Enhanced Th17 Phenotype in Uninfected Neonates Bornfrom Viremic HIV-1-Infected Pregnant Women

Joana Hygino & Morgana M. Vieira & Landi V. Guillermo & Renato G. Silva-Filho &

Carmen Saramago & Agostinho A. Lima-Silva & Regis M. Andrade &

Arnaldao F. B. Andrade & Rodrigo M. Brindeiro & Amilcar Tanuri &Vander Guimarães & Cleonice Alves de Melo Bento

Received: 30 September 2010 /Accepted: 21 October 2010 /Published online: 7 December 2010# Springer Science+Business Media, LLC 2010

Abstract Our objective was to evaluate the in vitrofunctional profile of T cells from uninfected neonates bornfrom HIV-1-infected pregnant women who controlled (G1)or not (G2) the virus replication. We demonstrated that thelymphoproliferation of T cell to polyclonal activators washigher in the G2 as compared with G1. Nevertheless, nodetectable proliferative response was observed in responseto HIV-1 antigens in both neonate groups. Cytokine dosagein the supernatants of these polyclonally activated T cellcultures demonstrated that, while IL-10 was the dominantcytokine produced in G1, Th17-related cytokines weresignificantly higher in G2 neonates. The higher Th17phenotype tendency in G2 was related to high production ofIL-23 by lipopolysaccharide-activated monocyte-deriveddendritic cells from these neonates. Our results demonstrated

immunological disorders in uninfected neonates born fromviremic HIV-1-infected mothers that can help to explain whysome of these children have elevated risk of clinical morbidityand mortality due to pathological hypersensitivity.

Keywords HIV. cytokines . neonates . pregnant women .

IL-10 and IL-17

Introduction

The growing frequency of HIV infection among fertilewomen, as well as the development of strategies for verticaltransmission avoidance, has enhanced the number ofuninfected neonates born from infected mothers [1]. Manyworks suggest that during pregnancy, maternal infectiousdiseases could have complex consequences to the fetusdevelopment [2–4], but few studies have evaluated theeffect of maternal HIV infection on the functional develop-ment of both T cells and dendritic cells of the HIV-exposeduninfected children. This kind of study is highly necessaryin the case of HIV infection because according to presentclinical recommendations, uninfected children have nospecial follow-up since vertical transmission is discarded.Given the immaturity of the fetal immune system, anyexternal influence during the intra-uterine life could bringlong-term consequences to the children’s health.

A large body of evidence suggests that the main forcedriving immune deficiency in HIV-1 infection is related tochronic immune activation, which is characterized byproduction of high levels of inflammatory cytokines, suchas IL-1β and TNF-α. These cytokines not only supportHIV-1 replication but also have been implicated in manyHIV-related immune adverse effects, particularly the highlevels of activation-induced cell death [5].

This work was supported by the Fundação de Amparo à PequisaCarlos Chagas Filho.

J. Hygino :M. M. Vieira : L. V. Guillermo :R. G. Silva-Filho :C. Saramago :A. A. Lima-Silva : R. M. Andrade :C. A. de Melo Bento (*)Department of Microbiology and Parasitology,Federal University of the State of Rio de Janeiro,Frei Caneca 94, 20.261-040,Rio de Janeiro, Rio de Janeiro, Brazile-mail: cbento@unirio.br

