Investigation of ER Stress in Respiratory Diseases with in Vitro and in Silico Models

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Examensarbete för masterexamen
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2022
Författare
Reijer, Jessica
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Chronic respiratory diseases are amongst the most common causes of death worldwide. Two of these are chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). COPD is the third leading cause of death worldwide and characterized by irreversible respiratory airflow limitations, with cigarette smoke as one of the most common causes of disease. IPF is characterized by irreversible scarring of the distal lung and often advances into respiratory failure and death within five years of diagnosis. Endoplasmic reticulum (ER) stress has been observed in these respiratory diseases. The aim of this project was to investigate ER stress and the unfolded protein response (UPR) with in vitro and in silico models. The in vitro models included induction of UPR in primary human bronchial epithelial cells (HBECs) by exposure to well characterized ER stress inducers, tunicamycin and thapsigargin, as well as stimuli associated with chronic respiratory disease, H2O2 and cigarette smoke extract. Gene and protein readouts of the UPR pathway, that are characterized markers used to monitor ER stress and UPR, were established and cell viability examined. Gene expressions of for example BiP, CHOP, spliced XBP1 and PPP1R15A and protein expressions of eIF2α, phospho-eIF2α, BiP and ATF6 were induced in HBECs after exposure to the stimuli used. In the in silico model, an UPR gene signature was investigated in single cell RNA sequencing (scRNA-seq) datasets from patients with IPF and COPD, to link epithelial cell type specific transcriptomic markers of UPR to the diseases and investigate the chances in UPR response of different epithelial cell subtypes at gene expression level. Epithelial club cells and aberrant basaloid cells were the most abundant cell subtypes found in IPF and with the highest up-regulation of UPR. In COPD, UPR genes were mostly down-regulated across epithelial cell subtypes. This project was performed at AstraZeneca in the Bioscience COPD IPF Department within the Early Respiratory and Immunology R&D unit located in Gothenburg, Sweden.
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COPD, IPF, ER stress, Unfolded protein response, in vitro, in silico, human bronchial epithelial cells, qPCR, Western blot, scRNA-seq
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