Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1␤ secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1␤ in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1␤ was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1␤ and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1␤ by antigen-presenting cells in an NLRP3-and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1␤ in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1␤ through at least two separate mechanisms: by targeting pro-IL-1␤ for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.IL-1␤ is an important proinflammatory cytokine, released at sites of infection or injury, that regulates diverse physiological responses, including cellular recruitment, appetite, sleep, and body temperature (1). IL-1␤ is first produced as a proform in response to inflammatory stimuli, such as TLR ligands. This inactive precursor is cleaved into the bioactive (p17) molecule by caspase 1, following the activation of an inflammasome (2).Inflammasomes are molecular scaffolds that trigger the activation of caspase 1 and subsequent maturation of IL-1␤ and IL-18. Typically, inflammasomes are formed from at least one member of the cytosolic innate immune sensor family, the NOD-like receptors (NLRs), including NLRP1, NLRP3, and NLRC4, coupled with the adaptor apoptosis-associated specklike protein containing a caspase recruitment domain (ASC or PYCARD) and caspase 1 (2). The NLRP3 inflammasome is the best characterized to date and is activated by a number of endogenous and exogenous signals.Most studies in vitro employ TLR ligands, particularly LPS, to induce pro-IL-1␤ formation, but in many cases, this is not enough to stimulate inflammasome activation and secretion of the mature cytokine. Instead, a second signal is commonly required, and this can come from a number of endogenous and exogenous sources, including ATP and particulates, including uric acid crystals, amyloid-␤, silica, asbestos, synthetic microparticles, and alum (3-8). Extracellular ATP triggers the P2X7 ATP-gated ion channel, leading to K ϩ efflux and induces recruitment of the pannexin-1 membrane pore (9). This may then allow extracellular NLRP3 agonists to enter the cell and activate inflammasome assembly (9). Particulates have been proposed to act through one of two mechanisms. Uptake of particulates by phagocytes may lead to lysosomal damage and release of lysosomal products into the cytosol, which a...