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P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.<\/jats:p>","DOI":"10.1165\/rcmb.2024-0059oc","type":"journal-article","created":{"date-parts":[[2024,4,24]],"date-time":"2024-04-24T12:09:22Z","timestamp":1713960562000},"page":"207-218","update-policy":"https:\/\/doi.org\/10.1164\/crossmarkpolicies","source":"Crossref","is-referenced-by-count":17,"title":["<i>Pseudomonas aeruginosa<\/i>\n                    Lipid A Structural Variants Induce Altered Immune Responses"],"prefix":"10.1093","volume":"71","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2798-1382","authenticated-orcid":false,"given":"Casey E.","family":"Hofstaedter","sequence":"first","affiliation":[{"name":"Department of Microbial Pathogenesis"},{"name":"Medical Scientist Training Program"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ian 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