Session Type: Abstract Submissions (ACR)
Background/Purpose: Self-activating NLRP3 mutations are responsible for cryopyrin-associated periodic fever syndromes (CAPS), the most severe form of which is neonatal-onset multisystem inflammatory disease (NOMID). Spontaneous inflammatory responses initiated by NLRP3 mutations promote inflammasome-mediated IL-1beta processing and release, and can induce a rapid cell death in a process known as pyronecrosis that is dependent on cathepsin B activation. Emerging evidence suggests a role for mitochondria in activation of the inflammasome, and mitochondrial STAT3 has been implicated in regulating cellular respiration. The aim of this study was to determine whether STAT3 is involved in IL-1b release and pyronecrosis.
Methods: We used whole blood cells from NOMID and healthy donors, THP-1 cells with STAT3 expression knocked down, THP-1 cells with NLRP3 expression knocked down, and monocytes derived from NRLP3 deficient mice. Cells were stimulated with LPS in the presence of inhibitors of STAT3, followed by ATP. Cell supernatants were collected and incubated with IL-1beta-capturing beads. Cells were fixed and permeabilized. Then beads were added back to cells, and the mixture of cells with beads was stained with anti-IL-1beta, CD14, CD16, and CD83 antibodies and then evaluated by flow cytometry. LPS stimulated cells were also evaluated using immunofluorescent, electron microscopy and western blot analysis. In addition, cell metabolism pathways were analyzed using an XF extracellular flux analyzer.
Results: By flow analysis we found that a small population of monocytes, which undergoes pyronecrosis and is characterized by CD14hi/CD16low and intracellular CD83 expression, is responsible for the majority of IL-1beta production and release. Using confocal microscopy to visualize pyronecrosis, we provided and evidence that this process is NLRP3 dependent. Blockade of STAT3 function with inhibitors leads to complete inhibition of IL-1beta processing and release, as well as pyronecrosis in NOMID and ATP-stimulated healthy donor monocytes and in THP-1 cells. Similarly, STAT3 knockdown in THP-1 cells significantly inhibited IL-1beta release and pyronecrosis. Enhancement of the mitochondrial membrane potential in STAT3 knockdown cells bypasses the effect of STAT3 knockdown, and leads to increased IL-1beta processing and release, which was reversed with inhibitors of oxidative phosphorylation and glycolysis.
Conclusion: Taken together, these data suggest a previously unrecognized role for mitochondrial STAT3 in mediating NLRP3 effects on pyronecrosis and IL-1beta release, and provide a novel therapeutic target for NOMID and other NRLP3-mediated inflammatory diseases.
J. H. Edwan,
J. J. Chae,
R. T. Goldbach-Mansky,
R. A. Colbert,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/evidence-that-stat3-controls-nlrp3-inflammasome-dependent-release-of-il-1%ce%b2-and-pyronecrosis-through-regulation-of-mitochondrial-activity/