aac accepts, published online ahead of print on 30 ... · department of immunology and...

Chloroquine Modulates HIV-1 Induced Plasmacytoid Dendritic Cell IFNα:

Implication for T cell Activation

Running Title: Chloroquine Inhibits pDC Activation

Jeffrey A. Martinson1, Carlos J. Montoya

2, Xiomara Usuga

1, Rollie Ronquillo

1, Alan L. Landay

1,

Seema N. Desai1*

1. Department of Immunology and Microbiology, Rush University Medical Center, Chicago, IL-

USA

2. Group of Immunovirology-Biogenesis, University of Antioquia, Medellin-Colombia

Please address correspondence to:

Seema N. Desai, Ph.D.

Department of Immunology/Microbiology

Rush University Medical Center

1735 West Harrison St (Cohn Research Building Lab 641)

Chicago, IL 60612

Phone: 312 942 9292; FAX: 312 942-5206

[email protected]

Key Words: type 1 interferon, innate immunity, immune activation, endosomal inhibitors

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.01246-09 AAC Accepts, published online ahead of print on 30 November 2009

by guest on January 13, 2010 aac.asm

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Abstract

Plasmacytoid dendritic cell (pDC) mainly contribute to antiviral immunity through recognition

of microbial products and viruses via intracellular toll-like receptors (TLR) 7 or 9 that results in

the production of type I interferons. Though interferons reduce viral burden in acute phase of

infection their role in chronic phase is unclear. Presence of elevated plasma IFNα levels in

advanced HIV disease and its association with microbial translocation in chronic HIV infection

lead us to hypothesize that IFNα could contribute to immune activation. Blocking IFNα

production using chloroquine an endosomal inhibitor was tested in a novel in vitro model system

with the aim to characterize the effects of chloroquine on HIV-1 mediated TLR signaling, IFNα

production and T cell activation. Our results indicate that chloroquine blocks TLR mediated

activation of pDC and MyD88 signaling as shown by decrease in the downstream signaling

molecules IRAK-4, IRF-7 and inhibition of IFNα synthesis. Chloroquine decreased CD8 T cell

activation induced by AT-2 HIV-1 in PBMC cultures. In addition to blocking pDC activation,

chloroquine also blocked negative modulators of T cell response like IDO (indoleamine 2, 3-

dioxygenase) and PDL-1 (programmed death ligand-1). Our results indicate that TLR

stimulation and production of IFN-α by pDC contribute to immune activation, and blocking

these pathways using chloroquine may interfere with events contributing to HIV pathogenesis.

Our results suggests that a safe well tolerated drug such as chloroquine can be proposed as an

adjuvant therapeutic candidate along with HAART to control immune activation in HIV-1

infection.


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Introduction

Plasmacytoid dendritic cells (pDC) are an integral part of the innate and adaptive immune

systems that recognize pathogens through toll-like receptor (TLR) 7 and 9 (2, 34, 42, 43, 45, 73).

Since these TLRs are intracellular, their ligands require cellular uptake and endosomal

maturation to trigger NF-κB and MAP kinase-mediated signals through the MyD88-dependent

pathway. These TLR signals result in pDC activation/maturation as well as the production of

pro-inflammatory cytokines and a large amount of type 1 interferon (IFN-α/β) (17, 43, 46). PDC

also provide negative regulatory signals that modulate and establish immune tolerance (62).

Several candidate molecules expressed by pDC are implicated in negative modulation of T cell

responses including the production of indoleamine 2, 3 dioxygenase (IDO) and the expression of

PDL-1. IDO, the rate limiting enzyme involved in tryptophan catabolism, inhibits CD4 T-cell

proliferation (8, 10, 53) and enhances T regulatory cell generation 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

contribute 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, TLR9 (48) or by HIV itself (5) results in activation

of PDC and interferon-alpha (IFNα) production that contributes to pathogenesis in chronic HIV-

1 disease(14). IFNα is known to be associated with elevated LPS levels in chronic HIV infection

that may contribute to persistent immune activation (14).

Chloroquine, an anti-malarial drug, has immunomodulatory properties and is used in the

treatment of autoimmune disorders (28, 69, 81). It is also commonly used in vitro to study the

role of endosomal acidification in cellular processes, such as the TLR activation pathways in


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pDC induced by HIV-1 (5, 9, 25, 35, 71). It is a weak base that accumulates within the

endosome of cells leading to an elevated vacuolar pH thereby inhibiting endosomal maturation

and nucleic acid binding to TLR7 and TLR9 (68). HIV-1 activates pDC through TLR7 (5) which

makes chloroquine a candidate for preventing 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 effects of chloroquine on HIV-1

mediated stimulation of pDC and its potential effect on modulating regulators of T cell function

including IFNα induced T cell activation. Our results show that chloroquine inhibits pDC

activation/maturation, up-regulation of MyD88 pathway signaling molecules IRF-7 and IRAK-4,

IFN-α production, IDO synthesis, and PDL-1 expression. We also observed that CD8+ T cell

activation induced by IFN-α can be modulated by chloroquine through its ability to decrease

IFNα production by pDC. The central role that TLRs play in innate and adaptive immunity

makes them an ideal target for therapeutic intervention using chloroquine. We propose that in

HIV infection, chronic TLR stimulation and production of IFNα by pDC contributes to immune

activation and immune cell dysfunction, and that blocking or modulation of these pathways with

chloroquine may interfere with the events of HIV pathogenesis.

