HIV-1 gp120 inhibits TLR9-mediated activation and IFN-{alpha} secretion in plasmacytoid dendritic cells

Abstract

Plasmacytoid dendritic cells (pDCs) play a central role in innate and adaptive immune responses against viral infections. pDCs secrete type I IFNs and proinflammatory cytokines upon stimulation by either TLR7 or TLR9. Throughout the course of HIV infection, the production of type-I IFNs is profoundly impaired, and total pDC cell counts in peripheral blood correlates inversely with viral load and positively with CD4(+) T cell count. The origin of these defects is unclear. pDCs express CD4, CCR5, and CXCR4, the primary receptor and coreceptors, respectively, for the HIV envelope; yet little is known concerning the effects of the viral envelope on these cells. Here, we show that exposure of pDCs to gp120 results in the suppression of activation of these cells. This suppression is specific for TLR9-mediated responses, because TLR7-mediated responses are unaffected by gp120. gp120 also suppressed TLR9-mediated induction of proinflammatory cytokines and expression of CD83, a marker of DC activation. Finally, gp120 suppressed pDC-induced cytolytic activity of natural killer cells. Taken together, these data demonstrate that the direct interaction of HIV-1 gp120 with pDCs interferes with TLR9 activation resulting in a decreased ability of pDCs to secrete antiviral and inflammatory factors that play a central role in initiating host immune responses against invading pathogens.

Fig. 1.

IFN-α production induced by TLR9-ligands, but not by a TLR7-ligand is inhibited by both monomeric and trimeric gp120. (a) pDCs were treated for with CpG (5 μg/ml), R5-gp120 (100 nM), or mock-treated with PBS. IFN-α in the supernatants was detected by using human IFN-α multisubtype ELISA. (b) IFN-α produced from freshly isolated pDC treated with CpGs (500 ng/ml) in presence or absence of R5-gp120 monomer or trimer (10 nM), SIV monomer (10 nM), or control recombinant protein (10 nM). (c) IFN-α produced from pDCs treated with the TLR 7 ligand Imiquimod (1 μg/ml) in the presence or absence of R5-gp120 monomer or trimer (gp120tr) (10 nM). (d) IFN-α produced from pDCs exposed to HSV-2 for 18 h in the presence or absence of HIV gp120. (e) IFN-β produced from pDCs exposed to HSV-2, or treated with CpG, in the presence or absence of gp120. Error bars represent SDs calculated from replicates. Results shown are representative of at least five independent experiments

Fig. 2.

gp120 suppresses TLR9-mediated up-regulation of CD83, but not CD86, on pDCs. pDCs were treated with CpGs in the presence or absence of R5-gp120 monomer or trimer and stained with PE anti-CD83 (a) or CD86 (b) mAbs and analyzed by flow-cytometry. Mean fluorescence intensities (MFI) were averaged from three different experiments. Error bars represent SDs calculated on them.

Fig. 3.

Secretion of inflammatory cytokines induced by TLR9 stimulation in pDCs is inhibited by gp120. Concentrations of TNF-α, IL6, and IP10 in the supernatants of pDC cultures with CpGs in the presence or absence of R5-gp120 monomer or trimer were detected by using BD Cytometric Bead Array (CBA) cell signaling flex sets. SDs were calculated from replicates. Results shown are representative of at least five independent experiments.

Fig. 4.

NK cell cytotoxicity induced by TLR9-stimulated pDCs is inhibited by gp120. pDCs were treated with CpGs in the presence or absence of an R5 gp120 monomer (a) or trimer (b). Stimulated pDCs were added to homologous NK cell cultures. NK cells cultured in the absence of pDCs were included as a control. PKH67-K562 target cells were added to cultures at ratios: 1:2, 1:1, and 2:1 (NK:K562). Frequency of cell killing was determined by flow cytometric measurement of the number of K562 cells staining positively for PKH67-green and propidium iodide. NK cytotoxicity is reported as the percentage of dead K562 cells calculated by subtracting in each condition the percentage of dead cells of the untreated control (K562 cultured without NK cells). SDs were calculated on the replicates.

Fig. 5.

gp120 binds pDCs through both CD4-dependent and Ca²⁺-dependent interactions. (a) Freshly isolated pDC were stained with anti-CD4, anti-CCR5, and anti-CXCR4 mAbs or an isotype control mAb. (b) Freshly isolated pDC were preincubated with an unlabeled anti-CD4, gp120-blocking mAb (Leu3a), and stained with biotin-gp120- streptavidin-PE in the presence or absence of Ca²⁺.

Fig. 6.

gp120 binds BDCA-2 COS-7-transfected cells. (a) BDCA-2 expression on BDCA-2-transfected COS-7 cells vs. mock-transfected cells. gp120 binding to mock-transfected and BDCA-2-transfected cells in the presence or absence of Ca²⁺. (b) gp120-trimer binding to BDCA-2-transfected cells. Results are representative of three independent experiments.