Abstract Rationale Endoplasmic reticulum (ER) stress activation and consequent production of ER chaperones are prominent features of pulmonary fibrosis (PF). Mucin 5B (MUC5B) plays a critical role in mucosal protection and hydration in the respiratory tract. Overexpression of MUC5B is a known risk factor for the development of PF, particularly due to its association with mucus overproduction and the formation of mucus plugs which can exacerbate lung damage. Research indicates that during PF, several ER stress markers, including protein disulfide isomerases (PDIs), are indeed overproduced. Earlier study from our laboratory reported that one of the PDIs, PDIA3, is involved in catalysis of disulfide bonds in key players of lung fibrosis including pro-fibrotic growth factors secreted phosphoprotein 1 (SPP1). By retrospective analysis of proteomics and sc-RNA-seq data sets we found several PDIs including PDIA3 is increased in MUC5B producing cells in IPF. In this study we are elucidating the relationship of PDIA3 and MUC5B in the pathogenesis of PF and the therapeutic potential of the inhibition of PDIA3 on MUC5B expression and improvement of PF. Methods The association of PDIA3 and MUC5B in lung fibrosis was studied by analyses of available RNA-seq and human proteomics data set. A fibrotic cocktail (FC) was developed to study MUC5B and other fibrotic markers in cell culture models. The Bleomycin (BLM) model of lung fibrosis was used to test whether epithelial PDIA3 expression promotes MUC5B production and pulmonary fibrosis in vivo. Two PDI inhibitors Punicalagin (Pun) and LOC14 were used to inhibit PDIA3 in this study. The resulting changes in lung phenotypes were analyzed by histology, immunofluorescence/immunohistochemical staining as well as by RNA and protein expression. Results Analysis of Human IPF single cell RNA-seq data sets showed an increase in PDIA3 in MUC5B+ cells. Total lung RNA-seq shows that an increase in PDIA3 decreases lung function in IPF patients. Proteomics data set shows an increase in PDIA3 in the MUC5B-producing cells located in the honeycomb area. Stimulation of Human Bronchial Epithelial Cells (HBECs) with FC increases MUC5B expression which was reduced after Pun or LOC14 treatment. PDIA3 ablated A549 cells also show decreases in MUC5B expression. The administration of PUN/ LOC14 post 14 days after BLM instillation decreases MUC5B and lung fibrosis in mice. Conclusion Our study demonstrates an association of PDIA3 and MUC5B in pulmonary fibrosis. Inhibition of PDIA3 leads to decrease expression of MUC5B indicating PDIA3 as a potential therapeutic target for PF. This abstract is funded by: R21AI183025-01A1; ALA emerging pathogen award and pathology and lab medicine, UVM
Kumar et al. (Fri,) studied this question.