w w w . e l s e v ie r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Original
article
Classical
and
alternative
macrophages
have
impaired
function
during
acute
and
chronic
HIV-1
infection
Leonardo
J.
Galvão-Lima
a,
Milena
S.
Espíndola
a,
Luana
S.
Soares
a,
Fabiana
A.
Zambuzi
a,
Maira
Cacemiro
a,
Caroline
Fontanari
a,
Valdes
R.
Bollela
b,
Fabiani
G.
Frantz
a,∗aUniversidadedeSãoPaulo,FaculdadedeCiênciasFarmacêuticas,LaboratóriodeImunologiaeEpigenética,SãoPaulo,SP,Brazil bUniversidadedeSãoPaulo,HospitaldasClínicasdeRibeirãoPreto,DivisãodeDoenc¸asInfecciosas,SãoPaulo,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received25May2016 Accepted3October2016
Availableonline29November2016
Keywords: HIV-1
Innateimmuneresponse HAART
Macrophages
a
b
s
t
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a
c
t
Objectives:ThreedecadesafterHIVrecognitionanditsassociationwithAIDSdevelopment, manyadvanceshaveemerged–especiallyrelatedtopreventionandtreatment. Undoubt-edly,thedevelopmentofHighlyActiveAntiretroviralTherapy(HAART)dramaticallychanged thefutureofthesyndromethatweknowtoday.Inthepresentstudy,weevaluatetheimpact ofHighlyActiveAntiretroviralTherapyonmacrophagefunctionanditsrelevancetoHIV pathogenesis.
Methods:PBMCswereisolatedfrombloodsamplesandmonocytes(CD14+cells)were puri-fied.Monocyte-DerivedMacrophages(MDMs)wereactivatedonclassical(MGM-CSF+IFN-␥)or
alternative(MIL-4+IL13)patternsusinghumanrecombinantcytokinesforsixdays.Afterthis
period,Monocyte-DerivedMacrophageswerestimulatedwithTLR2/Dectin-1orTLR4 ago-nistsandweevaluatedtheinfluenceofHIV-1infectionandHighlyActiveAntiretroviral Therapyonthereleaseofcytokines/chemokinesbymacrophages.
Results:ThedatawereobtainedusingMonocyte-DerivedMacrophagesderivedfromHIV naïveorfrompatientsonregularHighlyActiveAntiretroviralTherapy.Classically Monocyte-DerivedMacrophagesobtainedfromHIV-1infectedpatientsonHighlyActiveAntiretroviral TherapyreleasedhigherlevelsofIL-6andIL-12evenwithoutPAMPsstimuliwhencompared tocontrolgroup.Ontheotherhand,alternativeMonocyte-DerivedMacrophagesderived fromHIV-1infectedpatientsonHighlyActiveAntiretroviralTherapyreleasedlower lev-elsofIL-6,IL-10,TNF-␣,IP-10andRANTESafterLPSstimuliwhencomparedtocontrol group.Furthermore,healthyindividualshaveacomplexnetworkofcytokines/chemokines releasedbyMonocyte-DerivedMacrophagesafterPAMPstimuli,whichwasdeeplyaffected inMDMsobtainedfromnaïveHIV-1infectedpatientsandonlypartiallyrestoredinMDMs derivedfromHIV-1infectedpatientsevenonregularHighlyActiveAntiretroviralTherapy.
∗ Correspondingauthor.
E-mailaddress:[email protected](F.G.Frantz). http://dx.doi.org/10.1016/j.bjid.2016.10.004
1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Conclusion: Ourtherapyprotocolswerenoteffectiveinrestoringthefunctionalalterations inducedbyHIV,especiallythosefoundonmacrophages.Thesefindingsindicatethatwestill needtodevelopnewapproachesandimprovethecurrenttherapyprotocols,focusingonthe reestablishmentofcellularfunctionsandprevention/treatmentofopportunisticinfections. ©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction
SincethediscoverythatHIVwasthecauseoftheAIDSand the establishment of this condition as the main cause of deathassociatedwith infectiousdiseases,recentdata esti-matethatover35millionpeoplearoundtheworldareliving withHIV.1 During the last three decades, therehave been
manyadvancesinthe understandingofviralcycle replica-tionandpathogenesis-relatedmechanisms.Asaresultseveral antiretroviral drugs were developed. Specifically, the com-binedHighlyActiveAntiretroviralTherapy(HAART)resulted in the reduction of deaths related to AIDS, helped con-trolling the viral spread and increased the quality of life andthe lifeexpectancyofHIV-1 infectedpatients.2–4
How-ever,manychallengesstillhamperthedevelopmentofnew and more effective therapeutic approaches to functional cure.
