Prolonged NLRP3 inflammasome activation enhances the secretion of autophagy‐derived vesicles containing LC3II in murine dendritic cells

Abstract

Autophagy provides a brake on overall NLRP3 inflammasome activation while also promoting non‐classical Il‐1β export in the early phases of inflammasome activation. The mechanism of autophagy dependent non‐classical secretion in response to NLRP3 inflammasome activation is unclear. To investigate this process, bone marrow derived dendritic cells from wild‐type C57BL/6 mice were primed with LPS and then stimulated with ATP to activate P2X7R channels and the downstream NLRP3 inflammasome. Cell lysates and extracellular (EC) fractions were then processed for western blot analysis of LC3II, a marker of the autophagosome. A prolonged ATP stimulus resulted in increased secretion of both non‐lipidated LC3I and lipidated LC3II. To characterize secreted LC3II‐containing vesicles, EC samples were fractionated by differential centrifugation to isolate total, microvesicle‐free, and exosome‐free EC supernatants. Western blot analysis revealed that the majority of LC3II is present in the total EC supernatant, less is present in the microvesicle‐free EC supernatant, and LC3II is not present in the exosome‐free EC supernatant. LCI levels were equivalent in all EC fractions. Thus, prolonged NLRP3 inflammasome activation enhances the secretion of autophagy‐derived vesicles that are similar in size to microvesicles or exosomes. (Support: NIH‐R01‐GM36387)