The circadian clock in pulmonary fibrosis: a gain in circadian rhythmicity and new therapeutic opportunities

  • Johannes Meijer

Student thesis: Phd


Introduction: Idiopathic pulmonary fibrosis is a lethal and progressive lung disease with limited treatment options. There is indication in the literature that there is an interaction between pulmonary fibrosis and the circadian clock, though this interaction is poorly understood. An increased understanding of this mechanism may lead to new therapeutic opportunities such as pharmacological modulation of the circadian clock to combat pulmonary fibrosis. The aim of this thesis is to investigate the effect of the circadian clock on pulmonary fibrosis. Firstly, the effect of pulmonary fibrosis on circadian disruption was investigated and the development of the methodology is described. Secondly, a cell type specific ablation of the circadian clock machinery was used to investigate whether this circadian disruption was driven by a specific cell type. And finally, the effect of pharmacological targeting of the circadian clock on (markers of) pulmonary fibrosis was investigated. Method: Precision-cut lung slices were generated from mPER2::luc mice after oropharyngeal bleomycin challenge (3U/kg; 16-21 days). Circadian dynamics in the form of mPER2::luc bioluminescence was recorded via real-time live-tissue microscopy. Bmal1fl/fl mice were crossed with Ccsp-icre and Pdgfrb-cre mice in order to knock out Bmal1 in club cells and (myo)fibroblasts/pericytes respectively. The effect of REV-ERB ligands on myofibroblast differentiation and other markers of fibrosis was investigated in human cell line (MRC-5), human primary lung fibroblasts of IPF and non-fibrotic patients, and in human and murine precision-cut lung slices. Results: The alveolar spaces in non-fibrotic lung parenchyma exhibited no or low amplitude circadian oscillations in precision-cut lung slices of wild type mice. In fibrotic lung parenchyma, however, alveolar spaces exhibited high amplitude circadian oscillations. Lung slices of fibrotic Ccsp-Bmal1-/- mice showed a loss in circadian amplitude around the bronchioles, but the gain of amplitude in fibrotic alveolar spaces was not attenuated. Within lung slices of fibrotic Pdgfrb-Bmal1-/- mice, the gain of circadian amplitude in fibrotic alveolar spaces compared to non-fibrotic alveolar spaces was abolished. REV-ERB ligands were able to inhibit myofibroblast differentiation or markers of fibrosis in cell line and primary human lung fibroblasts and precision-cut lung slices of murine and human origin. siRNA mediated knock-down of NR1D1 (gene encoding for REV-ERBα) decreases the efficacy of the putative REV-ERB ligand GSK4112 to inhibit myofibroblast differentiation. Conclusion: Pulmonary fibrosis induces circadian disruption in the form of an induction of amplitude in alveolar spaces of fibrotic lung parenchyma. This effect was lost in Pdgfrb-Bmal1-/- mice, demonstrating that this effect depends on the circadian rhythmicity of (myo)fibroblasts/pericytes. Pharmacological targeting of the circadian clock via REV-ERB ligands inhibited myofibroblast differentiation in various models. Pharmacological modulation of the circadian clock is therefore a promising therapeutic opportunity to combat pulmonary fibrosis.
Date of Award1 Aug 2020
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorDavid Ray (Supervisor), John Blaikley (Supervisor) & Hannah Durrington (Supervisor)


  • precision-cut lung slices
  • organotypic culture
  • circadian rhythms
  • pulmonary fibrosis

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