Aspergillus fumigatus Cell Wall Promotes Apical Airway Epithelial Recruitment of Human Neutrophils.

Aspergillus fumigatus is a ubiquitous fungal pathogen capable of causing multiple pulmonary diseases, including invasive aspergillosis, chronic necrotizing aspergillosis, fungal colonization, and allergic bronchopulmonary aspergillosis. Intact mucociliary barrier function and early airway neutrophil responses are critical for clearing fungal conidia from the host airways prior to establishing disease. Following inhalation, Aspergillus conidia deposit in the small airways, where they are likely to make their initial host encounter with epithelial cells. Challenges in airway infection models have limited the ability to explore early steps in the interactions between A. fumigatus and the human airway epithelium. Here, we use inverted air-liquid interface cultures to demonstrate that the human airway epithelium responds to apical stimulation by A. fumigatus to promote the transepithelial migration of neutrophils from the basolateral membrane surface to the apical airway surface. Promoting epithelial transmigration with Aspergillus required prolonged exposure with live resting conidia. Swollen conidia did not expedite epithelial transmigration. Using A. fumigatus strains containing deletions of genes for cell wall components, we identified that deletion of the hydrophobic rodlet layer or dihydroxynaphthalene-melanin in the conidial cell wall amplified the epithelial transmigration of neutrophils, using primary human airway epithelium. Ultimately, we show that an as-yet-unidentified nonsecreted cell wall protein is required to promote the early epithelial transmigration of human neutrophils into the airspace in response to A. fumigatus Together, these data provide critical insight into the initial epithelial host response to Aspergillus.

Evaluation of an in vitro experimental platform of human polarized intestinal epithelial monolayers for the hazard assessment of insecticidal proteins.

Previous work demonstrated the utility of using human-derived intestinal epithelial cell (IEC) lines cultured as polarized monolayers on Transwell® filters to differentiate between hazardous and non-hazardous proteins. The current study seeks to further resolve appropriate concentrations for evaluating proteins of unknown hazard potential using the IEC experimental platform and leverages these parameters for evaluating the potential toxicity of insecticidal proteins characteristic of those expressed in genetically modified (GM) agricultural biotechnology crops. To establish optimal test protein concentrations, effects of several known hazardous (C. perfringens epsilon toxin, Listeriolysin O, Phaseolus vulgaris erythroagglutinin, E. coli Shiga toxin 1, C. difficile Toxin B and wheat germ agglutinin) and non-hazardous (Ara-h2, ß-lactoglobulin, fibronectin and Rubisco) proteins on IEC barrier integrity and cell viability were evaluated at concentration ranges. Two insecticidal proteins (AfIP-1A and AfIP-1B) were evaluated for effects in the IEC assay, a seven-day insecticidal bioassay, and assessed in a high-dose 14-day acute oral toxicity study in mice. The results obtained from the human in vitro IEC assay were consistent with results obtained from an in vivo acute oral toxicity study, both demonstrating that the combination of AfIP-1A and AfIP-1B do not exhibit any identifiable harmful impacts on mammalian cells.

Pepsin Triggers Neutrophil Migration Across Acid Damaged Lung Epithelium.

Pepsin represents a potential biomarker for extraesophageal reflux disease when detected in airways, however a direct role for pepsin in lung dysfunction has not been clearly established. Children experiencing gastroesophageal and extraesophageal reflux are often prescribed proton pump inhibitors (PPIs) to reduce gastric acid associated damage to esophageal and airway mucosa. The potential of pepsin and gastric fluid, from children that were either on or off PPI therapy, to cause inflammation and damage using a human in vitro co-culture model of the airway mucosa was evaluated herein. Exposure of the airway model to acidic solutions caused cellular damage and loss of viability, however, acid alone did not disrupt barrier integrity or instigate neutrophil trans-epithelial migration without pepsin. Gastric fluid from patients on PPI therapy exhibited only a slightly higher pH yet had significantly higher concentrations of pepsin and elicited more barrier disruption and neutrophil trans-epithelial migration compared to gastric fluid from patients off PPIs. Inflammatory and damaging responses observed with gastric fluid from patients on PPIs were largely driven by pepsin. These results indicate the potential for PPI usage to raise concentrations of pepsin in gastric fluid, which may enhance the pathological impact of micro-aspirations in children with extraesophageal reflux.