A. F. B. AndradeDepartment of Microbiology, State University of Rio de Janeiro,Rio de Janeiro, Brazil

R. M. Brindeiro :A. TanuriDepartment of Genetics, Federal University of Rio de Janeiro,Rio de Janeiro, Brazil

V. GuimarãesFernando Figueiras Institute, Oswaldo Cruz Institute,Rio de Janeiro, Brazil

J Clin Immunol (2011) 31:186–194DOI 10.1007/s10875-010-9485-3

This immune hyperresponsiveness observed in chroni-cally HIV-1-infected patients contrasts drastically with theimmunomodulatory events associated with normal preg-nancy. A successful pregnancy depends on an immunolog-ical tolerance state to the fetal allograft, coordinated bymaternal regulatory T cells [6, 7]. During normal pregnan-cy, the production of high levels of both anti-inflammatorycytokines IL-10 and TGF-β by regulatory T cells takesplace at the maternal–fetal interface, in order to support thegrowth and development of the fetus [8–11]. On the otherhand, maternal inflammatory response following infectiousdiseases has been pointed as a pivotal event able to inducethe breakdown of this fetus tolerance and consequently tobring a wide range of obstetric complications, affectingplacenta, membranes, and fetus [12–14]. In this scenario,maternal pro-inflammatory cytokines can promote fetaldamage directly, by activating embryotoxic maternal T cell,and/or indirectly, by inhibiting the activity of maternalregulatory T cells [10, 12, 15–17]. Furthermore, prematurecontact with pro-inflammatory cytokines may have adverseeffects on the development and function of the immunesystem from apparently normal neonates [4, 15, 18].

The human neonate is highly susceptible to infectionsdue to a combination of immaturity of the immune systemand antigenic inexperience [19, 20]. Immature naïvelymphocytes are normally the dominant phenotype in thenewborns [21]. These naïve cells are deficient in producingthe Th1 phenotype cytokines IL-2 and IFN-γ. Some workshave suggested that deficient cellular immune response innewborns is often related to functional deficiency ofdendritic cells (DCs) [22, 23]. The cord blood-derivedDCs, when compared to their adult counterparts, expresslower levels of MHC class II, ICAM-1, CD80, and CD86and secrete reduced levels of IL-12 and IFN-α in responseto different pathogen-associated molecular patterns [24].Therefore, due to these immune particularities, extrinsicevents can easily modulate the functional development ofthe immune system in early times of human life. In thiscontext, our study aimed to evaluate the in vitro functionalT cells profile in response to HIV-1-specific and non-specific stimuli in groups of uninfected neonates born frompregnant women who controlled or not the plasma viralload (PVL).

Methods

Study Design

In our study, 90 HIV-1-infected pregnant women whocontrolled or not the PVL were recruited between 2004 and2008 from the obstetrics service of three public hospitals inRio de Janeiro State. As control for pregnancy-related

immune events, a total of 20 healthy HIV-1-non-infectedpregnant women, matched by age and gestational time,were recruited from the same services.

Immediately after a term delivery by elective cesareansection, 20 mL of the umbilical cord blood of uninfectedneonates (n=90) born from HIV-1-seropositive pregnantwomen were drawn into tubes containing heparin. Thesamples were stratified into two groups, G1 and G2,according to the maternal PVL. The G1 and G2 groupscontained, respectively, samples collected from neonatesborn from mothers with undetectable (determined as <50copies of HIV-1-RNA/mL, n=60) and detectable (>50copies of HIV-1 RNA/mL, n=30) PVL. The non-infectedstatus of the neonate samples was confirmed by nucleicacid technique (PCR) for env, nef, and vif HIV-1 provirusgenes 6 weeks after birth. Pregnant women with otherinfections besides HIV-1, evidence of fetal intra-uterinegrowth restriction, previous uterine cervix surgery, or whosmoked or used illegal substances were not included in thestudy, since these factors may cause adverse pregnancyoutcomes. Furthermore, infants with a birth weight of lessthan 2,500 g or with any medical problem at birth wereexcluded. As control, a total of 20 HIV-non-exposedneonates, born from healthy mothers by elective cesareansection, were included in the study (G0). Written informedconsent was obtained from all mothers. The study wasapproved by the Ethical Committee for Research on HumanSubjects of the Federal University of the State of Rio deJaneiro (UNIRIO).

