LevelsofplasmaIFN(A),TNF(B),IL-6(C),IL-10(D),andCCL2(E),aswellasplacentaltissuelysateCCL2(F)areindicated.Thedatashownare y
g
derived from 10 experimental start days (three to six mice per experiment), which yielded a total of 45 mice. The Kruskal–Wallis test showed u
e
significanceacrossthegroupsforIFN-g(p=0.0120)andIL-6(p=0.0219),butpairwiseposthoctestsdidnot.*p,0.05,**p,0.01.ns,not st o
n
statisticallysignificant. N
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impacts the antioxidant response in the placenta and promotes ThemostremarkableantioxidantresponseintheP.chabaudi b
e
oxidativestress(8,9,11).Ourresultscorroboratethosestudies. AS–infected placenta was Sod3, the expression of which was r 1
7
InP.chabaudiASinfectioninB6mice,weobservedincreased highly elevated at alltested time points.Although the source of , 2
expressionofNrf2inconceptuses.ThisincreaseinNrf2mRNA thesetranscriptsintheplacentawasnotidentified,itisnoteworthy 01
8
was coincident with peak parasitemia, andcorrespondedto the that this enzyme plays an important role in scavenging ROS
daybeforeabortionbeginsinP.chabaudiAS–infectedmice.This extracellularly(41).InPM,wheresequesteredparasites(42)and
upregulation was observed together with increased expression responding immune cells (19, 20, 43) are likely to represent a
ofantioxidantgenesSod1,Sod2,Sod3,andCat.Expressionlevels potent source of ROS, extracellular SOD may be critical to
for most of these genes returnednearly to baseline on ED10. A counterbalance high levels of oxidative stress. Further studies
different pattern for placental catalase activity was observed in willberequiredtodeterminetheextenttowhichthisparticular
BALB/c mice infected at midgestation with P. berghei NK65: enzymemaybeacriticalplayerintheresponsetoPM,aswellas
relativetouninfectedmiceduringascendingparasitemia,catalase cellularsources.
activitywasreduced(9).Althoughthebasisforthisdiscrepancyis Regardlessoftheobservedsignificantupregulationofantiox-
notclear,thereare multipleobviousdifferencesinstudy design idantgeneexpression,intensestainingforHNE(lipidperoxida-
betweenthatandthepresentwork.ItisnoteworthythatP.berghei tion) in the placentas of infected mice suggests that the local
ANKA infection in B6 mice was associated with a significant antioxidantresponsewasinadequatetoprotecttheplacentafrom
decrease in Cat and Sod1 expression in the brain (40). Thus, infection-induced oxidative stress. Given that excessive or un-
different parasite species may mediate variable oxidative re- controlledproductionofHNEinterfereswithcellularsignaling,
sponsesinmice. andaroleinthepathogenesisofseveraldiseases,includingsevere
https://doi.org/10.4049/immunohorizons.1700002