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Plasmacytoid dendritic cells (pDC) contribute to antiviral immunity mainly through recognition of microbial products and viruses via intracellular Toll-like receptor 7 (TLR7) or TLR9, resulting in the production of type I interferons (IFNs). Although interferons reduce the viral burden in the acute phase of infection, their role in the chronic phase is unclear. The presence of elevated plasma IFN-α levels in advanced HIV disease and its association with microbial translocation in chronic HIV infection lead us to hypothesize that IFN-α could contribute to immune activation. Blocking of IFN-α production using chloroquine, an endosomal inhibitor, was tested in a novel in vitro model system with the aim of characterizing the effects of chloroquine on HIV-1-mediated TLR signaling, IFN-α production, and T-cell activation. Our results indicate that chloroquine blocks TLR-mediated activation of pDC and MyD88 signaling, as shown by decreases in the levels of the downstream signaling molecules IRAK-4 and IRF-7 and by inhibition of IFN-α synthesis. Chloroquine decreased CD8 T-cell activation induced by aldrithiol-2-treated HIV-1 in peripheral blood mononuclear cell cultures. In addition to blocking pDC activation, chloroquine also blocked negative modulators of the T-cell response, such as indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PDL-1). Our results indicate that TLR stimulation and production of IFN-α by pDC contribute to immune activation and that blocking of 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 highly active antiretroviral therapy to control immune activation in HIV-1 infection.
Plasmacytoid dendritic cells (pDC), which recognize pathogens through Toll-like receptor 7 (TLR7) and TLR9, are an integral part of the innate and adaptive immune systems (2, 34, 42, 43, 45, 73). Since these TLRs are intracellular, their ligands require cellular uptake and endosomal maturation to trigger NF-κB and mitogen-activated protein kinase-mediated signals through the MyD88-dependent pathway. These TLR signals result in pDC activation/maturation and in the production of proinflammatory cytokines and a large amount of type 1 interferon, or alpha/beta interferon (IFN-α/β) (17, 43, 46). pDC also provide negative regulatory signals that modulate and establish immune tolerance (62). Several molecules expressed by pDC, including indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PDL-1), are implicated in the negative modulation of T-cell responses. 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 and TLR9 (48), or by HIV itself (5), results in pDC activation and IFN-α production, which contribute to pathogenesis in chronic HIV-1 disease (14). IFN-α is known to be associated with elevated lipopolysaccharide (LPS) levels in chronic HIV infection, which may contribute to persistent immune activation (14).
Chloroquine, an antimalarial 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 pDC induced by HIV-1 (5, 9, 25, 35, 71). Chloroquine is a weak base that accumulates within the endosomes of cells, leading to an elevated vacuolar pH and 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 downstream effects on T-cell activation and function.
The goal of the present study was to characterize the effects of chloroquine on the HIV-1-mediated stimulation of pDC and its potential effect on modulating regulators of T-cell function, including IFN-α-induced T-cell activation. Our results show that chloroquine inhibits pDC activation/maturation, upregulation of the MyD88 pathway signaling molecules IFN regulatory factor 7 (IRF-7) and interleukin-1 receptor-associated kinase 4 (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 IFN-α 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 IFN-α by pDC contribute 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.
The following fluorochrome-conjugated mouse 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); and CD4-PE-Texas red from CalTag (Carlsbad, CA). Unconjugated rabbit anti-human polyclonal antibodies included those against IRF-7 (H-246; Santa Cruz Biotechnology, Santa Cruz, CA) and IRAK-4 (Invitrogen, Carlsbad, CA), as well as the anti-human IDO polyclonal rabbit antibody directed against the DLIESGLRERVEKLNMLC peptide (60), from David Munn (Medical College of Georgia, Augusta). 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 nonspecific 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 oligodeoxynucleotide (ODN) 2336 or 2216 was provided by Coley Pharmaceutical Group (Pfizer, Wellesley, MA). Noninfectious Aldrithiol-2 (AT-2)-treated HIV-1Ada (R5-tropic) and HIV-1MN (X4-tropic) preparations, along with matched control microvesicles, SUPT1-CCR5 and CEMX174 (T1), isolated from uninfected cell cultures (6), were kindly provided by Jeff Lifson of the AIDS Vaccine Program, National Cancer Institute (Frederick, MD). Recombinant IFN-α2a (rIFN-α2a) was obtained from PBL (Piscataway, NJ), and the 64G12 monoclonal antibody against the human IFN-α receptor (anti-IFN-αR) was provided by Michael Tovey of the Laboratory of Viral Oncology (Villejuif, France).
