The Journal of Immunology

AIDS Progression Is Associated with the Emergence ofIL-17–Producing Cells Early After Simian ImmunodeficiencyVirus Infection

Laure Campillo-Gimenez,* Marie-Christine Cumont,† Michele Fay,‡ Hassen Kared,*

Valerie Monceaux,† Ousmane Diop,x Michaela Muller-Trutwin,{ Bruno Hurtrel,† Yves Levy,*,‖

John Zaunders,# Michel Dy,** Maria C. Leite-de-Moraes,** Carole Elbim,*,††,1 and

Jerome Estaquier*,†,‖,1

IL-17 is a potent effector cytokine involved in inflammatory response and antimicrobial defense. We report that SIV infection of

rhesus macaques (RMs) results in the emergence of IL-17–expressing cells during the acute phase. This subpopulation appears at

day 14 postinfection concomitantly with an increase in TGF-b and IL-18 expression. This subset, which exhibits phenotypic

markers of NK T cells (NKT), rather than Th17 CD4 cells, persists during the chronic phase and is higher in noncontrollers SIV-

infected RMs compared with controllers SIV-infected RMs. In contrast, in the nonpathogenic model of SIVagm infection of

African green monkeys, no change in the level of IL-17–expressing cells is observed in lymphoid organs. Consistent with the

emergence of TGF-b and IL-18 during the acute phase in SIV-infected RMs, but not in SIV-infected African green monkeys, we

demonstrate that in vitro TGF-b and IL-18 induce the differentiation and expansion of IL-17+NKT+. Altogether, these results

demonstrate that IL-17–producing NKT are associated with the pathogenesis of SIV in RMs and suggest that TGF-b and IL-18

play a role in their development. The Journal of Immunology, 2010, 184: 984–992.

Immunopathology mediated by inappropriate or poorly con-trolled effector T cell responses has typically been viewed inthe context of the Th cells (Th1 and Th2) paradigm. More

recently, a CD4+ T cell subset characterized by the production ofIL-17 and crucially involved in certain autoimmune, allergic, andinflammatory diseases was identified (1–4). Invariant NK T cells(NKT), which have a role in antitumor immune responses andantiviral immunity (5), have also been recently reported to pro-duce IL-17 in the context of inflammatory diseases (6, 7).TGF-b and inflammatory cytokines together induce the de-

velopment of Th17 cells from CD4+ T cells in mice and humans (8–10). In the immune system, TGF-b affects multiple cell lineages byeither promoting or opposing their differentiation, survival, and

proliferation (11). At present, TGF-b ismainly viewed as an immunesuppressive cytokinebecauseTGF-b is a critical factor for regulatoryT cells, and its loss is associated with a fatal lymphoproliferativedisease (12). TGF-b regulates the components of adaptive immunity,such as T cells, as well as innate immunity, such as NK cells (13). Incontrast, an inflammatory cytokine environment inhibits the gener-ation of regulatory T cells and instead leads to the differentiation ofTh17 cells. Inflammatory cytokines are strongly induced in cells ofthe innate immune system following engagement of specific pattern-recognition receptors such as TLRs and C-type lectin receptors.Thus, in addition to IL-6, TNF-a and IL-1 have been proposed tohave an additional role in the amplification of Th17 responses (14).The elevation of TGF-b (15–17) plus an inflammatory envi-

ronment may suggest the possible induction of Th17 population inthis context. However, recent reports indicated a lower frequencyof Th17 CD4+ T cells at mucosal and systemic sites during HIVinfection and SIV infection (18–21). Given the role of IL-17 incontrolling commensal bacteria, it has been proposed that de-pletion of Th17 may participate in the disruption of the mucosalbarrier. However, the absence of Th17 CD4+ during the acutephase is not associated with an increase in LPS translocation (22),which suggests the possibility of additional IL-17+–expressingcells that control intestinal flora translocation compensating forthe defect in Th17 CD4+ T cells. However, whether the loss ofIL-17–expressing cells is also true in intact secondary lymphoidtissues and whether other cells, such as NKT expressing IL-17,emerge during SIV infection is presently unknown.