Classically,theimmunedysfunctionobservedduringHIV infectionisdirectlyassociatedwithanintensereductionof CD4+ Tcells in peripheralblood and other tissues includ-ing intestinalmucosa. Inrecentyears several studies have demonstrated thecontribution ofinnateimmunitytoviral pathogenesisandAIDSdevelopment.5–7Thispaperwillfocus
ontheroleofmacrophages,butotherauthorshaveexplored the relevance of dendritic cells and neutrophils on HIV pathogenesis.8–10
Monocyte-Derived Macrophages (MDMs) play a central role in the immune response, orchestrating the develop-ment ofinnate and adaptive immunity topathogens, and further, these MDMs can be infected chronically by HIV.11
Besidesremainingviable,HIVinfectedMDMscanbe differ-entlyactivatedanddevelopseveralfunctionalimpairments, withreducedphagocyticandintracellularkillingactivity,thus allowingfortheoccurrenceofopportunisticinfections.12–14
Intherecentyears,Chiharaandcolleagueshave demon-strated that HIV-1 proteins including gp120, Tat, and Nef induce macrophage polarization towards the classi-cal pathway13 and, according to Cassol and colleagues,
enablemacrophagesupportforincreasedviralreplication.15
Currently,few papershave evaluated the role of antiretro-viraldrugs on functional immunerecovery afterthe onset of regular therapy protocols. In our study we evaluated the influence of HIV-1 infection on cytokine/chemokine releasebyclassically(MGM-CSF+IFN-␥)oralternatively(MIL-4+IL13)
activated MDMs after PAMPs stimuli on MDMs derived from treatment-naïve patients or from those on regular HAART.
Material
and
methods
Casuisticandselectioncriteria
Eighttreatment-naïveHIV-1infectedpatientsonearlystage ofdiseasewererecruitedforthisstudy.Inaddition,15 HIV-1infectedpatientsonregularHAART(HIV-1+HAART)forat leastsixmonthsand15healthyblooddonorswereincluded ascontrols(Table1).Informedconsentwasobtainedfromall participantsincludedinthestudy.Allproceduresperformed inthestudyinvolvinghumanparticipantswereinaccordance withtheethicalstandardsoftheinstitutionaland/ornational researchcommitteeandwiththe 1964Helsinki declaration and itslateramendmentsorcomparableethicalstandards. ThepresentstudywasapprovedbytheClinical Hospitalof FMRP/USPEthicsCommittee(#9817/2012).
IsolationPBMCs,purificationofCD14+cells,andMDMs activation
Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-PaqueTM PLUS (GE Healthcare), following the
manufacturer’sinstructions.TheobtainedPBMCswere quan-tified and the monocytes (CD14+ cells)were purified using positive selection with magnetic system microbeads (Mil-tenyiBiotec).Afterelution,theCD14+cellswereculturedin 48-wellplates with RPMI1640mediumcontaining 1L/mL gentamicin, supplementedwith 10% ofFBS (Gibco Labora-tories) and incubated at37◦C,5%CO2. Toinduce classical
(MGM-CSF+IFN-␥) or alternative (MIL-4+IL13) MDMs activation,
CD14+cells wereculturedforsixdaysinmedium contain-ing GM-CSF (20ng/mL; R&D Systems)and IFN-␥ (10ng/mL; Millipore) or IL-4 and IL-13(50ng/mL each; R&D Systems), respectively.RestingMDMs(M0)wereculturedwithstandard medium without supplementation ofcytokines. Every two daysinculture,freshmediumcontainingtherespective con-ditions(M0,MGM-CSF+IFN-␥orMIL-4+IL13)wasaddedtokeepcell
viability.
Quantificationofcytokinesandchemokineson supernatantafterPAMPsstimuli
Aftertherespectiveactivation,MDMswerestimulatedwith 10ng/mL of LPS (Sigma–Aldrich) or 100g/mL of -glucan extracted from Saccharomyces cerevisiae (Calbiochem, Merck Millipore)during24h.Afterthisperiod,thesupernatantwas collectedandninecytokinesandchemokinesreleased(IL-1,
Table1–Generalcharacteristicsofpatientsincludedinthestudy.
Groupsevaluated Control(n=15) HIV-1+(n=8) HIV-1+under
HAART(n=15) Characteristics
Age(years) 28.4 35.7 46.5
Infectiontime/diagnosis(months) – 20.8 108.7
TimeelapsedbetweendiagnosisandstartofregularHAART(inmonths) – – 22.5
TimeunderregularHAART(months) – – 59.7
CD4+Tcellnadir(cells/mm3) – 382.5 155
CD8+Tcellnadir(cells/mm3) – 1011.1 483.1
CD4+/CD8+Tcellratioondiagnosis – 0.37 0.32
CD4+TcellcountafterregularHAART(cells/mm3) – – 535.4
CD8+TcellcountafterregularHAART(cells/mm3) – – 926.6
CD4+/CD8+TcellratioafterregularHAART – – 0.57
Serumviralload(copies/mL) – 41,868.25 <50a
HAART,HighlyActiveAnti-RetroviralTherapy.
a AllselectedpatientshadtotaladherencetotheHAART.
IL-6, IL-10,IL-12, IFN-␣2,TFN-␣,IP-10, MCP-1and RANTES) were quantified using a multiplex assay (HCYTOMAG-60K, Milliplex® kit, Merck Millipore, Germany) with Luminex® MagpixTM technology (Austin, TX, USA). The assay was
performed following the manufacturer’s instructions. The cytokines/chemokinesconcentrationswerecalculatedby Mil-liplexAnalyst5.1®softwareusingastandardcurvewithcubic splinefitting(logscale).
Statisticalanalysis
All data are presented as medianwith interquartile range and the classical statistical analysis was performed using Kruskal–Wallis test followed by Dunn’s test between all columns pairs. For correlation analysis was used Spear-man’s two-tailed test and the networks of correlations of cytokines/chemokines released was built using Cytoscape software (v.3.2.0), classifying the interactions according to thefollowingratio:negative(r<0);weak(r<0.36);moderate (0.36<r<0.67); or strong(r>0.67). In bothanalyses, p≤0.05 wasconsideredsignificant.
Results
HIV-1patientsunderregularuseofHAARTpresentmore pronouncedimpairmentofcytokines/chemokinesreleased fromMDMsafterPAMPsstimuli
The present data show that classical MDMs derived from both treatment-naïve HIV-1 infected patients and HIV-1+HAARTgroupsexpressedverysimilarlevelsofcytokines and chemokines after PAMPs stimuli. In spite of that, we observedanincreaseofIL-6andIL-12releasedby unstimu-latedMDMsderivedfromHIV-1+HAARTpatientscompared tocontrolpatients(Fig.S1).Ontheotherhand,alternatively activatedMDMsobtainedfromtheanalyzedgroupsshoweda lowerlevelofIL-6,IL-10,TNF-␣,IP-10,andRANTESreleasedby HIV-1+HAARTafterLPSstimulicomparedtocontrolpatients (Fig.S2).
MDMsobtainedfromtreatment-naïveHIV-1infected patientspresentseveralimpairmentsin
cytokines/chemokinesexpressionafterPAMPsstimuliand thesealterationswerenotrestoredwithantiretroviral therapy
To fully exploretheimpact ofHAART onthe development oftheMDM-dependentimmuneresponse,weexaminedthe expressionofcytokines/chemokinesfromclassicallyor alter-nativelyactivatedMDMsderivedfromtreatment-naïveHIV-1 infectedpatients,HIV-1+HAARTorcontrolgroupsafterPAMP stimuli.
Atbaseline(withoutPAMPstimuli),classicalMDMsderived from controlpatientsexpressedafewpositivecorrelations, especially strong between IL-1↔IL-6↔IL-10 (forming the ‘elementary triangle’) and moderate between IL-1↔IL-10 andTNF-␣↔IL-12↔IFN-␣2↔MCP-1(Fig.1A).However,these interactions changedwhenweanalyzedthe MDMcytokine expression patterns obtained from treatment-naïve HIV-1 infectedpatients.Inthesepatientstheelementarytriangleis preserved,intensifyingthecorrelationbetweenIL-1↔IL-10 and forming a new strong correlation amongst IL-12↔ IP-10↔IL-10 (Fig. 1B), suggesting a ‘primed’ proinflammatory profileofMDMsobtainedfromtreatment-naïveHIV-1infected patients. In HIV-1+HAART patients without PAMP stimuli, the elementary triangle was similar to the control group, with an increase of new moderate positive correlations (IL-10↔IL-12; TNF-␣↔RANTES) and,interestingly, a nega-tive(IL-1↔RANTES)correlation(Fig.1C).Nevertheless,the main changes observed amongst the groups with respect to classical MDMs’ function occurred after PAMP stimuli. Thecomplex networkobservedafterLPSstimulion MDMs derived from thecontrol group(Fig.1D)showed decreased interactions among cytokines from treatment-naïve HIV-1 infectedpatients,particularlyaffectingIL-6,IL-12,TNF-␣and IP-10(Fig.1E).Decreasedcytokinecorrelationswerestrongly accentuatedinMDMsobtainedfromHIV-1+HAARTpatients; in contrast, only six of 23 interactions obtained from the controlgroupwerepreserved(Fig.1F).Similarly,after recog-nitionof-glucan,classicalMDMsderivedfromthecontrol group formeda complex correlationnetwork (Fig. 1G) that
Fig.1–HIV-1infectioninducesdamageoncytokineandchemokinenetworkformedbyclassicalMDMsthatarenot restoredevenunderHAART.Theanalysisofcorrelationbetweenthecytokinesandchemokinesreleasedbyunstimulated, LPS-or-glucan-stimulatedclassicalMDMswasperformedusingSpearman’stwo-tailedtest.Thenetworkwasbuiltusing Cytoscapesoftware(v3.2.0).Solidlinesindicatestrongcorrelationsanddash-dotlinesarerepresentativeofmoderate correlations.Alltheblacklinesrepresentpositivecorrelationsbetweenthetargetsandtheredlinesarerepresentativeof negativecorrelations.
wasprogressivelylosingthe‘elementarytriangle’andother important correlations seen in the treatment-naïve HIV-1 infectedandHIV-1+HAARTgroups–preservingonlyfour inter-actions vs 23 obtained in the control group (Fig. 1H and I).
