Summary Nlrp3 inflammasome activation occurs in response to numerous agonists but the specific mechanism by which this takes place remains unclear. All previously evaluated activators of the Nlrp3 inflammasome induce the generation of mitochondrial reactive oxygen species (ROS), suggesting a model in which ROS is a required upstream mediator of Nlrp3 inflammasome activation. Here we have identified the oxazolidinone antibiotic, linezolid, as a Nlrp3 agonist that activates the Nlrp3 inflammasome independently of ROS. The pathways for ROS-dependent and ROS-independent Nlrp3 activation converged upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. Cardiolipin bound to Nlrp3 directly and interference with cardiolipin synthesis specifically inhibited Nlrp3 inflammasome activation. Together these data suggest that mitochondria play a critical role in the activation of the Nlrp3 inflammasome through the direct binding of Nlrp3 to cardiolipin.

Exposure to particulate crystals can induce oxidative stress in phagocytes, which triggers NLRP3 inflammasome-mediated interleukin 1β (IL-1β) secretion to initiate undesirable inflammatory responses that are associated with both autoinflammatory and metabolic diseases. Although mitochondrial reactive oxygen species (ROS) play a central role in NLRP3 inflammasome activation, how ROS signal assembly of the NLRP3 inflammasome remains elusive. Here, we identify liposomes as novel activators of NLRP3 inflammasome and further demonstrate that liposome-induced inflammasome activation also requires mitochondrial ROS. Moreover, we found that stimulation with liposomes/crystals induced ROS-dependent calcium influx via the TRPM2 channel and that macrophages deficient in TRPM2 displayed drastically impaired NLRP3 inflammasome activation and IL-1β secretion. Consistently, Trpm2−/− mice were resistant to crystal-/liposome-induced IL-1β-mediated peritonitis in vivo. Together, these results identify TRPM2 as a key player that links oxidative stress to the NLRP3 inflammasome activation. Therefore, targeting TRPM2 may be effective for the treatment of NLRP3 inflamamsome-associated inflammatory disorders.

The ability to repair membrane damage is conserved across eukaryotic cells and is necessary for the cells to survive a variety of physiological and pathological membrane disruptions. Membrane repair is mediated by rapid Ca(2+)-triggered exocytosis of various intracellular vesicles, such as lysosomes and enlargeosomes, which lead to the formation of a membrane patch that reseals the membrane lesion. Recent findings suggest a crucial role for dysferlin in this repair process in muscle, possibly as a Ca(2+) sensor that triggers vesicle fusion. The importance of membrane repair is highlighted by the genetic disease, dysferlinopathy, in which the primary defect is the loss of Ca(2+)-regulated membrane repair due to dysferlin deficiency. Future research on dysferlin and its interacting partners will enhance the understanding of this important process and provide novel avenues to potential therapies.

Dilated cardiomyopathy is a life-threatening syndrome that can arise from a myriad of causes, but predisposition toward this malady is inherited in many cases. A number of inherited forms of dilated cardiomyopathy arise from mutations in genes that encode proteins involved in linking the cytoskeleton to the extracellular matrix, and disruption of this link renders the cell membrane more susceptible to injury. Membrane repair is an important cellular mechanism that animal cells have developed to survive membrane disruption. We have previously shown that dysferlin deficiency leads to defective membrane resealing in skeletal muscle and muscle necrosis; however, the function of dysferlin in the heart remains to be determined. Here, we demonstrate that dysferlin is also involved in cardiomyocyte membrane repair and that dysferlin deficiency leads to cardiomyopathy. In particular, stress exercise disturbs left ventricular function in dysferlin-null mice and increases Evans blue dye uptake in dysferlin-deficient cardiomyocytes. Furthermore, a combined deficiency of dystrophin and dysferlin leads to early onset cardiomyopathy. Our results suggest that dysferlin-mediated membrane repair is important for maintaining membrane integrity of cardiomyocytes, particularly under conditions of mechanical stress. Thus, our study establishes what we believe is a novel mechanism underlying the cardiomyopathy that results from a defective membrane repair in the absence of dysferlin. IntroductionDilated cardiomyopathy is the most common type of cardiomyopathy, a condition that can often progress into heart failure and sudden death. Many cases of dilated cardiomyopathy have a genetic etiology. Indeed, inherited forms of idiopathic dilated cardiomyopathy account for at least 30% of dilated cardiomyopathy cases. These are a major cause of severe heart failure and necessitate heart transplantation (1). A number of genes encoding sarcomeric and cytoskeletal proteins have been identified as being responsible for dilated cardiomyopathy: cardiac actin, cardiac troponin T, β-myosin heavy chain, α-tropomyosin, α-actinin, titin, metavinculin, and desmin (reviewed in ref. 2). Mutations in genes such as dystrophin (3, 4), α-sarcoglycan (5), β-sarcoglycan (6), γ-sarcoglycan (7), and δ-sarcoglycan (8, 9), which encode proteins involved in linking the cytoskeleton, sarcolemma, and extracellular matrix, have also been identified as causes of some familial dilated cardiomyopathy. Such mutations lead to the disruption of the cytoskeleton-sarcolemma-extracellular matrix link and thus render the sarcolemma more susceptible to contraction-induced injury (10-12).The plasma membrane provides a physical barrier between the extracellular and intracellular environments, and the maintenance of this barrier is crucial for cell survival; however, plasma-membrane disruption occurs physiologically in certain types of cells,