Abstract Rationale Bacterial pneumonia is characterized by cellular inflammation that disrupts the alveolar-capillary barrier, leading to respiratory failure and high mortality rates. Besides antibiotics, current therapies are primarily supportive, and no molecular targets are known that improve patient outcomes. Pseudomonas aeruginosa (P. aeruginosa) poses particular challenges as one of the leading causes of hospital-acquired pneumonia and inpatient mortality. At the cellular level, pulmonary endothelial cells play a central role in sensing infection and amplifying inflammatory responses by releasing cytokines and promoting neutrophil transmigration, ultimately worsening lung injury. Our previous work demonstrated that pharmacological activation of large conductance Ca²+-activated K+ (BK) channels protects mice against P. aeruginosa-induced pneumonia. Importantly, these protective effects were absent in endothelial cell-specific BK knockout (endo BK-KO) mice, suggesting that endothelial cells may play a critical role in BK-mediated protection. Based on these findings, this study investigates the anti-inflammatory effects of BK channel activation against P. aeruginosa infection in primary human lung endothelial cells. Methods Publicly available single-cell RNA seq datasets from the Human Lung Cell Atlas were mined for expression of the BK channel α subunit (Kcnma1) and validated using RT-PCR and ELISA in primary human lung microvascular endothelial cells (HLMEC, Cell Biologics) and human pulmonary artery endothelial cells (HPAEC, Lifeline). To assess the protective mechanisms of BK activation, cells were infected with P. aeruginosa (MOI 10-50) and treated with the BK activator NS1619 (30 μM). Inflammatory and functional endpoints included cytokine profiling (Luminex/Multiplex), intracellular calcium (iCa²+) measurements (Fluo-4 assays), and barrier dysfunction assessment using live trans-endothelial Electric Cell-Substrate Impedance Sensing (ECIS) technology. Neutrophil transmigration across mouse lung microvascular endothelial cell monolayers was performed using Transwell assays. Results Kcnma1 expression was analyzed in publicly available single-cell RNA seq data sets and experimentally validated in primary HPAEC and HLMEC by qPCR and ELISA. P. aeruginosa infection of endothelial cells increased iCa²+ levels, which was counteracted by BK channel activation with NS1619. BK activation also reduced P. aeruginosa- induced inflammatory cytokine secretion (IL-6, CCL-2, CCL11, G-CSF, GM-CSF, INF-γ, IL-1α, Il-1Ra, IL-2, IL-4, IL-8, IL-15, IL-18, M-CSF, MIP-1α, CCL5, TNF-α, TNF-β) from endothelial cells and protected against P. aeruginosa-induced endothelial barrier dysfunction. Furthermore, NS1619 suppressed P. aeruginosa-induced blood neutrophil transmigration across endothelial cells. Conclusions Our findings indicate that endothelial BK channels are key modulators of inflammation and barrier protection during P. aeruginosa-induced inflammation. Targeting BK channels may offer a novel targeted therapeutic approach to reduce P. aeruginosa-induced lung inflammation. This abstract is funded by: 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; 1.AHA 25POST1366363 (NM) AHA award DOI: https://doi.org/10.58275/AHA.25POST1366363.pc.gr.227247 (NM); 2.ALA CA- 1374253 (NM); NHLBI, R01HL175464 (AS) and R01HL146821 (AS)
Zyrianova et al. (Fri,) studied this question.