Elevated Concentrations of Milk β2-Microglobulin Are Associated

7/23/2019 Elevated Concentrations of Milk 2-Microglobulin Are Associated

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Elevated Concentrations of Milk2-Microglobulin Are Associatedwith Increased Risk of Breastfeeding Transmission of HIV1(Vertical Transmission Study)

Alain Mange ,, Edouard Tuaillon,,, Johannes Viljoen,,# NicolasNagot,, Sophie Bendriss,,

Ruth M. Bland,# Marie-Louise Newell,# Philippe Van de Perre,,,, and Je rome Solassol,,,

University of Montpellier 1, EA 2415, 641 av. du Doyen Gaston Giraud, 34093 Montpellier, FranceDepartment of Clinical Oncoproteomic, ICM Val dAurelle, 208 Avenue des Apothicaires, 34298 Montpellier, FranceUniversity of Montpellier 1, 5 Bd Henri IV, 34295 Montpellier, FranceDepartment of Bacteriology and Virology, CHU Arnaud de Villeneuve, 371, av. du Doyen Gaston Giraud, 34295 Montpellier, FranceINSERM, U1058, 60 Rue de Navacelles, 34394 Montpellier, France#Africa Centre for Health and Population Studies, University of KwaZulu-Natal, R618 en rouite to Hlabisa, Somkhele A2074 Rd,Mtubatuba 3935, South Africa

Department of Biopathology, CHU Arnaud de Villeneuve, 371 av. du Doyen Gaston Giraud, 34295 Montpellier, France

*S Supporting Information

ABSTRACT: There is increasing evidence to support a relationship between humanimmunodeciency virus (HIV-1) transmission through breastfeeding and milk hostfactors. We analyzed skim milk proteome to further determine the contribution of hostfactors to the risk of mother-to-child transmission of HIV-1. Quantitative massspectrometry analysis was performed on nine casecontrol pairs of HIV+ transmitter/nontransmitter mothers, and specic biochemical assays on two selected proteins wereassessed in an independent validation set of 127 samples. 33 identied proteins weredifferentially expressed between HIV+ transmitter and nontransmitter mothers. Amongthem,2-microglobulin was signicantly higher in the maternal transmitter than in thenontransmitter groups (pvalue = 0.0007), and S100A9 was signicantly higher in the

early maternal transmitter cases (before 4 months of age) compared with thenontransmitters (p value = 0.004). 2-Microglobulin correlated with milk and plasmaHIV viral load and CD4+ cell count, whereas S100A9 correlated with the estimated timingof infection of the infant through breastfeeding. Finally, 2-microglobulin concentration inmilk could accurately predict the risk of HIV-1 postnatal transmission by breastfeeding (pvalue < 0.0001, log-rank test). In conclusion,milk2-microglobulin and S100A9 are host factors that are found to be associated with mother-to-child transmission of HIV-1.

KEYWORDS: HIV transmission, milk, proteomics

INTRODUCTION

Human milk is key for infant nutrition. It also protects theintegrity of the infants gut by delivering immunological andnonimmunological innate factors with antimicrobial or anti-

inammatory properties. However, despite its benecial effects,breast milk canalsobe a vehicle for the transmission of viruses to theinfant, including cytomegalovirus, human T-lymphotropic virustype I, and human immunodeciency virus type 1 (HIV-1).17

Mother-to-child transmission of HIV has become a rare eventin well-resourced settings, but around 330000 children werenewly infected with HIV in 2010, with 90% of these in sub-Saharan Africa.8 The risk of postnatal transmission of HIV-1through breastfeeding, in the absence of targeted interventions,has been estimated to be 3.2 per 100 child-years of breast-feeding.9 Recent advances in prophylactic HIV-1 treatment haveled to signicant decreases in perinatal transmission of HIV-1

from mother to child.10 However, despite advances in HIV-1transmission prevention, mechanisms of HIV-1 transmissionthrough breastfeeding remains poorly understood. We, andothers, have shown that high levels of cell-free HIV-1 RNA as

well as cell-associated HIV-1 proviral DNA in breast milkincrease the risk of breastfeeding transmission, suggesting a roleof both cell-free and cell-associated HIV-1 in breastfeedingtransmission.6,1113 The length and feeding mode of breastfeed-ing have also been shown to determine the infants risk ofpostnatal transmission of HIV-1.9 Finally, host factors present in

breast milk may alsoplay important roles in an infants susceptibilityto infection withHIV-1. For example, erythropoietin in human milk

Received: June 18, 2013Published: October 21, 2013

Article

pubs.acs.org/jpr

2013 American Chemical Society 5616 dx.doi.org/10.1021/pr400578h| J. Proteome Res.2013, 12, 56165625
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has been suggested to be a protective agent against mother-to-childtransmission of HIV-1 through breastfeeding.14

Not all infants who are breastfed by HIV-1-infected mothersbecome infected, even in the absence of maternal or infantprophylactic treatment.15 Thus, identifying potential factors in

breast milk that are protective or that facilitate transmission ofHIV-1 is an important area of research to prevent mother-to-child in resource-poor settings. Proteins are a major contributorof immunologically active components in human milk. Human

breast milk has been analyzed over the past decade usingcomprehensive proteomic approaches, increasing the number of

identi

ed proteins.1619

Among them, proteins involved inimmunity and inammation have been estimated to be 24% ofthe proteins identied in human milk, signifying the critical rolethat milk plays in protecting infants from infection.18,19 However,despite recent progress in the inventory of proteins in humanmilk, there is still limited knowledge on the defense proteome. Inaddition, a global protein proling assay has, to our knowledge,not yet been investigated to specically identify host factorsassociated with HIV-1 transmission via breast milk. Here weconducted a casecontrol study to identify factors associated

with increased or decreased risk of HIV-1 transmission bybreastfeeding in a cohort of women from South Africa using aquantitative proteomic approach.

METHODS

Patient Selection

For this casecontrol study, breast milk samples were collectedfrom a nonrandomized intervention cohort (the VerticalTransmission Study - VTS) of womenattending nine antenatalclinics in KwaZulu-Natal, South Africa.5,20,21 This study aimed toexamine breastfeeding andHIV-1 transmission (inthe absence ofprophylactic HIV-1 treatment) in a community with a highprevalence of HIV-1 infection. The VTS and breast milk analyses

were approved by the Biomedical Research Ethics Committee of

the University of KwaZulu-Natal. Breast milk samples werecollected by gentle bimanual expression at 6 weeks post-partum and monthly thereafter and stored as whole breast milk(80 C). Postnataltransmission in infants wasdened as HIV-1acquisition (positive PCR) between 6 and 28 weeks of age in apreviously uninfected child. Venous samples were taken from

women at enrollment for plasma RNA load assessment and CD4cell count.

For the discovery set analysis, nine casecontrol pairs, withinformation on breast milk viral load and postnatal transmissionstatus, were selected from the VTS cohort. For cases, the last

breast milk sample available before the estimated timing of infanttransmission was considered for analysis. Controls were

Figure 1.Flow chart illustrating the main steps of the study.

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HIV-uninfected infants matched by infant age and timing ofbreast milk sample collection to samples in transmitting mothers.

For the validation set analysis, 127 unmatched case and controlsubjects were selected from the same VTS cohort: 71 breast milksamples from HIV-1 transmitting and 56 from nontransmittingmothers. Samples of the 71 HIV-1 transmitters were collected

before the childs rst HIV-positive test result (n= 46) or at thesame time (n =25)astherst HIV-positive test result if an earliersample was not available.

Several fractionationmethods to process whole breast milkhave been described;22 here we used a combination of lipidremoval and casein depletion, as previously described.23Aliquotsof skimmed milk samples were stored at 80 C. The proteinconcentration was measured in triplicate using the Micro BCAKit (Pierce).

iTRAQ Reagent Labeling and Mass Spectrometry Analysis

The experimental design used for this study is illustrated inFigure 1 and Supplementary Table S1 in the SupportingInformation. Isobaric tags for relative and absolute quantitation(iTRAQ) labeling and mass spectrometry analysis wereperformed as previously described.24 In brief, proteins of eachsample were digested using trypsin before iTRAQ labeling.Labeled peptides were separated using the Agilent 3100OFFGEL Fractionator (Agilent) and analyzed by 2D nanoliquid chromatography tandem mass spectrometry (nanoLCMS/MS) using a MALDI time-of-ight (MALDI TOF/TOF)4800 mass spectrometer (AB Sciex). Protein identication andquantication was performed with the ProteinPilot Software2.0.1 (AB Sciex). The user-dened search parameters were asfollows: (1) sample type, iTRAQ 8-plex (Peptide Labeled); (2)

cysteine alkylation, MMTS; (3) digestion, trypsin; (4) instru-ment, 4800; (5) special factors, none; (6) species,Homo sapiens;(7) ID focus, biological modication; (8) database, UniProtKB/Swiss-Prot FASTA (release version 2013_08); and (9) searcheffort, Thorough ID. A reversed decoy database from the sameUniProt database was used to estimate the local false discoveryrate (FDR) using the Proteomics System PerformanceEvaluation Pipeline (PSPEP) tool.25 Proteins with one or morepeptides with a high condence score (>99%) and a low FDR(estimated local FDR of 5%) were considered positivelyidentied. The relative quantication was based on the ratio ofthe areas under the reporter peaks from the paired casecontrolsamples. The following criteria were required to select a protein

for further analysis: two or more unique peptides with a highcondence score(>99%), apvalue forprotein quantication assigned

by the ProteinPilot software


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Supplementary Table S1 in theSupporting Information). A totalof 416, 517, and 546 nonredundant proteins were identied bythe three iTRAQ labelings and 2D nanoLCMS/MS,respectively. A total of 212 nonredundant proteins wererepeatedly identied in all nine casecontrol pairs usingstringent criteria including one or more peptides with a >99%condence score and 5% local FDR. This represents 27% of thetotal proteins identied in all experiments. Interestingly, amongthem, 58 proteins had not previously been identied in human

milk. MS and MS/MS analysis results are summarized in theSupporting Information. (See Supplemental Tables S2, S3, andS4 in theSupporting Information.)

The biological processes and the molecular functions of all ofthe 212 proteins identied in the breast skimmed milk wereclassied using a GO classication system. The subcellulardistribution of identied proteins was 39% cytoplasmic proteins,30% extracellular space proteins, 21% membrane proteins, and10% nucleus proteins (Figure 2A). Skim proteins were associated

with a broad range of pathways, molecular functions, andbiological processes (Figure2B and Supplemental Table S5 inthe Supporting Information). Functionally, 46% of proteins

were involved in immune response, defense, and inammation

(Figure2B). The other proteins were associated with variouscellular functions including cellular metabolism, signaling, lipidtransport and metabolism, complement and coagulation cascade,response to nutrient level, or regulation of cell growth and celladhesion (Figure2B).

Identication and Characterization of the DifferentiallyExpressed Proteins

To identify the differentially expressed proteins related to

postnatal HIV-1 transmission through breastfeeding, wecompared protein proles of each casecontrol pair. First, weselected proteins that correspond to our biological selectioncriteria: (1) proteins identied and quantied in all three iTRAQlabelings; (2) proteins identied with at least two peptides with ahigh condence; and (3) proteins with a differential expression ofat least 1.3-fold relative to the control sample and apvalue


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Table2.

GeneOntologyCategoryEnrichedintheDifferentiallyExpressed

ProteinsintheBreastMilkCase/ControlSamples

ontologicalcategories

p

value

genesymbol

EnrichmentbyGeneGoProcessMaps

1.

inammation:complementsystem

4.6

65

104

CLU

2.

immuneresponse:antigenpresentation

2.8

84

103

B2M,

CALR,

AZGP1

3.reproduction:feedingandneurohorm

onesignaling

3.7

99

103

LALBA,

XDH,

CSN2

4.proteinfolding:responsetounfolded

proteins

4.9

40

103

CALR

5.

immuneresponse:phagosomeinantigenpresentation

7.1

57

103

B2M,

CALR,

CFL1

6.proteolysis:ECM

remodeling

8.8

13

103

SERPINA3,

CLU,

SPP1

7.

inammation:amphoterinsignaling

2.1

26

102

CFL1,

S100A8,

S100A9

8.proteinfolding:ERandcytoplasm

2.1

45

102

CALR

9.

inammation:IL-6signaling

2.1

74

102

HP,

SERPINA3,

ORM1

10.

immuneresponse:phagocytosis

2.5

13

102

MRC1L1,

CALR,

CFL1,

MFGE8

EnrichementbyGOProcesses

1.responsetostimulus

1.7

79

1012

LALBA,

IGHM,

B2M,

XDH,

MRC1L1

,FABP3,

HP,

SERPINA3,

ENO1,

KRT1,

IGHG1,

CALR,

PEBP1,

UGP2,

CFL1,

ORM1,

PPIA,

LYZ,

ALDOA,

CSTB,

S100A8,

S100A9,

MFGE8,

AZG

P1,

CLU,

SPP1,

TF,

IGHA2

2.responsetostress

8.3

95

1012

LALBA,

B2M,

HP,

SERPINA3,

KRT1,

CALR,

PEBP1,

CFL1,

ORM1,

PPIA,

LYZ,

ALDOA,

S100A8,

S100A9,

ENO1,

CLU,

FABP3,

SPP1,

TF

3.responsetowounding

2.8

24

1011

HP,

SERPINA3,

PEBP1,

CFL1,

ORM1

,PPIA,

LYZ,

ALDOA,

S100A8,

S100A9,

ENO1,

CLU,

FABP3,

SPP1,

TF

4.

