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(Unpublished)
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: |
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| ETD Committee: |
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| 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: |
22 Nov 2018 06:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/20071 |
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