Cord Blood Mononuclear Cell Cultures and Stimuli

The umbilical cord blood mononuclear cells (UCMC) wereobtained by centrifugation on Ficoll–Hypaque densitygradients. The mononuclear cells in the buffy coat werecollected and washed three times in Hank’s solution. Theviable UCMC were adjusted to 1×107/mL and cryopre-served or to 1×106/mL and cultured in 96-well flat-bottomed microtiter plates with 200 μl of RPMI 1640added with 2 mM of L-glutamine (GIBCO, Carlsbad, CA,USA), 10% of fetal calf serum (FCS), 20 U/mL ofpenicillin, 20 μg/mL of streptomycin, and 20 mM ofHEPES buffer. In the experiments evaluating the impact ofmonocyte-derived DCs supernatants on T cell cytokineprolife, we used cryopreserved UCMC. Briefly, UCMC(1×107 cells/mL) were frozen on liquid nitrogen in 10% ofdimethyl sulfoxide and 90% of FCS in aliquots of 1 mL.After thawed, the viability and recovery were more than90%, as determined by using trypan blue exclusion.

For some experiments, UCMC-derived monocytes wereisolated by negative selection with magnetic beads coatedwith a combination of anti-CD19, anti-CD3, anti-CD34,anti-CDw123, and anti-CD56 mAbs (Dynal Biotech, Great

J Clin Immunol (2011) 31:186–194 187

Neck, NY, USA). Evaluation by flow cytometry showedthat the efficiency of both procedures was about 94% forCD14+ cells (data not shown).

In order to measure HIV-1-specific response, UCMC(1×106/mL) were kept for 7 days in the presence of p24protein (p24HIV-1Gag, Sigma Co) at 1 μg/mL or a cocktailof immunodominant synthetic envelope peptides (ppHIV-1env: T1, T2, TH4, P18, MN, P18 IIIB) at 1 μM eachpeptide, with our without recombinant human IL-2 at 20 U/mL (BD systems, Minneapolis, MN, USA). The concen-trations of p24 protein (p24HIV-1Gag) and ENV-derivedpeptide were established in our laboratory as the dose thatinduced CD8+ T cell proliferation in acutely HIV-1 infectedpatients. In order to induce T cell polyclonal activation,whole or CD19-depleted UCMC cultures (1×106/mL) weremaintained for 3 days with plate-bound anti-CD3 mAb(OKT3, 1 μg/mL) plus anti-CD28 (1 μg/mL). We chosethis time (3 days) because it represents the peak of T cellproliferation in response to these polyclonal activators. Allcells were cultured at 37°C in a humidified 5% CO2

incubator. Finally, the presence of lipopolysaccharide (LPS)contamination in the plasma, as well as in the supernatantsfrom activated T cells, was discarded by limulus amebocytelysate test that reached to values <0.01 EU/mL byturbidimetric technique.

Generation of Monocyte-Derived Dendritic Cells

Purified neonatal-derived monocytes (1×106/mL), obtainedas described above, were initially cultured in the presence of100 ηg/mL GM-CSF and 25 ηg/mL IL-4 (BD systems,Minneapolis, MN, USA). Three days after, the cells weretreated with LPS (100 ηg/mL; from Escherichia coli 0111:B4 strain, Invitrogen) for additional 24 h. Evaluation by flowcytometry showed that the efficiency of this procedure wasabout 93.8% for CD11c+ cells (data not shown). In someexperiments, 100 μL of the supernatants from LPS-activatedmonocyte-derived DCs were collected and assayed for theability to modulate cytokine secretion by autologous poly-clonally activated CD19-depleted UCMC cultures. All cellswere cultured at 37°C in a humidified 5% CO2 incubator.