Materials and Methods

Monoclonal antibodies and reagents

The following fluorochrome conjugated mouse anti-human monoclonal antibodies were used in

these studies: BD Biosciences (BD; San Jose, CA): Lin1-FITC cocktail (CD3, CD14, CD16,

CD20, CD56), CD123 PerCP-Cy5.5, HLA DR APC, biotin-HLA DR, HLA DR FITC, CD86

APC, CD83 PE, CD38 PE, CD3 Pacific Blue, CD8 APC-H7; eBiosciences (San Diego, CA):


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PDL-1 APC; and CalTag (Carlsbad, CA): CD4 PE-Texas Red. Unconjugated rabbit anti-human

polyclonal antibodies included IRF-7 (H-246; Santa Cruz Biotechnology; Santa Cruz, CA),

IRAK-4 (Invitrogen; Carlsbad, CA) and the anti-human IDO polyclonal rabbit antibody directed

against the DLIESGLRERVEKLNMLC peptide (60) from Dr. David Munn (Medical College of

Georgia; Augusta, GA). Fluorochrome conjugated secondary reagents included streptavidin-

Pacific Blue (Invitrogen) and donkey anti-rabbit PE (Jackson ImmunoResearch Laboratories;

West Grove, PA). The BD Cytofix/Cytoperm™ kit was used for intracellular staining of IRF-7,

IRAK-4, and IDO. Cells were treated with the FcγR blocking reagent (Miltenyi Biotec; Auburn,

CA) and/or normal donkey serum (Jackson ImmunoResearch) to prevent non-specific antibody

binding to the cells.

The synthetic small molecule imidiazoquinoline, 3M-011 (TLR7/8) was provided by 3M

Pharmaceuticals (St. Paul, MN). Endotoxin-free A-class CpG ODN 2336 or 2216 were provided

by Coley Pharmaceutical Group (Pfizer Wellesley, MA). Non-infectious aldithriol-2 (AT-2)

treated HIV-1Ada (R5-tropic) and HIV-1MN (X4-tropic) viral preparations along with matched

control microvesicles, SUPT1-CCR5 and CEMX174 (T1) isolated from uninfected cell cultures

(6) were kindly provided by Dr. Jeff Lifson from the AIDS Vaccine Program, National Cancer

Institute (Frederick, MD). Recombinant IFNα2a (rIFNα2a) was obtained from PBL

(Piscacaway, NJ) and the 64G12 monoclonal antibody against the human interferon-alpha

receptor (anti-IFNαR) was provided by Michael Tovey from the Laboratory of Viral Oncology

(Villejuif, France).

TLR agonists and AT-2 HIV-1 for stimulation of PBMC


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The TLR agonists were used at the following concentrations; TLR7/8 agonist (3M-011; 3.0 µM)

and the TLR 9 agonist A-class CpG ODN (2336 or 2216; 4 µg/ml) for in vitro stimulation of

PBMC. It is now known that endocytosis of HIV-1 activates plasmacytoid dendritic cells via

Toll-like receptor-viral RNA interactions (5), we stimulated PBMC using non-infectious

aldithriol-2 (AT-2) treated HIV-1Ada (R5-tropic) and HIV-1MN (X4-tropic) as more than 99% of

HIV-1 particles detected in the circulation are non infectious (35, 64). AT-2 HIV-1Ada (AT-2

Ada) and HIV-1MN (AT-2 MN) were used at a concentration of 500 ng/ml of p24 capsid

equivalent. The concentration of HIV-1 (500ng/mL) used in this study is within the range found

in the plasma of infected individuals, children and adults which is known to vary depending on

stage of HIV disease (44, 66). Optimal concentrations of the TLR agonists (32, 54, 55) and AT-

2 HIV-1 (54) were determined previously. Media and matched microvesicle controls, MV-

CCR5 and MV-CEMX served as the negative controls in these experiments.

Chloroquine for inhibition of endosomal acidification and blocking pDC TLR signaling

To study the effect of chloroquine on regulating the endosomal TLR7 and TLR9 pathways in

response to inactivated HIV-1 or TLR agonists we used a concentration of 100 µM chloroquine

based on a previous report evaluating HSV viral activation of pDC (56). For the T cell activation

studies we used a concentration of chloroquine (0.5-5 µM) which is the achievable physiological

concentration in blood as described in the Medsafe datasafe database [www.medsafe.govt.nz]

(13). PBMC were pre-incubated for 1 hour in media containing chloroquine (diphosphate salt)

(InvivoGen, San Diego, CA) which was present throughout the cell culture.

Cell culture for pDC activation ± chloroquine


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Heparinized peripheral blood was collected via venipuncture from healthy HIV-seronegative

volunteers recruited from Rush University Medical Center (Chicago, IL) after informed consent.

Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation

using lymphocyte separation medium (Cambrex, Gaitherburg, MD). PBMC (1x106/ml) were

cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% heat-

inactivated fetal bovine serum (Invitrogen-Gibco), 100 U/ml penicillin, 100 µg/ml streptomycin

(Sigma, St. Louis, MO) and 2 mM L-glutamine (Sigma) in 6-well polystyrene tissue culture

plates in a humidified 370C 5% CO2 incubator. PBMC were pre-incubated with media

containing 100 µM of chloroquine or media alone for 1 hour prior to stimulation with TLR

agonists or AT-2 inactivated HIV-1. Cells were cultured for 20 hours, harvested and evaluated

for pDC activation/maturation and intracellular IDO, IRF-7 and IRAK-4 protein expression

using flow cytometry. Supernatants were stored at -200C for IFNα cytokine analysis by ELISA.

Cell culture for T cell activation ± chloroquine

PBMC or enriched CD8+ T cell subsets (indirect isolation, Miltenyi (Auburn, CA) Microbead

Isolation Kit) were pre-incubated for 1 hour with media containing chloroquine or 30 minutes

with the 64G12 anti-IFNαR blocking antibody (10 µg/ml). AT-2 HIV-1 or 1000 U/ml rIFNα2a

was added and cells were cultured for 20 hours, harvested and evaluated for T cell activation by

measuring CD38 surface expression on CD4+ and CD8

+ cells by flow cytometry. Logical gating

was used to identify the CD4 (CD3+/CD4

+) and CD8 (CD3

+/CD8

+) T cell subsets. T cell

activation is expressed as CD38 mean fluorescence intensity (CD38-MFI) for each parent T cell

subset.


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Cell culture for pDC IDO activity ± Chloroquine

PBMC pre-incubated (1 hour) with media containing 100 µM chloroquine or media alone were

cultured with TLR agonists or AT-2 inactivated HIV-1 in 96-well Tissue Culture plates (1x106

cell/well; 0.250 ml/well) in a humidified 370C 5% CO2 incubator. Cells were cultured for 20

hours, harvested and evaluated for pDC PDL-1 expression by flow cytometry. Supernatants

were stored at -200C for evaluation of IDO activity in fluorometric assays to detect tryptophan

and kynurenine in the pathway of L-tryptophan catabolism.

Flow cytometric analysis

Chloroquine treated or untreated PBMC from control or TLR agonist and AT-2 HIV-1

stimulated cultures were harvested, washed and resuspended in PBS containing 0.5% bovine

serum albumin and 0.1% sodium azide (FACS buffer). Non-specific antibody binding to Fc

receptors was blocked by pre-incubation of the cells with the Fcγ-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, washed with FACS buffer, fixed with 2% formaldehyde and stored at 40C until

analysis. Cells for intracellular IRF-7, IRAK-4 and IDO staining were surface stained to identify

pDC (Lin1-/CD123

+/HLA DR

+), washed with FACS buffer, blocked with normal donkey serum

(Jackson ImmunoResearch) and processed with BD Cytofix/Cytoperm™ solution. Cells were

then washed in Perm/Wash™ buffer and stained with the appropriate unconjugated polyclonal

rabbit anti-human IRF-7, IRAK-4, or IDO antibodies. Cells were washed in Perm/Wash™

buffer and stained with donkey anti-rabbit PE secondary antibody (Jackson ImmunoResearch).

Finally, the cells were washed twice in Perm/Wash™ buffer, fixed with 1% formaldehyde, and


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stored at 40C until analysis. All samples were evaluated within 24 hours of staining using a

FACSCalibur™ or LSRII flow cytometer.

Logical gating was used to identify the pDC (Lin1-/CD123

+/HLA DR

+) population. PDC

activation (pDC-CD86) and maturation (pDC-CD83) marker expression are expressed as a

percentage of the pDC parent population. Logical gating was used to identify the CD8

(CD3+/CD8

+) T cell population. Mean fluorescence intensity (MFI) was used to evaluate IRF-7,

IRAK-4, IDO, and PDL-1 levels within the parent pDC population and CD38 level within the

parent T cell subsets.

Measurement of IFNα by ELISA

Commercial ELISA kits were used to measure the concentration of IFNα (PBL Biomedical

Laboratories; Piscataway, NJ) in cell culture supernatants. ELISA assays were performed

according to the manufacturer’s guidelines.

IDO activity: kynurenine and tryptophan fluorescence assays

Depletion of tryptophan and accumulation of the immunosuppressive catabolite kynurenine is a

measure of IDO activity. Detection of tryptophan and kynurenine in the culture supernatants

was performed using fluorometric assays (7, 59). In brief, duplicate eppendorf tubes containing

cell culture supernatants or stock kynurenine solution (Sigma; 25–0.39 µg/ml) were incubated

with 30% trichloroacetic acid (TCA, Sigma) for 30 minutes in 500C water bath. The tubes were

allowed to cool to room temperature and then centrifuged at 2400 rpm for 10 minutes. An

aliquot from each tube was transferred into individual wells of a flat bottom polystyrene 96-well

plate. Freshly made 2% Erlich’s reagent (Sigma) in glacial acetic acid was added to each well


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and the plate was incubated at room temperature for 10 minutes in the dark. Finally the plate

was then read on a BioTek Synergy HT plate reader (BioTek; Winooski, VT) at 490 nm

wavelength. A kynurenine (0 –25 µg/ml) standard curve was used to obtain the kynurenine

concentration in the culture supernatants.

