Saccharomyces cerevisiae β-Glucan Training Induces a Nonclassical PGE2−High/NO−Low Macrophage Phenotype in Response to Pseudomonas aeruginosa Exopolysaccharide

Exopolysaccharide (EPS), a major constituent of Pseudomonas aeruginosa biofilms, protects bacteria from M1-macrophage-mediated clearance while promoting chronic inflammation. This study investigated how Saccharomyces cerevisiae β-glucan (BG)-induced macrophage training reshapes subsequent inflammatory and antimicrobial responses to EPS. Peritoneal macrophages from C57BL/6 mice were trained in vitro with BG and subsequently stimulated with EPS purified from a clinical P. aeruginosa isolate obtained from a patient with severe cystic fibrosis. Cytokines, prostaglandin E₂ (PGE₂), and nitric oxide (NO) were quantified, and global proteomic profiling was performed. BG training amplified EPS-induced secretion of TNF-α, IL-6, and additional pro-inflammatory mediators. Trained macrophages also showed markedly increased PGE₂ production, minimal NO release, and reduced phagocytic activity. Proteomic analyzes confirmed upregulation of PGE₂-biosynthetic enzymes and suppression of inducible NO synthase, along with enhanced expression of antimicrobial and immunoregulatory factors, including platelet factor 4, antileucoproteinase, C1q components, and selected chemokines. These data reveal a previously uncharacterized macrophage state—neither M1 nor M2—emerging specifically from BG training and defined by high PGE₂ and low NO production in response to EPS. S. cerevisiae BG training reprograms EPS-stimulated macrophages toward a distinct, non-classical trained-immunity phenotype characterized by elevated PGE₂ and suppressed NO production. This newly defined phenotype represents a novel form of trained immunity and highlights the dual proinflammatory and immunoregulatory roles of macrophage-derived PGE₂. These findings suggest that targeted macrophage reprogramming may offer a promising therapeutic strategy for mitigating P. aeruginosa biofilm-driven chronic inflammation, including in cystic fibrosis.