Lung epithelial transcriptional responses to bleomycin and effects of IRE1α or TGFβ inhibition

This experiment sought to characterize the epithelial-specific transcriptional response in the bleomycin model of lung fibrosis, and the contribution of IRE1a and the TGFb-activating integrin avb6 to this response. "Ribotag" mice were crossed to ShhCre mice, both on the C57BL/6 background to generate experimental cohorts. Mice used for the experiment conditionally expressed HA-tagged ribosomal protein Rpl22 in the epithelium. Mice were exposed to intranasal bleomycin (3 U/kg) on day 1 and subsequently treated with the IRE1a kinase inhibitor KIRA8 (50 mg/kg daily), or the anti-integrin beta6 antibody 3G9 (10 mg/kg on day 1 and day 4). Treatment controls were either drug vehicle (3% ethanol, 7% Tween80, and 90% saline) or the inert antibody Axum8 in saline). Lungs were harvested on day 7 after bleomycin exposure, flash-frozen, and later homogenized under native conditions with cycloheximide. An aliquot of the “input” homogenate was removed and total RNA purified by Qiagen RNeasy Mini and is representative of the whole-lung transcriptome/translatome. The remaining homogenate was subjected to immunoprecipitation of HA-tagged polysomes, followed by RNA purification by Qiagen RNeasy Micro, and is representative of the lung epithelial transcriptome/translatome. Factorial design. Principal treatment groups are: Saline/Control (6 mice), Bleomycin/Control (9 mice), Bleomycin/KIRA8 (5 mice) and Bleomycin/3G9 (4 mice). Each mouse has a corresponding whole lung and epithelial library.