chloroquine modulates hiv-1-induced plasmacytoid …aac.asm.org/content/54/2/871.full.pdf ·...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2010, p. 871–881 Vol. 54, No. 20066-4804/10/$12.00 doi:10.1128/AAC.01246-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Chloroquine Modulates HIV-1-Induced Plasmacytoid Dendritic CellAlpha Interferon: Implication for T-Cell Activation�

Jeffrey A. Martinson,1 Carlos J. Montoya,2 Xiomara Usuga,1 Rollie Ronquillo,1Alan L. Landay,1 and Seema N. Desai1*

Department of Immunology and Microbiology, Rush University Medical Center, Chicago, Illinois,1 andGroup of Immunovirology-Biogenesis, University of Antioquia, Medellin,Colombia2

Received 9 September 2009/Returned for modification 1 October 2009/Accepted 23 November 2009

Plasmacytoid dendritic cells (pDC) contribute to antiviral immunity mainly through recognition of microbialproducts and viruses via intracellular Toll-like receptor 7 (TLR7) or TLR9, resulting in the production of typeI interferons (IFNs). Although interferons reduce the viral burden in the acute phase of infection, their role inthe chronic phase is unclear. The presence of elevated plasma IFN-� levels in advanced HIV disease and itsassociation with microbial translocation in chronic HIV infection lead us to hypothesize that IFN-� couldcontribute to immune activation. Blocking of IFN-� production using chloroquine, an endosomal inhibitor, wastested in a novel in vitro model system with the aim of characterizing the effects of chloroquine on HIV-1-mediated TLR signaling, IFN-� production, and T-cell activation. Our results indicate that chloroquine blocksTLR-mediated activation of pDC and MyD88 signaling, as shown by decreases in the levels of the downstreamsignaling molecules IRAK-4 and IRF-7 and by inhibition of IFN-� synthesis. Chloroquine decreased CD8T-cell activation induced by aldrithiol-2-treated HIV-1 in peripheral blood mononuclear cell cultures. Inaddition to blocking pDC activation, chloroquine also blocked negative modulators of the T-cell response, suchas indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PDL-1). Our results indicate that TLRstimulation and production of IFN-� by pDC contribute to immune activation and that blocking of thesepathways using chloroquine may interfere with events contributing to HIV pathogenesis. Our results suggeststhat a safe, well-tolerated drug such as chloroquine can be proposed as an adjuvant therapeutic candidatealong with highly active antiretroviral therapy to control immune activation in HIV-1 infection.

Plasmacytoid dendritic cells (pDC), which recognize patho-gens through Toll-like receptor 7 (TLR7) and TLR9, are anintegral part of the innate and adaptive immune systems (2, 34,42, 43, 45, 73). Since these TLRs are intracellular, their ligandsrequire cellular uptake and endosomal maturation to triggerNF-�B and mitogen-activated protein kinase-mediated signalsthrough the MyD88-dependent pathway. These TLR signalsresult in pDC activation/maturation and in the production ofproinflammatory cytokines and a large amount of type 1 inter-feron, or alpha/beta interferon (IFN-�/�) (17, 43, 46). pDCalso provide negative regulatory signals that modulate andestablish immune tolerance (62). Several molecules expressedby pDC, including indoleamine 2,3-dioxygenase (IDO) andprogrammed death ligand 1 (PDL-1), are implicated in thenegative modulation of T-cell responses. IDO, the rate-limit-ing enzyme involved in tryptophan catabolism, inhibits CD4T-cell proliferation (8, 10, 53) and enhances T-regulatory cellgeneration and suppressor cell function (16). The PD-1–PDL-1 interaction results in decreased T-cell proliferation,cytokine production, and cell-mediated cytotoxicity (72). TLR-mediated induction of PDL-1 expression on pDC may contrib-ute to T-cell exhaustion and dysfunction through the PD-1–PDL-1 pathway. Despite a decrease in the number of

circulating pDC in HIV-1-infected individuals (3, 24, 26, 73,74), their stimulation by microbial products via TLR7 andTLR9 (48), or by HIV itself (5), results in pDC activation andIFN-� production, which contribute to pathogenesis in chronicHIV-1 disease (14). IFN-� is known to be associated withelevated lipopolysaccharide (LPS) levels in chronic HIV infec-tion, which may contribute to persistent immune activation(14).

Chloroquine, an antimalarial drug, has immunomodulatoryproperties and is used in the treatment of autoimmune disor-ders (28, 69, 81). It is also commonly used in vitro to study therole of endosomal acidification in cellular processes, such asthe TLR activation pathways in pDC induced by HIV-1 (5, 9,25, 35, 71). Chloroquine is a weak base that accumulates withinthe endosomes of cells, leading to an elevated vacuolar pH andthereby inhibiting endosomal maturation and nucleic acidbinding to TLR7 and TLR9 (68). HIV-1 activates pDCthrough TLR7 (5), which makes chloroquine a candidate forpreventing HIV-1-induced activation and subsequent down-stream effects on T-cell activation and function.

The goal of the present study was to characterize the effectsof chloroquine on the HIV-1-mediated stimulation of pDCand its potential effect on modulating regulators of T-cell func-tion, including IFN-�-induced T-cell activation. Our resultsshow that chloroquine inhibits pDC activation/maturation, up-regulation of the MyD88 pathway signaling molecules IFNregulatory factor 7 (IRF-7) and interleukin-1 receptor-associ-ated kinase 4 (IRAK-4), IFN-� production, IDO synthesis, andPDL-1 expression. We also observed that CD8� T-cell activa-tion induced by IFN-� can be modulated by chloroquine

* Corresponding author. Mailing address: Department of Immunol-ogy/Microbiology, Rush University Medical Center, 1735 West Harri-son St. (Cohn Research Building, Lab 641), Chicago, IL 60612. Phone:(312) 942-9292. Fax: (312) 942-5206. E-mail: [email protected].

