Effects Of Aspergillus Fumigatus On The Structure And Function Of Airway Epitheliums

Aspergillus fumigatus (A. fumigatus) is an ubiquitous and important indoor and outdoor allergen. Its spore is so small that it is easy to be inhaled into the airway, and it can also release some proteases. More and more attention is paid to the effects of A. fumigatus on the onset and development of asthma. Airway epitheliums play the most important effects on the allergens sensitization and the devolopment of asthma. Therefore, we detect the effects of A. fumigatus on airway epitheliums using animal asthma model and cultured cells in vitro, in order to explore in further the possible mechanism of A. fumigatus on the onset and development of asthma.Chapter 1 Effects of A. fumigatus spore on the structure and function of airway epitheliums of normal and asthma ratsObject:Explore the effects of A. fumigatus spore on the structure and function of airway epitheliums of normal and asthma rats. Methods: Male Wistar rats were sensitized to ovalbumin (OVA) and challenged with aerosol OVA. All rats were divided into A and B groups. A and B group was to investigate the effects of A.fumigatus spores on asthmatic rats before or after undergoing OVA aerosol challenged, respectively. The parameters associated with Muc5ac and GM-CSF in BALF and variation of airway resistances to Ach in all rats were measured.Results: Compared with normal rats, rats only treated with A.fumigatus spores have significantly the enhanced variation of airway resistances to Ach, increased Muc5ac and GM-CSF in BALF. Compared with asthmatic rats, asthmatic rats treated with A.fumigatus spores, the enhanced variation of airway resistances to Ach and increased Muc5ac and GM-CSF in BALF. Conclusion: A.fumigatus spores enhanced airway responsibility, increased Muc5ac and GM-CSF production. With our previous results, A.fumigatus spores may cause the onset of asthma and aggravate the symptoms of asthma.Chapter 2 Effects of A.fumigatus extract (AFE) on human bronchial epitheliumsObject: Explore the effects and possible mechanism of AFE on human bronchial epitheliums.Methods: Prepare and identification of AFE. To detect the effects of AFE on trans-epithelium electric resistance (TER), Tight Junction proteins occludin and ZO-1, cell wound closure ability in vitro and the production of Muc5ac and GM-CSF by human bronchi epitheliums (16HBE-14o). Using heat-treated AFE, protease-activated receptor-2 agonist and antagonist to explore the possible mechanisms.Results: AFE had significant proteolytic activity and serine protease activity, heat-treated AFE (65℃,30min) lost all proteolytic activity and the serine protease activity could be inhibited entirely with 2mg/L aprotinin. 160mg/L AFE had no effect on the cell viability and cell supernatant LDH level.When added on basal side of monolayer of polar 16HBE, A. fumigatus extracts reduced rapidly epithelial TER in 6 min, followed by recovery in subsequent several minutes, and reduced epithelial TER gradually in later 4 hours. However, if AFE was added to only apical side of the monolayer, the above concentration of AFE could not cause a rapid decrease at 6 min, but could decrease TER gradually, which were positively related to exposure time or the concentration of AFE. AFE led to a progressive cleavage of occludin, NOT zonula occludens-1, which allowing A. fumigatus allergen to cross the epithelial barrier. These changes could be blocked by aprotinin, a serine protease inhibitor. Protease-activated receptor-2 (PAR-2) agonist peptide (SLIGLV- NH2) could mimic the rapid effect of A. fumigatus extracts on epithelial TER at 6 min. Heat-treated AFE, which lost proteolytic activity, could not compromise the permeability of airway epithelium.20-40mg/L AFE could decrease significantly 16HBE wound closure ability in vitro, migration, attachment and spreading. HT-AFE had no effect on cell repair ability.With 8-24mg/LAFE exposure, cell produced more Muc5ac and GM-CSF and expressed more mRNA, compared with normal control group (P<0.01), which were positively related to exposure time or the concentration of AFE. Aprotinin and PAR-2 antagonist (FSLLRY-NH2) inhibited the effect of AFE on Muc5ac and GM-CSF production by 16HBE. PAR-2 activating peptide (SLIGLV- NH2) could stimulate cells release more GM-CSF. Heat-treated AFE, which lost protease activities, exerted no effect on Muc5ac and GM-CSF production and mRNA expression.Conclusion: 40-160mg/L AFE increased the permeability of airway epithelium, by activating PAR-2 and cleaving directly occludin, which allowing allergens to cross the epithelial barrier. 20-40mg/L AFE depending on its protease activity, could damage cell repair ability. 8-24mg/L AFE, depending on its protease activity, activated PAR-2, caused airway epithelial cell to produce and release more Muc5ac and GM-CSF, which may contribute to deterioration of asthma. These results suggest that A.fumigatus could contribute to the onset and development of asthma by the above actions. PAR-2 and protease inhibitor will be the possible therapeutic target and method in preventment and treatment of asthma in future.