Abstract Rationale Pseudomonas aeruginosa (P. aeruginosa) pneumonia is characterized by an inflammatory cytokine storm, cellular inflammation, metabolic dysregulation, and compromised alveolar-capillary barrier, leading to respiratory failure and high mortality. Beyond antibiotics, current treatments remain supportive, and no molecular targets are known to improve outcomes. Our previous work demonstrated that pharmacological activation of large-conductance Ca²+-activated K+ (BK) channels protects mice against P. aeruginosa pneumonia by reducing pro-inflammatory and promoting anti-inflammatory cytokine responses in bronchoalveolar lavage fluid (BALF) and plasma. However, it remains unknown whether alterations in specific metabolic pathways are associated with BK channel-mediated protection against P. aeruginosa infection. Therefore, this study investigates alterations in BALF and plasma metabolites following treatment of P. aeruginosa-infected mice with the pharmacological BK channel activator NS1619. Methods We employed Metabolon’s Global Metabolomics Platform to assess the effect of the BK channel activator NS1619 on BALF and plasma metabolomes in P. aeruginosa-infected mice (20 × 106 CFU) compared to controls. NS1619 (0.66 mg/kg) was intratracheally injected once-daily, and BALF and plasma were collected after 72 hours. A customized computational analysis pipeline was developed using R, Venny, and MetaboAnalyst, integrating metabolite profiling, cross-comparison of overlapping metabolites, network analysis, and pathway enrichment to identify key inflammatory, metabolic, and cellular pathways influenced by NS1619. Results Our LC/MS and GC/MS analyses identified 474 metabolites in BALF and 1,077 in plasma. Using a cutoff of log2FC ≤ -1 or ≥ 1 (p 0.05), we identified 117 BALF and 34 plasma metabolites that were elevated following P. aeruginosa infection, of which 65 BALF and 2 plasma metabolites were reduced by NS1619. Conversely, 19 BALF and 33 plasma metabolites decreased after P. aeruginosa infection, of which 10 BALF and 5 plasma metabolites increased with NS1619 treatment. Network analysis revealed distinct clusters of metabolites that were either reduced or increased by NS1619. The top 10 reduced hub metabolites included uracil, uridine 5′-monophosphate, uric acid, linoleic acid, 1-methylhistidine, eicosapentaenoic acid, docosahexaenoic acid, itaconic acid, L-kynurenine, and sphingomyelin (SM). The top 10 decreased hubs were D-ribose, inosine, xanthine, trimethylamine N-oxide, triethanolamine, NADH, sodium, magnesium, adenosine triphosphate, and guanosine monophosphate. Notably, itaconate, heme, and kynurenine, known regulators of oxidative stress and inflammation, were significantly modulated by NS1619. Conclusions The protective effects of BK channel activation against P. aeruginosa infection are associated with specific alterations in plasma and BALF metabolite profiles. Targeting the identified hub metabolites may represent a promising therapeutic approach to mitigate P. aeruginosa-induced lung injury. This abstract is funded by: NHLBI R01HL175464 (AS); NHLBI R01HL146 (AS); 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; 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)
Boehmer et al. (Fri,) studied this question.