Characterization of giant multinucleated cells phenotype and formation in gouty tophus

 

Charles Leroy, Anne Dupont, Mylène Zarka, Nghia Pham, Huy Nguyen D.q, Augustin Latourte, Thomas Bardin, Pascal Richette, Hang Korng Ea

 

Affiliation(s):

Inserm U1132 Bioscar, Université Paris Cité, Paris, France

 

 

Background: Gout, characterized by monosodium urate (MSU) deposits and calcium pyrophosphate deposition diseases (CPP), are responsible for recurrent inflammatory flares. Crystalline deposits of UMS are accompanied by adjacent bone destruction. The accumulation of UMS can induce the formation of a tophi organized into lobules containing the crystals surrounded by a fibrous and inflammatory reaction with the presence of multinucleated giant cells (GMCs) resulting from the fusion of macrophages. The concomitant presence of CPP crystals within the tophi is described in the literature.

Objectives: Study the formation of GMCs induced by MSU and CPP crystals and characterize GMCs phenotype inside tophus.

Methods: In vitro, the ability of synthetic MSU and CPP crystals to induce GMCs formation was compared to that of RANKL using the murine macrophage line (RAW 264.7). In vivo, the crystals were injected subcutaneously and the formation of CGMs studied 15 days later. Tophi collected from gout patients were included and then studied by HE, TRAP staining and cathepsin K and integrin-β3 immunostaining. The spatial transcriptome was performed via Nanostring's GeoMx kit on histological tophi section.

Results: In vitro, MSU and CPP crystals induced the fusion of RAW 264.7 macrophages and the formation of GMCs from 4 days of stimulation. The number of GMCs increased over time to reach a plateau after 7 days of stimulation. The kinetics of GMCs formation were equivalent to those observed under RANKL stimulation. But the GMCs induced by RANKL were larger than those induced by the crystals. These GMCs expressed Tartrate-Resistant Acid Phosphatase (TRAP) and had an actin ring similar to that seen in osteoclasts. Histological analysis of the tophi showed that CGMs had high expression of TRAP and cathepsin K. The CGMs also had a ring-organized expression of integrin- β3. In vivo in mice, subcutaneous injection of MSU and CPP induced a positive TRAP gigantocellular reaction similar to that observed in tophi. Spatial transcriptomic analysis confirmed that GMCs surrounding tophi strongly expressed osteoclastic markers (ACP5, CTSK, MMP9, SPP1) and lysosomal storage proteins (CTSK, CTSB, ATP6AP2, ATP6V1B2).

Conclusions: The GMCs induced by MSU crystals and surrounding the tophi have a phenotype close to that of osteoclasts, could be involved in the bone destruction associated with tophaceous deposits.