The tryptophan fluorescence assay was performed as follows: Stock L- tryptophan

(Sigma; 1.25 – 20 µg/ml) or cell culture supernatants were incubated in 20% TCA in a 96 well

plate (duplicate wells/sample) for 1 hour at 40C. The plates were centrifuged at 2400 rpm for 15

minutes and supernatants were transferred into polypropylene tubes. The supernatants were then

incubated with a mixture of 10% TCA, 1.8% in PBS formaldehyde and 6 mM FeCL3 at 900C for

2 hours. The tubes were allowed to cool to room temperature and aliquots from each tube were

transferred into individual wells of a flat bottom polystyrene 96-well plate. The plates were read

on a BioTek Synergy HT fluorescence plate reader (Ex=360/40; Em = 460/40; sensitivity =70).

The tryptophan (1.25–20 µg/ml) standard curve was used to obtain the tryptophan concentration

in the culture supernatants. IDO activity is expressed as the kynurenine: tryptophan ratio.

Statistical analysis

Results are expressed as the mean ± standard error of the mean (SEM). Differences between

groups were performed using an unpaired Student’s t-test (confidence level of 95%) with

GraphPad PRISM software v4.03. p values of <0.05 were considered statistically significant.

Results

AT-2 HIV-1 induced pDC activation/maturation is down-regulated by chloroquine


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We investigated the ability of chloroquine, an inhibitor of endosomal acidification, to abrogate

HIV-1 induced pDC activation (pDC-CD86) and maturation (pDC-CD83). PBMC, treated in the

presence or absence of chloroquine (100 µM), were stimulated with non-infectious HIV AT-2-

Ada, AT-2-MN, or control microvesicles overnight. Both CD86 and CD83 were significantly

up-regulated 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 media control (Fig 1

A,B). AT-2 Ada induced pDC activation markers (% pDC expressing CD86) decreased from

50.5 ± 9.8% to 11.1 ± 19.8% (p=0.017, Fig 1A) and pDC maturation markers (% pDC

expressing CD83) decreased from 64.6 ± 11.1% to 12.6 ± 6.8% (p=0.016, Fig 1B) in the

chloroquine treated cultures. Similarly, AT-2 MN induced pDC activation markers decreased

from 49.3 ± 5.4% to 8.2 ± 2.5% (p=0.002, Fig 1A) and maturation markers decreased from 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 pDC activation/maturation

(CD86: Media 1.23 ± 0.54%, CEMX-MV 1.65 ± 0.38%, CCR5-MV 2.89 ± 1.29% or CD83:

Media 4.42 ± 2.94%, CEMX-MV 5.24 ± 4.31%, CCR5-MV 3.03 ± 1.97%) compared to media

controls.

TLR signaling and IFNα production by pDC is inhibited by chloroquine

HIV-1 induces the secretion of type-1 interferons (IFNα/β) by pDC partly through TLR 7 (5).

TLR7 recognition of ssRNA (HIV-1 virus) depends on the kinase activity of interleukin-1

receptor-associated 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

chloroquine on AT-2 HIV-1 induced TLR signaling by measuring downstream transcription


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molecules IRAK-4 and IRF-7 and subsequent interferon production in PBMC from five donors.

AT-2 MN significantly up-regulated IRAK-4 MFI (p=0.034) while IRF-7 MFI was significantly

increased by both 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 up-regulation of

IRAK-4 in pDC compared with AT-2 HIV-1 stimulated cultures in the absence of chloroquine

(Fig 2A). Further down-stream in the TLR7 induced MyD88 signaling pathway, the

transcription factor, IRF-7, was also decreased in AT-2 Ada (p=0.004) and AT-2 MN (p=0.025)

stimulated cultures treated with chloroquine (Fig 2A) compared to AT-2 HIV-1 cultures in the

absence of chloroquine. Control microvesicles, CCR5-MV and CEMX-MV, did not induce

significant pDC IRAK-4 (Media 18.40 ± 2.42, CEMX-MV 16.25 ± 0.95, CCR5-MV 16.50 ±

1.19) or IRF-7 (Media 17.60 ± 1.50, CEMX-MV 17.80 ± 1.69, CCR5-MV 16.40 ± 0.60)

expression compared to media controls.

Modulation of the TLR7 signaling pathway via chloroquine should be reflected in the

production of IFNα. Therefore, we evaluated AT-2 HIV-1 stimulated culture supernatants for

IFNα using a commercially available ELISA assay. Supernatants from either the AT-2 Ada

(p=0.001) or AT-2 MN (p=0.0002) stimulated cultures contained significantly higher

concentrations of IFNα compared to the media control (Fig 2B). Chloroquine completely

abrogated the production of IFNα in PBMC cultures stimulated with AT-2 Ada (p=0.001) or AT-

2 MN (p= 0.0002) (Fig 2B). To confirm the ability of chloroquine to prevent IFNα secretion via

TLR7 or TLR9 signaling of pDC we used synthetic TLR7/8 (3M-011) and TLR 9 (CpG A)

agonists. Supernatants from the TLR9 CpG A (p=0.003) or TLR7/8 (p=0.026) agonist

stimulated cultures contained significantly higher concentrations of IFNα compared to the media


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control (Fig 2C). As observed in the AT-2 HIV-1 stimulated cultures, neither TLR9 CpG A

agonist (p=0.003) nor the TLR7/8 agonist (p=0.026) induced IFNα production in the presence of

chloroquine (Fig 2C). Taken together, these results demonstrate that chloroquine inhibits TLR

signaling as evidenced by a decrease in the IRAK-4 and IRF-7 signaling molecules along the

MyD88 specific pathway and the subsequent production of IFNα by preventing endocytosis and

the subsequent endosomal degradation of HIV-1 required for TLR signaling by pDC.