Materials and MethodsAnimals and virus infection

Twenty-four rhesus macaques (RMs; Macaca mulatta) were inoculated i.v.with the pathogenic SIVmac251 strain (10 50% animal infectious doses).All the animals were challenged with the same batch of virus, titrated invivo in RMs, and stored in liquid nitrogen. Six African green monkeys

*Institut National de la Sante et de la Recherche Medicale (U 955), Faculte deMedecine de Creteil, ‖Assistance Publique Hopitaux de Paris, Hopital Henri Mondor,Creteil; †Unite de Physiopathologie des Infections Lentivirales, {Unite de Regulationdes Infections Retrovirales, Institut Pasteur; ‡Universite Paris 7 Denis Diderot, Fac-ulte de Medecine, Site Bichat; **Unite Mixte de Recherche 8147, Faculte de Mede-cine Rene Descartes, Paris V, Hopital NeckerParis, Cedex 15; ††Centre de Recherchedes Cordeliers, Universite Pierre et Marie Curie–Paris 6, UMR S 872, Paris, France;xInstitut Pasteur, Dakar, Senegal; and #Centre for Immunology, St. Vincent’s Hospi-tal, Darlinghurst, New South Wales, Australia

1C.E. and J.E. contributed equally to this work.

Received for publication July 17, 2009. Accepted for publication November 12,2009.

L.C.-G. was supported by a grant from Ministere de la Recherche et de l’Enseigne-ment of Paris XI University. Funding from the Agence Nationale de Recherches surle SIDA and Fondation pour la Recherche Medicale to J.E. supported this work.

Address correspondence and reprint requests to Dr. Jerome Estaquier, INSERM (U955), Faculte de Medecine de Creteil, Hopital Henri Mondor, 8 Rue du GeneralSarrail, 94010 Creteil, France. E-mail address: estaquier@yahoo.fr

Abbreviations used in this paper: AGM, African green monkey; Ax, axillary; FSC,forward light scatter; LN, lymph node; Mes, mesenteric; NKT, NK T cell; RM,rhesus macaque; SIV+ RM, rhesus macaque infected with the pathogenic SIVmac251strain; SSC, side scatter.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902316

(AGMs) of sabaeus species were experimentally infected with 300 50%tissue culture-infective dose of SIVagm.sab92018 strain. As previouslydescribed (23), this virus derives from a naturally infected AGM and hasnever been cultured in vitro. Animals were demonstrated as being sero-negative for simian T leukemia virus type-1), SRV-1 (type D retrovirus),herpes B viruses, and SIVmac. Animals were housed and cared for incompliance with existing French regulations (Institut Pasteur, Paris,France). The organs were collected and frozen in isopentane cooled inliquid nitrogen. Organs were cut into 4-mm sections on a cryostat, and thesections were stored at 280˚C until use (16).

Viral quantification

RNA was extracted from plasma of SIV-infected monkeys using the TRIReagent BD Kit (Molecular Research Center, Cincinnati, OH). Real-timequantitative RT-PCR was used to determine viral loads as previously de-scribed (24). Productively infected cells were assessed in lymph nodes(LNs) by in situ hybridization using a [35S]-labeled RNA probe derivedfrom the SIVmac nef gene, and the frequency of SIV infected cells wasmeasured by limiting dilution PCR as described (24).

Immunohistochemical analysis

Tissues were prepared for immunohistochemistry as described (16). Briefly,cryostat sections (4 to 5 mm) were fixed in acetone for 10 min and stored at220˚C until use. For staining, tissue sections were fixed in 2% para-formaldehyde, permeabilized with Triton 0.05%, then incubated withmAbs against IL-17 (eBioscience, San Diego, CA), and TGF-b (9016,R&D Systems, Minneapolis, MN), for 2 h, followed by biotinylated goat

anti-mouse Ab and streptavidin peroxidase complex (Vectastain ABC kit,Vector Laboratories, Burlingame, CA). Analyses were performed on fourdifferent slides, in a blind manner, by two investigators using a Nikon-FXAmicroscope (Nikon, Melville, NY) and the Optilab Pro 2.5 software(Graftek Imaging, Austin, TX). The numbers of cells counted were dividedby the surface of the entire tissue section. The results were expressed ascell numbers/2 mm2 sections. Semiquantitative analyses were also de-termined by measuring the level of staining (numbers of pixels of the entiretissue section) divided by the total surface. The results were expressed asa number of positive cells.

