Cartilage Trauma Induces Necroptotic Chondrocyte Death and Expulsion of Cellular Contents

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 higher levels of RIPK3 and phospho-MLKL than uninjured controls. Primary murine chondrocytes stimulated in vitro with the TNFα and AKT-inhibitor alongside the pan-caspase inhibitor Z-VAD-fmk exhibited a significant loss of metabolic activity and viability, accompanied by an increase in MLKL phosphorylation, which was rescued by further treatment of chondrocytes with necrostatin-1. Transmission electron microscopy demonstrated morphological features of necroptosis in chondrocytes following 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 and inhibited in vitro, indicating that both necroptosis and its consequential release of immunostimulatory cellular contents are potential therapeutic targets in post-traumatic arthritis treatment.

RIP1 Perturbation Induces Chondrocyte Necroptosis and Promotes Osteoarthritis Pathogenesis via Targeting BMP7

“…Although the possible involvement of chondrocyte necroptosis in OA has been suggested, direct evidences are still lacking. Recent studies immunohistochemically analyzed the expression of necroptosis markers RIP3, MLKL, and p-MLKL to prove the existence of necroptosis in degenerated human and murine cartilage (Riegger and Brenner, 2019;Stolberg-Stolberg et al, 2020). However, it remains unclear whether chondrocyte necroptosis is the inducer of cartilage destruction or its byproduct, and the expression status of another important necroptosis marker, RIP1, in OA clinical samples has not been assessed.…”

“…Necroptosis is mediated by necrosome, a supermolecular complex which contains receptor-interacting protein kinase 1 and 3 (RIP1, RIP3), and its direct substrate mixed-lineage kinase domain-like protein (MLKL), targeting the complex to appropriate downstream effectors in the necroptosisinducing process (Cho et al, 2009;He et al, 2009;Wang et al, 2014). Previous studies have discovered a possible link between necroptotic process and cartilage injury depending on oxidative stress and cytokine release in OA, and the TRIM24-RIP3 axis was proposed to promote OA chronicity by modulating the expression of catabolic factors (Riegger and Brenner, 2019;Jeon et al, 2020;Stolberg-Stolberg et al, 2020). However, the involvement of RIP1 during OA pathogenesis still lacks direct evidence.…”

The role of necroptosis in disease and treatment

“…According to recent reports, significant accumulation of oxidative stress and cytokine release in OA cartilage, implies a complex relationship between cartilage injury and necroptotic processes. 119 , 120 Cheng et al. found that RIPK1 expression was markedly increased in OA cartilage, both in OA patients and experimental rat models.…”

Extracellular Vesicles in Musculoskeletal Pathologies and Regeneration

“…Trauma-induced cell death compromises direct necrosis and programmed cell death, such as apoptosis and necroptosis (Zhao et al, 2015 ; Riegger and Brenner, 2019 ; Stolberg-Stolberg et al, 2020 ). Apoptotic cells were found to secrete apoptotic exosome-like vesicles (AEVs), expressing typical markers such as CD63, lysosomal-associated membrane protein 1 (LAMP1) and stress-associated heat shock proteins 70 (HSP70) (Park et al, 2018 ).…”