� Published ahead of print on 30 November 2009.

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through its ability to decrease IFN-� production by pDC. Thecentral role that TLRs play in innate and adaptive immunitymakes them an ideal target for therapeutic intervention usingchloroquine. We propose that in HIV infection, chronic TLRstimulation and production of IFN-� by pDC contribute toimmune activation and immune cell dysfunction, and thatblocking or modulation of these pathways with chloroquinemay interfere with the events of HIV pathogenesis.

MATERIALS AND METHODS

Monoclonal antibodies and reagents. The following fluorochrome-conjugatedmouse anti-human monoclonal antibodies were used in these studies: Lin1-fluorescein isothiocyanate (FITC) cocktail (CD3, CD14, CD16, CD20, CD56),CD123-peridinin chlorophyll protein (PerCP)-Cy5.5, HLA DR-allophycocyanin(APC), biotin-HLA DR, HLA DR-FITC, CD86-APC, CD83-phycoerythrin(PE), CD38-PE, CD3-Pacific Blue, and CD8-APC-H7 from BD Biosciences(BD; San Jose, CA); PDL-1–APC from eBiosciences (San Diego, CA); andCD4-PE-Texas red from CalTag (Carlsbad, CA). Unconjugated rabbit anti-human polyclonal antibodies included those against IRF-7 (H-246; Santa CruzBiotechnology, Santa Cruz, CA) and IRAK-4 (Invitrogen, Carlsbad, CA), as wellas the anti-human IDO polyclonal rabbit antibody directed against the DLIESGLRERVEKLNMLC peptide (60), from David Munn (Medical College ofGeorgia, Augusta). Fluorochrome-conjugated secondary reagents includedstreptavidin-Pacific Blue (Invitrogen) and donkey anti-rabbit–PE (Jackson Im-munoResearch Laboratories, West Grove, PA). The BD Cytofix/Cytoperm kitwas used for intracellular staining of IRF-7, IRAK-4, and IDO. Cells weretreated with the Fc�R blocking reagent (Miltenyi Biotec, Auburn, CA) and/ornormal donkey serum (Jackson ImmunoResearch) to prevent nonspecific anti-body binding to the cells.

The synthetic small molecule imidiazoquinoline, 3M-011 (TLR7/8), was pro-vided by 3M Pharmaceuticals (St. Paul, MN). Endotoxin-free A-class CpG oli-godeoxynucleotide (ODN) 2336 or 2216 was provided by Coley PharmaceuticalGroup (Pfizer, Wellesley, MA). Noninfectious Aldrithiol-2 (AT-2)-treatedHIV-1Ada (R5-tropic) and HIV-1MN (X4-tropic) preparations, along withmatched control microvesicles, SUPT1-CCR5 and CEMX174 (T1), isolatedfrom uninfected cell cultures (6), were kindly provided by Jeff Lifson of the AIDSVaccine Program, National Cancer Institute (Frederick, MD). RecombinantIFN-�2a (rIFN-�2a) was obtained from PBL (Piscataway, NJ), and the 64G12monoclonal antibody against the human IFN-� receptor (anti-IFN-�R) wasprovided by Michael Tovey of the Laboratory of Viral Oncology (Villejuif,France).

TLR agonists and AT-2-treated HIV-1 for stimulation of PBMC. The TLRagonists were used at the following concentrations for in vitro stimulation ofperipheral blood mononuclear cells (PBMC): TLR7/8 agonist (3M-011), 3.0 �M;TLR9 agonist (A-class CpG ODN 2336 or 2216), 4 �g/ml. It is now known thatendocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like recep-tor–viral RNA interactions (5), so we stimulated PBMC using noninfectiousAldrithiol-2 (AT-2)-treated HIV-1Ada (R5 tropic) and HIV-1MN (X4 tropic),since more than 99% of HIV-1 particles detected in the circulation are nonin-fectious (35, 64). AT-2-treated HIV-1Ada and HIV-1MN (referred to below asAT-2 Ada and AT-2 MN, respectively) were used at a concentration of 500 ng/mlof p24 capsid equivalent. The concentration of HIV-1 (500 ng/ml) used in thisstudy is within the range found in the plasma of infected individuals (includingchildren and adults), which is known to vary depending on the stage of HIVdisease (44, 66). Optimal concentrations of the TLR agonists (32, 54, 55) andAT-2 HIV-1 (54) have been determined previously. Media and matched mi-crovesicle controls, MV-CCR5 and MV-CEMX, served as the negative controlsin these experiments.

Chloroquine for inhibition of endosomal acidification and blocking of pDCTLR signaling. To study the effect of chloroquine on the regulation of theendosomal TLR7 and TLR9 pathways in response to inactivated HIV-1 or TLRagonists, we used a 100 �M concentration of chloroquine based on a previousreport evaluating the activation of pDC by herpes simplex virus (HSV) (56). Forthe T-cell activation studies, we used a concentration of chloroquine (0.5 to5 �M) equivalent to the achievable physiological concentration in blood asdescribed in the Medsafe database (www.medsafe.govt.nz) (13). PBMC werepreincubated for 1 h in a medium containing chloroquine (diphosphate salt)(InvivoGen, San Diego, CA), which was present throughout the cell culture.

