Proteomic Analysis of Transbronchial Biopsies to Discover Novel Biomarkers for Early Identification of Chronic Lung Allograft Dysfunction

Abstract

Background: Chronic lung allograft dysfunction (CLAD) is a major contributor to poor long-term survival after lung transplantation (LTx). There is a paucity of validated tissue biomarkers which limits the early detection of CLAD. The aim of this study was to discover novel tissue proteins in CLAD.

Methods: A longitudinal cohort study analyzed 15 tissue specimens from 2 groups of bilateral LTx recipients; those with CLAD (n = 3) and those without CLAD (n = 3). In both groups, transbronchial biopsies (TBBx) were retrieved from 2 timepoints; stable surveillance at 90 d after transplant, and during episodes of acute lung allograft dysfunction. In the CLAD cohort, additional tissue from explant CLAD lungs collected at retransplantation was analyzed. Proteomics analysis and immunohistochemistry were used to identify and validate differentially expressed proteins.

Results: Tissue upregulation of a number of proteins including SerpinB1, SerpinH1, Cofilin 1, MUC1, COL15A1, COL4A4, and Coronin1B was found in recipients with CLAD. This finding was present when comparing CLAD onset and explant pathology to stable surveillance among recipients with CLAD and evident when compared with recipients without CLAD. Most of the upregulated tissue proteins in patients with CLAD had collectively critical roles in leukocytes migration and activation, inflammation, free radicals production and oxidative stress, epithelial-mesenchymal transition, myofibroblasts activation, and excessive deposition of extracellular matrix, which in turn enhance the risk of lung fibrosis and graft rejection. We also found exclusive expression of HLA-DQB1, JCHAIN, SAP18, FUCA1, MZB1, G3BP2, and BTF3 in CLAD cases, indicating they could be specific biomarkers of CLAD.

Conclusions: This study identifies distinct proteomes that are linked to CLAD development and consequently may be a useful indicator for identifying LTx patients at higher risk of CLAD.

FIGURE 1.

CONSORT diagram describing the study procedures (left side) and histopathologic features of TBBx in different groups (right side). TBBx sections of stable surveillance (A), CLAD onset (B), and explant pathology in patients who developed CLAD (C). TBBx sections of stable surveillance (D) and ALAD in patients without diagnosis of CLAD (E). All images taken at 40× magnification. ALAD, acute lung allograft dysfunction; CLAD, chronic lung allograft rejection; FFPE, formalin-fixed paraffin-embedded; KEGG, Kyoto Encyclopedia of Genes and Genomics; LC-MS, liquid-chromatography mass spectrometry; LTx, lung transplantation; TBBx, transbronchial biopsy.

FIGURE 2.

Proteomic homology and Volcano plot of DEPs in CLAD patients. A, Venn diagram showing the overlap of expressed proteins between all groups. A total of 862 (41.16%) proteins were found to be expressed in all 3 groups. The protein homology between stable surveillance with CLAD onset (B) and explant pathology was 50.1% (966 shared proteins) (D) and 53.75% (895 shared proteins), respectively. Volcano plot of DEPs between stable surveillance and CLAD onset (C), stable surveillance and explant pathology (E). Volcano plot depicts the log2 fold change (x-axis) vs -log10 Q value (y-axis, representing the probability that the protein is differentially expressed). P < 0.05 and fold change ≥1.25 were set as the significant threshold (red line) for differential expression. Dots in green denote significantly upregulated proteins which passed the screening threshold. Black dots present non-significantly DEPs. CLAD, chronic lung allograft rejection; DEPs, differentially expressed proteins.

FIGURE 3.

GO enrichment analysis of DEPs at the time point of CLAD onset compared with stable surveillance in patients with diagnosis of CLAD. The GO analysis for biological process (A), cellular component (B), and molecular function enriched by DEPs (C). The enrichment score is the ratio of the number of DEPs annotated to this pathway term to the total number of proteins annotated to this pathway term. A higher enrichment factor indicates greater intensiveness, a lower P value means greater intensiveness. The dot size represents the number of DEPs annotated to the pathway. CLAD, chronic lung allograft rejection; DEPs, differentially expressed proteins; GO, gene ontology.

FIGURE 4.

GO enrichment analysis of DEPs in explant tissues compared with stable surveillance in patients with diagnosis of CLAD. The GO analysis for biological processs (A), cellular component (B), and molecular function enriched by DEPs (C). The enrichment score is the ratio of the number of DEPs annotated to this pathway term to the total number of proteins annotated to this pathway term. A higher enrichment factor indicates greater intensiveness, a lower P-value means greater intensiveness. The dot size represents the number of DEPs annotated to the pathway. CLAD, chronic lung allograft rejection; DEPs, differentially expressed proteins; GO, gene ontology.

FIGURE 5.

Proteomic homology and Volcano plot of DEPs between patients with and without a diagnosis of CLAD. A, Venn diagram showing a high overlap (71.07%) of expressed proteins between the time points of ALAD and stable surveillance. B, Volcano plot of DEPs between ALAD and stable surveillance showed a mild difference. C, The protein homology between ALAD and CLAD onset was 67.53%. E, Venn diagram showing a mild overlap (57.37%) of expressed proteins between the time points of stable surveillance in CLAD and non-CLAD groups. F, Volcano plot of DEPs between the time points of stable surveillance in CLAD and non-CLAD groups showed a significant difference. Volcano plot depicts the log2 fold change (x-axis) vs -log10 Q value (y-axis, representing the probability that the protein is differentially expressed). P < 0.05 and fold change ≥1.25 were set as the significant threshold (red line) for differential expression. Dots in green denote significantly upregulated proteins which passed the screening threshold. Black dots present non-significantly DEPs. D, Volcano plot of DEPs between ALAD and CLAD onset showed a significant difference. ALAD, acute lung allograft dysfunction; CLAD, chronic lung allograft rejection; DEPs, differentially expressed proteins.

FIGURE 6.

Representative immunohistochemistry staining of target proteins in TBBx of LTx patients. IHC staining confirmed the presence and upregulation of SerpinB1 (green color; A), Coronin 1B (blue color; B), and Serpin H1 (purple color; C) and Cofilin 1 (Green color) in TBBx of CLAD and non-CLAD groups. Staining intensity of Serpin B1, Coronin 1B, Serpin H1, and Cofilin 1 was higher in CLAD onset and explant TBBx compared with non-CLAD cases, whereas the staining intensity in stable surveillance state was mild either in CLAD or non-CLAD cases. Original magnification ×63. CLAD, chronic lung allograft rejection; IHC, immunohistochemistry; LTx, lung transplantation; TBBx, transbronchial biopsies.

FIGURE 7.

Proposed mechanisms of upregulated proteins in CLAD development. Our proteomics analysis revealed upregulation of several proteins (orange color) that are collectively involved in leukocytes migration and activation to the allograft, inflammasome formation, ROS production and oxidative stress, apoptosis, EMT process, excessive ECM deposition, collagen biosynthesis, and fibrosis which in turn enhance the risk of CLAD onset and graft rejection. CLAD, chronic lung allograft rejection; ECM, extracellular matrix; EMT, epithelial–mesenchymal transition; EOS, eosinophilia; ROS, reactive oxygen species.