Inparallel,thebaselineanalysisofalternativeMDM net-work correlations revealed that the complex correlations (includingtheelementarytriangle)seeninthecontrolgroup wasnotpresentintreatment-naïveHIV-1infectedpatients (onlysevenof20previousinteractionsremained)andwasonly partiallyrestoredinHIV-1+HAARTpatients(with10of20 cor-relationsobserved),reducingtheIL-10andTNF-␣correlations andincreasingmoderateinteractionswithIL-12(Fig.2A–C). AfterstimuliwithLPSand-glucan,asimilarpatternof cor-relationwas observedamongst the groups, represented by complexinteractionsinthecontrolgroup(mainlybasedon TNF-␣;Fig. 2D and G) and reduced numbers and intensity ofcorrelationsfoundinMDMsfrom treatment-naïveHIV-1 infectedpatients(Fig.2EandH).Additionally,theanalysisof supernatantsobtainedfromHIV-1+HAARTpatientsafterLPS and-glucanstimuliresultedincorrelationssimilartothose foundinthecontrolgroupaswellastwonewnegative corre-lationsamongRANTES↔IL-1↔IP-10after-glucanstimuli (Fig.2FandI).
Discussion
Classically, the term “macrophage” refers to a set of ter-minally differentiated cells with low proliferation capacity that havedifferentnames accordingtotheir localization.16
Despite the commonname,macrophagesdonotrepresent ahomogeneouspopulationandcanoriginatefromtwo dis-tinctembryologicalsources.Thefirst waveofmacrophages is generated during early embryogenesis and is derived from Yolk Sac progenitors, giving rise to physiological tis-suemacrophages(namedmicroglia,Langerhanscells,Kupffer cells andalveolarmacrophages) withthemarkedabilityto self-renewintheperiphery.17–19Ontheotherhand,the
sec-ond wave of macrophages is derived from Hematopoietic StemCells(HSCs)thatbecomecirculatingbloodmonocytes, whichaftermigrationtoperipheraltissuesdifferentiateinto MDMs.20
Under physiological conditions, MDMs display a full spectrum of activation in different tissues, reflecting the microenvironment milieu in which they find themselves.21
TherecognitionofPAMPs(e.g.,LPS)and/orproinflammatory cytokines inducestheclassical macrophage(M1) activation pathway, related to Th1 CD4+ T cell activation, while the
Fig.2–HIV-1infectioninducesdamageoncytokineandchemokinenetworksformedbyalternativeMDMsthatarenot restoredevenunderHAART.Theanalysisofcorrelationbetweenthecytokinesandchemokinesreleasedbyunstimulated, LPS-or-glucan-stimulatedalternativeMDMswasperformedusingSpearman’stwo-tailedtest.Thenetworkwasbuilt usingCytoscapesoftware(v3.2.0).Solidlinesindicatestrongcorrelationsanddash-dotlinesarerepresentativeofmoderate correlations.Alltheblacklinesrepresentpositivecorrelationbetweenthetargetsandtheredlinesarerepresentativeof negativecorrelations.
recognitionofglucocorticoids,immunecomplexes,orothers cytokines(e.g.,IL-4,IL-10andIL-13)inducesthealternative macrophage(M2)activationpathway,relatedtoTh2CD4+T cellactivation.22,23
Althoughembryologicaldistinct,bothmacrophagesubsets can productively beinfected and contribute to HIV patho-genesisduringallstagesofinfection.24Thefirstevidenceof
HIV-infectedmacrophageswerepublishedinthe1980s,25,26
butitwasnotuntilrecentlythisfieldhasbeenbetterexplored. In this context, our knowledge of macrophage function is basedmainlyonstudiesofMDMsandnewstudiesare crit-icallyneededtoclosethelargegapofknowledgerelatingto YSDMsfunctionduringHIVinfection(Fig.3).
Overtheyears,differentauthorsdemonstratedthatonly afewmonocytes foundonperipheralbloodareinfectedby HIV-1,27,28 whileothersstudiesdemonstratedthatthevirus
candirectlyinfectbonemarrowprogenitors.29–31Despitethis
paradox,aftermigrationtoperipheraltissues,monocytes dif-ferentiate into macrophages and, even though expressing severalfactorsrestrictingviralreplication,32theseMDMsare
chronicallyinfectedandactasanimportantviralreservoir, allowingHIVreplicationandspreadtoothercells(including CD4+Tcells)duringthenormaltimecourseofinfection.11,33
ThisviralreplicationstrategyiscriticaltoHIVpathogenesis andgivesthesemacrophagesa‘Trojanhorse’status,thatcan
bedirectlyassociatedwiththedevelopmentofcomorbidities, suchasimmunosenescenceandinflammatory/cardiovascular diseases,andtheprogressiontoAIDS.34,35
Herein we have shown the increase of IL-6 and IL-12 released from classical MDMs derived from HIV-1+HAART. Thismay beassociatedtoprolonged timeofinfection and higherbaselineactivationofmacrophagesinducedby micro-bialtranslocationfromguttosystemiccirculation,acommon phenomenon found in HIV infected patients.36 As
conse-quenceoftheseevents,amassivereleaseofproinflammatory cytokines (also known as ‘cytokine storm’) takes place in plasma and the increaseofbaseline levelsofcellular acti-vation status. Interestingly, alternatively activated MDMs obtainedfromHIV-1+HAARTpatientsreleaselowerlevelsof IL-6, IL-10, TNF-␣,IP-10, and RANTES compared tocontrol patientsafterLPSstimuli.
