Analysing disease-specific variants of thymic stromal lymphopoietin (TSLP) in asthma

Abstract

TSLP is a regulator of Th2 immune responses and is linked to allergic disease. TSLP has been shown to be up-regulated in the epithelium in severe asthma, but regulation of TSLP and its splice variants in bronchial fibroblasts (HBFs) is relatively unexplored. We tested the hypothesis that TSLP is expressed in HBFs in a disease severity-dependent manner. HBFs were derived from bronchial biopsies of non-asthmatic, mild asthmatic and severe asthmatic donors (n=5-7 per group). Cells were stimulated with tumour necrosis factor (TNF)-α and/or interleukin (IL)-4. TSLP mRNA was measured by RT-qPCR at 8h, and TSLP protein release by ELISA at 24h; TSLP biological activity was assessed by TSLP-receptor/STAT-5 promoter/luciferase reporter gene assay (RGA). Semi-quantitative analysis of TSLP splice variant expression was performed using primers specific for long form (lf) or short form (sf) TSLP. Regardless of disease status, TSLP mRNA expression was induced by TNF-α; IL-4 alone did not induce TSLP, but augmented expression in the presence of TNF-α. However, stimulation of TSLP protein release decreased as asthma severity increased (non-asthmatic > mild > severe), as determined by both ELISA and RGA. Analysis of TSLP isoforms showed that sfTSLP was detected in all HBFs but the amount of lfTSLP decreased as asthma severity increased; this correlated with TSLP detection levels by ELISA. Western blots of cell lysate from HEK293T cells transfected with lfTSLP or sfTSLP expression vectors suggested that the anti-TSLP antibodies used only detected lfTSLP. HBFs from severe asthmatic donors preferentially make sfTSLP which may not influence Th2 responses. Further information about the role of TSLP in HBFs is required.