Interactions among human immunodeficiency virus (HIV)-1, interferon-gamma and receptor of activated NF-kappa B ligand (RANKL): implications for HIV pathogenesis

Abstract

We reported recently that exposure of human T cells to soluble HIV-1 envelope glycoprotein gp120 induced biologically active tumour necrosis factor (TNF)-alpha-related cytokine receptor of activated NF-kappaB ligand (RANKL), the primary drive to osteoclast differentiation and bone resorption. Furthermore, certain anti-HIV protease inhibitors linked clinically to accelerated bone loss in HIV disease blocked the physiological control of RANKL activity by interferon (IFN)-gamma through inhibition of degradation of the RANKL nuclear adapter signalling protein, TNF receptor associated protein 6 (TRAF6). We now report a series of reciprocal interactions among HIV-1, RANKL and IFN-gamma. RANKL augmented HIV replication in acutely and chronically infected cells of T lymphocyte and monocyte lineage, effects which occurred at a transcriptional level in conjunction with activation of NF-kappaB. TNF-alpha and RANKL were markedly synergistic in induction of HIV. Low pharmacological levels of IFN-gamma (0.75-3 ng/ml) suppressed RANKL-driven enhancement of HIV replication, as did L-T6DP-1, a cell-permeable peptide inhibitor of TRAF6. In contrast, HIV replication induced by TNF-alpha and phorbol ester were not inhibited, and in some cases augmented, by IFN-gamma. We conclude that a positive feedback loop exists between RANKL production and HIV replication, which may be relevant to both the pathophysiology of HIV-linked osteopenia and control of HIV growth. This pathway appears distinct from those of other cytokine activators of HIV, with respect to its utilization of TRAF6 and its suppression by IFN-gamma. These data raise the possibility that TRAF-specific inhibitory peptides, alone or in conjunction with IFN-gamma, could be used to regulate HIV activation in vivo.

Fig. 1

RANKL stimulates HIV-1 replication in acutely infected PHA-activated PBMCs and chronically infected U1 monocytic cells. (a) PHA-activated PBMCs were exposed to HIV-1 isolate IIIB for 2 h, washed, and cultured; 12 h later they were treated with RANKL (25 ng/ml). Production of HIV p24 Gag antigen in the culture supernatants was evaluated on day 7. RANKL augmented HIV replication (*P < 0·004 for controlversus RANKL-treated cultures), an effect blocked by RANK-Fc (100 ng/ml) and OPG (10 ng/ml) (P < 0·01 and < 0·02 for RANKL versus RANKL + RANK-Fc or OPG, respectively). (b) Enhancement of HIV replication in acutely infected PBMCs was dose-dependent, with HIV p24 antigen measured on day 7 post-HIV exposure. Similar results were obtained in three independent experiments (*indicates statistically significant differences at P < 0·05 between control and RANKL treated cultures). (c) U1 cells (2 × 10⁶/ml), chronically infected with two integrated copies of HIV-1, were incubated in the presence or absence of RANKL (25 ng/ml) and the p24 Gag concentration of culture supernatants determined 48 h later. RANK-Fc (100 ng/ml) was used to block RANKL-specific effects. (*P < 0·03 for control versus RANKL treated cultures.) (d) RANKL induction of HIV replication in PHA-PBMCs acutely infected with CXCR4 (IIIB) or CCR5 (Bal and p13) co-receptor utilizing HIV-1 isolates. (e) RANKL up-regulates HIV replication in purified CD3⁺ primary T cells. CD3⁺ cells were positively selected from PHA-PBMC then infected with HIV-1 IIIB as described in (a). (*P < 0·015 for control versus RANKL cultures). Each bar depicts the mean ± s.d. of quadruplicate samples.

Fig. 2

RANKL synergizes with IL-2 in the activation of HIV replication. Resting PBMCs were exposed to culture medium alone or with RANKL (25 ng/ml), IL-2 (32 U/ml), or RANKL plus IL-2 for 48 h, followed by a 2 h exposure to HIV-1 IIIB. Cells were washed and cultured for 12 days, with HIV p24 Gag antigen measured on days 4, 8 and 12 post viral exposure. Points represent means ± s.d. of triplicate measurements (*P < 0·007 for IL-2 versus IL-2 + RANKL-treated cultures) (• IL2 + RANKL; ▪ IL-2 alone; ▴ unstimulated; RANKL alone).

Fig. 3

RANKL induces HIV-1 LTR-driven reporter gene transcription in T cells via NF-κB activation. (a) CD4+ U937 monocytic cells were transfected with HIV-1 LTR-CAT reporter plasmids containing intact or mutated κB sites, as described in Materials and methods; 36 h post-transfection, cells were untreated or exposed to RANKL (25 ng/ml). Cell extracts were prepared 24 h later and CAT assays performed by ELISA. (*P < 0·004 for control versus RANKL-treated cultures transfected with intact HIV-LTR). (b) EMSAs were carried out with nuclear extracts prepared from resting PBMCs treated with either medium (lane A), RANKL (10 ng/ml, lanes B and C), or PMA (10 ng/ml, lane D) for 30 min. A competition experiment was performed with a 50 molar excess of unlabelled κB oligonucleotide (lane B).

Fig. 4

Anti-TRAF6 peptide blocks RANKL-mediated induction of HIV from chronically and acutely infected cells. Chronically HIV-1 infected U1 cells (2 × 10⁶) (a) or PHA-activated PBMCs inoculated with HIV-1 IIIB for 2 h (b) were exposed to anti-TRAF6 peptide L-T6DP1 (30 µm) (TR6I), capable of inhibiting TRAF6 signalling and NFκB activation in murine osteoclasts, or anti-TRAF6 peptide L-T6DP2 (30 µm) (TR6II), which does not have this capacity, in the presence and absence of RANKL (25 ng/ml). HIV-1 p24 Gag antigen was assessed in U1 cultures at 48 h and in PBMC cultures at 7 days. (*P < 0·04 and *P < 0·0047 for RANKL versus RANKL + TR6I peptide in U1 and PBMC cultures, respectively.)

Fig. 5

RANKL synergizes with TNF-α in enhancement of HIV-1 replication. PHA-activated PBMCs were exposed to HIV-1 for 2 h, washed, then cultured with increasing concentrations of TNF-α alone (open bars) or in the presence of 25 ng/ml RANKL (hatched bars). After 6 d, HIV p24 Gag levels were determined. Data are expressed as the mean ± s.d. of three independent samples. Synergy was observed at >500 pg/ml of TNF-α.

Fig. 6

Effects of IFN-γ on induction of HIV from chronically infected monocytic cells mediated by RANKL versus TNF-α. U1 cells (2 × 10⁶) were exposed to RANKL (25 ng/ml) (a) or TNF-α (500 ng/ml) (b), either alone or in the presence of varying concentrations of IFN-γ. HIV-1 p24 Gag antigen production was measured in culture supernatants 48 h later (a: *P < 0·01; **P < 0·04; b: P < 0·01 for all concentrations tested).