Basedontheseresults,wehypothesizedthatthe reduc-tionofproinflammatorycytokinescanbeasecondarydamage induced by the chronicity of HIV infection and establish-mentofviralreservoirs,whichcouldbereprogrammingthe macrophage function, or even could be induced by regu-lar antiretroviral therapy. Despite the relevance of HAART in controlling viral load and restoring CD4+ T cells count andCD4+/CD8+TcellratioinHIVinfectedpatients,regular HAARTwouldimpairmacrophagefunctionafterPAMPstimuli
Fig.3–RoleofdistinctmacrophagepopulationsondevelopmentofHIV-1pathogenesis.Despitebeingontogenically distinct,YSDMsandMDMshaveacriticalroleinthemaintenanceofviralreplication,reflectedinthedevelopmentof chronicHIV-1pathogenesis.MDMsarederivedfromtheterminaldifferentiationofmyeloidbonemarrowprogenitorcells andseveralstudieshavedemonstratedthatMDMsobtainedfromHIV-1patientshavefunctionalimpairmentsinvitro.12–14
Althoughmonocytesarepoorlyinfectedinthebloodstream,HIV-1caninfectdirectlyCD34+progenitorcellsandinduce functionalimpairmentsduringhematopoiesis.29–31Sometissuemacrophagesarederivedfrommonocytedifferentiationof
peripheraltissuesorfromYolkSacprogenitorsseededduringtheearlystepsofembryogenesis.17–19Bothofthesesubsets
canbechronicallyinfectedandcausethespreadofvirustoothercells.Inparallel,microgliaandotherCNS(asperivascular andmeningeal)macrophagesaredirectlyderivedfromYSprogenitorsandcontributetodevelopmentofneuronaldeath andHANDfoundmainlyonuntreatedHIVpatients.24,33
inthesepatients,whichmayberelatedtoglobalreductionof proinflammatorystatusfoundinHIV-infectedpatients.These hypothesesaresupportedbythegoodtherapeuticadherence andthelateinfectionstageontheonsetoftherapyof HIV-1+HAARTpatients (Table1), suggestingthat thesefailures areduetoprolongedtimeofHIVinfectionand alsobythe treatmentperse.
Takentogether,ourdatarevealthatalternativelyactivated MDMsaremoreadverselyaffectedafterHIV-1infectionthan classicallyactivatedMDMsandtheantiretroviraltherapydid notcompletely restore the MDMs functionalityafter PAMP stimuli.These results are consistent with previous reports demonstratingthatalternativelyactivatedmacrophagesare more affected after contact with HIV-1 proteins. Further-more,thiscontactinducesthereprogrammingofalternative towardsclassicalMDMspattern,andthisimbalancemaybe associatedwiththespreadofviralinfection13andthefailure
ofTcell-dependentimmuneresponsesthatfurthercontribute todevelopmentofAIDS.37
Undoubtedly, the developmentofantiretroviral drugsin the1990sandtheimprovementoftherapeuticprotocolshave changedthecourseofHIV-1infectioninthelast20years.38,39
Currently,theFDAhasapproved26antiretroviralsfortreating HIVinfection,includingcombinedtherapiesthattarget dif-ferentstepsofviralentryorthereplicationcycle.40Usually
afterafewweeksonHAART,theviralloadandthe inflamma-torymediatorsdecreasedrasticallyinplasma,oftenfallingto undetectablelevelsandstabilizing theCD4+Tcellscounts. However,evenundercontinuoustherapy,patientsmaintain HIV-1replicationindifferenttissuereservoirsthatare respon-sibleforthelongtermpersistenceofviralinfection.41Based
on the above positive results, the WHO now recommends antiretroviraltherapiesbemadeavailabletoallpatients diag-nosedandlivingwithHIV.42Relatedly,ourgroupandothers
Fig.4–HIV-1infectioninducesseveralimpairmentsonclassicalandalternativeMDMsthatwerenotfullyrestoredeven underregularHAART.AfterinfectionbyHIV-1,MDMsactivationisinfluencedbyinteractionwithviralproteinsand responsibleforsustainingviralreplicationoverdifferentstagesofinfection.ClassicalandalternativelyactivatedMDMs presentfunctionalimpairments–asreductioninphagocyticandintracellularkillingactivityandthegeneralprofileof cytokines/chemokinereleasedafterrecognitionofantigenicstimuli.EvenunderregularHAART,MDMsobtainedfrom HIV-1+patientsarechronicallyinfectedandrepresentanimportantviralreservoir.Thefunctionalchangesfoundinthese cellsareonlypartiallyrestored,impairingthedevelopmentofappropriateimmuneresponsesagainstpathogens,allowing theoccurrenceofopportunisticinfections.