Cell culture for pDC activation with or without chloroquine. Heparinizedperipheral blood was collected via venipuncture from healthy HIV-seronegative

volunteers recruited from Rush University Medical Center (Chicago, IL) afterinformed consent. PBMC were isolated by density gradient centrifugation usinglymphocyte separation medium (Cambrex, Gaithersburg, MD). PBMC (1 �106/ml) were cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) supple-mented with 10% heat-inactivated fetal bovine serum (Invitrogen-Gibco), 100U/ml penicillin, 100 �g/ml streptomycin (Sigma, St. Louis, MO) and 2 mML-glutamine (Sigma) in 6-well polystyrene tissue culture plates in a humidified37°C, 5% CO2 incubator. PBMC were preincubated with a medium containing100 �M chloroquine or the medium alone for 1 h prior to stimulation with TLRagonists or AT-2-inactivated HIV-1. Cells were cultured for 20 h, harvested, andevaluated for pDC activation/maturation and intracellular IDO, IRF-7, andIRAK-4 protein expression using flow cytometry. Supernatants were stored at�20°C for IFN-� cytokine analysis by an enzyme-linked immunosorbent assay(ELISA).

Cell culture for T-cell activation with or without chloroquine. PBMC orenriched CD8� T-cell subsets (indirect isolation; Miltenyi [Auburn, CA] mi-crobead isolation kit) were preincubated for 1 h with a medium containingchloroquine or for 30 min with the anti-IFN-�R blocking antibody 64G12 (10�g/ml). AT-2 HIV-1 or 1,000 U/ml rIFN-�2a was added, and cells were culturedfor 20 h, harvested, and evaluated for T-cell activation by measuring CD38surface expression on CD4� and CD8� T cells by flow cytometry. Logical gatingwas used to identify the CD4 (CD3� CD4�) and CD8 (CD3� CD8�) T-cellsubsets. T-cell activation is expressed as CD38 mean fluorescence intensity(CD38-MFI) for each parent T-cell subset.

Cell culture for pDC IDO activity with or without chloroquine. PBMC prein-cubated (1 h) with a medium containing 100 �M chloroquine or the mediumalone were cultured with TLR agonists or AT-2-inactivated HIV-1 in 96-welltissue culture plates (1 � 106 cells/well; 0.250 ml/well) in a humidified 37°C, 5%CO2 incubator. Cells were cultured for 20 h, harvested, and evaluated for pDCPDL-1 expression by flow cytometry. Supernatants were stored at �20°C forevaluation of IDO activity in fluorometric assays to detect tryptophan andkynurenine in the pathway of L-tryptophan catabolism.

Flow cytometric analysis. Chloroquine-treated or untreated PBMC from con-trol or TLR agonist- and AT-2 HIV-1-stimulated cultures were harvested,washed, and resuspended in phosphate-buffered saline (PBS) containing 0.5%bovine serum albumin and 0.1% sodium azide (FACS buffer). Nonspecific anti-body binding to Fc receptors was blocked by preincubation of the cells with theFc�-receptor-blocking reagent (Miltenyi Biotec). PBMC for pDC activation/maturation and PDL-1, along with PBMC and purified CD4� and CD8� T cells,for T-cell activation were surface stained with the appropriate antibodies, washedwith FACS buffer, fixed with 2% formaldehyde, and stored at 4°C until analysis.Cells for intracellular IRF-7, IRAK-4, and IDO staining were surface stained toidentify pDC (Lin1� CD123� HLA DR�), washed with FACS buffer, blockedwith normal donkey serum (Jackson ImmunoResearch), and processed with BDCytofix/Cytoperm solution. Cells were then washed in Perm/Wash buffer andstained with the appropriate unconjugated polyclonal rabbit anti-human IRF-7,IRAK-4, or IDO antibodies. Cells were washed in Perm/Wash buffer and stainedwith a PE-conjugated donkey anti-rabbit secondary antibody (Jackson Immu-noResearch). Finally, the cells were washed twice in Perm/Wash buffer, fixedwith 1% formaldehyde, and stored at 4°C until analysis. All samples were eval-uated within 24 h of staining using a FACSCalibur or LSRII flow cytometer.

Logical gating was used to identify the pDC (Lin1� CD123� HLA DR�)population. The expression of pDC activation (pDC-CD86) and maturation(pDC-CD83) markers is measured as percentages of the pDC parent population.Logical gating was used to identify the CD8 (CD3� CD8�) T-cell population.MFI was used to evaluate IRF-7, IRAK-4, IDO, and PDL-1 levels within theparent pDC population and the CD38 level within the parent T-cell subsets.

Measurement of IFN-� by ELISA. Commercial ELISA kits were used tomeasure the concentration of IFN-� (PBL Biomedical Laboratories, Piscataway,NJ) in cell culture supernatants. ELISAs were performed according to themanufacturer’s guidelines.

IDO activity: kynurenine and tryptophan fluorescence assays. Depletion oftryptophan and accumulation of the immunosuppressive catabolite kynurenineare measures of IDO activity. Detection of tryptophan and kynurenine in theculture supernatants was performed using fluorometric assays (7, 59). In brief,duplicate Eppendorf tubes containing cell culture supernatants or stock kynuren-ine solution (Sigma; 25 to 0.39 �g/ml) were incubated with 30% trichloroaceticacid (TCA; Sigma) for 30 min in 50°C water bath. The tubes were allowed to coolto room temperature and were then centrifuged at 2,400 rpm for 10 min. Analiquot from each tube was transferred to individual wells of a flat-bottompolystyrene 96-well plate. Freshly made 2% Ehrlich’s reagent (Sigma) in glacialacetic acid was added to each well, and the plate was incubated at room tem-perature for 10 min in the dark. Finally the plate was read on a BioTek (Wi-

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nooski, VT) Synergy HT plate reader at a wavelength of 490 nm. A kynurenine(0 to 25 �g/ml) standard curve was used to determine the kynurenine concen-trations in the culture supernatants.

