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A4484 - TREK1 Deficiency Abrogates LPS-Induced Alveolar Macrophage Inflammasome Priming and Activation
Author Block: C. N. Immanuel1, B. Teng1, B. Dong1, J. Kennedy1, J. Tatum1, C. Luellen1, E. A. Fitzpatrick2, S. Cormier3, A. Schwingshackl4, C. M. Waters5; 1Department of Physiology, University of Tennessee Health Science Center, Memphis, TN, United States, 2Univ of Tennessee Hlth Sci Ctr, Memphis, TN, United States, 3Dept of Pediatrics, University of Tennessee Health Science Center, Memphis, TN, United States, 4Dept of Pediatrics Div of Critical Care Med, Univ of California at Los Angeles, Los Angeles, CA, United States, 5Department of Physiology/PPG, University of Tennessee Health Science Center, Memphis, TN, United States.
RATIONALE: Acute respiratory distress syndrome is a severe form of lung injury often requiring oxygen and ventilation support. Previously, we found that hyperoxia (HO) exposure of mice deficient in the 2-pore potassium (K+) channel TREK1 led to increased lung injury compared to control mice and that TREK1 deficiency was associated with increased IL-1β release in alveolar macrophages (AM) after prolonged LPS exposure. In this study we determined if this increased inflammation in TREK1KO AMs was due to aberrant inflammasome activity. METHODS: Using a LPS+/-ATP model of inflammasome priming and activation in wt, TREK1KO, and TREK1-inhibited, and TREK1-activated AMs and bone marrow derived macrophages (BMDMs), we measured IL-1β, NLRP3, caspase-1, and TNFα using western blot and ELISA; and localized TREK1, NLRP3, ASC, caspase-1, and K+ using immunostaining. We then measured inflammasome activity following LPS stimulation in TREK1KO macrophages in the presence of a TRPV2 channel inhibitor. We performed experiments in BMDMs and conformed key findings with AMs. Additionally, we used an LPS model in which we collected bronchial alveolar lavage (BAL) at three and twenty-four hours after LPS-exposure. RESULTS: To delineate how TREK1 augmented the NLRP3-inflammasome, we studied both priming and activation steps. In response to LPS priming, both deficiency and inhibition of TREK1 caused decreased expression of pro-IL-1β and NLRP3 compared to wt BMDMs. Additionally, TREK1 activation abrogated NLRP3, pro-caspase-1, and pro-IL-1β expression with LPS priming. When TREK1 inhibition occurred during activation, IL-1β and cleaved caspase-1 (casp1p20) were decreased. Both TREK1 inhibition and deficiency caused elevated potassium efflux after short term LPS + ATP. However, with prolonged LPS we found increased casp1p20, IL-1β, as well as ASC-speck formation in TREK1-deficient and inhibited BMDMs, compared with wt. Inhibition of TRPV channels dampened the increased inflammatory reaction of ASC oligomerization and IL-1β secretion in the TREK1-deficient BMDMs after prolonged LPS exposure. CONCLUSION: Taken together, these results suggest that despite decreased NLRP3-inflammasome priming and potassium-associated activation, the protective effect of TREK1 is related to TRPV channel associated ASC-speck formation.