Proliferation Assay

Cultures containing approximately 1×106/mL of UCMCwere maintained for 3 days in the presence of anti-CD3plus anti-CD28 or for 7 days with HIV-1 antigens (ppHIV-1env or p24HIV-1Gag). The cellular proliferation wasmeasured by [3H] thymidine incorporation, added tocultures at 0.5 μCi/well 8 h before the end of the incubationtime. In some wells, IL-2 was added at the beginning of thecultures. The cells were harvested in glass fiber filters in anautomatic cell harvester and radioactive incorporation was

measured using a liquid-scintillation counter. The resultswere shown as stimulation indexes (SI), that is, the meancounts per minute (cpm) of stimulated UCMC divided bythe mean cpm of unstimulated cells from the same patientgroup. A SI >3 was considered to be a positive response.

Cytokine Determination

The plasma, obtained by umbilical blood centrifugation, andthe supernatants, collected from different neonatal cellcultures, were submitted to cytokine measurement by OptEIAELISA kits (BD, Pharmigen, San Diego, CA, USA), accord-ing to the manufacturer’s protocol. Briefly, each ELISA wasperformed using pairs of mAbs directed to human IL-1β, IL-6, IL-10, IL-4, TNF-α, IFN-γ, and IL-17. For supernatantscollected from LPS-stimulated monocyte-derived DCs, thepresence of IL-12 and IL-23 was assayed by using the capture/biotinylated detection Abs for human IL-12p70 (clone 20C2/C8.6) or IL-23 (clone eBio473p19/C8.6) purchased from BDBiosciences and eBioscience, respectively. The reaction wasrevealed with streptavidin horseradish peroxidase, using3,3′,5,5′-tetramethylbenzidine as substrate. Recombinant hu-man cytokine ranging from 10 to 500 pg/mL was used toconstruct standard curves.

Statistical Analysis

For each given variable, the nonparametric Mann–WhitneyU test was applied to determine whether the groups werestatistically different. The Student’s test was applied toverify the impact of B cell depletion and LPS-activatedmonocyte-derived DCs supernatants on cytokine profile ofpolyclonally activated T cell cultures. The significance inall experiments was defined as p<0.05.

Results

Characteristics of the HIV-1 Infected Pregnant Womenand Their Uninfected Newborns

The characteristics of the pregnant women are presented inTable I, including antiretroviral therapy, last PVL, andCD4+ T cell count, evaluated 24–48 h before delivery. Allwomen were antiretroviral therapy naïve before pregnancy.Of the 60 patients who controlled the PVL (G1), most ofthem (46 out of 60, 76%) received antiretroviral therapywith either zidovudine (AZT, n=16) or zidovudine pluslamivudine plus nelfinavir (AZT+3TC+NFV, n=30) thathad been introduced in the period of 20–30 weeks ofgestation. In this G1 group, the mean value of PVLimmediately before starting antiretroviral therapy was6,137±8,520 copies of RNA per milliliter (ranged 0–

188 J Clin Immunol (2011) 31:186–194

17,800), and all of them reached undetectable PVL 1 monthafter beginning of the treatment. As it is accepted that themajority of HIV-1 vertical transmission occurs in lategestational times, maternal viral loads were also determinedfrom peripheral blood collected just before the electivecesarean delivery (Table I). Among G2 patients, only fourout of 30 (13%) were under antiretroviral therapy. Althoughantiretroviral drugs were available at the site, many pregnantwomen had not been treated because they sought medicalattention at the obstetric service just before delivery. All HIV-1-infected pregnant women received continuous intravenousinfusion of AZT during the elective caesarian section.Importantly, although the mean values of CD4+ T cell countswere lower in the G2 group mothers (p<0.05), no patienthad levels of less than 400 CD4+ cells/mm3.

Concerning CD4+ and CD8+ cell counts in neonates, wedid not observe any statistical difference between the threegroups, although the children from G2 had a tendency topresent lower CD4 cell counts (p=0.057; Table I). Further-more, although all the babies were born to term, the G2neonates had also a tendency to lower birth weight whencompared with G1 neonates (3,415±353×3,015±511 g; p=0.058; Table I).

Lymphoproliferative Response and Cytokine Profilein UCMC Cultures from HIV-1-Exposed Non-infectedNeonates

The first immune event analyzed in our study was theneonatal T cell proliferation induced by a combination of

polyclonal activators (anti-CD3 plus anti-CD28). As shownin Fig. 1, while no significant difference was observed inthe extent of lymphoproliferative response of the cellcultures from control (G0) and G1 groups, both of themwere significantly lower than the level of proliferationdetected in the G2-activated cell cultures. This higher T cellresponsiveness, however, was not related to HIV-1-antigenssensitization, since the cultures treated with P24 protein or acombination of peptides from HIV-1 envelope did not

Table I Characteristics of the pregnant women and their neonates

G0e (n=20) G1f (n=60) G2g (n=30)

Maternal age in years (SD) 28.8 (6.1) 29 (3.1) 27.7 (5.4)

Mean gestational age at delivery (SD) 37.4 (0.91) 37.6 (0.85) 37.1 (0.96)

Antiretroviral therapy (%)a ND 76 13

Maternal mean CD4 cell count at delivery (SD)b 841.3±315 766.6±237* 650±207*

Neonate mean CD4 cell count (SD)c,d 2,961±816 2,796±731 2,314±876

Maternal mean CD8 cell count at delivery (SD)b 615±220* 1,131±432* 1,106±407*

Neonate mean CD8 cell count (SD)c,d 1,196±536 1,161±612 1,306±551

Mean newborns weight in grams (SD) 3,696±236 3,415±353 3,015±411

ND no data

*p<0.05a AZT alone or a combination of AZT/NVP or AZT/3TC/NFV was introduced after 20–30 weeks of gestationb The peripheral T cell counts were obtained from medical recordsc The non-infected status of the neonate samples was confirmed by nucleic acid technique (PCR) for env, nef, and vif HIV-1 provirus genes 6 weeks after birthd The umbilical T cell counts were obtained from medical recordse Healthy HIV-uninfected pregnant womenf HIV-1-infected pregnant women with PVL below the limit of detection (<50 copies of RNA per milliliter)g HIV-1-infected pregnant women with detectable PVL ranging from 700 to 32,000 copies of RNA per milliliter

GO G1 G20

20

40

60

80

p=0.641

p< 0.001

p< 0.001

Sti

mu

lati

on

Ind

exes

Fig. 1 Analysis of the lymphoproliferative response of UCMC fromuninfected HIV-1-exposed neonates. UCMC cultures (1×106/mL),obtained from HIV-1-non-exposed (G0, n=20) or HIV-1-exposed non-infected neonates born from mothers who controlled (G1, n=60) ornot (G2, n=30) the plasma viral load, were stimulated with anti-CD3(1 μg/mL) plus anti-CD28 (1 μg/mL). Three days later, theproliferation was determined by the level of [3H] thymidine and theresults are shown as mean ± SD of stimulation indexes. A SI>3 wasconsidered to be a positive response. All the p values are indicated atthe figure

J Clin Immunol (2011) 31:186–194 189

achieve stimulation indexes higher than 3, even in thepresence of IL-2 (G0, 1.75±0.436; G1, 1.46±0.458; G2,2.3±0.153).

Another immune event analyzed was the cytokine profilein cultures of polyclonally activated mononuclear cellsfrom the different experimental groups. Of note, nodetectable spontaneous release of cytokines was observed(data not shown). As demonstrated in Fig. 2a, the anti-inflammatory cytokine IL-10 was the dominant cytokinedetected in the supernatants from polyclonally activated G0and G1 cultures. Furthermore, any statistical difference wasobserved concerning cytokine profile among G1 neonates

born from treated or naïve HIV-1-infected pregnant women(data not shown). On the other hand, in cultures from G2neonates, the production of IFN-γ and mainly Th17-relatedcytokines (IL-1β, IL-6, TNF-α, and IL-17) was signifi-cantly higher if compared to the other two groups.Furthermore, the augmented inflammatory profile in G2neonate group could be favored by the lower IL-10production in these cell cultures (Fig. 2a). No differencein the level of IL-4 release was observed in any neonate cellculture (Fig. 2a). Even in lower levels, the same cytokineprofile was observed in G1 and G2 after 24 and 48 h (datanot shown). Finally, the elimination of B cells, by depleting

