New insights into NET formation after contact of polymorphonuclear neutrophils to Aspergillus fumigatus

M. Hasenberg1, S.Wolke2,3, A. Brakhage2,3,M. Gunzer1

Author address: 

1 Institute for Molecular and Clinical Immunology, Otto-von-Guericke-University, MAGDEBURG, Germany, 2Leibniz Institute for Natural Product Research and Infection Biology Hans-Knoell-Institute (HKI), JENA, Germany, 3Friedrich-Schiller-University

Abstract: 

Since their discovery in 2004 nucleic extracellular traps (NETs) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. Recently it was found by us and others that neutrophil granulocytes release NETs also upon contact to the filamentous fungus Aspergillus fumigatus. In the present study we aimed to characterize this process in more detail focusing on the kinetics of NET-formation as well as clarifying the responsible cell-biological mechanisms. By the use of several microscopic techniques (Scanning electron microscopy, fluorescence widefield microscopy and confocal microscopy) we initially demonstrated the generation of NET like structures after coincubation of A. fumigatus germlings and freshly isolated murine or human PMN in vitro. The analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of NETs up to 3 hours later. Moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. Therefore we added the NADPH-oxidase inhibitor DPI to the cell coincubation and found clearly reduced NET formation. By fluorescence staining of reactive oxygen species we could demonstrate that ROS are released prior to NET detection. Currently we aim to visualize NET formation in a murine in vivo infection model by 2-photonmicroscopy. First results show NET like structures around sources of infection in explanted lungs of fungi treated animals. To summarize our data, we found rapid NET formation as a commonly observed immune response of neutrophil granulocytes contacting A. fumigatus in vitro and in vivo. Consistent with studies on different pathogens this mechanism seems to be ROS-dependent. The time course of NET-formation and the ROS-dependency in vivo has to be investigated by us in future. Furthermore we focus on establishing an innovative infection model to perform intravital 2-photon-imaging in the respiratory organs of mice.
2009

abstract No: 

P204

Full conference title: 

4th Trends in Medical Mycology
    • TIMM 4th (2012)