부산대학교 도서관

Background: Severe asthma accounts for 3-6% of all asthma patients (Hekking et al., 2015; Backman et al., 2018; Larsson et al., 2018). Despite being a small percentage of all asthma patients, severe asthma accounts for a disproportionate amount of asthma healthcare costs (Bahadori et al., 2009). For these patients their disease remains uncontrolled despite being on maximum therapy, which includes high doses of glucocorticoids (GCs). There are currently five approved biologics that target allergic asthma, driven by type 2 inflammation, and these novel treatments have reduced the dependency of patients on GCs and their number of exacerbations, and improved their quality of life as well as lung function. However this overlooks patients who are not eligible for type-2 biologics and as such asthma still has a heavy reliance on GC therapy. Long-term GC use is associated with significant adverse events. Moreover, the mechanisms underlying GC non-responsiveness remain incompletely understood. In addition, current therapeutics in asthma, which do not suffice, target the smooth muscle and inflammatory components of the disease, given the lack of complete benefit with therapeutics, it is likely that other mechanisms are at play contributing to the pathobiology of disease. The majority of 'omics' research in severe asthma and GC responsiveness assesses total mRNA levels of patients with asthma, ignoring post-transcriptional processes influencing translation of mRNA to protein. We have previously shown the expression of two RNA binding proteins, ZFP36L1 and ZFP36L2, to be decreased in airway epithelial cells at the translational level in severe asthma. The regions targeted by these RNA binding proteins (RBPs), AU-rich elements, are enriched on mRNAs encoding inflammatory mediators, such as those upregulated in asthma. There is also some evidence of ZFP36L1 and ZFP36L2 being regulated by GCs. Our hypothesis is that ZFP36L1 and ZFP36L2 deficiency leads to impaired clearance of target mRNAs contributing to the inflammation present in the airways of patients with severe asthma, and that they modulate the effects of GCs. Methods: We performed quantitative PCR and western blot to assess RNA and protein expression, respectively, in bronchial epithelial cells (BECs) to establish time and dose dependent effects of GCs and inflammatory stimuli. To establish the effects of ZFP36L1/L2 on bronchial epithelium, these RBPs were depleted employing siRNAs. BECs depleted of ZFP36L1/L2 were stimulated with type 1 (IL-1, TNF) and/or type 2 cytokines (IL-4, IL-13) , and/or GCs to establish GC-dependent effects in bronchial epithelium. We performed Frac-seq to assess the genome wide role of ZFP36L1/L2 in GC-mediated responses in BECs. RNA immunoprecipitation and co-immunoprecipitation were conducted to explore the RNA and protein interactions, respectively, between ZFP36L1/L2 and transcripts of interest such as IL6, VEGF, TGFBR2, CCL5, to name a few, as well as protein-protein interactions with the glucocorticoid receptor. Results: We confirmed downregulation of ZFP35L1/L2 levels in the U-BIOPRED dataset and the Wessex severe asthma cohort. We also demonstrate that dexamethasone modulates ZFP36L1 and ZFP36L2 expression at both mRNA and protein levels, in a time and dose-dependent manner. GCs currently used widely in the clinic, namely fluticasone, hydrocortisone and prednisolone can also modulate ZFP36L1/L2 levels. ZFP36L1 expression was increased early on by GCs and decreased over time, while ZFP36L2 was induced and sustained over time. We show a direct interaction between ZFP36L1 and ZFP36L2 with several mRNAs encoding inflammatory factors, namely IL6, IL8, GMCSF, VEGF, as well as with ZPF36L1 and ZFP36L2 transcripts themselves. ZFP36L1/L2 binding of ZFP36L2, GILZ and TGFBR2 transcripts was modulated by GCs. Frac-seq in primary bronchial epithelial cells depleted of ZFP36L1 and ZFP36L2 and treated with dexamethasone demonstrated that GCs act differently in distinct subcellular compartments, and GC responses are in part mediated by ZFP36L1 and ZFP36L2, suggesting downregulation of ZPF36L1 and ZFP36L2 impairs GC-mediated effects. Dexamethasone treatment led to changes in transcription, as previously shown; however, it also showed a major switch in the association of mRNAs to monosomes. ZFP36L1/L2 had a predominant effect on active translation and modulated molecules involved in epithelial integrity, suggesting a novel role for these RBPs as regulators of epithelial structure and which may contribute to airway remodelling in severe asthma. Conclusion: In summary, the results reported in this thesis highlight an important role for ZFP36L1 and ZFP36L2 in GC-mediated effects and pose ZFP36L1/L2 levels as a possible contributor to GC resistance in the epithelium in severe asthma patients. Together this work provides a rationale for exploring post-transcriptional mechanisms of GC regulation to potentially enable therapeutic intervention that could result in reduced GC-dependence and/or achieve GC responsiveness in patients currently resistant to GC. It also highlights ZFP36L1 and ZFP36L2 as a potential biomarker for GC responsiveness and highlights their role in epithelial structure.