Proteomic analysis of transbronchial biopsy tissue reveals a distinct proteome and mechanistic pathways in high-grade eosinophilic inflammation after lung transplantation

Abstract

Background: Eosinophilic (EOS) inflammation is associated with acute cellular rejection (ACR) and an increased risk of chronic lung allograft dysfunction (CLAD) after lung transplantation (LTx); however, the underlying mechanisms remain unclear. We aimed to identify potential biomarkers and molecular mechanisms behind EOS inflammation after LTx. Methods: A cross-sectional comparative proteome analysis of transbronchial biopsy (TBBx) tissue was performed using formalin-fixed paraffin-embedded (FFPE) tissue from 18 LTx recipients. The cohort was comprised of isolated EOS inflammation (n=6), ACR (n=6) and these were compared with stable controls (n=6). EOS TBBx were defined as ≥10 eosinophil per high power field without ACR. ACR TBBx was defined as ISHLT A-grade >/=2. Control biopsies were defined as those without ACR, EOS or positive BAL microbiology at 120 days after LTx. Peptides were extracted from TBBx and subjected to liquid-chromatography mass spectrometry. Differentially expressed proteins (DEPs) were identified and quantified using bioinformatic tools and then candidate biomarkers validated by immunohistochemistry (IHC) staining. Results: Using a cut-off of Q<0.05 and a fold change of >1.6 for the variation in expression, 61 DEPs were identified in EOS TBBx, 56 of which were upregulated and 5 were downregulated. High-protein overlap (74.84%) was found between ACR and EOS groups. Several proteins such as Serpins, CFL-1, MIF, DDX3X, CCT8, Gal-3, Coro1A, Collagens and Mucins were upregulated in EOS TBBx. IHC staining validated alterations in the expression of target proteins. Bioinformatic analysis further revealed that most DEPs in EOS TBBx are related to leukocytes migration and activation, inflammasome formation, free radical production and oxidative stress, epithelial mesenchymal transition and excessive deposition of extracellular matrix. Conclusions: This study discovers and validates specific proteomic signatures that link EOS with ACR and elucidates mechanisms of injurious allograft inflammation. A number of novel therapeutic targets and potential early biomarkers are identified for allograft inflammation which require future diagnostic and prognostic validation.