Signaling mechanism of HIV-1 gp120 and virion-induced IL-1beta release in primary human macrophages

Abstract

HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of proinflammatory cytokines, including IL-1beta from macrophages, that are implicated in the pathogenesis of HIV-associated dementia. However, the signal transduction pathways involved have not been fully defined. Previously, our laboratory reported that soluble gp120 activates multiple protein kinases in primary human monocyte-derived macrophages, including the Src family kinase Lyn, PI3K, and the focal adhesion-related proline-rich tyrosine kinase Pyk2. In this study we showed that gp120 induces IL-1beta release from macrophages in a time- and concentration-dependent manner through binding to the chemokine receptor CCR5 and coupling to G(i)alpha protein. Using pharmacological inhibitors and small interfering RNA gene knockdown, we demonstrated that concomitant activation of Lyn, Pyk2, and class IA PI3K are required for gp120-induced IL-1beta production. By coimmunoprecipitation and immunofluorescence confocal microscopy, we showed that CCR5 activation by gp120 triggered the assembly of a signaling complex involving endogenous Lyn, PI3K, and Pyk2 and is associated with PI3K and Pyk2 translocation from the cytoplasm to the membrane where they colocalized with Lyn. Finally, we demonstrated that virion-associated gp120 induced similar response, as structurally intact whole virions also triggered IL-1beta release and re-localization of PI3K and Pyk2. This study identifies a novel signaling mechanism for HIV-1-induced IL-1beta production by primary human macrophages that may be involved in the neuropathogenesis of HIV-associated dementia.

FIGURE 1

HIV-1 gp120 induces IL-1β release by primary human macrophages. A, MDMs were treated without (gray bars) or with (black bars) gp120 (20 nM) for indicated times. B, MDMs were stimulated for 16 h with indicated concentrations of gp120. C, gp120 was subjected to heat inactivation (ΔHI) for 30 min or incubation with anti-gp120 IgG or control human IgG for 1 h prior to exposure to MDMs. D, MDMs were pretreated without or with the CCR5 antagonist (M657, 1 μM) for 1 h, or inhibitors for G_i (PTX, 100 ng/ml) or G_s (NF449, 200 nM) for 2 h, prior to stimulate with gp120. E, MDMs from donors lacking CCR5 (CCR5Δ32) or wild type (WT) were stimulated with gp120, LPS (0.1 ng/ml) or CCL4 (MIP-1β, 10 nM). Cell culture supernatants were collected and IL-1β levels were quantitated by ELISA. (*, P<0.05; **, P<0.01; ***, P<0.001).

FIGURE 2

gp120-induced IL-1β production in macrophages requires activation of Src family kinase Lyn. A, MDMs were pretreated for 1 to 2 h with the broad-spectrum SFK inhibitor PP2 or its inactive analog PP3 (10 μM), a Lyn-specific peptide inhibitor KRX-123.302 or its negative control peptide KRX-107.110 (10 μM), or control vehicle alone, then stimulated with gp120. B, MDMs were transfected with non-targeting control or Lyn-specific siRNA prior to stimulation without (gray bars) or with (black bars) gp120. Inset, immunoblots (IB) showing Pyk2, PI3Kp85, Lyn, Hck and β-actin protein levels in siRNA-transfected MDMs from representative parallel transfection. (***, P<0.001).

FIGURE 3

IL-1β release by gp120-stimulated macrophages requires PI3K activation. A, MDMs were pretreated for 1 to 2 h with the broad PI3K inhibitor LY294002 (10 μM), wortmannin (100 nM) or control vehicle alone prior to exposure to gp120. B, MDMs were treated with pharmacologic inhibitors specific for class IA PI3K (PI-103; 40 nM) or class IB (AS605240; 10 nM), or both, or control vehicle only, prior to stimulation with gp120. C, MDMs were transfected with non-targeting control or PI3Kp85-specific siRNA prior to stimulation without (gray bars) or with (black bars) gp120. Inset, immunoblots (IB) showing Pyk2, PI3Kp85, PI3Kp101, Lyn, Hck and β-actin protein levels in siRNA-transfected MDMs from representative parallel transfection. (*, P<0.05; **, P<0.01; ***, P<0.001).

FIGURE 4

Macrophage IL-1β release triggered by gp120 requires the proline-rich tyrosine kinase Pyk2. A, MDMs were pretreated for 15 to 60 min with the upstream Pyk2 inhibitor dantrolene (10 μM), AG17 or its inactive analog AG43 (both 20 μM), or control vehicle alone prior to stimulation with gp120. B, MDMs were pretreated for 1 h with the CaMKII inhibitor KN62 (1 μM), GSK3 inhibitor BIO (10 nM), or control vehicle alone prior to stimulation with gp120. C, MDMs were transfected with non-targeting control or Pyk2-specific siRNA prior to stimulation without (gray bars) or with (black bars) gp120. To monitor protein expression, immunoblots (IB) were performed in parallel for Pyk2, PI3Kp85, Lyn, Hck and β-actin on the same batch of transfected MDMs used or IL-1β ELISA. (***, P<0.001).

FIGURE 5

Lyn, PI3K and Pyk2 physically associated to form a multi-kinase signaling complex in response to HIV-1 gp120 stimulation. MDMs were treated without or with gp120 (20 nM) for 10 min prior to cell lysis. Cell lysates were immunoprecipitated (IP) with antibody specific for (A) Lyn, (B) PI3Kp85, (C) Pyk2, (D) Hck, or with control IgG. Immune complexes were washed, resolved on SDS-PAGE and subjected to immunoblot (IB) with antibodies specific for Pyk2, PI3Kp85, Lyn and Hck. Whole cell lysates (WCL) of unstimulated MDMs served as a positive control for immunoblotting.

FIGURE 6

HIV-1 gp120 triggers PI3K and Pyk2 redistribution and co-localization with Lyn in primary MDMs. MDMs were pretreated without or with the CCR5 antagonist M657 (1 μM) for 1 h prior to stimulation with gp120 for 10 min. MDMs were fixed, permeabilized and triple-labeled with Lyn, PI3Kp85 and Pyk2 antibodies before examination by confocal microscopy. Subcellular distribution of Lyn (green; A, E, I, M), PI3K (blue; B, F, J, N) and Pyk2 (red; C, G, K, O) are shown in the single channel images. Co-localization of Lyn, PI3K and Pyk2 are indicated by arrowheads shown in the merge images (white; D, H, L, P).

FIGURE 7

Virion-associated gp120 induces CCR5-mediated redistribution of PI3K and Pyk2, co-localization with Lyn and IL-1β release in primary MDMs. MDMs were stimulated without or with HIV-1 pseudotype virions carrying envelope from the R5 strain BaL, or lacking envelope (bald virus), using 10 ng of p24^gag antigen content per virus. In parallel, MDMs were treated with the CCR5 antagonist M657 (1 μM) prior to exposure to BaL pseudotype virion. A, After 10 min of virion exposure, MDMs were fixed, permeabilized and triple-labeled with Pyk2, PI3Kp85 and Lyn antibodies before examination by confocal microscopy. B, Cell culture supernatants were collected after 16 h of virion stimulation and IL-1β levels were quantitated by ELISA. (***, P<0.001).

FIGURE 8

Model for signaling mechanism mediating gp120-induced IL-1β release in primary human macrophages. Binding of monomeric or virion-associated HIV-1 gp120 to macrophage CCR5 triggers G_i-mediated PI3Kp85 and Pyk2 re-localization to the membrane and formation of a signaling complex with Lyn. Activation of this complex then leads to IL-1β production, likely through the action of downstream MAP kinases and nuclear transcription factors (NTFs).