{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T21:02:12Z","timestamp":1791838932412,"version":"4.3.4"},"reference-count":44,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T00:00:00Z","timestamp":1765756800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"\n Background<\/jats:title>\n \n Pseudomonas aeruginosa(P.aeruginosa)<\/jats:italic>\n employs c-di-GMP to govern biofilm formation and virulence, enhancing bacterial adhesion and immune evasion. In pleural infections, c-di-GMP simultaneously promotes invasion and modulates neutrophil extracellular traps (NETs), though its mechanistic role remains undefined. While c-di-GMP\u2019s pathogenic contributions are established, its specific regulation of NETs formation during pleural infection has not been explored. Our study investigates c-di-GMP-driven NETs generation in murine infection models.\n <\/jats:p>\n <\/jats:sec>\n \n Method<\/jats:title>\n \n C57BL\/6J mice were infected with\n P.aeruginosa<\/jats:italic>\n strains expressing high (PAO1\u25b3wspF), wild-type (PAO1), or low (PAO1\/plac-yhjH) c-di-GMP levels to establish pleural infection.\n In vitro<\/jats:italic>\n , c-di-GMP levels and biofilm formation of strains were verified using pcdrA-gfp reporter, crystal violet staining, and confocal microscopy.\n In vivo<\/jats:italic>\n , pleural histopathology was observed via HE staining, confocal microscopy and scanning electron microscopy (SEM); RNA sequencing (RNA-seq) of mouse pleural lavage fluid cells was performed, followed by Mfuzz clustering, KEGG\/GO enrichment analysis, and protein-protein interaction (PPI) network construction to identify key regulatory genes. Candidate genes (e.g.,\n Cybb<\/jats:italic>\n ,\n Mapk14<\/jats:italic>\n ,\n Pik3cd<\/jats:italic>\n ) were validated by qRT-PCR. NETs formation was quantified via immunofluorescence, and measurements of cell-free DNA (cf-DNA) and myeloperoxidase-DNA (MPO-DNA) complexes in pleural lavage fluid.\n <\/jats:p>\n <\/jats:sec>\n \n Result<\/jats:title>\n \n In vitro<\/jats:italic>\n , PAO1\u25b3wspF showed the highest c-di-GMP level and biofilm formation, followed by PAO1 and PAO1\/plac-yhjH.\n In vivo<\/jats:italic>\n , infected mice exhibited thoracic purulent secretions and parietal pleura thickening (vs. PBS control), with no obvious morphological differences among the three infected groups. RNA-seq and Mfuzz clustering identified 5 continuously downregulated gene clusters in infected mice; KEGG enrichment showed 48 genes enriched in the \u2018neutrophil extracellular trap formation\u2019 pathway. PPI network analysis screened 10 hub genes (including\n Cybb<\/jats:italic>\n ,\n Mapk14<\/jats:italic>\n ,\n Pik3cd<\/jats:italic>\n ), whose differential expression was confirmed by qRT-PCR (p&lt;0.05). NETs detection revealed a c-di-GMP-dependent trend: PAO1\u25b3wspF induced the strongest NETs response (highest immunofluorescence intensity, cf-DNA, and MPO-DNA levels), followed by PAO1 and PAO1\/plac-yhjH.\n <\/jats:p>\n <\/jats:sec>\n \n Conclusions<\/jats:title>\n \n This work reveals how\n P.aeruginosa<\/jats:italic>\n \u2019s c-di-GMP controls NETs formation in murine pleural infections, bridging bacterial virulence mechanisms with host immune responses. By identifying key regulatory genes, it establishes groundwork for targeted anti-biofilm therapies against chronic infections.\n <\/jats:p>\n <\/jats:sec>","DOI":"10.3389\/fimmu.2025.1610266","type":"journal-article","created":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T06:29:44Z","timestamp":1765780184000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Regulation of neutrophil extracellular trap formation by c-di-GMP in Pseudomonas aeruginosa-induced pleural 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