The TLR agonists were used at the following concentrations for in vitro stimulation of peripheral blood mononuclear cells (PBMC): TLR7/8 agonist (3M-011), 3.0 μM; TLR9 agonist (A-class CpG ODN 2336 or 2216), 4 μg/ml. It is now known that endocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like receptor-viral RNA interactions (5), so we stimulated PBMC using noninfectious Aldrithiol-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 noninfectious (35, 64). AT-2-treated HIV-1Ada and HIV-1MN (referred to below as AT-2 Ada and AT-2 MN, respectively) were used at a concentration of 500 ng/ml of p24 capsid equivalent. The concentration of HIV-1 (500 ng/ml) used in this study is within the range found in the plasma of infected individuals (including children and adults), which is known to vary depending on the stage of HIV disease (44, 66). Optimal concentrations of the TLR agonists (32, 54, 55) and AT-2 HIV-1 (54) have been determined previously. Media and matched microvesicle controls, MV-CCR5 and MV-CEMX, served as the negative controls in these experiments.
To study the effect of chloroquine on the regulation of the endosomal TLR7 and TLR9 pathways in response to inactivated HIV-1 or TLR agonists, we used a 100 μM concentration of chloroquine based on a previous report evaluating the activation of pDC by herpes simplex virus (HSV) (56). For the T-cell activation studies, we used a concentration of chloroquine (0.5 to 5 μM) equivalent to the achievable physiological concentration in blood as described in the Medsafe database (www.medsafe.govt.nz) (13). PBMC were preincubated for 1 h in a medium containing chloroquine (diphosphate salt) (InvivoGen, San Diego, CA), which was present throughout the cell culture.
Heparinized peripheral blood was collected via venipuncture from healthy HIV-seronegative volunteers recruited from Rush University Medical Center (Chicago, IL) after informed consent. PBMC were isolated by density gradient centrifugation using lymphocyte separation medium (Cambrex, Gaithersburg, MD). PBMC (1 × 106/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 37°C, 5% CO2 incubator. PBMC were preincubated with a medium containing 100 μM chloroquine or the medium alone for 1 h prior to stimulation with TLR agonists or AT-2-inactivated HIV-1. Cells were cultured for 20 h, harvested, and evaluated for pDC activation/maturation and intracellular IDO, IRF-7, and IRAK-4 protein expression using flow cytometry. Supernatants were stored at −20°C for IFN-α cytokine analysis by an enzyme-linked immunosorbent assay (ELISA).
PBMC or enriched CD8+ T-cell subsets (indirect isolation; Miltenyi [Auburn, CA] microbead isolation kit) were preincubated for 1 h with a medium containing chloroquine or for 30 min with the anti-IFN-αR blocking antibody 64G12 (10 μg/ml). AT-2 HIV-1 or 1,000 U/ml rIFN-α2a was added, and cells were cultured for 20 h, harvested, and evaluated for T-cell activation by measuring CD38 surface expression on CD4+ and CD8+ T 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.
PBMC preincubated (1 h) with a medium containing 100 μM chloroquine or the medium alone were cultured with TLR agonists or AT-2-inactivated HIV-1 in 96-well tissue 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 pDC PDL-1 expression by flow cytometry. Supernatants were stored at −20°C for evaluation of IDO activity in fluorometric assays to detect tryptophan and kynurenine in the pathway of l-tryptophan catabolism.
Chloroquine-treated or untreated PBMC from control 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 antibody binding to Fc receptors was blocked by preincubation 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 4°C 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 a PE-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch). Finally, the cells were washed twice in Perm/Wash buffer, fixed with 1% formaldehyde, and stored at 4°C until analysis. All samples were evaluated 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 the parent pDC population and the CD38 level within the parent T-cell subsets.
Commercial ELISA kits were used to measure the concentration of IFN-α (PBL Biomedical Laboratories, Piscataway, NJ) in cell culture supernatants. ELISAs were performed according to the manufacturer's guidelines.
