Role of REV-ERBalpha in the regulation of lung inflammation

Abstract

The clock-controlled nuclear receptor REV-ERBalpha has emerged as a critical regulator of multiple pathways involved in metabolism, development and immunity. Recent evidence has highlighted a major role for the clock in epithelial cells regulating lung inflammation, mediated by control of neutrophil chemokine expression. In this thesis, I examined the role of REV-ERBalpha in pulmonary immunity, using in-vivo gene targeting and nebulised lipopolysaccharide (LPS), a model for gram-negative bacterial infection, ex-vivo cell biology approaches and in vitro cell models.Initial studies of Rev-Erbalpha knock-out mice revealed an increase in pulmonary neutrophilia and inflammation upon aerosolised LPS challenge. Moreover, by selectively deleting the REV-ERBalpha DNA binding domain (DBD) in the mouse bronchial epithelium, I observed exaggerated inflammatory responses to LPS and augmented CXCL5 secretion. Interestingly, a dual deletion of REV-ERBalpha DBD and REV-ERBβ in mouse bronchial epithelium had a more dramatic effect on neutrophil recruitment and chemokine secretion than deletion of just the REV-ERBalpha DBD; in both basal and bacterial challenged conditions. Ex-vivo analysis revealed bronchial epithelial cells and macrophages both responded to novel REV-ERBalpha synthetic ligand GSK1362 but displayed divergent inflammatory responses in presence of this compound. Finally, I observed a striking loss of REV-ERBalpha protein upon pro-inflammatory challenge. Further analysis revealed this degradation was dependent on the 26S proteasome and driven by sumoylation and ubiquitination of REV-ERBalpha. However, by using novel REV-ERB ligand GSK1362, these post-translational modifications were blocked and the protein protected from degradation. Collectively, my results propose a new model for a central role for REV-ERBalpha in conferring clock control to lung neutrophilic inflammation. I have also identified a feed-forward circuit activated by inflammatory stimuli, leading to suppression of the endogenous anti-inflammatory REV-ERBalpha protein. Finally, I have discovered a novel mechanism for small-molecule regulation of REV-ERBalpha, operating via suppression of endogenous protein ubiquitination process. These observations implicate REV-ERBalpha as a novel therapeutic target in human inflammatory disease.

Date of Award	1 Aug 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Ray (Supervisor) & Andrew Loudon (Supervisor)