Massive HIV infection abrogates osteoclastogenesis by influencing CCR5 and CD9 expression

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

Brisbane

Conferencia; 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 bone loss. The membrane CCR5 and tetraspanins play an essential role in bone-destructive conditions through the functional regulation of osteoclasts that could be altered 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 (30 ng/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 and replication 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 bone slices.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) occurred between 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; HIV-high: 10.8±3.4). Besides, two CCR5-ligands (TAK-779, and recombinant-AD8-gp120), and HIV-VSV-G infections (R5-independent cell entry) significantly impaired osteoclastogenesis, as well. OC precursors and early OC challenged with high (but not low) HIV inoculum triggered a significant increase in membrane CCR5 (1056.0±97.6MFI) and CD9 expression (4196.0±277.2MFI). The number of osteoclasts formed on bone slices correlated directly with bone resorption (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.