Depletion of tryptophan and accumulation of the immunosuppressive catabolite kynurenine are measures 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 to 0.39 μg/ml) were incubated with 30% trichloroacetic acid (TCA; Sigma) for 30 min in 50°C water bath. The tubes were allowed to cool to room temperature and were then centrifuged at 2,400 rpm for 10 min. An aliquot from each tube was transferred to individual wells of a flat-bottom polystyrene 96-well plate. Freshly made 2% Ehrlich's reagent (Sigma) in glacial acetic acid was added to each well, and the plate was incubated at room temperature for 10 min in the dark. Finally the plate was read on a BioTek (Winooski, VT) Synergy HT plate reader at a wavelength of 490 nm. A kynurenine (0 to 25 μg/ml) standard curve was used to determine the kynurenine concentrations in the culture supernatants.
The tryptophan fluorescence assay was performed as follows. Stock l-tryptophan (Sigma; 1.25 to 20 μg/ml) or cell culture supernatants were incubated in 20% TCA in a 96-well plate (duplicate wells/sample) for 1 h at 4°C. The plates were centrifuged at 2,400 rpm for 15 min, and supernatants were transferred to polypropylene tubes. The supernatants were then incubated with a mixture of 10% TCA, 1.8% formaldehyde (prepared in PBS), and 6 mM FeCl3 at 90°C for 2 h. 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 (excitation wavelength, 360/40 nm; emission wavelength, 460/40 nm; sensitivity, 70 nm). The tryptophan (1.25 to 20 μg/ml) standard curve was used to determine the tryptophan concentrations in the culture supernatants. IDO activity is expressed as the kynurenine-to-tryptophan ratio.
Results are expressed as means ± standard errors of the means (SEM). The significance of differences between groups was determined using an unpaired Student t test (with a confidence level of 95%) with GraphPad Prism software, version 4.03. P values of <0.05 were considered statistically significant.
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 noninfectious HIV 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. (Fig.11 A and B). Levels of AT-2 Ada-induced pDC activation markers (expressed as the percentage of pDC expressing CD86) decreased from 50.5% ± 9.8% to 11.1% ± 19.8% (P = 0.017) (Fig. (Fig.1A),1A), and those of pDC maturation markers (percentage of pDC expressing CD83) decreased from 64.6% ± 11.1% to 12.6% ± 6.8% (P = 0.016) (Fig. (Fig.1B)1B) in the chloroquine-treated cultures. Similarly, levels of AT-2 MN-induced pDC activation markers decreased from 49.3% ± 5.4% to 8.2% ± 2.5% (P = 0.002) (Fig. (Fig.1A),1A), and those of maturation markers decreased from 69.0% ± 13.9% to 14.3% ± 10.6% (P = 0.035) (Fig. (Fig.1B)1B) in chloroquine-treated cultures. Control microvesicles, CCR5-MV and CEMX-MV, did not induce significant pDC activation/maturation (CD86 levels were 1.23% ± 0.54% in the medium, 1.65% ± 0.38% in CEMX-MV, and 2.89% ± 1.29% in CCR5-MV; CD83 levels were 4.42% ± 2.94% in the medium, 5.24% ± 4.31% in CEMX-MV, and 3.03% ± 1.97% in CCR5-MV) compared to levels of the markers in medium controls.
HIV-1 induces the secretion of type 1 interferons (IFN-α/β) by pDC, partly through TLR 7 (5). TLR7 recognition of single-stranded RNA (ssRNA) (HIV-1) 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 levels of the downstream transcription molecules IRAK-4 and IRF-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 by both AT-2 MN (P = 0.011) and AT-2 Ada (P = 0.006) (Fig. (Fig.2A).2A). Chloroquine treatment prevented AT-2 Ada (P = 0.004)- and AT-2 MN (P = 0.005)-induced upregulation of IRAK-4 in pDC compared with AT-2 HIV-1-stimulated cultures in the absence of chloroquine (Fig. (Fig.2A).2A). Further downstream in the TLR7-induced MyD88 signaling pathway, levels of the transcription factor IRF-7 ertr also decreased in AT-2 Ada (P = 0.004)- and AT-2 MN (P = 0.025)-stimulated cultures treated with chloroquine (Fig. (Fig.2A)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 (MFI, 18.40 ± 2.42 in the medium, 16.25 ± 0.95 in CEMX-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, andV 16.40 ± 0.60 in CCR5-M) expression compared to that in medium controls.