The tryptophan fluorescence assay was performed as follows. Stock L-trypto-phan (Sigma; 1.25 to 20 �g/ml) or cell culture supernatants were incubated in20% TCA in a 96-well plate (duplicate wells/sample) for 1 h at 4°C. The plateswere centrifuged at 2,400 rpm for 15 min, and supernatants were transferred topolypropylene tubes. The supernatants were then incubated with a mixture of10% TCA, 1.8% formaldehyde (prepared in PBS), and 6 mM FeCl3 at 90°C for2 h. The tubes were allowed to cool to room temperature, and aliquots from eachtube were transferred into individual wells of a flat-bottom polystyrene 96-wellplate. The plates were read on a BioTek Synergy HT fluorescence plate reader(excitation wavelength, 360/40 nm; emission wavelength, 460/40 nm; sensitivity,70 nm). The tryptophan (1.25 to 20 �g/ml) standard curve was used to determinethe tryptophan concentrations in the culture supernatants. IDO activity is ex-pressed as the kynurenine-to-tryptophan ratio.

Statistical analysis. Results are expressed as means standard errors of themeans (SEM). The significance of differences between groups was determinedusing an unpaired Student t test (with a confidence level of 95%) with GraphPadPrism software, version 4.03. P values of 0.05 were considered statisticallysignificant.

RESULTS

AT-2 HIV-1-induced pDC activation/maturation is down-regulated by chloroquine. We investigated the ability of chlo-roquine, an inhibitor of endosomal acidification, to abrogateHIV-1-induced pDC activation (pDC-CD86) and maturation(pDC-CD83). PBMC, treated in the presence or absence ofchloroquine (100 �M), were stimulated with noninfectiousHIV AT-2 Ada, AT-2 MN, or control microvesicles overnight.Both CD86 and CD83 were significantly upregulated on pDC(Lin1� CD123� HLA DR�) exposed to AT-2 Ada (CD86, P �0.008; CD83, P � 0.006) or AT-2 MN (CD86, P � 0.001;CD83, P � 0.010) compared to the medium control (Fig. 1Aand B). Levels of AT-2 Ada-induced pDC activation markers(expressed as the percentage of pDC expressing CD86) de-creased from 50.5% 9.8% to 11.1% 19.8% (P � 0.017)(Fig. 1A), and those of pDC maturation markers (percentageof pDC expressing CD83) decreased from 64.6% 11.1% to12.6% 6.8% (P � 0.016) (Fig. 1B) in the chloroquine-treatedcultures. Similarly, levels of AT-2 MN-induced pDC activationmarkers decreased from 49.3% 5.4% to 8.2% 2.5% (P �0.002) (Fig. 1A), and those of maturation markers decreasedfrom 69.0% 13.9% to 14.3% 10.6% (P � 0.035) (Fig. 1B)in chloroquine-treated cultures. Control microvesicles,CCR5-MV and CEMX-MV, did not induce significant pDCactivation/maturation (CD86 levels were 1.23% 0.54% in themedium, 1.65% 0.38% in CEMX-MV, and 2.89% 1.29%in CCR5-MV; CD83 levels were 4.42% 2.94% in the me-dium, 5.24% 4.31% in CEMX-MV, and 3.03% 1.97% inCCR5-MV) compared to levels of the markers in mediumcontrols.

TLR signaling and IFN-� production by pDC are inhibitedby chloroquine. HIV-1 induces the secretion of type 1 inter-ferons (IFN-�/�) by pDC, partly through TLR 7 (5). TLR7recognition of single-stranded RNA (ssRNA) (HIV-1) de-pends on the kinase activity of interleukin-1 receptor-associ-ated kinase 4 (IRAK-4), the interferon regulatory factor 7(IRF-7) transcription factor, and the adaptor molecule MyD88(2, 38, 39, 47). In this study, we tested the effects of chloro-quine on AT-2 HIV-1-induced TLR signaling by measuringlevels of the downstream transcription molecules IRAK-4 andIRF-7 and subsequent interferon production in PBMC from

five donors. AT-2 MN significantly upregulated IRAK-4 MFI(P � 0.034), while IRF-7 MFI was significantly increased byboth AT-2 MN (P � 0.011) and AT-2 Ada (P � 0.006) (Fig.2A). Chloroquine treatment prevented AT-2 Ada (P � 0.004)-and AT-2 MN (P � 0.005)-induced upregulation of IRAK-4 inpDC compared with AT-2 HIV-1-stimulated cultures in theabsence of chloroquine (Fig. 2A). Further downstream in theTLR7-induced MyD88 signaling pathway, levels of the tran-scription factor IRF-7 ertr also decreased in AT-2 Ada (P �0.004)- and AT-2 MN (P � 0.025)-stimulated cultures treatedwith chloroquine (Fig. 2A) compared to AT-2 HIV-1 culturesin the absence of chloroquine. Control microvesicles,CCR5-MV and CEMX-MV, did not induce significant pDCIRAK-4 (MFI, 18.40 2.42 in the medium, 16.25 0.95 inCEMX-MV, and 16.50 1.19 in CCR5-MV) or IRF-7 (MFI,17.60 1.50 in the medium, 17.80 1.69 in CEMX-MV, andV16.40 0.60 in CCR5-M) expression compared to that inmedium controls.

