Cartilage Trauma Induces Necroptotic Chondrocyte Death and Expulsion of Cellular Contents

Stolberg-Stolberg Josef, Sambale Meike, Hansen Uwe, Schäfer Alexandra, Raschke Michael, Bertrand Jessica, Pap Thomas, Sherwood Joanna

Abstract

Necroptotic cell death is characterized by an activation of RIPK3 and MLKL that leads to plasma membrane permeabilization and the release of immunostimulatory cellular contents.High levels of chondrocyte death occur following intra-articular trauma, which frequently leads to post-traumatic osteoarthritis development. The aim of this study is to assess necroptosis levels in cartilage post-trauma and to examine whether chondrocyte necroptotic mechanisms may be investigated and modified in vitro. Fractured human and murine cartilage, analysed immunohistochemically for necroptosis marker expression, demonstrated significantly higherlevels of RIPK3 and phospho-MLKL than uninjured controls. Primary murine chondrocytesstimulated in vitro with the TNFα and AKT-inhibitor alongside the pan-caspase inhibitor Z-VAD-fmkexhibited a significant loss of metabolic activity and viability, accompanied by an increase inMLKL phosphorylation, which was rescued by further treatment of chondrocytes with necrostatin-1.Transmission electron microscopy demonstrated morphological features of necroptosis in chondrocytesfollowing TNFα and Z-VAD-fmk treatment. Release of dsDNA from necroptotic chondrocytes was found to be significantly increased compared to controls. This study demonstrates that cartilage trauma leads to a high prevalence of necroptotic chondrocyte death, which can be induced andinhibited in vitro, indicating that both necroptosis and its consequential release of immunostimulatorycellular contents are potential therapeutic targets in post-traumatic arthritis treatment.