Cytokine assays

Blood from monkeys was collected in sterile EDTA-treated vacuum tubesand immediately centrifuged at 400 g for 15 min at 4˚C to avoid cytokinesynthesis in vitro. IL-6, TNF-a, and IL-1b were detected simultaneously inplasma by using the human inflammatory cytokine cytometric bead arraykit (BD Pharmingen, San Diego, CA), which has been validated for cy-tokine measurement in RMs and AGMs. The cytometric bead arrayworking range was 20–5000 pg/ml for each cytokine. IL-18 in plasma wasmeasured by using an ELISA kit (MBL Biomedical, Clinisciences), andstimulation of cells from healthy monkeys demonstrated no difference inIL-18 expression between RM and AGM. We also used ELISA to mea-sure type I IFN (PBL interferon source, Clinisciences) validated for bothspecies.

PBMCs from monkeys were isolated from peripheral blood by densitygradient centrifugation LymphoPrep (PAA Laboratories, Pasching, Austria)and cultured in RPMI 1640 supplemented with 10% FCS, 1% glutamine,1% pyruvate, and 1% antibiotics. Up to 500,000 PBMCs were cultured

FIGURE 1. In situ expression of IL-17 and

TGF-b in peripheral tissues during SIV in-

fection Dynamics of IL-17 (A) and TGF-b (B)

expression in peripheral LNs from SIV-

infected RMs during the acute phase. Ax and

Mes LNs from RMs were stained using anti–

IL-17 and anti–TGF-b Abs at days 0, 7, 11, 14,

and 60 postinfection. At each time point, three

monkeys were sacrificed. Magnification 340

and 3200 for the enlarged image (IL-17 in-

dicated by black arrow). White arrows indicate

neutrophils. C, Dynamics of IL-17 and TGF-b

expression in peripheral Ax LNs from AGM.

Ax LNs were stained using anti–IL-17 and

anti–TGF-b Abs at days 0, 7, 14, and 60

postinfection. Magnification 320 and 3200

for the enlarged image. White arrows indicate

neutrophils. D, SIV RNA+ cells were detected

in LNs of SIV-infected RMs (n = 3 at each time

point) and SIVagm-infected AGMs (n = 6). E–

G represent the quantification of IL-17– and

TGF-b–expressing cells as well as neutrophils

in LNs of SIV+ RMs (three RM were sacrificed

at each time point) and AGMs (n = 6). H,

Correlation between IL-17 cell numbers, viral

replication, and TGF-b cell numbers in Ax

LNs at day 14. Each symbol represents one

individual (n = 12).

The Journal of Immunology 985

overnight. Cells were incubated in the presence of brefeldin A (5 mg/mlfor the last 12 h) before being stained for FITC–anti-CD4 and PerCP–anti-CD3 (BD Biosciences, San Jose, CA), then washed twice in PBS, andfurther incubated with PE–anti-IFN-g or PE–anti-TNF-a and APC–anti-IL-17A. Cells were also stained with PE-CD8 (RPA-T8), PE-CD56(MY31), PE-HLA-DR (G46-6), FITC-CD161 (DX12), PE-CD25 (M-A251), PE-CD27 (M-T271), and PE-CD95 (DX2) from BD Biosciences;PE-CXCR6 (56811) and PE-CCR6 (53103) from R&D Systems; PE-CD127 (R34.34) and FITC–anti-Va24 (clone C15) from Beckman Coulter(Fullerton, CA); and PE–anti-human invariant NKT (clone 6B11, BDPharmingen), PerCP–anti-CD4, and APC–anti-IL-17A. We also used anti-human FITC–anti-CD45RA (clone 2H4, Beckman Coulter, Fullerton, CA)and PE–anti-CD62L (clone SK11, BD Biosciences). Samples were ana-lyzed on an FACScalibur cytometer with CellQuest software (CellQuest,Tampa, FL).

In vitro differentiation of NKT

Human PBMCs were isolated from peripheral blood by density gradientcentrifugation on LymphoPrep (PAA Laboratories). Up to 500,000 PBMCswere stimulated with Con A (1 mg/ml) and then incubated with either TGF-b alone (5 ng/ml), IL-18 alone (50 ng/ml), or TGF-b plus IL-18 for 5 d at37˚C with 5% CO2 in RPMI 1640 with 5% of FCS. At day 6, cells wereactivated with PMA (100 ng/ml) plus ionomycin (300 ng/ml) for 5 h in thepresence of brefeldin A (5 mg/ml). This experiment was conducted usinghuman cells and human cytokines to preclude the possibility that thesecytokines might not have cross-species reactivity.

