Date of Degree
Access restricted until 07/03/2020
PhD (Doctor of Philosophy)
Molecular and Cell Biology
Sutterwala, Fayyaz S.
First Committee Member
Sutterwala, Fayyaz S.
Second Committee Member
Cassel, Suzanne L.
Third Committee Member
Weiss, Jerrold P.
Fourth Committee Member
Colgan, John D.
Fifth Committee Member
Grumbach, Isabella M.
Sixth Committee Member
Pattern recognition receptors coordinate innate immune responses by sensing infection or injury. Nucleotide-binding, leucine rich repeat, and pyrin domain-containing protein 3 (NLRP3) is a cytosolic PRR which perceives diverse pathogenic and sterile insults. NLRP3 orchestrates inflammatory signaling responses by forming inflammasomes with the adaptor protein apoptosis-associated speck like protein with a caspase recruitment domain (ASC) and the cysteine protease caspase-1. Assembly of the intracellular macromolecular inflammasome complex culminates in proximity-induced autocatalysis of caspase-1. Caspase-1 activation promotes cell death by pyroptosis and activation and secretion of proinflammatory cytokines interleukin (IL)-1β and IL-18. While NLRP3-mediated inflammation protects against bacterial, fungal, viral, and parasitic infections, aberrant NLRP3 activation is implicated in numerous inflammatory diseases and heritable syndromes. Mechanistically, inflammasome activation requires a preliminary NF-κB-activating priming step (signal 1) and a subsequent NLRP3-specific stimulus (signal 2). While there is enormous molecular diversity among NLRP3-specific agonists, this second signal appears to engage a common pathway involving cation flux. Furthermore, NLRP3 associates with mitochondria and mitochondrial damage is implicated in NLRP3 activation, although the precise role for mitochondria in inflammasome assembly remains controversial. We previously demonstrated that the mitochondrial phospholipid cardiolipin binds to NLRP3 and is critical for NLRP3 inflammasome activation.
Here, we further investigated how mitochondria contribute to NLRP3 activation. We found that liposomes containing molar concentrations of cardiolipin that resemble mitochondrial cardiolipin levels can induce NLRP3-dependent caspase-1 autoactivation. Unexpectedly, we discovered that caspase-1 binds directly to cardiolipin, causing inflammasome-independent caspase-1 complex formation and autocatalysis at higher cardiolipin densities. Finding that caspase-1 and NLRP3 are independently capable of binding to cardiolipin, we more thoroughly examined the association of inflammasome components with mitochondria. Normally confined within mitochondrial inner membranes, cardiolipin relocates to outer membranes of stressed mitochondria. We found that reactive oxygen species (ROS) produced in response to signal 1 facilitate cardiolipin externalization to the outer membrane during priming. We also determined that this coincides with ROS-dependent recruitment of NLRP3 and caspase-1 to the outer membrane of mitochondria at priming. In contrast, we found that NLRP3 activation by the signal 2 agonist nigericin induces calcium-dependent recruitment of the adaptor ASC to mitochondria and caspase-1 activation. Finally, to determine what type of mitochondrial damage was necessary to promote NLRP3 inflammasome activation, we examined how different NLRP3 agonists affect mitochondria. We found substantial variability in the extent of mitochondrial damage induced among different NLRP3 agonists. Collectively, our findings illustrate that mitochondria serve as innate immune signaling platforms through multiple stages of NLRP3 inflammasome activation. Further, paralleling lipid A interactions with caspase-11, we have demonstrated that caspase-1 is capable of binding to the phospholipid cardiolipin.
Cardiolipin, Caspase-1, Inflammasome, Mitochondria, NLRP3, SMOC
xviii, 158 pages
Includes bibliographical references (pages 136-158).
Copyright © 2018 Eric Isaac Elliott
Elliott, Eric Isaac. "Regulation of NLRP3 inflammasome activation by mitochondria." PhD (Doctor of Philosophy) thesis, University of Iowa, 2018.
Available for download on Friday, July 03, 2020