D17-05 Protective Role of the TREK-1 (K2P2.1) Potassium Channel Against Pseudomonas Aeruginosa Pneumonia

Abstract Background and Rationale Pseudomonas aeruginosa (PA) lung infections significantly contribute to inpatient morbidity and mortality rates in bacterial pneumonia patients despite antibiotic therapies and oxygen supplementation. However, besides these interventions, currently no molecular targets exist that translate into improved patient outcomes. In previous studies we found a potentially protective role for TREK-1 potassium channels against viral lung infections, but whether this also applies to bacterial pneumonia remains unknown. Therefore, in this study we use a live PA pneumonia model to test the hypothesis that TREK-1 activation causes protective epithelial and endothelial cell membrane hyperpolarization, which inhibits voltage-gated calcium channel opening and downstream Ca2+-dependent inflammatory mediator secretion. Methods We intratracheally (i.t.) infected mice with PA (CFU: 20x106 ) and once signs of infection (hypothermia) developed, administered once daily two structurally-different pharmacological TREK-1 activators (ML335, BL1249) for 72 hours. We quantified bronchoalveolar lavage fluid (BALF) cellular infiltration, oxidative stress (ROS, H2O2, SOD, catalase) and inflammatory cytokine levels, and changes in BALF and plasma metabolites. To further dissect the electrochemical signaling processes occurring during PA infection, we infected primary human alveolar epithelial and endothelial cells with PA (MOI: 10) for 6 hours, with or without delayed treatment with TREK-1 activators (ML335, BL1249) to mimic a therapeutic regimen. We then quantified alterations in (a) TREK-1 expression using qPCR and ELISA, (b) membrane potential using high-throughput FLIPR assay, (c) intracellular Ca2+ (iCa2+) levels using Fluo-4-based assays, (d) biochemical and physiological markers of injury (pro-inflammatory cytokine secretion, oxidative stress levels) using ELISA and fluorometric assays, respectively. Results PA infection resulted in downregulation of TREK-1 expression in mouse lungs tissue and in primary lung epithelial and endothelial cells. Pharmacological activation of residual TREK-1 channels reduced PA-induced cellular infiltration (macrophages and neutrophiles) and inflammatory cytokine levels in BALF. Similarly, TREK-1 activation reduced oxidative stress markers in BALF, as evidenced by recovery of superoxide-dismutase levels in PA treated mice, and reduced ROS and hydrogen peroxide production. Metabolomic studies in mouse BALF and plasma indicate that i.t. PA infection decreases the GSH/GSSG ratio via an NADPH-dependent pathway, which can be counteracted by TREK-1 activation. At the cellular level, epithelial and endothelial PA infection causes membrane depolarization, raises iCa2+ levels, and promotes iCa2+-dependent oxidative stress and inflammatory cytokine secretion (IP-10, MCP-1, IL-6), which can be counteracted by TREK-1 activation. Conclusion TREK-1 channel activation can activate anti-inflammatory protective responses in PA infection and thus represents a potential new target for therapeutic intervention. This abstract is funded by: NHLBI R01HL175464 (AS); NHLBI R01HL146821 (AS); American Heart Association 25POST1366363 (NM); AHA award DOI: https://doi.org/10.58275/AHA.25POST1366363.pc.gr.227247 (NM) ; American Lung Association CA- 1374253 (NM) ; Department of Defense: W81XWH2210040 (T.Z.) ; American Heart Association: 23CDA1052265 (T.Z.), AHA award DOI: https://doi.org/10.58275/AHA.23CDA1052265.pc.gr.168003;