G0 G1

G1

G1 G1G20

1000

2000

3000

4000 p < 0.0001

IL-1β

p = 0.304

p = 0.0103

Cyt

oki

nes

(ρg

/mL

)

G0 G2

p < 0.0001

IL-6

p = 0.960

p < 0.0031

G0 G2

p= 0.0042

TNF-α

p= 0.621

p= 0.0404

G2 G1 G2

G2 G2 G20

1000

2000

3000

IL-4

p= 0.183p= 0.684

p= 0.201

Cyt

oki

ne

(ρg

/mL

)

G0 G1G2G0 G1

p= 0.002

IFN-γ

p= 0.870

p= 0.015

G0 G1 G0 G1

p= 0.0001

IL-10

p= 0.851

p= 0.0035

p < 0.0001

IL-17

p = 0.246

p < 0.0001

A

G0 G0 G1 G2G0 G1 G2G0

0100200300400500600700800900

1000IL-1β TNF-α IL-6 IL-10

p< 0.0001

p= 0.0013

p< 0.0001

p= 0.0017

p=0.187 p=0.487

p=0.475

B

p< 0.0001p< 0.0001

p= 0.0003

p= 0.0351

Cyt

oki

ne

(ρg

/mL

)

p=0.932

Fig. 2 Cytokine profile in the plasma and polyclonally activatedumbilical cord mononuclear cells obtained from uninfected neonatesborn from HIV-1-infected mothers. In a, UCMC cultures (1×106/mL),obtained from HIV-1-non-exposed (G0, n=20) or HIV-1-exposed non-infected neonates born from mothers who controlled (G1, n=60) ornot (G2, n=30) the plasma viral load, were stimulated with anti-CD3(1 μg/mL) plus anti-CD28 (1 μg/mL). Three days later, the super-

natants were collected and the cytokine content quantified by ELISA.The horizontal bars within boxes correspond to the median, box limitscorrespond to 25th and 75th percentiles, and vertical lines indicaterange. In b, we did the in vivo cytokines dosage, expressed as mean ±SD, from plasmas obtained from each neonate group. All the p valuesare indicated at the figure

190 J Clin Immunol (2011) 31:186–194

CD19+ cells from UCMC, did not modify the release ofIFN-γ and IL-17 in cell cultures for all groups (data notshown).

In line with the high pro-inflammatory profile observedin G2 cultures, higher levels of systemic TNF-α, IL-6, andIL-1β were detected in the umbilical cord blood of G2neonates (Fig. 2b). As observed in the activated culturessupernatants, no statistical difference was detectedconcerning systemic IL-4 production (data not shown).The plasma levels of IFN-γ and IL-17 were not detectablein any neonate group.

Cytokine Response in LPS-Activated Monocyte-DerivedDCs Cultures and the Effect of Their Supernatantson Th1/Th17 Cytokine in the Group of HIV-1-ExposedUninfected Neonates

The effect of LPS on the cytokine profile of monocyte-derived DCs response was evaluated after 24 h ofstimulation. As shown in Fig. 3, the production of IL-12and, mainly, of IL-23 was significantly higher in thesupernatants from G2 cell cultures, while IL-10 wasdetectable in low levels in this group.

In order to test the bioactivity of IL-23 and IL-12 releaseby LPS-activated monocyte-derived DCs, we analyzed theircapacity to enhance, respectively, IL-17 and IFN-γ release.In this regard, supernatants collected after 24 h of DCsactivation with LPS were added at the beginning of CD19-depleted UCMC cultures and then stimulated with anti-CD3 and anti-CD28 for 3 days. Despite the elevation ofIFN-γ and IL-17 production after addition of LPS-activatedDCs supernatants had been observed in all studied groups,the major impact was on IL-17 release in polyclonallyactivated T cell cultures from G2 neonates (Fig. 4).