Modulation of the TLR7 signaling pathway via chloroquineshould be reflected in the production of IFN-�. Therefore, weevaluated AT-2 HIV-1-stimulated culture supernatants forIFN-� using a commercially available ELISA. Supernatantsfrom either AT-2 Ada (P � 0.001)- or AT-2 MN (P � 0.0002)-stimulated cultures contained significantly higher concentra-tions of IFN-� than the medium control (Fig. 2B). Chloro-quine completely abrogated the production of IFN-� in PBMCcultures stimulated with AT-2 Ada (P � 0.001) or AT-2 MN(P � 0.0002) (Fig. 2B). To confirm the ability of chloroquine toprevent IFN-� secretion via TLR7 or TLR9 signaling of pDC,we used synthetic TLR7/8 (3M-011) and TLR9 (CpG A) ago-nists. Supernatants from the TLR9 CpG A (P � 0.003) orTLR7/8 (P � 0.026) agonist-stimulated cultures contained sig-nificantly higher concentrations of IFN-� than the mediumcontrol (Fig. 2C). As observed for the AT-2 HIV-1-stimulatedcultures, neither the TLR9 CpG A agonist (P � 0.003) nor theTLR7/8 agonist (P � 0.026) induced IFN-� production in thepresence of chloroquine (Fig. 2C). Taken together, these re-sults demonstrate that chloroquine inhibits TLR signaling, asevidenced by decreases in the levels of the IRAK-4 and IRF-7signaling molecules along the MyD88-specific pathway and inthe subsequent production of IFN-�, by preventing endocyto-sis and the subsequent endosomal degradation of HIV-1 re-quired for TLR signaling by pDC.

Chloroquine downregulates IFN-�-induced T-cell activa-tion. T-cell activation, a predictor of HIV disease progression,can be measured by the elevated expression of the cell surfacemarker CD38 (31, 52, 75) on CD8 T cells. We tested the abilityof physiologic concentrations of chloroquine (0.5 to 5 �M) tomodulate IFN-� production and its subsequent effect on T-cellexpression of CD38 during exposure to noninfectious AT-2HIV-1 or exogenous rIFN-�2a. Using a commercially availableELISA, we observed a dose-dependent decrease in IFN-� con-centrations in the supernatants of AT-2 HIV-1-stimulatedPBMC cultures (n � 3) treated with 0.5, 1, or 5 �M concen-trations of chloroquine (Fig. 3A). At a concentration of 5 �Mchloroquine, AT-2 HIV-1 Ada (P � 0.007)- and AT-2 HIV-1MN (P � 0.002)-stimulated PBMC supernatants containedsignificantly reduced levels of IFN-� compared to those inAT-2-HIV-1-stimulated supernatants without chloroquine.Since IFN-� production in response to AT-2 MN (268.4 35.1

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pg/ml) was lower than that in response to AT-2 Ada (1,113 120.6 pg/ml), we studied the effect of 5 �M chloroquine onAT-2 Ada-stimulated PBMC and purified T cells. Flow cyto-metric analyses revealed significant increases in the CD38-MFIon CD8� T cells (Fig. 3B) after exposure of PBMC to AT-2Ada (P � 0.006) and exogenous rIFN-�2a (P � 0.005) overthat for the medium control. Chloroquine (5 �M) decreasedCD38-MFI on CD8 T cells 23.4% in AT-2 Ada-stimulatedPBMC (Fig. 3B). In contrast, blocking of the IFN-� receptorwith monoclonal antibody 64G12 significantly downregulatedthe CD38-MFI on CD8� T cells (P � 0.014; 43% decrease inCD38-MFI) in PBMC stimulated with exogenous rIFN-�.

Next, we utilized Miltenyi bead-enriched CD8� T cells to eval-

uate the role of IFN-� or HIV-1 in inducing CD8� T-cellactivation (n � 3 experiments). CD38 expression (CD38-MFI)was significantly increased on CD8� cells exposed to exoge-nous rIFN-�2a (P � 0.047) but not on CD8� cells exposed toAT-2 Ada HIV-1 compared to that in the medium (Fig. 3C).Blocking of the IFN-� receptor by pretreatment of enrichedCD8� cells with the 64G12 monoclonal antibody partially de-creased CD38-MFI, by 22.3%, in rIFN-�2a-stimulated cultures(Fig. 3C). As expected, chloroquine did not inhibit rIFN-�2a-induced CD8� T-cell activation. These data suggest that theuse of chloroquine to block IFN-� produced by pDC duringHIV-1 infection may play a critical role in reducing chronicT-cell activation.

FIG. 1. Chloroquine abrogates AT-2 HIV-1-induced DC activation and maturation. PBMC were stimulated with AT-2 HIV-1 for 20 h in thepresence or absence of 100 �M chloroquine and were then analyzed by flow cytometry. Logical gating was used to identify the pDC population(Lin1� HLA DR� CD123�) for analyses. (A) Representative flow cytometric analysis and summary of the pDC activation marker CD86.(B) Representative flow cytometric analysis and summary of the pDC maturation marker CD83. Results are expressed as mean percentages of pDCexpressing the marker SEM (n � 3).



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AT-2 HIV-1-induced pDC IDO expression and IDO trypto-phan catabolism are downregulated by chloroquine. pDC pro-duce indoleamine 2,3-dioxygenase (IDO), a key enzyme intryptophan degradation along the kynurenine pathway that hasbeen implicated in decreased CD4 T-cell proliferative capacityin HIV-1 infection (10). In this study, PBMC from five indi-

viduals were tested for the effect of chloroquine on AT-2HIV-1-induced intracellular IDO expression in the pDC pop-ulation. Intracellular IDO levels in pDC (pDC-IDO MFI)were upregulated following overnight stimulation with AT-2Ada or AT-2 MN (Fig. 4A; representative examples shown).pDC expression of IDO was significantly upregulated in cul-

FIG. 2. Chloroquine abrogates the TLR-activated MyD88-dependent signaling pathway and IFN-� production in pDC. PBMC were stimulatedwith AT-2 HIV-1 or the TLR9 (CpG A) and TLR7/8 (3M-011) agonists for 20 h in the presence or absence of 100 �M chloroquine. Logical gatingwas used to identify the pDC population (Lin1� HLA DR� CD123�) for analyses. (A) Representative flow cytometric analysis and summary ofthe expression of the IRAK-4 and IRF-7 signaling molecules in pDC stimulated with AT-2 HIV-1 with or without 100 �M chloroquine. (B andC) Summary of IFN-� (pg/ml) concentrations in culture supernatants from PBMC cultures stimulated with AT-2 HIV-1 (B) or with a syntheticTLR9 (CpG A) or TLR7/8 (3M-011) agonist (C) with or without 100 �M chloroquine. Results are expressed as means SEM (n � 5).