Statistical analysis

Data are reported as means 6 SEM. Comparisons were based on ANOVAand Tukey’s post hoc test, using Prism 3.0 software (GraphPad, San Diego,CA). Student t test and Mann-Whitney U test (Prism software, GraphPad)were also used to determine whether differences were significant if p ,0.05. Correlations were identified by means of the Spearman rank corre-lation coefficient (r). Differences were considered significant if p , 0.05.

ResultsEmergence of IL-17–expressing cells during primary SIVinfection

Byimmunochemistryanalyses,weclearlyobservedIL-17–expressingcells in axillary (Ax) andmesenteric (Mes) LNs of RMs infectedwiththe pathogenic SIVmac251 strain (SIV+ RM) (Fig. 1A). The quanti-fication of IL-17+–producing cells from RMs, killed at different timepoints postinfection (n = 3 RMs, at each time point), revealed a sig-nificant increase at day 14 (Fig. 1A, 1E) concomitantly with the peakof viral replication in these organs (Fig. 1D). These cells weremostlylocated within the paracortical T cell zone. TGF-b, assessed in thesame tissue sections, progressively increased and peaked at day 14(Fig. 1B, 1F). Thereafter, at day 60, IL-17– as well as TGF-b–expressing cells decreased both in Ax and Mes LNs but remainedhigher thanpreinfection.By analyzing agroupof 12monkeys (24, 25)at day 14, we found a positive correlation between the numbers ofIL-17–expressing cells and thenumbers ofTGF-b–expressingcells inAx LNs of SIV+ RM, whereas no correlation was observed with theextent of viral replication inLNs (Fig. 1H). Although SIVinfection ofRMs leads to progressive CD4+ T cell depletion and AIDS, SIVagminfection ofAGMis nonpathogenic despite levels of plasmaviral loadsimilar to those observed in SIVmac-infectedRMs (23, 26, 27). In thisstudy, in stark contrast toRMs,wedid not detect an increase in IL-17–and TGF-b–positive cells in peripheral LNs from six SIV-infectedAGMs (Fig. 1C, 1E, 1F). A role for IL-17 in the recruitment ofneutrophils in inflamed tissues hasbeenproposed (2). In this study,wefound, despite the absence of IL-17, the presence of neutrophils inLNs of SIV-infected AGMs at day 14, similar to that observed in AxLN of SIV-infected RMs (Fig. 1G), suggesting the possible role ofadditional cytokines (i.e., IL-8) in the recruitment of neutrophils innonpathogenic monkeys.These results demonstrate the differential expansion of IL-17–

expressing cells early postinfection in the pathogenic primatemodel of SIV infection in RMs.

Persistence of IL-17–expressing cells during the chronic phaseof SIV infection

We recently demonstrated during the chronic phase of SIV+ RMthat TGF-b expression is significantly increased in noncontrollersversus controllers (16). We thus quantified the expression of IL-17–expressing cells in Ax and Mes LNs of 10 SIV+ RMs (5 werenoncontrollers as previously described (16). Whereas no IL-17–expressing cells were found in a healthy monkey (#93750), weobserved increased levels of IL-17–expressing cells in Ax LNsfrom SIV+ monkeys; these levels were higher in noncontrollers (asshown in monkeys #94746 and #272) than in controllers (as shown

FIGURE 2. A, In situ expression of IL-17 in peripheral tissues during

chronic SIV infection. Analysis of IL-17 expression during the chronic

phase of SIV infection. Ax and Mes LNs tissues (A), ileum (C, upper

panels), and colon (C, lower panels) from SIV2 RMs (#93750, healthy

macaque) and SIV+ RM displaying distinct rate of progression toward

AIDS (controller #94422 and noncontrollers #94746 and #272). B,

Quantification was performed by analyzing SIV2 RMs (n = 4; gray box)

and 10 SIV-infected RMs for Ax and Mes LNs (five controllers, gray box,

versus five noncontrollers, black box) and six SIV-infected RMs for ileum

and colon (three controllers versus three noncontrollers) (D). Quantitation

was performed as in B; asterisk represents significant differences with

values observed preinfection: p , 0.05.