Discussion

The frequency of HIV vertical transmission has dramati-cally reduced with the introduction of antiretroviral treat-ment in combination with cesarean delivery and avoidanceof breastfeeding [1]. As consequence, the number of HIV-1exposed uninfected newborns is growing, and few studieshave evaluated the impact of HIV-1 exposure on functionalimmunological development of uninfected newborns. Ourresults demonstrated that the in vitro T cell proliferationinduced by polyclonal activators was higher in neonatesborn from HIV-1-infected women who did not control theirPVL, as compared to those born from HIV-1-seronegativeor virus-infected women with undetectable PVL. Althoughsome studies have documented the presence of HIV-1antigens in cord blood of HIV-negative infants born fromseropositive mothers [25] and also HIV-antigens-inducedIL-2 release by cord blood CD4 T cells in exposed neonates[26], the elevated immune responsiveness observed in ourcohort of neonates was not related to any virus sensitiza-tion, since no significant lymphoproliferative response wasobserved to either p24 protein or a pool of synthetic HIVenvelope peptides (ppHIVENV), even after addition ofoptimal doses of exogenous IL-2. Of note, both HIV-1-specific antigens used in our study elicited detectable T cellproliferation in the HIV-1-infected mothers (manuscript inpreparation). This last observation also discards anycontamination of maternal lymphocytes in infants cordblood.

Some immunological abnormalities found on the cordblood T lymphocytes phenotype seem to persist for longtime [27], and our study suggests that these immuneabnormalities can be amplified or linked to disturbanceson cytokine network. High production of pro-inflammatory

G0 G1 G2 G0 G1 G2 G0 G1 G20

250

500

750

1000

G0 G1 G2

IL-12 IL-23 IL-10

p=0.037

p= 0.0012

p< 0.001

p=0.135

p= 0.863

p= 0.922

Cyt

oki

nes

(ρ

g/m

L)

p=0.007

p< 0.0001

p< 0.001

Fig. 3 The production of cytokines by LPS-activated monocyte-derived DCs from uninfected neonates born from HIV-1-infectedmothers. Viable monocyte-derived DCs (1×106/mL) were then kept inthe presence of LPS (100 ηg/mL) for 24 h, and the supernatants werecollected and submitted to ELISA technique to quantify IL-10, IL-23,and IL-12 release. At the figure, G0 (n=20) represents healthy HIV-1-

non-exposed newborns, while G1 (n=30) and G2 (n=30) indicateHIV-1-exposed non-infected neonates born from mothers whocontrolled or not the plasma viral load, respectively. The values areexpressed as mean ± SD, and the level of significance is indicated atthe figure

J Clin Immunol (2011) 31:186–194 191

cytokines was detected in the plasma and in the super-natants of polyclonally activated T lymphocyte in G2neonates. Among the cytokines evaluated by our group,the most interesting results were those concerning Th17-related cytokines. The Th17-mediated response is charac-terized by production of high levels of IL-17 (also calledIL-17A) as well as IL-1β, IL-6, TNF-α, IL-21, and IL-22[28]. Although we did not measure IL-21 and IL-22, weobserved that, in general, neonates had a higher tendency toperform Th17 response, as compared to the other Thphenotypes. Nevertheless, this event was markedly strongerin G2 neonates. The IL-23 is critically involved in themaintenance and amplification of IL-17-secreting T cell[29], and in this study, the higher Th17 tendency observedin G2 neonates also correlated with higher IL-23 release inLPS-activated myeloid DCs.

