GE Hayes, P Wang, DW Denning, P Bowyer


Inhalation of A.fumigatus spores represents the primary mode of human infection, disease manifestations being dependent on the underlying state of the host immune system. The bronchial epithelium is increasingly recognised as the first line of defence against inhaled Aspergillus however the transcriptional/translational responses of the epithelium are poorly defined. We describe the change in the transcriptional response of the bronchial epithelium to challenge with A. fumigatus over 12 hours establishing key biological pathways and processes involved in the early epithelial response to infection.
Confluent monolayers of the transformed epithelial cell line 16HBE were challenged with live A. fumigatus conidia. RNA was harvested at 0.5 hours, 3 hours, 6 hours, 9 hours and 12 hours and sequenced using the Illumina TruSeq® platform. Data analysis was undertaken using a Tophat2 – DESEQ2 pipeline then by Ingenuity Pathway Analysis using a filter of p<0.05, any log2fold change, and comparison of infected samples to the corresponding uninfected time point control.
11,022 genes were statistically viable for analysis. At 0.5 hours 23 genes were differentially regulated with fold changes between -1.625 and +1.113. All genes mapped to processes involved in normal cellular function and processing. At three hours the transcriptional response changed little with 24 genes being upregulated, most genes involved in maintenance of the basal cell state.
At 6 hours the transcriptional response changed significantly with 305 genes upregulated and 152 downregulated. Network analysis at this time point demonstrates strong upregulation of pathways involved in organisation of the cytoskeleton (79 genes), organisation of the cytoplasm (84 genes) and endocytosis (22 genes). Further interrogation demonstrates genetic upregulation in microtubule dynamics, the formation of lamellipodia, and the organisation and formation of actin filaments. This strongly suggests changes occur in the cytoskeleton of the bronchial epithelium in response to germinating conidia, perhaps facilitating conidial internalisation. Further canonical pathway analysis demonstrates epithelial adherens junction signalling and epidermal growth factor signalling, known to be involved in MAPK and subsequent JAK-STAT signalling, are enriched in the data set at this time point. No genes were significantly up or down regulated at 9 hours.
At 12 hours 31 genes were upregulated, 62 were down regulated. Genes mapped to pathways involved in cellular apoptosis, recognition of cell damage and reduction in chemotaxis of myeloid and phagocytic cells (activation Z scores of -2.159 and -1.709 respectively).
At no time point was a prominent inflammatory transcriptional response demonstrated. Network analysis of upstream regulators across the time points has demonstrated a prominent role for MAPK driven signalling pathways at 3, 6 and 12 hours.
The transcriptional response of the bronchial epithelium to challenge with A. fumigatus is greatest at 6 hours. Associated changes in the cytoskeleton and the cytoplasm may suggest a phagocytic epithelial response, or represent cellular responses to early damage to the monolayer. The lack of a significant transcriptional inflammatory response at any time point may suggest the bronchial epithelium presents a muted and permissive inflammatory response to early Aspergillus infection allowing latent reservoirs of infection to develop.


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7th Advances Against Aspergillosis conference