Modulation of the TLR7 signaling pathway via chloroquine should be reflected in the production of IFN-α. Therefore, we evaluated AT-2 HIV-1-stimulated culture supernatants for IFN-α using a commercially available ELISA. Supernatants from either AT-2 Ada (P = 0.001)- or AT-2 MN (P = 0.0002)-stimulated cultures contained significantly higher concentrations of IFN-α than the medium control (Fig. (Fig.2B).2B). Chloroquine completely abrogated the production of IFN-α in PBMC cultures stimulated with AT-2 Ada (P = 0.001) or AT-2 MN (P = 0.0002) (Fig. (Fig.2B).2B). To confirm the ability of chloroquine to prevent IFN-α secretion via TLR7 or TLR9 signaling of pDC, we used synthetic TLR7/8 (3M-011) and TLR9 (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 IFN-α than the medium control (Fig. (Fig.2C).2C). As observed for the AT-2 HIV-1-stimulated cultures, neither the TLR9 CpG A agonist (P = 0.003) nor the TLR7/8 agonist (P = 0.026) induced IFN-α production in the presence of chloroquine (Fig. (Fig.2C).2C). Taken together, these results demonstrate that chloroquine inhibits TLR signaling, as evidenced by decreases in the levels of the IRAK-4 and IRF-7 signaling molecules along the MyD88-specific pathway and in the subsequent production of IFN-α, by preventing endocytosis and the subsequent endosomal degradation of HIV-1 required for TLR signaling by pDC.
T-cell activation, a predictor of HIV disease progression, can be measured by the elevated expression of the cell surface marker CD38 (31, 52, 75) on CD8 T cells. We tested the ability of physiologic concentrations of chloroquine (0.5 to 5 μM) to modulate IFN-α production and its subsequent effect on T-cell expression of CD38 during exposure to noninfectious AT-2 HIV-1 or exogenous rIFN-α2a. Using a commercially available ELISA, we observed a dose-dependent decrease in IFN-α concentrations 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. (Fig.3A).3A). At a concentration of 5 μM chloroquine, AT-2 HIV-1 Ada (P = 0.007)- and AT-2 HIV-1 MN (P = 0.002)-stimulated PBMC supernatants contained significantly reduced levels of IFN-α compared to those in AT-2-HIV-1-stimulated supernatants without chloroquine. Since IFN-α production in response to AT-2 MN (268.4 ± 35.1 pg/ml) was lower than that in response to AT-2 Ada (1,113 ± 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 significant increases in the CD38-MFI on CD8+ T cells (Fig. (Fig.3B)3B) after exposure of PBMC to AT-2 Ada (P = 0.006) and exogenous rIFN-α2a (P = 0.005) over that for the medium control. Chloroquine (5 μM) decreased CD38-MFI on CD8 T cells 23.4% in AT-2 Ada-stimulated PBMC (Fig. (Fig.3B).3B). In contrast, blocking of the IFN-α receptor with monoclonal antibody 64G12 significantly downregulated the CD38-MFI on CD8+ T cells (P = 0.014; 43% decrease in CD38-MFI) in PBMC stimulated with exogenous rIFN-α.
Next, we utilized Miltenyi bead-enriched CD8+ T cells to evaluate the role of IFN-α or HIV-1 in inducing CD8+ T-cell activation (n = 3 experiments). CD38 expression (CD38-MFI) was significantly increased on CD8+ cells exposed to exogenous rIFN-α2a (P = 0.047) but not on CD8+ cells exposed to AT-2 Ada HIV-1 compared to that in the medium (Fig. (Fig.3C).3C). Blocking of the IFN-α receptor by pretreatment of enriched CD8+ cells with the 64G12 monoclonal antibody partially decreased CD38-MFI, by 22.3%, in rIFN-α2a-stimulated cultures (Fig. (Fig.3C).3C). As expected, chloroquine did not inhibit rIFN-α2a-induced CD8+ T-cell activation. These data suggest that the use of chloroquine to block IFN-α produced by pDC during HIV-1 infection may play a critical role in reducing chronic T-cell activation.