986 IL-17–PRODUCING NKT CELLS AND SIV/HIV INFECTION

in monkey #94422) (Fig. 2A, 2B). In Mes LNs, the levels of IL-17–expressing cells in SIV+ RMs were also higher compared withhealthy monkeys (Fig. 2A, 2B), a result that is consistent with theexpression of TGF-b (16). We also studied IL-17 expression inGALTs discriminating small and large intestine (ileum and colon,respectively). We found that SIV infection induces increasednumbers of IL-17+ cells in the ileum that is higher in non-controllers (monkeys #94746 and #272) than in controllers(monkey #94422) (Fig. 2C, 2D) and higher than that observed inhealthy macaques (#93750). Moreover, whereas we detected largenumbers of IL-17+ cells in the colon of healthy RMs (#93750),which may reflect the presence of commensal bacteria (28), weobserved a progressive depletion of these cells in animals pro-gressing toward AIDS (Fig. 2C, 2D). In Indian RMs, which aremore rapid progressors than Chinese RMs, it has also been shownthat there are differential dynamics between large and small in-testines (19). Thus, in large intestine, the depletion is higher.Altogether,our tissueanalysesdemonstrategreaterexpressionof IL-

17 cells associated with the progression to AIDS, except in the colon.

The IL-17+ subpopulation exhibits phenotypic markers of NKT

In addition to classical Th17 cells, NKT have been recently reportedto produce IL-17 (6, 7, 29). Therefore, we next investigated the

phenotype of cells that ex vivo spontaneously produced IL-17 inSIV+ RM. We also stimulated the cells with PMA/ionomycin thatmeasure the pool of IL-17–expressing cells. Our data revealed thatex vivo cells (CD4low) from SIV+ macaques spontaneously pro-duced IL-17 compared with cells from healthy macaques. In con-trast, invitro stimulation revealed that healthymacaques have a poolof effector IL-17+CD4+ cells higher than SIV+RMs, consistent withrecent published data (18–20). Most importantly, in vitro stimula-tion induced the loss of this subset, which produced spontaneousIL-17 (data not shown). Therefore, in studying the spontaneousproduction of this cytokine, we found in PBMCs of SIV+RMs an IL-17–producing population that is higher in noncontrollers than incontrollers at day 60 (Fig. 3A). As shown in Fig. 3A and 3C, the IL-17–producing subset expressed low levels of CD4 and CD3 mole-cules. These cells were negative for CD8 and CD56, a marker usedto detect macrophages in primates. We then examined whether thissubset exhibits phenotypic characteristics of NKT. We found thatIL-17+–expressing cells from peripheral blood of noncontrollers areNKT+Va24+ and also express CD161+ and CCR6+ molecules (Fig.3C), which have been reported to characterize this type of cell, al-though other cell types express these markers (30, 31). We dem-onstrated that IL-17+CD4low cells spontaneously produced TNF-aand IFN-g (Fig. 3B), consistent with the fact that freshly isolated

FIGURE 3. Identification of IL-17–producing cells

in SIV+ RMs. A, Flow cytometric analyses of IL-17–

producing cells in healthy RMs (Healthy) and con-

troller versus noncontroller SIV+ RMs at day 60. Cells

were gated on forward light scatter (FSC) and side

scatter (SSC). IL-17–producing cells from peripheral

blood displayed low CD4 and CD3 expression. B,

Expression of IFN-g and TNF-a versus IL-17 in

CD4bright and CD4low subpopulations. C, Dot plot

showing the expression of isotype controls, CD56,

CD4, CD8, CD161, CCR6, and NKT. D, Expression

of the CD45RA/CD62L markers on IL-17+ (R2),

CD4+ (R1), and CD4- (R3), respectively, from pe-

ripheral blood of noncontroller SIV+ RMs at day 60.

The Journal of Immunology 987

NKT from histologically tumor-bearing human tissues, were morepotent producers of TNF-a and IFN-g (32). We also analyzed theexpression of CD62L and CD45RAmarkers. Instead of being eitherCD62Lbright or CD62Lneg and either CD45RAbright or CD45RAneg

cells like CD4+ or CD42 T cells from peripheral blood of non-controllers (Fig. 3D), this population, as previously described (33),is mostly CD45RAint and CD62Lint (Fig. 3D). Gating on IL-17+

NKT+ comparedwith CD4+ T cells fromperipheral blood,we foundthat these cells were negative for CD127 and CD25; half of themexpressed HLA-DR and CXCR6 but they expressed CD27 (TNFreceptor) at a low level and a higher level of CD95 (death receptor)(Fig. 4). We also analyzed the phenotype of freshly isolated IL-17+

cells in Ax LNs and the spleen of noncontrollers, and we found thatIL-17–expressing cells were NKT+, CD161+, CCR6+, and CD82

but, unlike peripheral blood, were all positive for HLA-DR (Fig. 5).During the acute phase,we found that the percentage of the IL-17+

