Invasive aspergillosis is a life-threatening infection in immunocompromised patients. Activation of innate immune cells is crucial for the successful control of invasive aspergillosis. Monocytes play an important role in the cellular defense against Aspergillus fumigatus by attacking defined differentiation states of the fungus and by the release of pro-inflammatory cytokines and chemokines. In order to get a better understanding on pathways involved in these defense mechanism, genome-wide expression profiling was performed after incubation of CD14+ selected monocytes with A. fumigatus conidia and hyphae for 3, 6 and 9 hours using the Affymetrix HG-U133A array. Moreover, phagocytosis of conidia was analyzed by immunofluorescence. Expression of defined cytokines and chemokines was confirmed by Real-time PCR and on protein level by ELISA. Phagocytosis of conidia was observed during the first 3 hours (up to 2-3 conidia / monocyte). The conidia became swollen after 6 hours. By genome-wide expression profiling, ~600 genes were differentially regulated after 3 hours of incubation with hyphae and ~200 after incubation with conidia. At 6 and 9 hours of incubation, the number of differentially regulated genes showed no difference (~400 genes by both stimuli). Hyphae but not conidia strongly induced the expression of chemokines attracting neutrophils and T-lymphocytes as well as their receptors already after 3 hours, indicating that phagocytosis of conidia does not immediately induce expression of inflammatory genes. Pentraxin-3 was upregulated after 3 but not after 6 hours by both conidia and hyphae. PTX-3 binds to A. fumigatus and plays a role in initiating phagocytosis. Genes encoding for inflammatory cytokines and chemokines as well as their receptors were found to be up-regulated at 6 and 9 hours by both stimuli. Urokinase plasminogen activator (uPA), which plays an essential role in regulating fibrinolysis and thereby monocyte/macrophage recruitment to sites of inflammation, was also found to be upregulated after stimulation with both stimuli. ELISA experiments confirmed these results on protein level, demonstrating a steady increase of chemokine and cytokine concentrations in culture supernatants from 3 to 9 hours. In conclusion, A. fumigatus differentially regulates a wide number of genes expressed by human monocytes, which play a crucial role in immune response during infection. The expression of these genes is dependent on the differentiation and germination state of the fungus and the incubation period.
Full conference title:
46th American Society for Haematology
- ASH 46th (2004)