Immune Response of Human Polymorphnuclear Neutrophils to Aspergillus Fumigatus

Abstract

Invasive fungal infections with Aspergillus fumigatus show an increasing incidence due to the growing number of severely immunocompromised patients. Polymorphonuclear neutrophils (PMNs), as part of the innate immune system, are key players in antifungal immune responses, recognizing fungal pathogens at an early step of infection. Besides phagocytic mechanisms, PMNs kill pathogens by the release of reactive oxygen species (ROS). Human PMNs were isolated from blood of healthy donors using Biocoll separation. Cells were co-cultivated with conidia, germlings and hyphae of the clinical strain ATCC 9197. The oxidative burst was determined in a kinetic measurement quantifying dichlorfluorescein production. Chemotaxis was analyzed by transwell assays. Furthermore, total RNA was extracted and gene expression profiling was performed using Affymetrix U133Plus2.0 arrays. We could demonstrate that A. fumigatus represents a strong stimulus releasing ROS, depending on the morphotype; germlings revealed high ROS release, whereas resting conidia showed low stimulation capacity. PMNs actively tracked germlings and directly attached to fungi as demonstrated by real-time microscopy. Transwell assays revealed that chemotaxis of PMN is strongly dependent on the fungal morphotype; PMN showed only weak chemotaxis in the presence of conidia, whereas in the presence of germlings, high chemotactic activity was achieved. Furthermore, after 6h co-cultivation of PMNs and A. fumigatus germ tubes, 195 (1.273) genes showed an at least 4fold (2fold) altered gene expression. We observed upregulation of genes (hemoxygenase, heat shock 70kDa protein, HSPA8, HSPA1B, HSP90AB1, Ferritin) involved in self-protection against radicals. Additionally, genes involved in inflammatory responses (IL-8, CCL3, CXCL2, IL1RN) were significantly upregulated. Luminex analysis was performed for TNF-a, IL-12, GM-CSF, IFN-γ, IL-6, IL-8, IL-10 and IL-1b to identify secreted cytokines, thereby confirming array data. In conclusion, A. fumigatus had substantial effects on the activation of human PMNs. Various defence strategies were initiated, including phagocytosis, ROS release and mobilization of other immune effector cells by secretion of chemoattractants.