Background:  Enhanced bone demineralization occurs in advanced HIV disease and may be accelerated by certain ART drugs. We reported that T-cell production of RANKL, the obligate cytokine for osteoclastogenesis, is induced by HIV-1 gp120, with positive feedback between RANKL secretion and HIV replication. We also found that low pharmacologic levels (1 to 5 μM) of HIV protease inhibitors (PI) ritonavir (RTV) and saquinavir (SQV), but not other ART drugs, increased RANKL-mediated osteoclasts differentiation and activity in vitro, and blocked the physiologic suppression of RANKL via degradation of TRAF6 by physiologic levels (1 ng/mL) of interferon-gamma (IFN-γ), without altering other proteasome functions. We sought to define the pathways involved in the marked specificity of these processes.

Methods:  Non-adherent peripheral blood mononuclear cells were prepared from a healthy donor and cultured in the presence of physiologic levels of RANKL (50 ng/mL), M-CSF (100 ng/mL), and IFN-γ (1 ng/mL) (control) for 24 hours or 5 days. Select cultures were exposed to RTV (5 μM), or therapeutic levels of IFN-γ (10 ng/mL), or both. RNA was amplified, biotin-labeled, and subjected to array hybridization using U133A 2.0 Affymetrix chips. Experiments were repeated 2 or 3 times, and supported by real-time polymerase chain reactions (RT-PCR). 

Results:  When expression of 470 genes was altered by RTV, and then re-affected by addition of IFN-γ, 24-hour results were most informative. Key genes for osteoclast gene transcription (NFATc1) and WNT signaling were decreased 3- to 33-fold by RTV, and reversed by IFN-γ, while genes for apoptosis, cytokines linked to suppression of osteoclast activity (interleukin [IL] -12, IL-18) and MAPK genes involved in osteoclast inhibition were increased 2.5- to 8-fold by RTV, and reversed by IFN-γ. Ephrin-B2 and -B3 genes and their signaling pathways, involved in osteoclast/osteoblast coupling, were enhanced by RTV and decreased by IFN-γ, adding a new dimension to effects of anti-HIV drugs on bone.

Conclusions:  Concentrations of RTV present in all protease inhibitor (PI) -boosted regimens significantly alter expression of genes critical to osteoclast differentiation, activity, and cooperation with osteoblasts, and can be corrected by IFN-γ. This suggests a means of possible therapeutic intervention in at least 1 metabolic complication of HIV PI therapy.