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LPS PROMOTES ASC‐SPECK FORMATION IN TREK‐1‐DEFICIENT MACROPHAGES
Author(s) -
Immanuel Camille N.,
Teng Bin,
Dong Brittany,
Gordon Elizabeth,
Luellen Charlean,
Cormier Stephania,
Fitzpatrick Elizabeth A.,
Schwingshackl Andreas,
Waters Christopher M.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.549.7
Subject(s) - inflammasome , chemistry , western blot , mg132 , caspase 1 , potassium channel , lipopolysaccharide , pharmacology , microbiology and biotechnology , atp sensitive potassium channel , biochemistry , apoptosis , proteasome inhibitor , biology , endocrinology , glibenclamide , receptor , gene , diabetes mellitus
BACKGROUND TREK‐1 is a 2‐pore potassium channel that we previously showed played a protective role against hyperoxia‐induced lung injury. To determine whether this protective effect was related to reduced inflammasome activation, we investigated LPS‐induced responses. Our preliminary studies with macrophages from TREK‐1−/− mice indicated a reduction in NLRP3 inflammasome priming and activation compared with wt mice, but we found that prolonged LPS exposure promoted increased IL‐1β release. We therefore investigatElied non‐canonical inflammasome pathways. METHODS We stimulated alveolar macrophages (AMs) and bone marrow derived macrophages (BMDMs) using both a short‐term LPS+/−ATP model of inflammasome priming and activation and a prolonged‐LPS exposure. wt, TREK‐1−/−, and NLRP3−/− AMs and BMDMs were pre‐treated with a TREK‐1‐inhibitor (SPADIN), a TREK‐1‐activator, a Pannexin inhibitor, a P2X channel inhibitor, an ASC‐oligomerization inhibitor, or a JNK‐inhibitor. We measured IL‐1β, NLRP3, caspase‐1, ASC, TNFα as well as caspase−8, −7, and −11, and phosphorylated‐JNK using western blot, immunostaining, and ELISA. We used a membrane permeable potassium indicator to measure potassium efflux during ATP exposure. Using a caspase‐luciferace assay, we then measured caspase‐activity +/− MG132 (a proteasome inhibitor). RESULTS ATP treatment of LPS‐primed TREK‐1‐inhibited and ‐deficient BMDMs exaggerated caspase‐7 and caspase‐11 activity compared to wt BMDMs, but both were dampened with co‐treatment with Pannexin and P2X inhibitors. Both TREK1 inhibition and deficiency caused elevated potassium efflux after short term LPS + ATP. With prolonged LPS exposure, both wt and TREK‐1−/− BMDMs contained more intracellular potassium than unstimulated wt BMDMs, with the LPS‐treated TREK‐1−/− BMDMs containing the highest concentration. Prolonged LPS exposure increased casp1‐p20, IL‐1β, and ASC‐speck formation in TREK‐1−/− BMDMs and AMs, compared with wt. Similarly, TREK‐1 inhibition with SPADIN increased ASC‐speck formation in both wt and NLRP3−/− BMDMs after prolonged LPS exposure. Additionally, MG132 completely blocked caspase activity in wt BMDMs treated with SPADIN after LPS treatment. JNK inhibition decreased ASC‐oligomerization in both wt and TREK‐1−/− BMDMs. However, prolonged LPS exposure led to increased caspase‐7 and JNK activity in TREK‐1−/− BMDMs compared to wt. Additionally, TREK‐1 inhibition increased caspase‐7 and caspase‐8 activity after LPS exposure in both wt and NLRP3−/− BMDMs. CONCLUSION These results suggest that the protective effect of TREK‐1 may be associated with regulation of apoptotic association ASC‐speck formation. Support or Funding Information Supported by NIH grants HL123540, HL131526, and HL118118, and by the Le Bonheur Children's Hospital Research Foundation. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .