Unraveling the signaling mechanisms behind Histoplasma capsulatum-induced neutrophil NETosis.

The release of neutrophil extracellular traps (NETs) - web-like structures composed of extracellular DNA and antimicrobial proteins - is a key innate immune response mechanism against different pathogens, including fungi like Histoplasma capsulatum, the primary etiological agent of histoplasmosis. Previously, we demonstrated that H. capsulatum yeasts induce NETs formation in human neutrophils through a lytic oxidative process involving reactive oxygen species (ROS), CD18, Src family kinases, and Syk, contributing to fungal killing. In this study, we further elucidated the intracellular signaling pathways involved. Pharmacological inhibitors or neutralizing antibodies were used to evaluate the involvement of different signaling pathways in the production of ROS and NETs by purified human neutrophils in response to H. capsulatum yeasts. Fluorimetric assays were used to detect NETs and ROS. NETs were visualized using confocal fluorescence microscopy. We found that NETs formation occurs gradually and involves CD18, PI3K and Akt signaling, and the enzymatic activities of myeloperoxidase and neutrophil elastase. Notably, NETs release does not require live fungi. Importantly, both NETs release and ROS generation depend on the class IA PI3K δ isoform, while the class IB PI3K γ is involved in NETs formation but is dispensable for ROS production. ROS generation specifically requires CD11b/CD18, PI3K δ, Akt, and myeloperoxidase, but not elastase activities, suggesting that ROS production occurs downstream of PI3K-Akt-myeloperoxidase signaling. These findings clarify key molecular events in neutrophil responses to H. capsulatum and may inform future therapeutic strategies for managing histoplasmosis.