NKT+ subset increased both in peripheral blood and in Ax LNs ofSIV+ RMs (n = 10); in contrast, the percentage of IL-17+CD4+ cellsdecreased both in the blood and peripheral LNs (Fig. 6A), consistentwith other recent reports (18–20).We also examined in the differentorgans the fraction of CD8, NKT, and CD4 cells expressing IL-17+

at the chronic phase. Six RMswere analyzed at necropsy. The levelsof the IL-17+NKT+ subset were higher in SIV+ RMs than in healthymonkeys and greater in Mes LNs and the spleen than in the Ax LNs

(Fig. 6B). These results are consistent with our recent reportshowing the same profile of TGF-b expression in these tissues (16).Our data also revealed that in splenic and Mes LN tissues, nochanges in IL-17+CD8+ cells were observed, whereas the fraction ofIL-17+CD4+ T cells increased due to the fact that the percentage ofCD4+ T cells in LNs decreased in SIV-infected RMs compared withhealthy macaques (Fig. 6B).Altogether, these data demonstrate that AIDS progression is

associated with the emergence of IL-17+NKT+ early after SIVinfection, and these cells persist in animals progressing morerapidly to AIDS.

Emergence of IL-17+ subpopulation is associated withincreased plasma levels of IL-18 and type I IFN in SIV+ RMs

Because an inflammatory environment concomitantly with exoge-nous TGF-b has been reported to induce the differentiation ofIL-17–expressing cells (14) and IL-18 is essential for NKT (34), wenext investigated the presence of proinflammatory cytokines duringprimary SIV infection. In addition to the pyrogenic cytokinesIL-1b, TNF-a, and IL-6, we also assessed the expression of IL-18,a proinflammatory cytokine and member of the IL-1 “superfamily”that is unable to induce fever (35), as well as a type I IFN, that hasbeen reported to be increased during SIV infection and favors NKT(36, 37). In SIV+ RMs, we found that type I IFN as well as IL-18plasma levels increased postinfection, reaching a peak at day 11 andday 14, respectively, and then decreased (Fig. 7A) (n = 12); we alsoobserved a moderate increase in IL-6 levels, whereas IL-1b andTNFa were undetectable. This peak of IL-18 in the sera is asso-ciated with increased expression of IL-18 in the LN tissues (Ax andMes LNs) (Fig. 7B) and is consistent with the absence of fever in

FIGURE 4. Phenotype of IL-17+ cells from peripheral blood. Flow

cytometric analyses of CD127, CD25, CD27, CD95, CXCR6, and HLA-

DR expression gating on IL-17+NKT+ (R1) and on CD4+ T cells (R2) of

noncontroller SIV+ RMs at day 60.

FIGURE 5. Phenotype of IL-17+–expressing cells from Ax LNs and

spleen. Flow cytometric analyses of NKT, CD8, CD161, CCR6, and HLA-

DR versus IL-17. Cells were gated on forward light scatter (FSC) and side

scatter (SSC).

988 IL-17–PRODUCING NKT CELLS AND SIV/HIV INFECTION

SIV-infected RMs during the acute phase. Most interestingly, de-spite similar levels of viral replication, IL-18 and type I IFN wereonly slightly increased in SIV-infected AGMs.Thus, we found cytokine profiles that were distinct in pathogenic

compared with nonpathogenic SIV infection.

In vitro TGF-b and IL-18 induces the differentiation andexpansion of IL-17+NKT+

Because IL-18 is essential for NKT (34) and because it has beenproposed that IL-6 is dispensable for IL-17+NKT (29), we thendetermined the involvement of IL-18 in the expansion and in theinduction of IL-17+NKT in SIV+ RMs. Thus, we investigatedwhether IL-18 concomitantly with TGF-b might induce in vitrothe differentiation of IL-17+NKT. PBMCs from human healthydonors were used to test the effect of these cytokines to avoid anypossible defect in cross-reactivity in monkeys. Cells were firstactivated with Con A and then incubated in the absence or pres-ence of TGF-b and IL-18. At day 6, the cells were restimulatedwith PMA plus ionomycin (Fig. 8A, 8B). We found that TGF-balone increased the proportion of the cells that differentiated intoIL-17+NKT as well as IL-17+CD4+ cells. Given the role of stim-ulated APCs in the release of inflammatory cytokines, it is possiblethat the initial Con A stimulation also contributed to the in vitrodifferentiation of IL-17+ cells. IL-18 alone also increased thepercentage of IL-17+NKT, whereas it had no effect on the per-centage of IL-17+CD4+ cells. The combination of both TGF-b andIL-18 enhanced the percentage of both IL-17+CD4+ and IL-17+