Choroquine down-regulates IFN-α induced T cell activation

T cell activation, a predictor of HIV disease progression, can be measured by the elevated

expression of the CD38 cell surface marker (31, 52, 75) on CD8 T cells. We tested the ability of

physiologic concentrations of chloroquine (0.5–5 µM) to modulate IFNα production and its

subsequent effect on T cell expression of CD38 during exposure to non-infectious AT-2 HIV-1

viruses or exogenous rIFNα2a. Using a commercially available ELISA assay we observed a

dose-dependent decrease in IFNα concentration in the supernatants of AT-2 HIV-1 stimulated

PBMC cultures (n=3) treated with 0.5, 1, or 5 µM concentrations of chloroquine (Fig 3A). At a

concentration of 5 µM Chloroquine, AT-2 Ada (p=0.007) and AT-2 MN (p=0.002) HIV-1

stimulated PBMC supernatants contained significantly reduced levels of IFNα compared to AT-

2-HIV-1 stimulated supernatants without chloroquine. Since IFNα production in response to

AT-2 MN (268.4 ± 35.1 pg/mL) was lower compared to AT-2 Ada (1113 ± 120.6 pg/mL), we

studied the effect of 5 µM chloroquine on AT-2 Ada stimulated PBMC and purified T cells.

Flow cytometric analyses revealed a significant increase in CD38-MFI on CD8+

T- cells (Fig 3B)

after exposure of PBMC to AT-2 Ada (p=0.006), and exogenous rIFNα2a (p=0.005) as

compared to the media control. Chloroquine (5 µM) decreased CD38-MFI on CD8 T cells


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23.4% in the AT-2 Ada stimulated PBMC (Fig 3B). In contrast, blocking of the IFNα-receptor

with the 64G12 monoclonal antibody significantly down-regulated the CD38-MFI on CD8+

T-

cells (p=0.014; 43% decrease CD38-MFI) in PBMC stimulated with exogenous rIFNα.

Next, we utilized Miltenyi bead enriched CD8+ T cells (n=3) to evaluate the role of IFNα

or HIV-1 to induce CD8+ T cell activation. CD38 expression (CD38-MFI) was significantly

increased on CD8+ cells exposed to exogenous rIFNα2a (p=0.047) but not on CD8

+ cells

exposed to AT-2 Ada HIV-1 as compared to media (Fig 3C). Blocking of the IFNα-receptor by

pre-treatment of enriched CD8+ cells with the 64G12 monoclonal antibody partially decreased

CD38-MFI by 22.3% in the rIFNα2a stimulated cultures (Fig 3C). As expected, chloroquine did

not inhibit rIFNα2a induced CD8+ T cell activation. These data suggest that using chloroquine

to block IFNα produced by pDC during HIV-1 infection may play a critical role in reducing

chronic T cell activation.

AT-2 HIV-1 induced pDC-IDO expression and IDO tryptophan catabolism is down-regulated by

chloroquine

PDC produce indoleamine 2, 3 dioxygenase (IDO), a key enzyme in tryptophan degradation

along the kynurenine pathway that has been implicated in decreased CD4 T cell proliferative

capacity in HIV-1 infection (10). In this study, PBMC from five individuals were tested for the

effect of chloroquine on AT-2 HIV-1 induced intracellular expression of IDO in the pDC

population. Intracellular levels of IDO in pDC (pDC-IDO mean fluorescence intensity; MFI)

were up-regulated following overnight stimulation with AT-2 Ada or AT-2 MN (Fig 4A,

representative examples shown). PDC-IDO expression was significantly up-regulated in the

cultures stimulated with AT-2 Ada (MFI = 448.0 ± 96.0; p=0.021) compared to the media (MFI


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= 170.8 ± 35.8) (Fig 4A). Chloroquine treatment abrogated AT-2 HIV-1induced pDC-IDO

expression to baseline MFI values similar to the media control cultures (Ada+CHL MFI = 126.4

± 30.1, p=0.010; MN+CHL MFI = 159.0 ± 34.0; Media MFI = 170.8 ± 35.8) (Fig 4A). These

results demonstrate that chloroquine can suppress TLR mediated IDO expression by pDC which

may prove beneficial in blocking tryptophan degradation.

Next, we evaluated IDO functional activity by measuring kynurenine (KYN) and

tryptophan (TRYP) levels in cell culture supernatants. Results are expressed as the KYN:TRYP

ratio, whereby, an increase in the KYN:TRYP ratio indicates active IDO catabolism of

tryptophan along the kynurenine pathway. The KYN:TRYP ratios (Fig 4B) were significantly

increased in the supernatants 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) as compared to the media control (0.589 ± 0.066).

