Dual organism transcriptomics of airway epithelial cells interacting with conidia of Aspergillus fumigatus

Abstract

Background: Given the complex nature of the responses that can occur in host-pathogen interactions, dual transcriptomics offers a powerful method of elucidating these interactions during infection. The gene expression patterns of Aspergillus fumigatus conidia or host cells have been reported in a number of previous studies, but each focused on only one of the interacting organisms. In the present study, we profiled simultaneously the transcriptional response of both A. fumigatus and human airway epithelial cells (AECs).

Methodology: 16HBE14o- transformed bronchial epithelial cells were incubated with A. fumigatus conidia at 37°C for 6 hours, followed by genome-wide transcriptome analysis using human and fungal microarrays. Differentially expressed gene lists were generated from the microarrays, from which biologically relevant themes were identified. Human and fungal candidate genes were selected for validation, using RT-qPCR, in both 16HBE14o- cells and primary AECs co-cultured with conidia.

Principal findings: We report that ontologies related to the innate immune response are activated by co-incubation with A. fumigatus condia, and interleukin-6 (IL-6) was confirmed to be up-regulated in primary AECs via RT-qPCR. Concomitantly, A. fumigatus was found to up-regulate fungal pathways involved in iron acquisition, vacuolar acidification, and formate dehydrogenase activity.

Conclusion: To our knowledge, this is the first study to apply a dual organism transcriptomics approach to interactions of A. fumigatus conidia and human airway epithelial cells. The up-regulation of IL-6 by epithelia and simultaneous activation of several pathways by fungal conidia warrants further investigation as we seek to better understand this interaction in both health and disease. The cellular response of the airway epithelium to A. fumigatus is important to understand if we are to improve host-pathogen outcomes.

Figure 1. Localization of A. fumigatus conidia within the airway epithelial cell monolayer.

GFP-expressing A. fumigatus conidia and primary AECs were co-incubated for 6 hours and treated with DAPI and monoclonal E-cadherin Alexa 594 antibody before visualization by confocal microscopy. Labeling of nuclei (blue) and the membrane tight junctional protein E-cadherin (red) allowed visualization of AECs. Some GFP-expressing A. fumigatus conidia (green) are found outside the cells, while others localize within the cell monolayer, in close association with AEC nuclei.

Figure 2. Relative mRNA expression levels of human genes obtained by RT-qPCR.

RNA was obtained from four co-incubations each of 16HBE14o- (grey bars) or primary normal bronchial epithelial cells (AECs) (white bars) with conidia of A. fumigatus. Height of each bar represents expression of gene in co-incubated condition relative to cells alone control (mean ± SE). (* p<0.05).

Figure 3. Relative mRNA expression levels of A. fumigatus genes as determined by RT-qPCR.

RNA was obtained from four co-incubations each of A. fumigatus conidia with 16HBE14o- (grey bars) or primary human bronchial epithelial cells (AECs) (white bars). Height of each bar represents expression of gene in co-incubated condition relative to conida alone control (mean ± SE). (* p<0.05).