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 IDO expression in the pDC population. Intracellular IDO levels in pDC (pDC-IDO MFI) were upregulated following overnight stimulation with AT-2 Ada or AT-2 MN (Fig. (Fig.4A;4A; representative examples shown). pDC expression of IDO was significantly upregulated in cultures 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. (Fig.4A).4A). Chloroquine treatment abrogated AT-2 HIV-1-induced pDC IDO expression to baseline MFI values similar to those in medium control cultures (MFI, 126.4 ± 30.1 [P = 0.010] for Ada cultures with chloroquine, 159.0 ± 34.0 for MN cultures with chloroquine, and 170.8 ± 35.8 for medium control cultures) (Fig. (Fig.4A).4A). These results demonstrate that chloroquine can suppress TLR-mediated IDO expression by pDC, an effect that 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; an increase in the KYN/TRYP ratio indicates active IDO catabolism of tryptophan along the kynurenine pathway. The KYN/TRYP ratios (Fig. (Fig.4B)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) over that for the medium control (0.589 ± 0.066). Chloroquine treatment resulted in decreased tryptophan catabolism in AT-2 Ada (P = 0.037)- and AT-2 MN (P = 0.020)-stimulated culture supernatants.
We also evaluated IDO activity in cultures stimulated with a synthetic TLR9 (CpG A) or TLR7/8 agonist (Fig. (Fig.4C).4C). The KYN/TRYP ratio was significantly increased in the supernatants 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 in those without chloroquine. KYN/TRYP ratios were elevated in supernatants stimulated with the TLR7/8 agonist over that for the medium control and were reduced in chloroquine-treated cultures, though no statistically significant differences were observed. These results demonstrate that increased IDO expression in pDC can be directly mediated by TLR7 and/or TLR9 stimulation and that the abrogation of the TLR signal by chloroquine may offer a means to regulate pDC IDO expression in HIV-1 infection.
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 PDL-1 expression on pDC over that for the medium control (Fig. 5A and B). We tested the ability of chloroquine to regulate AT-2 HIV-1- or synthetic TLR7/8 or TLR9 agonist-induced PDL-1 expression on pDC. The mean fluorescence 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 upregulation of PDL-1 on pDC in cultures stimulated with either AT-2 HIV-1 (P < 0.0001 for AT-2 Ada; P = 0.002 for AT-2 MN) or a TLR agonist (P = 0.0001 for TLR9 CpG A; P < 0.0001 for TLR7/8). These results demonstrate that chloroquine downmodulates the potential contribution of pDC to T-cell exhaustion/dysfunction through the PD-1/PDL-1 pathway by suppressing PDL-1 expression on pDC.
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 achieved in order to develop alternative therapeutic approaches. Among potential mechanisms, chronic activation of pDC by direct interaction with infectious and noninfectious HIV-1 particles contributes 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 demonstrate the potential immunoregulatory function of chloroquine to modulate HIV-1-induced pDC activation/maturation, IFN-α production, IDO expression/activity, and PDL-1 expression. We also show that production of IFN-α, 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 to inhibit the potential deleterious effects of chronic pDC activation. Our data show that interfering with TLR signaling reduces levels of 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 noninfectious particles contribute to HIV-induced immunopathogenesis (21, 36). We and others have shown that AT-2 HIV-1 at concentrations between 300 and 1,000 ng/ml induces pDC activation/maturation and production of IFN-α (27, 35, 36, 54, 82). The concentration of HIV (500 ng/ml) used in this study is within the range found in the plasma of infected individuals (both children and adults), which is known to vary depending on the stage of HIV disease (44, 66). Using 500 ng/ml of noninfectious AT-2-inactivated HIV-1 Ada or MN, we could activate the pDC IFN-α 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 IFN-α 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 upregulate the downstream IRAK-4 and IRF-7 signaling molecules within the MyD88 pathway. By showing that chloroquine decreases IFN-α production, it can be inferred that AT-2 HIV-1 utilizes TLR7 signaling in pDC and not the cytosolic antiviral 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 IFN-α by pDC in HIV-1 infection may also have important consequences for T-cell activation and survival (67). During acute HIV-1 infection, high plasma IFN-α titers provide a protective antiviral effect and help trigger the adaptive immune response (73). However, in late-stage disease, elevated serum IFN-α levels are an indicator of poor clinical prognosis (30, 80). We demonstrate that 100 μM chloroquine has an inhibitory effect on HIV-1-mediated production of IFN-α. It has been shown that the use of 100 μM chloroquine in the treatment of leukocytes 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 IFN-α production in HSV-infected systems (25, 50). However, in our study, at the declining concentrations of chloroquine (0.5 to 5 μM), we observed a dose-dependent decrease in HIV-1-induced IFN-α production. At our lowest tested concentration of chloroquine (0.5 μM), we observed a partial blockage of IFN-α production, which is in contrast to a published report (71) of complete blockage of IFN-α with a similar concentration of chloroquine (0.5 μM) used to block induction of pDC activation through TLR9. These differences in the concentration of chloroquine required for blocking IFN-α could be attributed to the 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 IFN-α production can be modulated with chloroquine at concentrations that are obtainable for therapeutic application.