Chloroquine treatment resulted in decreased tryptophan catabolism in the AT-2 Ada (Ada vs.

Ada-CHL p=0.037) and AT-2 MN (MN vs. MN-CHL p=0.020) stimulated culture supernatants.

We also evaluated IDO activity in cultures stimulated with synthetic TLR9 (CpG A) and

TLR7/8 agonists (Fig 4C). The KYN:TRYP ratio was significantly increased in the supernatants

of cultures stimulated with TLR9 (1.590 ± 0.314; p=0.014) as compared to the media control

(0.589 ± 0.066). KYN:TRYP ratios were significantly lower in TLR9 CpG A (p=0.026)

stimulated cultures containing chloroquine compared to cultures without chloroquine. The

KYN:TRYP ratio was elevated in the supernatants stimulated with TLR7/8 agonist compared to

media and were reduced in chloroquine treated cultures though no statistical differences were

observed. These results demonstrate that increased IDO expression in pDC can be directly

mediated by TLR7 and/or TLR9 stimulation and the abrogation of the TLR signal with

chloroquine may offer a means to regulate pDC-IDO expression in HIV-1 infection.


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AT-2 HIV-1 and TLR agonist induced PDL-1 expression on pDC is down-regulated by

chloroquine

Microbial persistence may be related to the exploitation of the PD-1 and PDL-1 pathway by

certain pathogens (72). PDL-1 is expressed constitutively 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 the

PDL-1 expression on pDC compared to the media control (Fig 5A,B). We tested the ability of

chloroquine to regulate AT-2 HIV-1 or synthetic TLR7/8 and TLR9 agonist induced PDL-1

expression on pDC. PDL-1 mean fluorescence intensity on pDC (pDC-PDL-1 MFI) was

significantly reduced in the presence of chloroquine (Fig 5A,B). Chloroquine completely

abrogated the up-regulation of PDL-1 on pDC in cultures stimulated with either AT-2 HIV-1 or

TLR agonists [AT-2 Ada (p<0.0001), AT-2 MN (p=0.002), TLR9 CpG A (p=0.0001), TLR7/8

(p<0.0001)]. These results demonstrate that chloroquine down-modulates the potential

contribution of pDC to T cell exhaustion/dysfunction through the PD-1/PDL-1 pathway by

suppressing PDL-1 expression on pDC.

Discussion

HIV infection is associated with the impairment of immune function with deficiencies observed

in nearly every type of immune cell. A better understanding of the process by which HIV

hijacks innate immune cell function could be explored for alternative therapeutic approaches.

Among potential mechanisms, chronic activation of pDC by direct interaction with infectious

and non-infectious HIV-1 particles contribute to the maintenance of immune cell dysfunction

and HIV-associated pathogenesis (8, 11, 21, 36). In this study, we used an in vitro model to


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demonstrate the potential immunoregulatory function of chloroquine to modulate HIV-1 induced

pDC activation/maturation, IFNα production, IDO expression/activity, and PDL-1 expression.

We also show that IFNα, an outcome of pDC activation, directly contributes to CD8+ T cell

activation. Using chloroquine, an inhibitor of endosomal fusion and acidification, we were able

to suppress HIV-1 mediated TLR signaling in pDC and inhibit the potential deleterious effects of

chronic pDC activation. Our data shows that interfering with TLR signaling reduces HIV-1

induced pDC activation products that are known to contribute to immune dysregulation in HIV-1

infection.

More than 99% of HIV-1 particles detected in the circulation are not productively

infectious (35, 64). These non-infectious particles contribute to HIV-induced

immunopathogenesis (21, 36). We and others have shown that AT-2 HIV-1 at concentrations

between 300-1000ng/mL induces pDC activation/maturation and production of IFNα (27, 35, 36,

54, 82). The concentration of HIV (500ng/mL) used in this study is within the range found in the

plasma of infected individuals, children and adults which is known to vary depending on stage of

HIV disease (44, 66). Using 500ng/mL of noninfectious AT-2 inactivated HIV-1 Ada and MN

virus we could activate pDC IFNα pathway. Activation of pDC by HIV-1 requires endocytosis

of the virion leading to endosomal TLR7 recognition of viral RNA (5, 58) and initiation of the

MyD88 pathway for the production of IFNα by pDC. Chloroquine, an inhibitor of endocytosis

and endosomal acidification/maturation, prevented HIV-1 mediated TLR7 signaling in pDC as

shown by the failure of pDC to up-regulate the down-stream IRAK-4 and IRF-7 signaling

molecules within the MyD88 pathway. By showing that chloroquine decreases IFNα production

it can be inferred that AT-2 HIV-1 utilizes TLR7 signaling in pDC and not the cytosolic anti-


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viral pathway mediated by RIG-1 recognition of ssRNA (41, 65). These data highlight the

importance of the endolysosomal pathway in HIV-1 mediated signaling of pDC.