recently have demonstrated that the adoption of HAART works to effectively reduce the viral load and the inflam-mationbiomarkersinplasma,43–45reducingboththeeffects
of the systemic inflammatory disease and the comorbidi-ties/mortalityassociatedwithHIVinfection.46,47
Despite these therapeutic successes, no currently used drugisabletorestoretheMDMfunctionalalterationsinduced byHIVordirectlystrengthentheimmuneresponseofinfected patients.Thesefindingsindicatethatwestillneedtodevelop newapproachesandimproveourcurrenttherapeutic proto-cols, focusingon reestablishmentofcellular functionsand prevention/treatmentofopportunisticinfections.
Therefore,theseobservationssuggestanimportanteffect of antiretroviral drugs on MDMs function, even without PAMPstimuliinvitro.UnderregularHAART,HIV-1+patients exhibitedapronouncedreductionofinflammatorymediators (includingsCD14,sCD163andTF)associatedwithmicrobial translocationand development ofcomorbiditiesassociated withinfection,48–50thusimprovingthelifeexpectancyofthese
patients. Recently, our group showed that treatment with
secondlinetherapyprotocolsdonotrestoretheimmune acti-vation status, despitethe reductionof viral load found in plasmasamples.43Inthepresentpaper,weexaminedMDMs
functionduringHIV-1infectionanddemonstratedan impor-tantreductionincytokine/chemokinereleasedbyclassically andalternativelyactivatedMDMsderivedfromHIV-1infected patients atbaselineand after recognitionofPAMPstimuli. Afterall,it itsunquestionabletherelevanceofHAART dur-ingHIVinfectionanditsbenefitstoavoidAIDSdevelopment. Severalrecentstudiesevidencedthatthebeginningof com-binedantiretroviraltherapyeveninnewlydiagnosedpatients has agreatepidemiologicalrelevance andis being encour-agedbyWHO.51Nonetheless,wecannotmissthepointthat
thecurrentHAARTprotocolsdonotrestoretheimpairments found in macrophages or in other immune cells obtained fromHIV-infectedpatients,andthesefunctionalchangesmay beassociatedtothedevelopmentofothernon-AIDSrelated comorbidities (Fig. 4). In Brazil, sincethe year of 2014, all HIVinfectedpatientsareentitledtostarttreatment irrespec-tiveofcomorbiditiesorCD4+Tcellcountingandviralload
level.52 Therefore,inthedatashowedhere,consideringthe
CD4+Tcellnadir,CD4+/CD8+Tcellratioondiagnosis,and thetimeelapsedsincediagnosisandstartofregulartherapy, theHIV-treatedpatientswereinalatestageofinfection com-paredtothetreatment-naïveHIV-1infectedgroup.Infact,the advancedstagesofHIVinfectionmayinduceseveral impair-ments onimmune responsethat could bereflected inour cytokinedata.Anotherpointtoconsideristhe immunosenes-cence.ItiswelldocumentedthatHIVinfectionisrelatedto prematureimmunosenescence53,54andtheprolongedtimeof
infectioncouldacceleratetheoccurrenceoffunctional alter-ationsinmacrophages.Thus,itiscrucialthatnewadjuvant therapiesshouldbedeveloped,consideringtheaimat restor-ingcellularfunctionwhileeliminatingviralreservoirs.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
Thefunding forthis work was provided by the São Paulo Research Foundation (FAPESP, grants #2011/12199-0 and #2012/02799-3)andCAPES.Theauthorsaregratefulto Hemo-centrodeRibeirãoPretoandHospitaldasClínicasdeRibeirão Preto–FMRP/USP.TheauthorsthankDr.JudithConnettfor herthoroughreadingofthemanuscript.
Appendix
A.
Supplementary
data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.bjid.2016.10.004.
r
e
f
e
r
e
n
c
e
s
1. WHO.HIVReporting:Globalupdateonthehealthsector responsetoHIV.Geneva:WorldHealthOrganization;2014. 2. CrumNF,RiffenburghRH,WegnerS,etal.Comparisonsof
causesofdeathandmortalityratesamongHIV-infected persons:analysisofthepre-,early,andlateHAART(highly activeantiretroviraltherapy)eras.JAcquirImmuneDefic Syndr.2006;41:194–200.
3. PalellaFJJr,BakerRK,MoormanAC,etal.Mortalityinthe highlyactiveantiretroviraltherapyera:changingcausesof deathanddiseaseintheHIVoutpatientstudy.JAcquir ImmuneDeficSyndr.2006;43:27–34.
4. GranichR,CrowleyS,VitoriaM,etal.Highlyactive antiretroviraltreatmentaspreventionofHIVtransmission: reviewofscientificevidenceandupdate.CurrOpinHIVAIDS. 2010;5:298–304.
