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DISSECTING THE MECHANISM OF NLRP3 INFLAMMASOME ACTIVATION IN INDIVIDUAL CELLS: THE ROLE OF REACTIVE OXYGEN SPECIES AND ORGANELLE DAMAGE

Heid, Michelle E. (2013) DISSECTING THE MECHANISM OF NLRP3 INFLAMMASOME ACTIVATION IN INDIVIDUAL CELLS: THE ROLE OF REACTIVE OXYGEN SPECIES AND ORGANELLE DAMAGE. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Inflammation underlies the pathology of numerous diseases. It can be initiated by macrophages through the secretion of pro-inflammatory cytokines, such as IL-18 and IL-1β, following the activation of a molecular complex called the inflammasome. Inflammasomes are a protein scaffolding complex consisting of three known components: a sensory NLR, such as NLRP3, ASC, and caspase-1. The NLRP3 inflammasome is activated by a diverse array of stimuli, including crystals, ATP, pore-forming toxins, such as tetanolysin O (TLO), and the potassium ionophore nigericin. Here, we have explored the mechanism of NLRP3 inflammasome activation using nigericin, TLO and ATP. We found that nigericin induced NLRP3 inflammasome activation in bone-marrow derived macrophages (BMDM) results in inflammasome dependent lysosomal membrane permeabilization (LMP), mitochondrial membrane permeabilization (MMP), and the processing and secretion of IL-1β. All of these events required mitochondrial reactive oxygen species (ROS). Through combining bulk biochemical assays with live cell analysis of individual cells, we provide a kinetic analysis and sequence of events resulting from nigericin stimulation of LPS primed BMDM. We recapitulated NLRP3 inflammasome activation in a dendritic cell line, using the novel system of D2SC-1 transduced with ASC. These transduced cells undergo a similar sequence of events as macrophages, confirming that this mechanism is a general result of NLRP3 inflammasome activation applicable to multiple cell types. Furthermore, we have found that ATP and TLO stimulation of BMDM results in a similar sequence of NLRP3 dependent events. ATP, but not nigericin, requires P2X7 for activation of the NLRP3 inflammasome. Surprisingly, inhibition of P2X4 blocked nigericin, but not ATP, induced, NLRP3-dependent IL-1β secretion in BMDM. Our work has demonstrated a central, common role for mitochondrial ROS in NLRP3 inflammasome activation and determined the kinetics of organelle crosstalk during inflammasome activation. These data place both the mitochondria and lysosomes in a critical position controlling NLRP3 inflammasome activation. Based on these results, we suggest mitochondrial ROS as a potential therapeutic target for treating NLRP3 inflammasome related diseases. Inhibition or scavenging of mitochondrial ROS would not only prevent the pro-inflammatory effects of IL-1β secretion in these patients, but also NLRP3 dependent organelle damage and the resulting cell death.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Heid, Michelle E.heidmich@pitt.eduHEIDMICH
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSalter, Russell Drds@pitt.eduRDS
Committee MemberKane, Lawrencelkane@pitt.eduLKANE
Committee MemberMcClane, Bruce A.bamcc@pitt.eduBAMCC
Committee MemberNau, Gerard Jgjnau@pitt.eduGJNAU
Committee MemberSt Croix, Claudette Mcls13@pitt.eduCLS13
Committee MemberWatkins, Simon C.swatkins@pitt.eduSWATKINS
Date: 22 November 2013
Date Type: Publication
Defense Date: 15 November 2013
Approval Date: 22 November 2013
Submission Date: 21 November 2013
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 145
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Immunology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: NLRP3 inflammasome
Date Deposited: 22 Nov 2013 15:10
Last Modified: 15 Nov 2016 14:15
URI: http://d-scholarship.pitt.edu/id/eprint/20071

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