humoralimmuneresponse

4.6

59

1011

IGHM,

KRT1,

CLU

5.

defenseresponse

1.4

97

1010

LALBA,

B2M,

HP,

SERPINA3,

KRT1,

ORM1,

LYZ,

S100A8,

S100A9,

CLU,

SPP1,

TF

6.

humoralimmuneresponsemediatedbyimmunoglobulin

6.5

61

1010

IGHM,

CLU

7.responsetobioticstimulus

6.6

43

1010

LALBA,

B2M,

HP,

ENO1,

CFL1,

ORM

1,

LYZ,

ALDOA,

S100A8,

S100A9,

CLU

8.complementactivation

1.3

04

109

KRT1,

CLU

9.plateletdegranulation

3.2

52

109

CFL1,

PPIA,

ALDOA,

CLU,

TF

10.proteinactivationcascade

5.9

93

109

KRT1,C

LU

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majority of the biological replicates. For the others proteins,there are some variations in the protein ratio in different casecontrol pairs, which reect biological variation (clinical

biological parameters, timing of breast milk collection, etc.)and interindividual variability. We did not nd a correlation

between these biological variation and any of the clinicalbiological parameters.

To further determine the roles of these differentially expressedproteins, we used the MetaCoreTM pathway mapping tool. Weinvestigated the enrichment of the 33 differentially expressedproteins across two different ontological categories usingGeneGo and GO processes. Table2shows the top 10 GeneGocellular process networks and the top 10 GO processes with thedifferentially expressed proteins. Results stressed the importanceof immune system response and inammation-related ontologyin mother-to-child HIV-1 transmission risk.

Assessment of2-Microglobulin and S100A9 ExpressionLevels in the Independent Validation Set

Because immune and inammatory responses are both highlyrelated to mother-to-child HIV transmission risk, we selectedamong the 33 differentially expressed proteins2-microglobulin

as a marker of immune activity and S100A9 for its associationwith inammation. Bothproteins were quantied in independentskimmed breast milk samples of controls (nontransmitter) andcases (transmitter women) by ELISA (Table3). Consistent withscreening results, 2-microglobulin concentrations were sig-nicantly higher (pvalue = 0.0007) in breast milk of maternaltransmitters (11.4g/mL) than in nontransmitters (9.1g/mL).However, for S100A9, no signicant difference was observed

between maternal transmitters (4.9 g/mL) and maternalnontransmitter controls (4.1 g/mL). Because the dynamics ofprotein expression in human milk vary highly during the courseof lactation, we determined expression of both these proteins

based on timing of breast milk sample collection since delivery.2-microglobulin concentration was signicantly higher in

transmitters compared with nontransmitters, and S100A9remained not signicantly different between both groups,whatever the period of lactation (Table3).

Correlation between2-Microglobulin and S100A9 andClinical-Biological Parameters Associated with HIV-1Transmission Risk

Many epidemiological or clinical factors associated with highviral replication or immune activation in transmitters couldmodify the risk of HIV transmission. Therefore, correlation statisticalanalyses were performed between both milk2-microglobulin andS100A9 concentration and several clinicalbiological parametersincluding maternal age, age of transmission, antenatal blood CD4+cell count, and HIV RNA viral load in plasma and milk (Table 4).Breast milk 2-microglobulin concentration was positively and

Table3.

2-MicroglobulinandS10

0A9ConcentrationAssessedinthe127

IndependentSamplesoftheVTSCohort

2-microglobulin

S100A9

controls

case

s

co

ntrols

cases

no.

median[range]

no.

med

ian[range]

p

value

no.

m

edian[range]

no.

median[range]

p

value

Allsample

56

9.1

2[5.3

423.9

9]

71

11.43

[5.8

931.2

1]

0.0

007

56

4.08[0.5

230.2

0]

71

4.9

1[0.0

646

.57]

NSa

TimeofSampleCollection

0to

Elevated Concentrations of Milk β2-Microglobulin Are Associated

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