IN VITRO, SARS-COV-2-EXPOSED MACROPHAGES DEPICT ENHANCED OSTEOCLAST DIFFERENTIATION

FRANCO SVIERCZ; PATRICIO JARMOLUK; CINTIA CEVALLOS; ALICIA LOPEZ; NICOLE FREIBERGER; M. VICTORIA DELPINO; JORGE QUARLERI

Congreso; LXXI REUNIÓN CIENTÍFICA ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI) 9 -11 de noviembre de 2023 Universidad Nacional de San Luis-San Luis; 2023

Background: The complications attributed to SARS-CoV-2 within the musculoskeletal system are progressively rising, even in asymptomatic patients. Certain studies have linked these complications to mature bone-resorbing osteoclasts (OC), and it is possible that their precursors (macrophages) are influenced by the virus creating a proinflammatory-environment that enhances their differentiation. The aim of this study was to analyze the effect of macrophage infection by SARS-CoV-2 and their subsequent differentiation into OC.Methods. Macrophages as 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 (MDM). Then, by adding RANKL (50 ng/mL) for 9 days mature OC were obtained. At 3 days of MDM differentiation, SARS-CoV-2 infections with Wuhan (Wh) and Omicron (BA.5) variants were performed using a MOI=0.1. Multinucleated and tartrate-resistant acid phosphatase (TRAP) positive cells with ≥3 nuclei were considered mature osteoclasts. Bone resorption activity was measured by light microscopy on bovine cortical bone slices. Using flow cytometry in cells detached, cell death (annexin-V/7-AAD) and mitochondrial ROS production (MitoSOX) were measured. SARS-CoV-2 replication was assessed at 3, 6, 9, and 12 dpi by measuring viral load in supernatants by RTqPCR, and by measuring intracellular SARS-CoV-2 nucleoprotein (N)-expressing cells (flow cytometry). Quantitative RT-PCR (qPCR) was used to detect RANK expression (as one of the key genes of osteoclast differentiation).Results: SARS-CoV-2 exposure of macrophages markedly increased (p