Chronic immune activation is a hallmark of HIV-1 pathogenesis. Elevated IFN-α levels in the plasma and lymphoid tissues, along with increased expression of interferon-inducible genes, in HIV-infected patients have been shown in previous reports to correlate with elevated T-cell activation, characteristic of HIV progression (14, 36, 37). In this study, the CD38 activation marker, commonly used to measure the T-cell activation state in HIV-infected patients (8, 20, 31, 75), was downmodulated in PBMC cultures when 5 μM chloroquine was utilized. Since cell separation procedures affect the uptake of chloroquine (29), we used a lower dose of chloroquine (5 μM) to study its effect on T-cell activation in enriched T cells and compared it 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 IFN-α production decreases T-cell activation. Using enriched CD8+ T cells, we demonstrate that increased CD38 expression is induced by exogenous rIFN-α2a and not by direct interaction with HIV-1, suggesting that pDC IFN-α certainly contributes to CD8+ T-cell activation. As previously reported (67), we did not find IFN-α-induced activation of CD4+ T cells (data not shown). The ability to block IFN-α-mediated CD8+ activation with the anti-IFN-αR monoclonal antibody (64G12) and not with chloroquine highlights the importance of modulating IFN-α-induced CD8+ T-cell activation. This is the first direct evidence that chloroquine blocks pDC IFN-α-mediated immune activation, and it is supported by a previous study demonstrating the ability of chloroquine to control TLR9 (CpG)- or LPS-induced proinflammatory cytokine production in mice (40).
HIV-1 also modulates immune function through the induction of negative regulatory signals that 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 the level of kynurenine metabolites in the culture supernatants, indicative of IDO-regulated tryptophan catabolism. As shown by other 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, the development of new treatment modalities (i.e., chloroquine or hydroxychloroquine) to reverse the induction of IDO levels found both in blood cells and in 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 antiviral 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 showing TLR-induced PDL-1 on pDC and IFN-α-induced PDL-1 on myeloid dendritic cells (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 antiretroviral therapy. In one study, a decrease in HIV loads was reported (77), while in another, a decrease in plasma p24 capsid antigen levels was observed in chloroquine-treated individuals, although no alterations in CD4+ T-lymphocyte counts were observed, compared to those for control group (76).
HIV-1 infection hijacks the innate immune response, directly contributing to disease pathogenesis. Chronic stimulation of pDC with noninfectious and infectious virions leads to their enhanced activation, production of the cytokine IFN-α, and upregulation of IDO and PDL-1, all of which are known to contribute directly 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 production of the immunoregulatory cytokine IFN-α, which in turn could reduce IFN-α-mediated immune activation and improve T-cell survival by blocking negative modulators such as IDO and PDL-1. Chloroquine is widely accessible, inexpensive, and well tolerated when administered over several years, making it a good candidate for adjuvant therapy along with highly active antiretroviral therapy (HAART) to control immune activation in HIV-1 infection. The findings presented in this study are particularly important and relevant, since it is now known that non-AIDS-defining illnesses, such as atherosclerosis, liver disease, and renal diseases, which occur despite effective HAART, contribute to mortality. Immune activation and inflammation are the key contributing factors linked to these comorbidities (49). The use of chloroquine as an adjuvant with HAART could be an effective and inexpensive approach to controlling immune activation and reducing the risk of comorbidities for HAART-treated HIV-infected individuals. The use of chloroquine for HIV-1-infected individuals in resource-rich and resource-poor countries needs to be further investigated.
Published ahead of print on 30 November 2009.