Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited therapeutics. ER stress and UPR are potential contributors to pulmonary fibrosis; however, the role of ER stress and UPR signaling in lung macrophages remains poorly understood. There is an unmet need to determine the mechanism(s) linking ER stress to macrophage-driven fibrosis and to develop effective therapeutics. Methods WT, Eif2ak3fl/fl and Eif2ak3-/-Cx3cr1creER mice were used for the studies. Briefly, Eif2ak3fl/fl and Eif2ak3-/-Cx3Cr1CreER mice were exposed to bleomycin followed by tamoxifen administration (75 mg/kg, intraperitoneally) at day 14. In a separate cohort, mice received tamoxifen injections prior to bleomycin exposure. Lungs and BAL cells were harvested on day 21 followed by hydroxyproline determination, histological analysis, immunoblotting, qPCR. Primary fibroblasts were isolated from lungs of bleomycin-injured mice and co-cultured with bone marrow derived macrophages (BMDM). The human RPAP2 gene was cloned into an empty V5-His-tag expression vector to investigate phosphatase activity in macrophages. Results Our data showed bleomycin activates ER stress in lung macrophages through PERK phosphorylation. BAL macrophages from Eif2ak3fl/fl displayed increased pro-fibrotic gene expression, while Eif2ak3-/-Cx3Cr1CreER mice showed reduced expression. BAL macrophages from Eif2ak3fl/fl mice showed increased RPAP2 expression. In contrast, Eif2ak3-/-Cx3Cr1CreER mice showed reduced expression suggesting potential involvement of RPAP2 in fibrosis progression. Co-culture studies with isolated fibroblasts from Eif2ak3fl/fl mice and BMDM macrophages showed increased pro-fibrotic gene expression in fibroblasts, while Eif2ak3-/-Cx3Cr1CreER fibroblasts showed significantly reduced expression. BMDM showed increased Tgfb1 gene expression cultured with Eif2ak3fl/fl fibroblasts, whereas BMDM cultured with Eif2ak3-/-Cx3Cr1CreER fibroblasts showed significantly reduced expression. Emerging evidence showed that PERK phosphorylation increased RPAP2 phosphatase expression to dephosphorylate IRE1α. RPAP2 phosphatase activity assay showed reduced IRE1α phosphorylation. Conclusion Bleomycin-induced injury mediated RPAP2 activity to dephosphorylate IRE1α in pulmonary fibrosis. Targeting PERK-RPAP2 axis may open novel therapeutics for the treatment of pulmonary fibrosis. This abstract is funded by: NIH
Pandey et al. (Fri,) studied this question.