These results point toward mitochondrial membrane potential as a novel therapeutic target for NLRP3-mediated inflammatory diseases. Using confocal microscopy of BMDM from NLRP3 knockout mice to visualize pyronecrosis, we provide evidence that this process is NLRP3 dependent.Ĭonclusion: These data suggest a previously unrecognized role for ATP5O in regulating mitochondrial membrane potential, which can regulate NLRP3 inflammasome activation. Decreasing the proton gradient with FCCP and 2DG significantly inhibits inflammasome activation, suggesting a requirement for an increased proton gradient across the mitochondrial membrane. Results: By flow analysis we provide evidence that an increase in the proton gradient induced by oligomycin results in a significant increase in ATP induced inflammasome activation, while knockdown of ATP5O, results in a significant reduction in inflammasome activation. LPS stimulated cells were also evaluated by immunofluorescence and western blot analysis. Viability of non-fixed cells was evaluated with 7AAD staining. Supernatants were incubated with IL-1β-capture beads, added back to fixed and permeabilized cells, and both were stained with antibodies against IL-1β, then evaluated by flow cytometry. IL-1β release and intracellular IL-1β were measured with a flow-based assay. Cells were stimulated with LPS followed by oligomycin and ATP. Methods: ATP5O expression was knocked down in THP-1 cells. Here we asked whether regulation of mitochondrial membrane potential by ATP50 and oligomycin plays a role in NLRP3 inflammasome activation. Proton gradients are equally central to all forms of photosynthesis, as well as to bacterial motility (via the famous flagellar motor, a rotary motor similar to the ATP synthase) and homeostasis. ATP5O appears to be part of the connector linking these two components and it confers sensitivity to oligomycin induced increase in the proton gradient. F-type ATPases consist of two structural domains, F(1) – containing the extramembraneous catalytic core, and F(0) – containing the membrane proton channel. ATP is produced from ADP in the presence of a proton gradient across the mitochondrial membrane by ATP synthase (F(1)F(0) ATP synthase). Emerging evidence suggests that mitochondria are involved in inflammasome activation. Although several models for inflammasome activation have been proposed the precise molecular mechanism, as well as the role of NRLP3 mutations, remains to be elucidated. NLRP3 is a key component of a multiprotein complex known as the inflammasome that mediates the maturation of the proinflammatory cytokine IL-1β, and can induce rapid cell death in a process known as pyronecrosis. Background/Purpose: Self-activating mutations in NLRP3 cause a spectrum of autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS).
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