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tures stimulated with AT-2 Ada (MFI, 448.0 96.0 [P �0.021]) relative to that in the medium (MFI, 170.8 35.8) (Fig.4A). Chloroquine treatment abrogated AT-2 HIV-1-inducedpDC IDO expression to baseline MFI values similar to those inmedium control cultures (MFI, 126.4 30.1 [P � 0.010] forAda cultures with chloroquine, 159.0 34.0 for MN cultureswith chloroquine, and 170.8 35.8 for medium control cul-tures) (Fig. 4A). These results demonstrate that chloroquinecan suppress TLR-mediated IDO expression by pDC, an effectthat may prove beneficial in blocking tryptophan degradation.

Next, we evaluated IDO functional activity by measuringkynurenine (KYN) and tryptophan (TRYP) levels in cell cul-

ture supernatants. Results are expressed as the KYN/TRYPratio; an increase in the KYN/TRYP ratio indicates active IDOcatabolism of tryptophan along the kynurenine pathway. TheKYN/TRYP ratios (Fig. 4B) were significantly increased in thesupernatants of cultures stimulated with AT-2 Ada (1.548 0.320; P � 0.019) or AT-2 MN (1.594 0.308; P � 0.013) overthat for the medium control (0.589 0.066). Chloroquinetreatment resulted in decreased tryptophan catabolism in AT-2Ada (P � 0.037)- and AT-2 MN (P � 0.020)-stimulated culturesupernatants.

We also evaluated IDO activity in cultures stimulated with asynthetic TLR9 (CpG A) or TLR7/8 agonist (Fig. 4C). TheKYN/TRYP ratio was significantly increased in the superna-tants of cultures stimulated with TLR9 (1.590 0.314; P �0.014) over that for the medium control (0.589 0.066). KYN/TRYP ratios were significantly lower in TLR9 CpG A (P �0.026)-stimulated cultures containing chloroquine than inthose without chloroquine. KYN/TRYP ratios were elevated insupernatants stimulated with the TLR7/8 agonist over that forthe medium control and were reduced in chloroquine-treatedcultures, though no statistically significant differences were ob-served. These results demonstrate that increased IDO expres-sion in pDC can be directly mediated by TLR7 and/or TLR9stimulation and that the abrogation of the TLR signal by chlo-roquine may offer a means to regulate pDC IDO expression inHIV-1 infection.

AT-2 HIV-1- and TLR agonist-induced PDL-1 expression onpDC is downregulated by chloroquine. Microbial persistencemay be related to the exploitation of the PD-1 and PDL-1pathway by certain pathogens (72). PDL-1 is expressed consti-tutively on pDC. AT-2 Ada (P 0.0001), AT-2 MN (P �0.002), TLR9 CpG A (P � 0.0001), and TLR7/8 (P 0.0001)significantly increased PDL-1 expression on pDC over that forthe medium control (Fig. 5A and B). We tested the ability ofchloroquine to regulate AT-2 HIV-1- or synthetic TLR7/8 orTLR9 agonist-induced PDL-1 expression on pDC. The meanfluorescence intensity of PDL-1 on pDC (pDC PDL-1 MFI)was significantly reduced in the presence of chloroquine (Fig.5A and B). Chloroquine completely abrogated the upregula-tion of PDL-1 on pDC in cultures stimulated with either AT-2HIV-1 (P 0.0001 for AT-2 Ada; P � 0.002 for AT-2 MN) ora TLR agonist (P � 0.0001 for TLR9 CpG A; P 0.0001 forTLR7/8). These results demonstrate that chloroquine down-modulates the potential contribution of pDC to T-cell exhaus-tion/dysfunction through the PD-1/PDL-1 pathway by sup-pressing PDL-1 expression on pDC.

DISCUSSION

HIV infection is associated with the impairment of immunefunction, with deficiencies observed in nearly every type ofimmune cell. A better understanding of the process by whichHIV hijacks innate immune cell function could be achieved inorder to develop alternative therapeutic approaches. Amongpotential mechanisms, chronic activation of pDC by directinteraction with infectious and noninfectious HIV-1 particlescontributes to the maintenance of immune cell dysfunction andHIV-associated pathogenesis (8, 11, 21, 36). In this study, weused an in vitro model to demonstrate the potential immuno-regulatory function of chloroquine to modulate HIV-1-in-

FIG. 3. Chloroquine decreases IFN-�-induced CD8� T-cell activa-tion. PBMC were stimulated with AT-2 HIV-1 (Ada or MN) for 20 hin the presence or absence of physiologically achievable concentrationsof chloroquine (0.5 to 5.0 �M). (A) IFN-� (pg/ml) concentrations insupernatants of AT-2 Ada- and MN-stimulated PBMC with or withoutchloroquine. (B) PBMC cultures stimulated with AT-2 HIV-1 Ada or1,000 U/ml rIFN-�2a were analyzed by flow cytometry for expressionof the CD38 activation marker in the CD8� T-cell subset (CD3�

CD8�) with or without 5 �M chloroquine or the anti-IFN-�R blockingmonoclonal antibody 64G12. (C) Miltenyi bead-purified CD8� T cellsstimulated with AT-2 HIV-1 or 1,000 U/ml rIFN-�2a were analyzed byflow cytometry for expression of the CD38 activation marker in theCD8� T-cell subset (CD3� CD8�) with or without 5 �M chloroquineor the anti-IFN-�R blocking monoclonal antibody 64G12. Results areexpressed as means SEM (n � 3).



