Massive HIV infection abrogates osteoclastogenesis by influencing CCR5 and CD9 expression

SVIERCZ FRANCO; JARMOLUK PATRICIO; CEVALLOS CINTIA; ADAMCZYK ALAN; FREIBERGER NICOLE; LOPEZ ALICIA; GUANO ALEX ; OSTROWSKI MATIAS; DELPINO M VICTORIA; QUARLERI JORGE

Congreso; IAS 2023, the 12th IAS Conference on HIV Science; 2023

Background: Mature bone-resorbing osteoclasts (OC) and their precursors (macrophages) are structurally and functionally affected by HIV-influencing boneloss. The membrane CCR5 and tetraspanins play an essential role in bone-destructive conditions through the functional regulation of osteoclasts that could bealtered directly by R5-tropic HIV infection.Methods: Macrophages (OC precursors) were obtained in cell culture from human monocytes isolated from buffy coats and differentiated with M-CSF (30ng/mL) for 6 days. Then, by adding RANKL (50 ng/mL) for 9 days mature OC were obtained. At 3 days of MDM differentiation, HIV infections (R5-tropic AD8,and BaL strains; pseudotyped pNLAD8-VSV-G strain) were performed using two inoculums (high:1pg/cell vs. low:0.01pg/cell). HIV Infection efficiency andreplication were assessed at 3, 6, 9, and 12 dpi by measuring intracellular p24-expressing cells (flow cytometry), and soluble p24 in cell supernatants (ELISA).Multinucleated tartrate-resistant acid phosphatase-positive cells with ≥3 nuclei were considered mature osteoclasts. Using flow cytometry in cells detached,cell-death (annexin-V/7-AAD) and CCR5/CD9 expression were measured. Bone resorption activity was measured by light microscopy on bovine cortical boneslices.Results: An HIV replication peak was found earlier with the high inoculum (42-fold change from 3 to 6 dpi), whereas this peak (30-fold change) occurredbetween 6 and 9 dpi with the low inoculum. At 12 dpi, both inoculums depicted similar infection efficiency (p24-expressing cells: 57.7±12.8% vs. 46.0±10.6%)and cell-death level (11.9±4.7 vs. 8.6±3.3). High but not low HIV-inoculum abrogates markedly OC number (x200; control:37.2±12.4; HIV-low:32.3±13.3; HIVhigh: 10.8±3.4). Besides, two CCR5-ligands (TAK-779, and recombinant-AD8-gp120), and HIV-VSV-G infections (R5-independent cell entry) significantlyimpaired osteoclastogenesis, as well. OC precursors and early OC challenged with high (but not low) HIV inoculum triggered a significant increase inmembrane CCR5 (1056.0±97.6MFI) and CD9 expression (4196.0±277.2MFI). The number of osteoclasts formed on bone slices correlated directly with boneresorption (by examinations of the resorbed area). When HIV replication was inhibited using nevirapine (1 mM), osteoclastogenesis was recovered.Conclusions: Osteoclasts differentiation is impaired by R5-tropic HIV strains when a massive replication and CCR5 blocking occurs in their precursors,altering both CCR5 and tetraspanin expression and its resorptive functions.