Airway Transcriptome Analysis and Immune Proteome Profiling of the Lung Allograft Reveals Novel Immune Signatures in Chronic Lung Allograft Dysfunction

Abstract

Purpose Chronic lung allograft dysfunction (CLAD) is the major limitation to long-term survival in lung transplant recipients (LTRs). The underlying biologic mechanisms that drive CLAD are poorly understood. To address the pathogenesis of CLAD at the molecular level, we performed RNA-seq analysis of airway brush samples and Meso Scale Discovery (MSD) multiplex cytokine and chemokine measurements in bronchial lavage (BAL) samples. Methods Total RNA was extracted from distal bronchial brush samples and sequenced using an Illumina instrument to obtain bulk RNA-seq data which were subsequently analyzed for differential gene expression (DGE) profiling with correction for a potential batch effect. Gene enrichment and gene and cell ontology analyses of DGE profile was done and used to predict canonical pathways and cellular upstream regular of activated signal pathways. False discovery rate (FDR) p<0.05 distinguished significantly overrepresented expression in CLAD samples. For upstream regulator analysis, a z-score score above 2.00 with p<0.05 was significant. BAL from corresponding LTRs were subject to the multiplex MSD cytokine and chemokine array. Mann-Whitney U test was used and any analytes with p<0.05 was considered to be significant. Results 21 CLAD and 18 stable control LTRs were included for RNA-seq analysis. Corresponding MSD measurements were performed on 17 CLAD and 11 control BAL samples. 1031 DEGs were overrepresented in CLAD samples. Gene analyses revealed enrichment of the Type-1 adaptive immune response and the inflammasome. 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) were identified as key immune pathways upregulated in CLAD. MSD protein multiplex analysis of BAL demonstrated significantly increased levels of the Type I cytokines IL-12/IL23p40 (p<0.01) and TNF-α (p<0.007), the inflammasome component, IL-1β (p<0.03) and the B cell regulatory cytokine IL-21 (p<0.001) in CLAD LTRs. Conclusion Transcriptome analyses and the lung allograft BAL immune proteome, provide novel insights into the immune activation pathways prevalent in CLAD versus controls. Further analyses may provide the rationale for testing select immune targets in CLAD and uncover distinct immune endotypes within CLAD. Chronic lung allograft dysfunction (CLAD) is the major limitation to long-term survival in lung transplant recipients (LTRs). The underlying biologic mechanisms that drive CLAD are poorly understood. To address the pathogenesis of CLAD at the molecular level, we performed RNA-seq analysis of airway brush samples and Meso Scale Discovery (MSD) multiplex cytokine and chemokine measurements in bronchial lavage (BAL) samples. Total RNA was extracted from distal bronchial brush samples and sequenced using an Illumina instrument to obtain bulk RNA-seq data which were subsequently analyzed for differential gene expression (DGE) profiling with correction for a potential batch effect. Gene enrichment and gene and cell ontology analyses of DGE profile was done and used to predict canonical pathways and cellular upstream regular of activated signal pathways. False discovery rate (FDR) p<0.05 distinguished significantly overrepresented expression in CLAD samples. For upstream regulator analysis, a z-score score above 2.00 with p<0.05 was significant. BAL from corresponding LTRs were subject to the multiplex MSD cytokine and chemokine array. Mann-Whitney U test was used and any analytes with p<0.05 was considered to be significant. 21 CLAD and 18 stable control LTRs were included for RNA-seq analysis. Corresponding MSD measurements were performed on 17 CLAD and 11 control BAL samples. 1031 DEGs were overrepresented in CLAD samples. Gene analyses revealed enrichment of the Type-1 adaptive immune response and the inflammasome. 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) were identified as key immune pathways upregulated in CLAD. MSD protein multiplex analysis of BAL demonstrated significantly increased levels of the Type I cytokines IL-12/IL23p40 (p<0.01) and TNF-α (p<0.007), the inflammasome component, IL-1β (p<0.03) and the B cell regulatory cytokine IL-21 (p<0.001) in CLAD LTRs. Transcriptome analyses and the lung allograft BAL immune proteome, provide novel insights into the immune activation pathways prevalent in CLAD versus controls. Further analyses may provide the rationale for testing select immune targets in CLAD and uncover distinct immune endotypes within CLAD.