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duced pDC activation/maturation, IFN-� production, IDO ex-pression/activity, and PDL-1 expression. We also show thatproduction of IFN-�, an outcome of pDC activation, directlycontributes to CD8� T-cell activation. Using chloroquine, aninhibitor of endosomal fusion and acidification, we were ableto suppress HIV-1-mediated TLR signaling in pDC and toinhibit the potential deleterious effects of chronic pDC activa-tion. Our data show that interfering with TLR signaling re-duces levels of HIV-1-induced pDC activation products that

are known to contribute to immune dysregulation in HIV-1infection.

More than 99% of HIV-1 particles detected in the circula-tion are not productively infectious (35, 64). These noninfec-tious particles contribute to HIV-induced immunopathogen-esis (21, 36). We and others have shown that AT-2 HIV-1 atconcentrations between 300 and 1,000 ng/ml induces pDC ac-tivation/maturation and production of IFN-� (27, 35, 36, 54,82). The concentration of HIV (500 ng/ml) used in this study is

FIG. 4. Chloroquine abrogates AT-2 HIV-1-induced IDO expression and activity in pDC. PBMC were stimulated with AT-2 HIV-1 for 20 hin the presence or absence of 100 �M chloroquine and were then analyzed by flow cytometry. (A) Representative flow cytometric analysis andsummary of AT-2 HIV-1-induced IDO expression (IDO MFI) in pDC (Lin1� HLA DR� CD123�) with or without 100 �M chloroquine. (B andC) Supernatants from PBMC cultures stimulated with AT-2 HIV-1 (B) or a TLR agonist (C) for 20 h with or without 100 �M chloroquine wereanalyzed for IDO activity using fluorometric assays to detect kynurenine and tryptophan. Results are expressed as the kynurenine-to-tryptophanratio. Results are expressed as means SEM (n � 5).



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within the range found in the plasma of infected individuals(both children and adults), which is known to vary dependingon the stage of HIV disease (44, 66). Using 500 ng/ml ofnoninfectious AT-2-inactivated HIV-1 Ada or MN, we couldactivate the pDC IFN-� pathway. Activation of pDC by HIV-1requires endocytosis of the virion, leading to endosomal TLR7recognition of viral RNA (5, 58) and initiation of the MyD88pathway for the production of IFN-� by pDC. Chloroquine, aninhibitor of endocytosis and endosomal acidification/matura-tion, prevented HIV-1-mediated TLR7 signaling in pDC, asshown by the failure of pDC to upregulate the downstreamIRAK-4 and IRF-7 signaling molecules within the MyD88pathway. By showing that chloroquine decreases IFN-� pro-duction, it can be inferred that AT-2 HIV-1 utilizes TLR7signaling in pDC and not the cytosolic antiviral pathway me-

diated by RIG-1 recognition of ssRNA (41, 65). These datahighlight the importance of the endolysosomal pathway inHIV-1-mediated signaling of pDC.

The production of IFN-� by pDC in HIV-1 infection mayalso have important consequences for T-cell activation andsurvival (67). During acute HIV-1 infection, high plasmaIFN-� titers provide a protective antiviral effect and help trig-ger the adaptive immune response (73). However, in late-stagedisease, elevated serum IFN-� levels are an indicator of poorclinical prognosis (30, 80). We demonstrate that 100 �M chlo-roquine has an inhibitory effect on HIV-1-mediated produc-tion of IFN-�. It has been shown that the use of 100 �Mchloroquine in the treatment of leukocytes in vitro results inintracellular concentrations comparable to those obtained invivo during chloroquine therapy (29). Our findings are similar

FIG. 5. Chloroquine abrogates PDL-1 expression in pDC. PBMC were stimulated with AT-2 HIV-1 for 20 h in the presence or absence of 100�M chloroquine and were then analyzed by flow cytometry. (A) Representative flow cytometric analysis and summary of AT-2 HIV-1-inducedPDL-1 expression on pDC (Lin1� HLA DR� CD123�) with or without 100 �M chloroquine. (B) Representative flow cytometric analysis andsummary of TLR agonist-induced PDL-1 expression on pDC (Lin1� HLA DR� CD123�) with or without 100 �M chloroquine. Results areexpressed as means SEM (n � 5).



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to those of other investigators using chloroquine to modulateIFN-� production in HSV-infected systems (25, 50). However,in our study, at the declining concentrations of chloroquine(0.5 to 5 �M), we observed a dose-dependent decrease inHIV-1-induced IFN-� production. At our lowest tested con-centration of chloroquine (0.5 �M), we observed a partialblockage of IFN-� production, which is in contrast to a pub-lished report (71) of complete blockage of IFN-� with a similarconcentration of chloroquine (0.5 �M) used to block inductionof pDC activation through TLR9. These differences in theconcentration of chloroquine required for blocking IFN-�could be attributed to the activation pathways. As previouslyreported, a higher concentration of chloroquine is necessary toblock TLR7 endosomal maturation and acidification comparedto TLR9 (51). Therefore, our results provide evidence thatHIV-1-induced IFN-� production can be modulated with chlo-roquine at concentrations that are obtainable for therapeuticapplication.

