Endocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like receptor-viral RNA interactions

Abstract

HIV-1 directly activates human plasmacytoid DCs (pDCs) by upregulating the expression of costimulatory and MHC molecules and maturation markers, increasing T cell stimulatory activity, and inducing the production of type I interferons and TNF-alpha. A consequence of this activation is the bystander maturation of myeloid DCs and overall enhancement of antigen-presenting function. However, little is known about the mechanism(s) of pDC activation by HIV-1. Here we demonstrate by in vitro studies that IFN-alpha production by pDC in response to HIV-1 requires at least 2 interactions between the cell and virus. Initially, envelope-CD4 interactions mediate endocytosis of HIV-1, as demonstrated through the use of inhibitors of binding, fusion, endocytosis, and endosomal acidification. Subsequently, endosomally delivered viral nucleic acids, particularly RNA, stimulate pDCs through TLRs, as activation is reproduced with purified genomic RNA but not viral RNA packaging-deficient HIV-1 and blocked with different inhibitory TLR ligands. Finally, by using genetic complementation, we show that TLR7 is the likely primary target. Viral RNA rather than DNA in early retrotranscripts appears to be the active factor in HIV-1 that induces IFN-alpha secretion by pDCs. Since the decline in pDCs in chronic HIV-1 infection is associated with high viral loads and opportunistic infections, exploiting this natural adjuvant activity of HIV-1 RNA might be useful in the development of vaccines for the prevention of AIDS.

Figure 1

pDC activation in response to HIV-1. BDCA-4⁺ pDCs were cultured in the presence of various doses of HIV-1_MN (ng p24^CA/ml). Supernatants were collected after 15 hours and assessed for the presence of IFN-α (ng/ml) and MIP-1α (pg/ml) by ELISA.

Figure 2

Inhibition of binding. BDCA-4⁺ pDCs were cultured overnight in the presence of HIV-1_MN or R848 and a neutralizing anti-gp120 mAb, b12 or 17b, versus an irrelevant human IgG1 (A); a neutralizing anti-CD4 antibody versus an irrelevant murine IgG1 (B); or AMD 3100 (C). The presence of IFN-α (ng/ml) in the supernatants was assessed by ELISA.

Figure 3

Inhibition of fusion with plasma membrane and endocytosis. (A) Flow cytometry–sorted pDCs were incubated overnight in the presence of HIV-1_MN and C34. BDCA-4⁺ pDCs were cultured for 15 hours with HIV-1_MN and DMA, CCD, or DMSO (B) and chloropromazine (Chlorpro) (C). As a read-out, IFN-α (ng/ml) and TNF-α (pg/ml) secretion and CD83 expression were measured by ELISA and flow cytometry, respectively. The percentage of positive cells is indicated in bold and the mean of fluorescence intensity underlined.

Figure 4

Inhibition of endosomal acidification/maturation. pDCs were enriched by BDCA-4 positive selection and cultured for 15 hours with HIV-1_MN. The inhibitory effects of chloroquine (A), quinacrine (B), NH₄Cl (C) and Baf A1 (D) were assessed. IFN-α (ng/ml) and TNF-α (pg/ml) concentrations were determined by ELISA. As controls, pDCs were cultured with R848, CpGA or CpGB ODNs.

Figure 5

HIV RNA stimulates pDCs but not MDCs. (A) Flow cytometry–sorted pDCs were exposed overnight to recombinant wild-type or mutant NC SSHS/SSHS HIV-1_NL4-3 at the doses of 900 or 300 ng p24^CA Eq/ml. As controls, pDCs were cultured with CpGA ODN or HIV-1_MN. (B) BDCA-4⁺ cells were stimulated with 10,000 times more copies of pNL4-3 than the amount present in 900 ng p24^CA/ml of HIV-1_NL4-3, in the circular (cir) and linearized (lin) forms and after formulation with DOTAP. Low doses of CpGA and CpGB ODNs (1 μg/ml) and R848 (1 μM) were used as controls. Flow cytometry–sorted blood pDCs (C) and MDCs (D) were treated overnight with HIV-1_MN RNA, microvesicle-derived RNA (Mv RNA), or with RNA40 and RNA41 mixed with DOTAP as controls. HIV-1_MN and Mv RNA were preincubated with RNase prior to formulation with DOTAP. Flow cytometry–sorted pDCs (E) and MDCs (F) were cultured overnight with 900 ng p24^CA/ml of wild-type HIV-1_NL4-3 or pseudotyped VSV-G HIV-1_NL4-3. Concentrations of IFN-α (ng/ml) produced by pDCs and TNF-α (pg/ml) produced by pDCs and MDCs were determined by ELISA. CD83 expression was measured by flow cytometry (the percentage of positive cells is indicated in bold and the mean of fluorescence intensity underlined).

Figure 6

Inhibition with some inhibitory TLR ligands. pDCs were stimulated overnight with 300 or 37.5 ng p24^CA Eq/ml HIV-1_MN and m^1,7o⁸Guo (A), 2088 ODN (B), (TTAGGG)₄ ODN (C), or GpCB ODN (D). As controls, CpGA and CpGB ODNs or R848 were used. IFN-α (ng/ml) and TNF-α (pg/ml) production were measured by ELISA.

Figure 7

TLR7 is the likely target of HIV in pDCs. HEK 293 cells expressing human TLR7 (A) or human TLR9 (B) and an NF-κB–luciferase reporter gene were pretreated (black bars) or not (white bars) with lipofectamine and stimulated overnight with HIV-1_MN at doses of 600, 300, and 150 ng p24^CA Eq/ml, R848, or CpGB ODN. Cells were lysed and assayed for luciferase gene activity. Data are expressed as luciferase activity in relative light units and are representative of 2 independent experiments.