Mitochondrial Calcium Uniporter (MCU)-dependent mitochondrial fragmentation triggers inflammation in vitro and in vivo

The NLRP3 inflammasome is a multiprotein complex comprising NLRP3, ASC, and caspase-1, primarily activated in macrophages via a two-signal mechanism. The first signal induces transcription of inflammasome components, while the second promotes NLRP3 oligomerization, leading to caspase-1 activation and subsequent processing of pro-inflammatory cytokines IL-1β and IL-18, as well as pyroptotic cell death via gasdermin D cleavage. Gout, driven by monosodium urate (MSU) crystal deposition, serves as a prototypical model of inflammasome-mediated inflammation. Recent evidence implicates mitochondria—and specifically mitochondrial calcium (Ca²⁺) homeostasis—as critical modulators of inflammasome activity. Mitochondrial Ca²⁺ uptake, mediated by the mitochondrial calcium uniporter (MCU), can promote reactive oxygen species (ROS) generation and mitochondrial permeability transition, both known triggers of NLRP3 activation. This study investigates the role of mitochondrial Ca²⁺ regulation in NLRP3 inflammasome activation using bone marrow-derived macrophages (BMDMs) from MCU⁺/⁻ and macMCU⁻/⁻ mice, which possess whole-body heterozygous and myeloid-specific MCU deletions, respectively. BMDMs were primed with lipopolysaccharide (LPS) and stimulated with ATP or MSU crystals. IL-1β secretion, mitochondrial morphology, and inflammasome-associated protein expression were assessed via ELISA, TMRM-based fluorescence imaging, and western blotting. In vivo MSU-induced peritonitis and joint inflammation models were also employed to assess inflammatory outcomes. Both MCU-deficient models exhibited reduced mitochondrial fragmentation and significantly lower IL-1β secretion following inflammasome activation. In vivo, MSU challenge led to attenuated ankle swelling and enhanced pain tolerance in MCU⁺/⁻ and macMCU⁻/⁻ mice, indicating diminished inflammatory responses. These findings highlight mitochondrial Ca²⁺ uptake as a key regulator of NLRP3 inflammasome activity and suggest that limiting mitochondrial Ca²⁺ influx may offer a protective mechanism against pathological inflammation.