Transcriptomic analyses reveal regional signatures in lung allograft recipients

Background and objective: Long term outcomes of allograft recipients are compromised by the development of chronic lung allograft dysfunction (CLAD) or bronchiolitis obliterans syndrome (BOS) which initiates in the lung periphery. We established baseline transcriptomic profiles of both the large and small airway epithelial cells (referred as LAEC and SAEC, respectively) to identify regional differences irrespective of initiating disease. Methods: We obtained matched primary LAEC and SAEC from lung allograft recipients (n=4, 42.5±4.2 years) and established primary cultures. Bulk RNA sequencing was performed to determine differentially expressed genes. Results: We observed differences in the transcriptional program between LAEC and SAEC Transcription factors (TF) were ranked within the top ten differentially regulated genes. The most abundant TF families included C2H2-ZF, homeobox and bHLH. Upstream regulator analyses identified homeobox genes being significantly in LAEC. Protein-protein interaction network analysis emphasised the role of TFs (ISL1, MSX1, HOXA1, GATA6, ZNF423) in airway modulation. Additionally, functional enrichment analysis revealed the activation of chemotaxis, metalloendipeptidase/metallopeptidase activity and pro-inflammatory signatures (IL17 signalling and RAGE), in LAEC, while SAEC were characterised by elevated expression of surfactant metabolism related genes. Moreover, alveolar and club cells-related genes were expressed in SAEC, suggesting a lower airway-specific signature. Conclusion: Our analysis shows robust transcriptional differences between LAEC and SAEC. We suggest a potential role for homeobox TF family as well as the activation of the immune system in the biology of LAEC. Conversely, we observed an alveoli-like transcriptional signature in SAEC, including gas-exchange signals and surfactant metabolism; pathways involved in lung homeostasis. Overall design: Matched samples from proximal and distal lung biopsies were obtained from lung allograft recipients during the routine surveillance bronchoscopy. Patients did not present symptomatology indicative of bronchiolitis obliterans syndrome (BOS), transplant rejection or infections at the time of sample collection. Specimens were obtained from four patients (3 females, age range 38-47) that underwent double lung transplantation. The reasons for lung transplantation were cystic fibrosis (CF; 3 cases), and congenital heart disease (CHD; 1 case). Both, large and small airway epithelial cells (referred as LAEC and SAEC) were grown as a monolayer to confluency and cells then harvested for transcriptomics analysis.