Profiling Early Molecular Responses in Primary Human Bronchial Epithelial Air-Liquid Interface Cultures Exposed to Aspergillus Fumigatus Conidia

A. Toor1, L. Culibrk2, G. K. Singhera1, W. Tam1, K. Moon2, A. Prudova2, L. J. Foster2, M. M. Moore3, D. R. Dorscheid1, S. J. Tebbutt1;

Author address: 

1Centre for Heart Lung Innovation/University of British Columbia, Vancouver, BC, Canada, 2University of British Columbia, Vancouver, BC, Canada, 3Simon Fraser University, Burnaby, BC, Canada.

Abstract: 

Rationale
Aspergillus fumigatus (A. fumigatus) is an opportunistic fungal pathogen that is widely distributed in nature through the release of conidia. Upon inhalation, fungal conidia (2-3μm) reach the terminal bronchioles and alveoli where they bind to and may be internalized by airway epithelial cells. This interaction may result in a range of life-threatening diseases in patients, collectively known as aspergillosis. The purpose of our research was to determine the early transcriptional and translational response of host airway cells to conidia. Previous in-vitro studies of the host-pathogen interaction have used airway epithelial cells grown in submerged cultures or primary nasal epithelial Air-Liquid Interface (ALI) 14-day old cultures. In the present study, we used primary human bronchial epithelial (HBE) cells grown for 21-28 days as ALI cultures to recapitulate in-vivo bronchial epithelium. We hypothesize that elucidating the early response of airway epithelial cells is important for understanding the pathogenesis of A. fumigatus that in turn, may improve patient outcomes.
Methods
Primary HBE cells grown as ALI cultures were incubated with and without A. fumigatus conidia for 6 hours at 37C. RNA was extracted and analyzed using nCounter PanCancer Immune Profiling Panel (NanoString nCounter™ platform) (n=5). Proteins were extracted and analyzed by LC-MS/MS using stable-isotope labeling for quantification (n=3). LIMMA (R package) was used to identify differentially abundant genes and proteins. Pathway analysis was conducted using enrichR.
Results
Differential abundance analysis revealed 56 genes and 153 proteins that were differentially abundant upon exposure to A. fumigatus conidia (P-value < 0.05). Of the 56 genes, 40 were up-regulated including genes involved in rearrangement of actin cytoskeleton (e.g., ANXA1, BST, HCK) and apoptosis, (e.g., CASP8, FADD, CTSS), and 16 were down-regulated including genes involved in the complement and coagulation cascades pathways (e.g., CFB, C3 and SERPING1). Of the 153 proteins differentially abundant upon exposure to conidia, 73 proteins were up-regulated. These included proteins involved in protein quality control in the endoplasmic reticulum, e.g., HSP90AA1, PRKCSH, CALR and UBQLN1. The 80 down-regulated proteins included the ribosomal proteins RPL3, RPS8, RPS5 and RPS18.
Conclusion
We developed an in-vitro dual-organism interaction model that closely mimics the exposure of pseudostratified mucocililary in-vivo epithelium to A. fumigatus. NanoString and shotgun proteomics identified key candidate genes and proteins involved in the early host response to conidia. These can now be validated in functional assays as potential mediators of A. fumigatus pathogenesis.

2018

abstract No: 

A5482 / P1262

Full conference title: 

The American Thoracic Society Conference 2018
    • ATS 2018