title: Multiple Cathepsins Promote Pro-IL-1β Synthesis and NLRP3-Mediated IL-1β Activation.
abstract: Sterile particles induce robust inflammatory responses that underlie the pathogenesis of diseases like silicosis, gout, and atherosclerosis. A key cytokine mediating this response is IL-1β. The generation of bioactive IL-1β by sterile particles is mediated by the NOD-like receptor containing a pyrin domain 3 (NLRP3) inflammasome, although exactly how this occurs is incompletely resolved. Prior studies have found that the cathepsin B inhibitor, Ca074Me, suppresses this response, supporting a model whereby ingested particles disrupt lysosomes and release cathepsin B into the cytosol, somehow activating NLRP3. However, reports that cathepsin B-deficient macrophages have no defect in particle-induced IL-1β generation have questioned cathepsin B's involvement. In this study, we examine the hypothesis that multiple redundant cathepsins (not just cathepsin B) mediate this process by evaluating IL-1β generation in murine macrophages, singly or multiply deficient in cathepsins B, L, C, S and X. Using an activity-based probe, we measure specific cathepsin activity in living cells, documenting compensatory changes in cathepsin-deficient cells, and Ca074Me's dose-dependent cathepsin inhibition profile is analyzed in parallel with its suppression of particle-induced IL-1β secretion. Also, we evaluate endogenous cathepsin inhibitors cystatins C and B. Surprisingly, we find that multiple redundant cathepsins, inhibited by Ca074Me and cystatins, promote pro-IL-1β synthesis, and to our knowledge, we provide the first evidence that cathepsin X plays a nonredundant role in nonparticulate NLRP3 activation. Finally, we find cathepsin inhibitors selectively block particle-induced NLRP3 activation, independently of suppressing pro-IL-1β synthesis. Altogether, we demonstrate that both small molecule and endogenous cathepsin inhibitors suppress particle-induced IL-1β secretion, implicating roles for multiple cathepsins in both pro-IL-1β synthesis and NLRP3 activation.
authors.affiliation: Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655;
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Orlowski
authors.affiliation: Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655;
authors.forename: Jeff D
authors.initials: JD
authors.lastname: Colbert
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01655;
authors.forename: Shruti
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; and.
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
authors.affiliation: Sandler Center for Drug Discovery, University of California, San Francisco, San Francisco, CA 94158.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Robertson
authors.affiliation: Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655; Kenneth.Rock@umassmed.edu.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Rock
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D058847:Inflammasomes / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D010641:Phenotype; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D058847:Inflammasomes; D053583:Interleukin-1beta; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C492716:Nlrp3 protein, mouse; D002403:Cathepsins