NKT as compared with each cytokine alone. To determine whetherthis increase was reflected by the expansion and/or differentiationof these subsets, we evaluated the numbers of these cells in cul-ture. Each cytokine, TGF-b and IL-18 alone, reduced the numbersof IL-172NKT+, leading to a relative increase in the percentage ofIL-17–expressing NKT (Fig. 8C); in contrast, TGF-b together withIL-18 induced the expansion of these IL-17+NKT. Whereas TGF-balone induced the expansion of IL-17+CD4+, the addition of IL-18in culture with TGF-b enhanced their expansion. Thus, increasedIL-18 expression concomitantly with elevated TGF-b in SIV+

RMs may contribute to the induction of the IL-17+NKT subset.

DiscussionRecently, several groups have shown a decline of Th17CD4+ T cellsduring HIVand SIV infection and proposed that the absence of thissubset may participate in the disruption of mucosal barrier integrity(18–21). In this study, our results demonstrated the early expansion

of IL-17–expressing cells in SIV+ RMs that is correlated with TGF-b expression. Thus, an innate IL-17 production by NKT is rapid andprecedes the adaptive Th17 response. The emergence of this IL-17+

NKT+ population in SIV-infected RMs, therefore, could compen-sate for the defect in Th17 CD4+ T cells (19, 21), preventing mi-crobial translocation (no LPS was detected during the acute phase(20) and the occurrence of a wasting syndrome early postinfection.Moreover, in chronically SIV-infected monkeys (19) as well as inHIV-infected Africans (38), however, microbial translocation wasnot correlated with immune activation and viral replication. Thus,microbial translocation might be a symptom of the defect of IL-17populations and not a direct cause of HIV-1 disease. The observa-tion that SIV-infected AGMs that do not progress to disease have noexpansion of IL-17–expressing cells is consistent with the resultsfrom another group (20) that found to be unchanged numbers ofIL-17 cells in peripheral blood in the nonpathogenic model of Sootymangabey. The possible difference in the previous reports could bethe origin of the monkeys used, given that we used RMs of Chineseorigin instead of RMs of Indian origin, the latter being more sus-ceptible to SIV infection (26), and we used freshly isolated cellsinstead of frozen samples to perform these assays. Another point isour observation that ex vivo activation induced the loss of thissubset, suggesting the hypothesis that cells might be more prone todie (activation-induced cell death) because they express CD95 andCD27. Finally, to our knowledge, no other groups have assessed theexpression of IL-17 in the tissues, and therefore this is the firstreport of this observation. Therefore, although we cannot excludethe possibility that other cells in the tissues express IL-17, our datastrongly suggest that most of the freshly isolated cells that producedIL-17 were NKT.It is now clear that IL-17 is associated with a number of human

autoimmune disorders by inducing excessive inflammation. IL-17

FIGURE 6. Dynamics of NKT+, IL-17+NKT+, and IL-17+CD4+ T cells.

A, Cells were analyzed from peripheral blood and LNs at days 0, 14, and 60

from SIV+ RMs (n = 10). The percentages of these subsets of cells, re-

spectively, are expressed as mean 6 SEM. B, Graphs show the fractions of

IL-17–expressing cells gating on CD8, NKT, and CD4 cells in Ax and Mes

LNs and the spleen of healthy (SIV2, n = 4) and chronically SIV-infected

RMs (SIV+) (n = 6). IL-17–producing cells are expressed as mean6 SEM.

FIGURE 7. Dynamics of proinflammatory cytokines during primary

infection in SIV-infected monkeys. A, Levels of IL-18, IL-6, IL-1b, TNF-

a, and type I IFN were measured in the sera of SIV+ RMs (n = 12) and in

SIV+ AGMs (n = 6) at days 0, 7, 11, 14, 21, 30, and 60 postinfection. B,

Expression of IL-18 in Mes and Ax LNs. Analyses were performed at days

0, 7, and 14 postinfection.

