Abstract Background/Aims Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory bone disease that, while most commonly affecting children and young people, can affect all age groups. Clinical presentations range from single bone lesions to chronic inflammation of multiple bones, known as chronic recurrent multifocal osteomyelitis (CRMO). Due to variable presentations and limited awareness among clinicians, diagnosis and treatment are frequently delayed. Untreated, CNO can result in chronic pain, arthritis (40%), pathological fractures (usually vertebrae, ∼10%), reduced mobility, and inflammation of additional tissues/organs (e.g., skin: 15-30%; gut: 10-15%), substantially impairing patients’ quality of life. Recently, we identified variants in the P2RX7 gene, encoding for a key regulator of NLRP3 inflammasome assembly (P2X7), across a large cohort of CNO patients. Rare variants in P2RX7 were present in ∼40% of N = 191 CNO patients versus 6% of N = 1873 healthy controls. Experiments in genetically modified THP-1 monocyte-derived macrophages suggested a key role for CNO-associated P2RX7 variants in increasing inflammasome activation, IL-1β and IL-18 release, while allowing prolonged macrophage survival. We aimed to understand how CNO-associated P2XR7 variants contribute to prolonged cell survival. This project investigated their effects on K+/Ca2+ mobilisation, reactive oxygen species (ROS) production, mitochondrial integrity, and cytokine expression in THP-1-derived macrophages. Methods Genetically modified THP-1 monocytes were differentiated into macrophage-like cells to investigate effects of CNO-associated P2RX7 variants (c.349CT, rare gain-of-function; c.920GA rare loss-of-function; c.489CT common gain-of-function). Activation of the NLRP3 inflammasome (ASC specks), K+ (ELISA) and Ca2+ flux (flow cytometry), expression of pro- and anti-inflammatory cytokines (MSD, Luminex assays), ROS production (2’,7’-dichlorofluorescein diacetate/DCFDA, Lucigenin assays, mitoSOX) and mitochondrial function (MitoTracker) were studied. Effects of small molecule P2X7 (A-804598) or NLRP3 (MCC950) inhibitors and mitoTEMPO (mitochondria-targeted antioxidant) were interrogated. Results Compared with cells expressing wild-type P2X7, THP-1-derived macrophages expressing the c.349CT or c.489CT variants showed increased K+ efflux and sustained Ca²+ influx, enhanced inflammasome activation, elevated secretion of IL-1β and IL-18, and reduced LDH release following prolonged stimulation (estimating pyroptosis). Prolonged survival associated with reduced ROS levels across all CNO-associated P2RX7 variants, decreased mitochondrial stress for the c.920GA loss-of-function variant (reduced superoxide production), and an increased number of functional mitochondria for the c.349CT and c.920GA variants. Co-culture with the P2X7 inhibitor A-804598 or mitoTEMPO, across all cell lines, led to reduced intracellular Ca²+ content, decreased inflammasome assembly, reduced ASC speck release and cytokine secretion (particularly IL-18), as well as reduced mitochondrial damage (lower mROS levels and mitochondrial volume). Notably, effects of A-804598 or mitoTEMPO were more pronounced when compared to treatment with the NLRP3 inhibitor MCC950. Conclusion Imbalanced K+/Ca2+ mobilization in THP-1-derived macrophages expressing CNO-associated P2RX7 variants associates with reduced mitochondrial stress and prolonged survival. Understanding the contribution of CNO-associated variation in P2RX7 will promote the development of target-directed treatments. Disclosure A. Charras: None. S.R. Hofmann: None. F. Schulze: None. S. Russ: None. J. Hawkes: None. C. Hedrich: Grants/research support; CMH receives unrestricted funding from Merck (MISP) to study lupus nephritis.
Charras et al. (Wed,) studied this question.
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