The production of IFNα by pDC in HIV-1 infection may also have important

consequences for T cell activation and survival (67). During acute infection HIV-1 high plasma

titers of IFNα provide a protective antiviral effect and help trigger the adaptive immune response

(73). However, in late-stage disease elevated levels of serum IFNα is an indicator of poor

clinical prognosis (30, 80). We demonstrate that 100µM chloroquine has an inhibitory effect on

HIV-1 mediated production of IFNα. It has been shown that 100µM chloroquine used in

treatment of leucocytes in vitro results in intracellular concentrations comparable to those

obtained in vivo during chloroquine therapy (29). Our findings are similar to those of other

investigators using chloroquine to modulate IFNα production in HSV infected systems (25, 50).

However, in our study at the declining concentrations of chloroquine (0.5-5 µM) we observed a

dose-dependent decrease in HIV-1 induced IFNα. At our lowest tested concentration of

chloroquine( 0.5 µM), we observed a partial blockage of IFNα which is in contrast to a published

report (71) of complete blockage of IFNα with similar concentration of chloroquine (0.5 µM)

used to induce pDC activation through TLR9. These differences in concentration of chloroquine

required for blocking IFNα could be attributed to their activation pathways. As previously

reported, a higher concentration of chloroquine is necessary to block TLR7 endosomal

maturation and acidification compared to TLR9 (51). Therefore, our results provide evidence

that HIV-1 induced IFNα can be modulated with chloroquine at concentrations that are

obtainable for therapeutic application.

Chronic immune activation is a hallmark of HIV-1 pathogenesis. Previous reports of

elevated IFNα levels in the plasma and lymphoid tissues along with increased expression of


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interferon-inducible genes in HIV-infected patients has been shown to correlate with elevated T

cell activation characteristic of HIV progression (14, 36, 37). In this study, CD38 activation

marker, commonly used to measure the T cell activation state in HIV-infected patients (8, 20, 31,

75), was down-modulated in PBMC cultures when utilizing 5µM chloroquine . As cell separation

procedures affect the uptake of chloroquine (29) we used lower dose of chloroquine (5µM) to

study its effect on T cell activation in enriched T cells and compared with a similar dose of

chloroquine in PBMC cultures. Chloroquine downmodulates T cell activation in AT-2 HIV-1

stimulated PBMC cultures and not in AT-2 HIV-1 stimulated enriched T cells suggesting that

inhibition of pDC IFNα decreases T cell activation. Using enriched CD8+ cells we demonstrate

that increased CD38 expression is induced by exogenous rIFNα2a and not by direct interaction

with HIV-1 suggesting that pDC IFNα certainly contributes to CD8+ T cell activation. As

previously reported(67), we did not find IFNα induced activation of CD4+ T cell (data not

shown). The ability to block IFNα mediated CD8+ activation with the anti-IFNαR monoclonal

antibody (64G12) and not chloroquine highlights the importance of modulating IFNα induced

CD8+ T cell activation. This is the first direct evidence of chloroquine blocking pDC IFN-α

mediated activation and is supported by a previous study demonstrating the ability of

chloroquine to control TLR9 (CpG) or LPS induced pro-inflammatory cytokine production in

mice (40).

HIV-1 also modulates immune function through the induction of negative regulatory

signals which directly affect the development of the adaptive immune response. One such

mechanism evaluated in our studies was the induction of the immunosuppressive enzyme, IDO

in pDC following HIV-1 stimulation. We also observed an increase in kynurenine metabolites in

the culture supernatants indicative of IDO regulated tryptophan catabolism. As shown by other


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in vitro studies HIV-1 induces IDO in pDC (10, 22, 57) which directly inhibits T cell

proliferation and function through tryptophan depletion and through the activity of kynurenine

metabolites (4, 12, 61). Another key regulatory function of IDO is the induction and stimulation

of T regulatory cells during HIV infection (18, 23, 33) which dampen HIV specific T cell

responses. Thus, development of new treatment modalities (i.e. chloroquine or

hydroxychloroquine) to reverse the induction of IDO levels found in both blood cells and

lymphoid tissues (4, 10, 61) may prove beneficial for the improvement of T cell proliferation and

function in addition to controlling aberrant immune activation in HIV infected individuals.

A second mechanism induced by HIV-1 infection to dampen anti-viral responses is the

PD-1/PDL-1 pathway (9, 19, 63, 79). Our data suggests that “non-infectious” HIV-1 (AT-2

HIV-1) exposure up-regulates PDL-1 ligand expression on pDC and is consistent with reports of

other investigators showing TLR induced PDL-1 on pDC and IFNα induced PDL-1 on mDC (1,

15, 78). Manipulation of the PD-1/PDL-1 pathway in HIV-1 infection may prove beneficial in

the restoration of virus specific CD8+ T cell responses by decreasing programmed death pathway

induced apoptosis.

In addition to the immunomodulatory effects observed in these studies chloroquine has

been shown in vitro to have direct inhibitory activity on newly produced HIV virions by altering

the glycosylation of the 2G12 epitope which is located on the gp120 envelope surface protein

required for virus infectivity (69, 70). Several clinical trials have been performed where

chloroquine or hydroxychloroquine was given to HIV infected individuals along with anti-

retroviral therapy. In one study a decrease in HIV viral load measurements was reported (77).

Whilst in another a decrease in plasma p24 capsid antigen levels was observed in chloroquine


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treated individuals while no alterations in CD4+ T lymphocyte counts were identified compared

to control group (76).