Chronic immune activation is a hallmark of HIV-1 patho-genesis. Elevated IFN-� levels in the plasma and lymphoidtissues, along with increased expression of interferon-induciblegenes, in HIV-infected patients have been shown in previousreports to correlate with elevated T-cell activation, character-istic of HIV progression (14, 36, 37). In this study, the CD38activation marker, commonly used to measure the T-cell acti-vation state in HIV-infected patients (8, 20, 31, 75), was down-modulated in PBMC cultures when 5 �M chloroquine wasutilized. Since cell separation procedures affect the uptake ofchloroquine (29), we used a lower dose of chloroquine (5 �M)to study its effect on T-cell activation in enriched T cells andcompared it with a similar dose of chloroquine in PBMC cul-tures. Chloroquine downmodulates T-cell activation in AT-2HIV-1-stimulated PBMC cultures and not in AT-2 HIV-1-stimulated enriched T cells, suggesting that inhibition of pDCIFN-� production decreases T-cell activation. Using enrichedCD8� T cells, we demonstrate that increased CD38 expressionis induced by exogenous rIFN-�2a and not by direct interac-tion with HIV-1, suggesting that pDC IFN-� certainly contrib-utes to CD8� T-cell activation. As previously reported (67), wedid not find IFN-�-induced activation of CD4� T cells (datanot shown). The ability to block IFN-�-mediated CD8� acti-vation with the anti-IFN-�R monoclonal antibody (64G12)and not with chloroquine highlights the importance of modu-lating IFN-�-induced CD8� T-cell activation. This is the firstdirect evidence that chloroquine blocks pDC IFN-�-mediatedimmune activation, and it is supported by a previous studydemonstrating the ability of chloroquine to control TLR9(CpG)- or LPS-induced proinflammatory cytokine productionin mice (40).

HIV-1 also modulates immune function through the induc-tion of negative regulatory signals that directly affect the de-velopment of the adaptive immune response. One such mech-anism evaluated in our studies was the induction of theimmunosuppressive enzyme IDO in pDC following HIV-1stimulation. We also observed an increase in the level ofkynurenine metabolites in the culture supernatants, indicativeof IDO-regulated tryptophan catabolism. As shown by other invitro studies, HIV-1 induces IDO in pDC (10, 22, 57), whichdirectly inhibits T-cell proliferation and function through tryp-tophan depletion and through the activity of kynurenine me-

tabolites (4, 12, 61). Another key regulatory function of IDO isthe induction and stimulation of T-regulatory cells during HIVinfection (18, 23, 33), which dampen HIV-specific T-cell re-sponses. Thus, the development of new treatment modalities(i.e., chloroquine or hydroxychloroquine) to reverse the induc-tion of IDO levels found both in blood cells and in lymphoidtissues (4, 10, 61) may prove beneficial for the improvement ofT-cell proliferation and function, in addition to controllingaberrant immune activation in HIV-infected individuals.

A second mechanism induced by HIV-1 infection to dampenantiviral responses is the PD-1/PDL-1 pathway (9, 19, 63, 79).Our data suggest that exposure to “noninfectious” HIV-1(AT-2 HIV-1) upregulates PDL-1 ligand expression on pDC,which is consistent with reports of other investigators showingTLR-induced PDL-1 on pDC and IFN-�-induced PDL-1 onmyeloid dendritic cells (1, 15, 78). Manipulation of the PD-1/PDL-1 pathway in HIV-1 infection may prove beneficial in therestoration of virus-specific CD8� T-cell responses by decreas-ing programmed death pathway-induced apoptosis.

In addition to the immunomodulatory effects observed inthese studies, chloroquine has been shown in vitro to havedirect inhibitory activity on newly produced HIV virions byaltering the glycosylation of the 2G12 epitope, which is locatedon the gp120 envelope surface protein, required for virus in-fectivity (69, 70). Several clinical trials have been performedwhere chloroquine or hydroxychloroquine was given to HIV-infected individuals along with antiretroviral therapy. In onestudy, a decrease in HIV loads was reported (77), while inanother, a decrease in plasma p24 capsid antigen levels wasobserved in chloroquine-treated individuals, although no alter-ations in CD4� T-lymphocyte counts were observed, comparedto those for control group (76).

HIV-1 infection hijacks the innate immune response, di-rectly contributing to disease pathogenesis. Chronic stimula-tion of pDC with noninfectious and infectious virions leads totheir enhanced activation, production of the cytokine IFN-�,and upregulation of IDO and PDL-1, all of which are known tocontribute directly to decreased T-cell survival, proliferation,and function. In this study, we have shown that chloroquineblocks TLR signaling in pDC, a critical step in its activationpathway. By blocking pDC activation, we observed a decreasein the production of the immunoregulatory cytokine IFN-�,which in turn could reduce IFN-�-mediated immune activationand improve T-cell survival by blocking negative modulatorssuch as IDO and PDL-1. Chloroquine is widely accessible,inexpensive, and well tolerated when administered over severalyears, making it a good candidate for adjuvant therapy alongwith highly active antiretroviral therapy (HAART) to controlimmune activation in HIV-1 infection. The findings presentedin this study are particularly important and relevant, since it isnow known that non-AIDS-defining illnesses, such as athero-sclerosis, liver disease, and renal diseases, which occur despiteeffective HAART, contribute to mortality. Immune activationand inflammation are the key contributing factors linked tothese comorbidities (49). The use of chloroquine as an adju-vant with HAART could be an effective and inexpensive ap-proach to controlling immune activation and reducing the riskof comorbidities for HAART-treated HIV-infected individu-als. The use of chloroquine for HIV-1-infected individuals in



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resource-rich and resource-poor countries needs to be furtherinvestigated.

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