5. ChangJJ,AltfeldM.Innateimmuneactivationinprimary HIV-1infection.JInfectDis.2010;202Suppl.2:S297–301. 6. MogensenTH,MelchjorsenJ,LarsenCS,PaludanSR.Innate
immunerecognitionandactivationduringHIVinfection. Retrovirology.2010;7:54.
7. RasaiyaahJ,TanCP,FletcherAJ,etal.HIV-1evadesinnate immunerecognitionthroughspecificcofactorrecruitment. Nature.2013;503:402–5.
8.Izquierdo-UserosN,LorizateM,McLarenPJ,TelentiA, KrausslichHG,Martinez-PicadoJ.HIV-1captureand transmissionbydendriticcells:theroleofviralglycolipids andthecellularreceptorSiglec-1.PLoSPathog.
2014;10:e1004146.
9.BowersNL,HeltonES,HuijbregtsRP,GoepfertPA,HeathSL, HelZ.ImmunesuppressionbyneutrophilsinHIV-1infection: roleofPD-L1/PD-1pathway.PLoSPathog.2014;10:e1003993. 10.SilvinA,ManelN.InnateimmunesensingofHIVinfection.
CurrOpinImmunol.2015;32:54–60.
11.KumarA,AbbasW,HerbeinG.HIV-1latencyin monocytes/macrophages.Viruses.2014;6:1837–60. 12.CassolE,CassettaL,AlfanoM,PoliG.Macrophage
polarizationandHIV-1infection.JLeukocBiol. 2010;87:599–608.
13.ChiharaT,HashimotoM,OsmanA,etal.HIV-1proteins preferentiallyactivateanti-inflammatoryM2-type macrophages.JImmunol.2012;188:3620–7.
14.LudlowLE,ZhouJ,TippettE,etal.HIV-1inhibitsphagocytosis andinflammatorycytokineresponsesofhuman
monocyte-derivedmacrophagestoP.falciparuminfected erythrocytes.PLoSONE.2012;7:e32102.
15.CassolE,CassettaL,RizziC,AlfanoM,PoliG.M1andM2a polarizationofhumanmonocyte-derivedmacrophages inhibitsHIV-1replicationbydistinctmechanisms.JImmunol. 2009;182:6237–46.
16.DaviesLC,JenkinsSJ,AllenJE,TaylorPR.Tissue-resident macrophages.NatImmunol.2013;14:986–95.
17.GinhouxF,GreterM,LeboeufM,etal.Fatemappinganalysis revealsthatadultmicrogliaderivefromprimitive
macrophages.Science.2010;330:841–5.
18.SchulzC,GomezPerdigueroE,ChorroL,etal.Alineageof myeloidcellsindependentofMybandhematopoieticstem cells.Science.2012;336:86–90.
19.GomezPerdigueroE,KlapprothK,SchulzC,etal.
Tissue-residentmacrophagesoriginatefromyolk-sac-derived erythro-myeloidprogenitors.Nature.2015;518:547–51. 20.HoeffelG,GinhouxF.Ontogenyoftissue-resident
macrophages.FrontImmunol.2015;6:486.
21.NovakML,KohTJ.Macrophagephenotypesduringtissue repair.JLeukocBiol.2013;93:875–81.
22.BiswasSK,MantovaniA.Macrophageplasticityand interactionwithlymphocytesubsets:cancerasaparadigm. NatImmunol.2010;11:889–96.
23.MurrayPJ,AllenJE,BiswasSK,etal.Macrophageactivation andpolarization:nomenclatureandexperimentalguidelines. Immunity.2014;41:14–20.
24.BurdoTH,LacknerA,WilliamsKC.Monocyte/macrophages andtheirroleinHIVneuropathogenesis.ImmunolRev. 2013;254:102–13.
25.HoDD,RotaTR,HirschMS.Infectionof
monocyte/macrophagesbyhumanTlymphotropicvirustype III.JClinInvestig.1986;77:1712–5.
26.NicholsonJK,CrossGD,CallawayCS,McDougalJS.Invitro infectionofhumanmonocyteswithhumanTlymphotropic virustypeIII/lymphadenopathy-associatedvirus
(HTLV-III/LAV).JImmunol.1986;137:323–9.
27.AlexakiA,LiuY,WigdahlB.CellularreservoirsofHIV-1and theirroleinviralpersistence.CurrHIVRes.2008;6:388–400. 28.SpivakAM,SalgadoM,RabiSA,O’ConnellKA,BlanksonJN. CirculatingmonocytesarenotamajorreservoirofHIV-1in elitesuppressors.JVirol.2011;85:10399–403.
29.FolksTM,KesslerSW,OrensteinJM,JustementJS,JaffeES, FauciAS.InfectionandreplicationofHIV-1inpurified progenitorcellsofnormalhumanbonemarrow.Science. 1988;242:919–22.
30.AlexakiA,WigdahlB.HIV-1infectionofbonemarrow hematopoieticprogenitorcellsandtheirroleintrafficking andviraldissemination.PLoSPathog.2008;4:e1000215.
