Purpose: Aspergillus fumigatus is a ubiquitous fungus that lives on decaying material. The fungus disseminates by producing asexual spores (conidia) that humans inhale every day. For immunocompromised patients this poses a great risk to develop invasive aspergillosis. One cause for the virulence of A. fumigatus are the various secondary metabolites on the conidial surface. A well studied example is the grey-greenish pigment dihydroxynaphthalene (DHN)-melanin. It is a known virulence factor inhibiting degradation of conidia in the phagolysosome of host cells. On the host side research has shown that the neutrophil granulocytes play a decisive role in fighting the infection. Infected mice that lack neutrophils die much earlier compared to mice lacking macrophages. This underlines the importance of studying neutrophil behavior.
Methods: The focus of this study lays on the motility of neutrophils. For this purpose we performed live-cell imaging with human neutrophils isolated from fresh blood. The videos were automatically analysed by a bioinformatical algorithm providing us with a huge amount of information extracted from a video. Our aim was to study the influence of the fungal pigment DHN-melanin on neutrophils. In two experiments we studied the effect of the dissolved monomer 1,8-DHN but also of the supernatant of germinating conidia on neutrophil motility. Also, we analysed the phagocytic activity of neutrophils when coincubated with fungal conidia. Again live-cell imaging videos were analysed regarding the time point of phagocytosis, contact with the conidia before and neutrophil motility.
Results: We could show that the algorithm successfully analysed the videos. In the concentration range of 2.5 nM to 25 nM of 1,8-DHN we observed the following neutrophil behavior: The motility had increased at 2.5 nM of 1,8-DHN. At 2.5 nM and 25 nM of 1,8-DHN the neutrophils moved in a straight line compared to the medium control where they moved in circles. When coincubated with conidia neutrophils were frequently in contact with conidia without phagocytosing them. Phagocytosis increased at later time points with less touching of the conidia beforehand.
Conclusion: The fungal pigment component 1,8-DHN influenced neutrophil motility even at extremely low concentrations (2.5 nM – 25 nM). An interesting observation is that 1,8-DHN makes neutrophils to move in a straight line instead a circle. This points towards a chemotactic response towards 1,8-DHN. Moreover we aim at elucidating whether the concentrations of 1,8-DHN reflect physiological conditions.
It has been known before that conidia release the protective melanin layer during swelling up which increases their recognition. Here we report the kinetics of this process. Moreover we show that neutrophils even touch conidia multiple times without phagocytosing them. In the course of time the conidia lost their protective layers and touching events decreased whereas phagocytosis increased.
Full conference title:
- AAA 8th (2018)