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Respiratory tract infections are a global health problem. The main causative agents of these infections are influenza A virus (IAV), <i>Staphylococcus aureus</i> (<i>S. aureus</i>), and <i>Streptococcus pneumoniae</i> (<i>S. pneumoniae</i>). Major research focuses on genetics and immune responses in these infections. Eicosanoids and other oxylipins are host-derived lipid mediators that play an important role in the activation and resolution of inflammation. In this study, we assess, for the first time, the different intracellular profiles of these bioactive lipid mediators during <i>S. aureus</i> LUG2012, <i>S. pneumoniae</i> TIGR4, IAV, and corresponding viral and bacterial co-infections of 16HBE cells. We observed a multitude of altered lipid mediators. Changes in the amount of 5-hydroxyeicosatetraenoic acid (5-HETE) were prominent for all bacterial infections. The infection with <i>S. pneumoniae</i> showed the strongest impact on bioactive lipid production and led to alterations in the amount of PPAR&#947; ligands and precursors of pro-resolving lipid mediators.