HIV-1 infection hijacks the innate immune response which directly contributes to disease

pathogenesis. Chronic stimulation of pDC with non-infectious and infectious virions leads to

their enhanced activation, state, production of the cytokine IFNα and an up-regulation of IDO

and PDL-1, all of which are known to directly contribute to decreased T cell survival,

proliferation, and function. In this study, we have shown that chloroquine blocks TLR signaling

in pDC, a critical step in its activation pathway. By blocking pDC activation we observed a

decrease in the immunoregulatory cytokine IFNα which in turn could reduce IFNα mediated

immune activation and improve T cell survival by blocking negative modulators like IDO and

PDL-1. Chloroquine is widely accessible, inexpensive and is well tolerated when administered

over periods of several years making it a good candidate for adjuvant therapy along with

HAART to control immune activation in HIV-1 infection. The relevance of the findings

presented in this study are particularly important as it is now known that non AIDS defining

illnesses such as atherosclerosis, liver disease, renal diseases that occur despite effective HAART

contribute to mortality. Immune activation and inflammation are the key contributing factors

linked to these co-morbidities (49). The use of chloroquine as an adjuvant with HAART could

be an effective and inexpensive approach to control immune activation and reduce the risk of co-

morbidities in HAART treated HIV infected individuals. The use of chloroquine in HIV-1

infected individuals in resource rich and resource poor countries need to be further investigated.


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Figure Legends

Figure 1. Chloroquine abrogates AT-2 HIV-1 induced DC activation and maturation.

PBMC were stimulated with AT-2 HIV-1 for 20 hours in the presence or absence of 100 µM

chloroquine and 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 pDC activation marker CD86 and B) Representative flow cytometric analysis

and summary of pDC maturation marker CD83. Results are expressed as the mean % expression

± SEM, n=3

Figure 2. Chloroquine abrogates TLR activated MyD88-dependent signaling pathway and

IFNα production in pDC.

PBMC were stimulated with AT-2 HIV-1 or the TLR9 (CpG A) and TLR7/8 (3M-011) agonists

for 20 hours in the presence or absence of 100 µM chloroquine. Logical gating was used to

identify the pDC population (Lin1-/HLA DR

+/CD123

+) for analyses. A). Representative flow

cytometric analysis and summary of IRAK-4 and IRF-7 signaling molecules expressed in pDC

stimulated with AT-2 HIV-1 ± 100 µM chloroquine. B) Summary of IFNα (pg/ml)

concentration in the culture supernatants from PBMC cultures stimulated with AT-2 HIV-1 and

C) synthetic TLR9 (CpG A) and TLR7/8 (3M-011) agonists ± 100 µM chloroquine. Results are

expressed as the mean ± SEM, n=5.

Figure 3. Chloroquine decreases IFNαααα induced CD8+ T cell activation.

PBMC were stimulated with AT-2 HIV-1 (Ada and MN) for 20 hours in the presence or absence

of physiologicically achievable concentrations of chloroquine (0.5 – 5.0 µM). A) IFNα (pg/ml)


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concentration in supernatants of AT-2 Ada and MN stimulated PBMC ± chloroquine. B). PBMC

cultures stimulated with AT-2 Ada HIV-1 or 1000 U/ml rIFNα2a were analyzed by flow

cytometry for expression of the CD38 activation marker in the CD8+ T cell subset (CD3

+/CD8

+)

± 5 µM chloroquine or the anti-IFNαR blocking monoclonal antibody, 64G12. C) Miltenyi bead

purified CD8+ T cells stimulated with AT-2 HIV-1 or 1000 U/ml rIFNα2a were analyzed by flow

cytometry for expression of the CD38 activation marker in the CD8+ T cell subset (CD3

+/CD8

+)

± 5 µM chloroquine or the anti-IFNαR blocking monoclonal antibody, 64G12. Results are

expressed as mean ± SEM, n=3.

Figure 4. Chloroquine abrogates AT-2 HIV-1 induced IDO (indoleamine 2-3-dioxygenase)

expression and activity in pDC.


chloroquine and analyzed by flow cytometry. A). Representative flow cytometric analysis and

summary of AT-2 HIV-1 induced IDO expression (IDO mean fluorescence intensity; IDO-MFI)

in pDC (Lin1-/HLA DR+/CD123+) ± 100 µM chloroquine. Supernatants from PBMC cultures

stimulated with B) AT-2 HIV-1 or C) TLR agonists for 20 hours ±100 µM chloroquine were

analyzed for IDO activity using fluorometric assays to detect kynurenine and tryptophan. Results

are expressed as the kynurenine: tryptophan ratio (KYN: TRYP). Results are expressed as mean

± SEM, n=5.

Figure 5. Chloroquine abrogates PDL-1 expression in pDC.


chloroquine and analyzed by flow cytometry. A). Representative flow cytometric analysis and


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summary of AT-2 HIV-1 induced PDL-1 expression on pDC (Lin1-/HLA DR+/CD123+) ± 100

µM chloroquine. B). Representative flow cytometric analysis and summary of TLR agonist

induced PDL-1 expression on pDC (Lin1-/HLA DR+/CD123+) ± 100 µM chloroquine. Results

are expressed as mean ± SEM, n=5.


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