There is a broad consensus among investigators that, duringthe pathogenesis of HIV infection, disease progression isclosely linked to the level of immune activation [5, 30]. InHIV-1-infected children, for example, some authors haveobserved a positive correlation between TNF-α/IL-10 ratioand disease progression [31]. Additionally, recent studiespublished by our group demonstrated a high tendency of G2mothers to produce pro-inflammatory cytokines associatedwith lower levels of IL-10, as compared to G0 and G1pregnant women [32, 33]. Interestingly, the cytokine profiledetected in neonates born from G1 patients who spontane-ously controlled their plasma viral load (elite suppressors)was very similar to that observed in the neonates born fromtreated G1 mothers (data not shown). This suggests that thesame dominant anti-inflammatory cytokine profile previouslydescribed in both treated and untreated G1 mothers issomehow passed to their neonates, which makes themimmunologically similar to G0 neonates.

Some studies have demonstrated a direct relationship ofthe severity of HIV infection and immune dysfunction inmothers, with the increase of clinical morbidity andmortality in their uninfected infants [11, 34, 35]. Amongthese HIV-exposed neonates, the most common cause ofinfant death and hospitalization is pneumonia and/or sepsis[36, 37], both clinical conditions associated with anpathologically enhanced pro-inflammatory reactions. In thiscontext, recent studies have demonstrated the involvementof Th17 cytokines in patients with severe sepsis [38, 39].Furthermore, given the major implication of IL-23/IL-17 inautoimmune diseases [40–43], the impact of the patholog-ical release of IL-23 in early life on the risk of futurehypersensitivity reactions needs to be investigated atclinical level.

Therefore, even born apparently normal, the abnormalimmune hyperresponsiveness of these neonates, probablydue to the fetal exposure to mother-derived increased levelsof pro-inflammatory cytokines, might potentially bringadverse effects to the fetal immune development. At themoment, we are dedicating our efforts to identify pheno-typically the cell subsets involved in this high responsive-ness and particularly the frequency of classical regulatoryCD4+ T cells (CD25hiFOXP3+CD127−CTLA-4+) in theseneonates.

In summary, our results describe an abnormally highpro-inflammatory responsiveness to T cell polyclonalactivators, particularly into the Th17 compartment, inuninfected neonates born from HIV-1-infected motherswith uncontrolled plasma viral load. Our observations risethe concern that, even being free from HIV infection, thesechildren are not immunologically normal, as they areassumed to be. This phenomenon can have impact on theimmune response to different antigens following infections

G0 G1 G20

500

1000

1500

2000 IFN- γ

p = 0.012 p = 0.011p < 0.0001

p = 0.531

p < 0.0001

p = 0.0011

G0 G1 G2

IL-17

p = 0.031

p = 0.0121

p < 0.0001

p = 0.761

p < 0.0001p< 0.0001

None LPS-activated DCSN

Cyt

oki

nes

(ρ

g/m

L)

Fig. 4 Impact of LPS-activated monocyte-derived DCs supernatantson IL-17 and IFN-γ secreting T cells obtained from uninfectedneonates born from HIV-1-infected mothers. CD19-depleted UCMCcultures (1×106/mL) were stimulated with anti-CD3 (1 μg/mL) plusanti-CD28 (1 μg/mL) in the absence (none) or presence of 100 μL ofsupernatants collected from LPS-activated monocyte-derived DCs(LPS-activated DCSN). Three days after, the IL-17 and IFN-γ in the

supernatant were assayed by ELISA. The values are expressed asmean ± SD, and the p values are indicated at the figure. At the figure,G0 (n=20) represents healthy HIV-1-non-exposed newborns, whileG1 (n=30) and G2 (n=30) indicate HIV-1-exposed non-infectedneonates born from mothers who controlled or not the plasma viralload, respectively

192 J Clin Immunol (2011) 31:186–194

or immunizations throughout children’s life. Longitudinalstudies on multiple cohorts of HIV-exposed uninfectedchildren will be needed to clarify these issues.

Conflict of Interest All authors declare that there are no conflicts ofinterest.

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