The Interaction Of Aspergillus Fumigatus And Airway Epithelial Cell

BacgroundIt is worthy of being noted that the exacerbation of asthma and the fatality rate exhibit seasonal and diurnal rhythm. A. fumigatus is a ubiquitous fungus with airborne conidia with a diameter of 2 - 3um, small enough to penetrate deeply into the airways and may reach the lung alveoli. Inhalation of conidia and adhesion to epithelial cells generally is followed by germination and formation of hyphae which is the major characteristic of invasive manifestation of aspergillosis. In patients with allergic bronchopulmonary aspergillosis it has been shown that aspergillus hyphae grow on and between epithelial cells suggesting that hyphal growth is dependent on the binding of spores to cell surface structures. Furthermore, sensitization to fungal antigens has been associated with more severe and life-threatening asthmatic reactions, especially in childhood asthma. The epithelium of the airways plays a key position to participate in local airway inflammation, which may thus contribute to the pathogenesis of airway disease. Recognition systems employed by airway epithelial cells to respond to microbial exposure may include the action of the toll-like receptors (TLRs). which are pattern recognition receptors that recognize conserved molecular patterns on microbes and link innate and adaptive immune systems. The production of cytokines by the airway epithelium such as IL-6 and IL-8 activated by environmental allergens may play a role in causing inflammation associated with respiratory diseases.ObjectiveTo study the interaction of airway epithelial cell line A549 and primary bronchial epithelial cell with fragments of mycelium, spores of A. fumigatus in vitro and to determine if toll-like receptors (TLRs) are involved in the process.MethodsA549 cells and the primary bronchial epithelial cells were exposed to fragments of A. fumigatus mycelium, zymosan. and inactivated A. fumigatus spores. Interleukin 6 (IL-6) and IL-8 released by A549 cells to the culture supernatant were measured by ELISA. Presence of TLR2 and TLR4 on A549 cells were studied by immuno-histochemisty. The phagocytosis of spores by A549 cells were determined by MGG staining.ResultsAfter 24h of incubation with the stimuli no apparent morphological change or detachment of epithelial cells was observed. After washing the plates twice with RPMI1640, most of the zymosan and HAF were washed away but UAF, WIAF and UWIAF and irradiated spores remained firmly attached to the epithelial cells. Non-irradiated spores started to germinate and after incubation for another 24h mycelium was growing while about one third of A549 cells were detached from the culture plate. Irradiated spores did not germinate after 24h and 48h of incubation and the morphology of A549 cells and the primary epithelial cells did not change apparently. It was observed that the majority of the spores remained attached to the outer surface of the epithelial cells while a smaller number of them apparently showed phagocytosis by epithelial cells. A549 cells spontaneously released IL-8 and IL-6 that increased in time. Firm binding of mycelium fragments of A. fumigatus to A549 cells had limited effects on the release of IL-6 and IL-8 by A549 cells. Irradiated A. fumigatus spores were partly internalised by A549 cells and inhibited A549 cells to produce IL-6. TNF-a pre-incubated A549 cells produced increased IL-6 after exposure to zymosan and WIAF. Immuno-histochemistry showed a negative staining for TLR2 and TLR4.ConclusionsThe low levels of cytokines by A549 cells after binding of either mycelium or spores of A. fumigatus may lead to insufficient recruitment of inflammatory cells to the infected site, which may result in the escape of detection by the immune defence...