The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.

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Alternative oxidases (AOXs) bypass respiratory complexes III and IV by transferring electrons from coenzyme Q directly to O2. They have therefore been proposed as a potential therapeutic tool for mitochondrial diseases. We crossed the severely myopathic skeletal muscle-specific COX15 knockout (KO) mouse with an AOX-transgenic mouse. Surprisingly, the double KO-AOX mutants had decreased lifespan and a substantial worsening of the myopathy compared with KO alone. Decreased ROS production in KO-AOX versus KO mice led to impaired AMPK/PGC-1α signaling and PAX7/MYOD-dependent muscle regeneration, blunting compensatory responses. Importantly, the antioxidant N-acetylcysteine had a similar effect, decreasing the lifespan of KO mice. Our findings have major implications for understanding pathogenic mechanisms in mitochondrial diseases and for the design of therapies, highlighting the benefits of ROS signaling and the potential hazards of antioxidant treatment.

Mitochondrial electron transport chain (ETC) function is linked to the generation of ATP, signaling molecules including reactive oxygen species (ROS), pyrimidines and tricarboxylic acid cycle metabolites¹. Mitochondrial electron transport is required for T cell proliferation^2,3,4. However, which mitochondrial ETC functions are necessary for each dynamic state of CD8⁺ T cell responses is unknown. Here we report that impairing mitochondrial complex III function, which diminishes respiration, proton pumping linked to ATP production and superoxide production, decreases peripheral naive numbers, antigen-induced CD8⁺ T cell proliferation and memory formation. Acute stimulation of mitochondrial complex III-deficient CD8⁺ T cells induced an exhausted-like phenotype. Expression of Ciona intestinalis alternative oxidase (AOX) in mitochondrial complex III-deficient CD8⁺ T cells restores respiration without generating ROS or proton pumping, and rescues proliferation and the exhausted phenotype but not naive or memory formation. Thus, T cell development, proliferation and memory formation have distinct requirements for mitochondrial complex III ROS.

Mitochondrial oxidative phosphorylation (OXPHOS) and cellular workload are tightly balanced by the key cellular regulator, calcium (Ca²⁺). Current models assume that cytosolic Ca²⁺ regulates workload and that mitochondrial Ca²⁺ uptake precedes activation of matrix dehydrogenases, thereby matching OXPHOS substrate supply to ATP demand. Surprisingly, knockout (KO) of the mitochondrial Ca²⁺ uniporter (MCU) in mice results in only minimal phenotypic changes and does not alter OXPHOS. This implies that adaptive activation of mitochondrial dehydrogenases by intramitochondrial Ca²⁺ cannot be the exclusive mechanism for OXPHOS control. We hypothesized that cytosolic Ca²⁺, but not mitochondrial matrix Ca²⁺, may adapt OXPHOS to workload by adjusting the rate of pyruvate supply from the cytosol to the mitochondria. Here, we studied the role of malate-aspartate shuttle (MAS)-dependent substrate supply in OXPHOS responses to changing Ca²⁺ concentrations in isolated brain and heart mitochondria, synaptosomes, fibroblasts, and thymocytes from WT and MCU KO mice and the isolated working rat heart. Our results indicate that extramitochondrial Ca²⁺ controls up to 85% of maximal pyruvate-driven OXPHOS rates, mediated by the activity of the complete MAS, and that intramitochondrial Ca²⁺ accounts for the remaining 15%. Of note, the complete MAS, as applied here, included besides its classical NADH oxidation reaction the generation of cytosolic pyruvate. Part of this largely neglected mechanism has previously been described as the “mitochondrial gas pedal.” Its implementation into OXPHOS control models integrates seemingly contradictory results and warrants a critical reappraisal of metabolic control mechanisms in health and disease.

Signaling from the mitochondrial respiratory chain is essential for acute vascular oxygen sensing in the lung.