The Journal of Immunology 989

exerts a potent effect against fibroblasts, epithelial cells, and en-dothelial cells and promotes the infiltration of inflammatory T cellsand neutrophils (39, 40). Thus, the early emergence of this novelpopulation expressing IL-17 could contribute to local tissue dam-age and favor viral dissemination. Despite this side effect, there isalso considerable evidence, in mice, that IL-17 is important in hostresponses to infection by Gram-negative bacteria, specificallyKlebsiella, Pseudomonas, Escherichia coli, Salmonella, andBordetella species (41, 42). Whereas we found higher levels ofIL-17–expressing cells in the ileum, our data demonstrated theabsence of IL-17–expressing cells in the colon of noncontrollersSIV+ RMs during the chronic phase. This could contribute to theabsence of bacterial control, consistent with the clinical observationthat animals progressing toward AIDS develop a wasting syndromecharacterized by a loss of weight and frequent diarrhea. These re-sults may be related to a distinct dynamic of IL-17+ cells in thesmall and large intestine of SIV-infected monkeys or a re-distribution of IL-17+ cells from the colon to the ileum.TGF-b and inflammatory cytokines together induce the de-

velopment of IL-17–expressingCD4+ T cells (8–10) aswell as NKT(6, 7). Our results demonstrate for the first time a role for IL-18 inthe genesis of IL-17+ cells consistent with its role in the emergenceof NKT (34). Caspase-1, a member of the cysteine protease familyinvolved in apoptosis, is involved in the maturation and secretion of

IL-18 (35). Therefore, the absence of IL-18 in SIV-infected AGMscould reflect the absence of apoptosis that we and others have de-scribed in this nonpathogenic primate model, compared withpathogenic primate models, during both the acute (26) and thechronic phase (24, 43–46). Thus, a vicious cycle may be generatedby the occurrence of apoptotic cells in the tissues of SIV+ RMs,favoring not only the expression of IL-18, but also, through theirphagocytosis by macrophages and dendritic cells, the production ofTGF-b and the absence of TNF-a and IL-1b (47), favoring theemergence of IL-17+NKT+ over IL-17+CD4+ T cells.However, how is the expansion of NKT early after SIV infection

generated? Viral danger signals through the type I IFN pathwaytriggerNKTactivation bymyeloid dendritic cells (48, 49). DNAandRNA viruses induce type I IFN through TLR7 and TLR9, and, inparticular, HIV has been shown to induce type I IFN (50). Recently,several groups (36, 37) have shown increased levels of type I IFNearly after SIV infection of RMs, whereas in nonpathogenic SI-Vagm infection, these levels are relatively low (23). Thus, the dy-namics of IL-17+NKT is consistent with the expression of type IIFN during the acute phase observed in SIV-infected RMs.In conclusion, we demonstrated the emergence of IL-17–pro-

ducing cells early after SIV infection. The levels of these cells,and their subsequent persistence in peripheral blood and in a largenumber of tissues, except colon, were associated with progression

FIGURE 8. Involvement of IL-18 in the expansion

of IL-17+NKT+. A, Human PBMCs were stimulated

with Con A (1 mg/ml) and then incubated with RPMI

1640 (Med), TGF-b (5 ng/ml), IL-18 (50 ng/ml), or

TGF-b plus IL-18 for 6 d at 37˚C. Cells were then

stimulated with PMA (100 ng/ml) and ionomycin (300

ng/ml) for 5 h at 37˚C in the presence of brefeldin A (5

mg/ml). Cells were stained with PE–anti-NKT, PerCP–

anti-CD4, and APC–anti-IL-17. Cells were gated on

live CD4+ T cells and NKT+ and dot plots show IL-17

expression. The cutoff reflects isotype controls. B,

Percentages of IL-17–expressing cells among NKT+

and CD4+ populations. Results are the mean 6 SD of

three independent xperiments. C, Cell numbers were

measured in parallel to A to determine the expansion of

NKT+IL-172, NKT+IL-17+, CD4+IL-172, and CD4+

IL-17+. One experiment is shown, out of three, giving

similar results.

990 IL-17–PRODUCING NKT CELLS AND SIV/HIV INFECTION

to AIDS. Moreover, our results show for the first time the ex-pansion of the IL-17+NKT+ subset in response to microbial in-fections. Our results strongly suggest the role of IL-18, in additionto TGF-b, in the differentiation of this novel subpopulation.

AcknowledgmentsWe thank J.M. Panaud (Institut Pasteur) for assistance with micro-

photographs.

DisclosuresThe authors have no financial conflicts of interest.

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