31.CarterCC,Onafuwa-NugaA,McNamaraLA,etal.HIV-1 infectsmultipotentprogenitorcellscausingcelldeathand establishinglatentcellularreservoirs.NatMed.
2010;16:446–51.
32.CobosJimenezV,BooimanT,deTaeyeSW,etal.Differential expressionofHIV-1interferingfactorsinmonocyte-derived macrophagesstimulatedwithpolarizingcytokinesor interferons.SciRep.2012;2:763.
33.KoppensteinerH,Brack-WernerR,SchindlerM.Macrophages andtheirrelevanceinHumanImmunodeficiencyVirusTypeI infection.Retrovirology.2012;9:82.
34.AbergJA.Aging,inflammation,andHIVinfection.TopAntivir Med.2012;20:101–5.
35.AnzingerJJ,ButterfieldTR,AngelovichTA,CroweSM,Palmer CS.MonocytesasregulatorsofinflammationandHIV-related comorbiditiesduringcART.JImmunolRes.2014;2014:569819. 36.BrenchleyJM,PriceDA,SchackerTW,etal.Microbial
translocationisacauseofsystemicimmuneactivationin chronicHIVinfection.NatMed.2006;12:1365–71.
37.HerbeinG,VarinA.ThemacrophageinHIV-1infection:from activationtodeactivation?Retrovirology.2010;7:33.
38.Lifeexpectancyofindividualsoncombinationantiretroviral therapyinhigh-incomecountries:acollaborativeanalysisof 14cohortstudies.Lancet.2008;372:293–9.
39.MontanerJS,LimaVD,BarriosR,etal.Associationofhighly activeantiretroviraltherapycoverage,populationviralload, andyearlynewHIVdiagnosesinBritishColumbia,Canada:a population-basedstudy.Lancet.2010;376:532–9.
40.AdministrationUSFaD.Antiretroviraldrugsusedinthe treatmentofHIVinfection;2015.Availablefrom:http://www. fda.gov/forpatients/illness/hivaids/treatment/ucm118915.htm [cited16.03.14].
41.Lorenzo-RedondoR,FryerHR,BedfordT,etal.Persistent HIV-1replicationmaintainsthetissuereservoirduring therapy.Nature.2016.
42.WHO.ConsolidatedguidelinesonHIVprevention,diagnosis, treatmentandcareforkeypopulations;2014.
43.EspindolaMS,LimaLJ,SoaresLS,etal.Dysregulatedimmune activationinsecond-lineHAARTHIV+patientsissimilarto
thatofuntreatedpatients.PLOSONE.2015;10:e0145261. 44.RajasuriarR,BoothD,SolomonA,etal.Biological
determinantsofimmunereconstitutioninHIV-infected patientsreceivingantiretroviraltherapy:theroleof interleukin7andinterleukin7receptoralphaandmicrobial translocation.JInfectDis.2010;202:1254–64.
45.WalletMA,RodriguezCA,YinL,etal.Microbialtranslocation inducespersistentmacrophageactivationunrelatedtoHIV-1 levelsorT-cellactivationfollowingtherapy.AIDS.
2010;24:1281–90.
46.DeeksSG,TracyR,DouekDC.Systemiceffectsof inflammationonhealthduringchronicHIVinfection. Immunity.2013;39:633–45.
47.DeeksSG,LewinSR,HavlirDV.TheendofAIDS:HIVinfection asachronicdisease.Lancet.2013;382:1525–33.
48.BurdoTH,LentzMR,AutissierP,etal.SolubleCD163madeby monocyte/macrophagesisanovelmarkerofHIVactivityin earlyandchronicinfectionpriortoandafteranti-retroviral therapy.JInfectDis.2011;204:154–63.
49.SandlerNG,WandH,RoqueA,etal.Plasmalevelsofsoluble CD14independentlypredictmortalityinHIVinfection.J InfectDis.2011;203:780–90.
50.MarchettiG,Cozzi-LepriA,MerliniE,etal.Microbial translocationpredictsdiseaseprogressionofHIV-infected antiretroviral-naivepatientswithhighCD4+cellcount.AIDS. 2011;25:1385–94.
51.WHO.In:OrganizationWH,editor.Guidelineonwhentostart antiretroviraltherapyandonpre-exposureprophylaxisfor HIV.2015/11/25ed.Geneva.2015.
52.BRASILMDS.In:DepartamentodeDSTAeHV,editor.Protocolo ClínicoeDiretrizesTerapêuticasparaManejodaInfecc¸ão peloHIVemAdultos.Brasília:MINISTÉRIODASAÚDE;2013. 53.DeeksSG.HIVinfection,inflammation,immunosenescence,
andaging.AnnuRevMed.2011;62:141–55.
54.NasiM,PintiM,DeBiasiS,etal.AgingwithHIVinfection:a journeytothecenterofinflammAIDS,immunosenescence andneuroHIV.ImmunolLett.2014;162PtB:329–33.