Chloroquine modulates HIV-1-induced plasmacytoid dendritic cell alpha interferon: implication for T-cell activation

Abstract

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-alpha levels in advanced HIV disease and its association with microbial translocation in chronic HIV infection lead us to hypothesize that IFN-alpha could contribute to immune activation. Blocking of IFN-alpha 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-alpha 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-alpha 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-alpha 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.

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 the presence or absence of 100 μM 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 pDC expressing the marker ± SEM (n = 3).

FIG. 2.

Chloroquine abrogates the TLR-activated MyD88-dependent signaling pathway and IFN-α production in pDC. PBMC were stimulated with 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 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 the expression of the IRAK-4 and IRF-7 signaling molecules in pDC stimulated with AT-2 HIV-1 with or without 100 μM chloroquine. (B and C) Summary of IFN-α (pg/ml) concentrations in culture supernatants from PBMC cultures stimulated with AT-2 HIV-1 (B) or with a synthetic TLR9 (CpG A) or TLR7/8 (3M-011) agonist (C) with or without 100 μM chloroquine. Results are expressed as means ± SEM (n = 5).

FIG. 3.

Chloroquine decreases IFN-α-induced CD8⁺ T-cell activation. PBMC were stimulated with AT-2 HIV-1 (Ada or MN) for 20 h in the presence or absence of physiologically achievable concentrations of chloroquine (0.5 to 5.0 μM). (A) IFN-α (pg/ml) concentrations in supernatants of AT-2 Ada- and MN-stimulated PBMC with or without chloroquine. (B) PBMC cultures stimulated with AT-2 HIV-1 Ada or 1,000 U/ml rIFN-α2a were analyzed by flow cytometry for expression of the CD38 activation marker in the CD8⁺ T-cell subset (CD3⁺ CD8⁺) with or without 5 μM chloroquine or the anti-IFN-αR blocking monoclonal antibody 64G12. (C) Miltenyi bead-purified CD8⁺ T cells stimulated with AT-2 HIV-1 or 1,000 U/ml rIFN-α2a were analyzed by flow cytometry for expression of the CD38 activation marker in the CD8⁺ T-cell subset (CD3⁺ CD8⁺) with or without 5 μM chloroquine or the anti-IFN-αR blocking monoclonal antibody 64G12. Results are expressed as means ± SEM (n = 3).

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 h in the presence or absence of 100 μM chloroquine and were then analyzed by flow cytometry. (A) Representative flow cytometric analysis and summary of AT-2 HIV-1-induced IDO expression (IDO MFI) in pDC (Lin1⁻ HLA DR⁺ CD123⁺) with or without 100 μM chloroquine. (B and C) Supernatants from PBMC cultures stimulated with AT-2 HIV-1 (B) or a TLR agonist (C) for 20 h with or without 100 μM chloroquine were analyzed for IDO activity using fluorometric assays to detect kynurenine and tryptophan. Results are expressed as the kynurenine-to-tryptophan ratio. Results are expressed as means ± SEM (n = 5).

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 μM chloroquine and were then analyzed by flow cytometry. (A) Representative flow cytometric analysis and summary of AT-2 HIV-1-induced PDL-1 expression on pDC (Lin1⁻ HLA DR⁺ CD123⁺) with or without 100 μM chloroquine. (B) Representative flow cytometric analysis and summary of TLR agonist-induced PDL-1 expression on pDC (Lin1⁻ HLA DR⁺ CD123⁺) with or without 100 μM chloroquine. Results are expressed as means ± SEM (n = 5).