Transcriptome Analysis of Airway Brushes in Lung Transplant Recipients with and without Chronic Lung Allograft Dysfunction

Abstract

Chronic lung allograft dysfunction (CLAD) in lung transplant recipients (LTRs) limits long-term survival compared to other solid organ transplants. Underlying mechanisms of CLAD are poorly understood. To address the pathogenesis of CLAD at the molecular level, we performed RNA-seq analysis of airway brush samples in LTRs with and without CLAD. LTRs with and without CLAD were evaluated for inclusion. CLAD was staged using ISHLT criteria. RNA was extracted from airway brush samples and sequenced to obtain bulk RNA-seq data. These were subsequently analyzed for differential gene expression (DGE) profiling using false-discovery rate (FDR) p-value of 0.05 and absolute log2 fold change > 2 as thresholds for significance. Gene enrichment and gene and cell ontology analyses of DGE profile was completed to predict canonical pathways and cellular upstream regulator of activated signal pathways where p<0.05 was significant. For subjects with multiple brush samples before and after the development of CLAD, a secondary DGE analysis was completed to evaluate changes in the transcriptome over time. DGE profiling by CLAD stage was also conducted. Results were compared to an external validation cohort, and the area under the curve using receiver-operator characteristic methods (AUC) was evaluated. 24 CLAD and 21 stable control LTRs were included for RNA-seq analysis. The majority of cells identified from brushing were epithelial cells (54%). 293 genes were deferentially expressed between CLAD and non-CLAD. Gene enrichment analyses revealed activation TNF- α (p<1.09E-41), IL-1β (p<5.24E-40), IL-1α (p<8.71E-24), STAT1 (p<7.96E-31), and IFN-γ (p<7.07E-45) in CLAD. Similar patterns of activation over time were observed in 2 LTRs with serial brushes before and after the development of CLAD, and the pattern of activation strengthened in those with CLAD 3 (n=5) versus earlier stages of CLAD. Comparison to the external validation cohort revealed a AUC = 0.73. Transcriptome analyses of bronchial brush samples revealed activation of Type-1 immune responses. Furthermore, this signature appears to strengthen over time as patients progress to CLAD and in later stages of CLAD. Further analyses may provide the rationale for testing select immune targets in CLAD and uncover distinct immune endotypes within CLAD.