NanoString Gene Expression Profiling in Chronic Lung Allograft Dysfunction (CLAD)

Abstract

CLAD, mainly manifest as bronchiolitis obliterans syndrome (BOS), is the leading cause of late death in lung recipients. BOS develops despite routine immunosuppression and its pathogenesis is poorly understood. The histological correlate of BOS is bronchiolitis obliterans (BO), or airways fibrosis. We quantified over 700 immune-related genes in BO affected airways to better understand immune responses active in end-stage BOS. Formalin fixed paraffin embedded lung tissue was procured at retransplantation for BOS (n=6). Unused donor tissue was used as normal control (n=5). Histologies of interest were isolated using laser capture microdissection (LCMD). Within each BOS lung, airways histologically affected by BO pathology, defined as intraluminal fibrous expansion, were isolated. Additionally, airways unaffected by evident fibrosis were isolated. Within each donor lung, histologically normal airways were isolated. Cellular RNA was extracted and multiplex gene expression analysis was performed using the NanoString PanCancer Immune Profiling Panel. Linear regression was used to compare gene expression among groups. The primary comparison was differential expression in BO affected vs. normal donor airways. We also compared expression in BO affected vs. unaffected airways within BOS explant lungs. P-values were corrected for multiple comparisons using Benjamini-Hochberg. Sixty-seven genes were significantly different between BO affected vs. normal donor airways, of which 52 had a fold change (FC) <0.5 or >2. Along with genes in adaptive immunity, several genes related to innate and humoral activation were significantly increased in BO affected airways. Among the innate immune genes with increased expression in BO were toll-like receptor 1 (FC 2.31) and its adaptor molecule CD14 (FC 5.26) in addition to the complement subcomponent C1s (FC 3.06), while those in humoral immunity included bone marrow stromal antigen 2 (FC 3.41), important to the growth and development of B-cells. No genes were significantly differentially expressed between BO affected vs. unaffected airways within BOS explant lungs. Our results suggest innate and humoral immune responses are active in airways affected by BO pathology, and also in airways without evident fibrosis. Ongoing analyses of BO lesions through LCMD will provide new insights into CLAD pathogenesis.