Regulation of basal and TNFa-induced fibroblast gene expressions by peptidylarginine deiminase 2

Objective: Peptidylarginine deiminases (PADs) modulate immune cell function and contribute to the development of rheumatoid arthritis (RA), which frequently co-occurs with interstitial lung disease (ILD). Fibroblasts exhibit organ-specific function and play pathogenic roles in RA and ILD. This study aims to examine the cell-autonomous role of PADs in regulating gene expression of fibroblasts from various organs. Methods: Fibroblasts of various organs and thioglycolate elicited peritoneal cells were harvested from wild type, PAD2-deficient, or PAD4-deficient mice. Primary human lung fibroblasts were subjected to CRISPR-mediated PAD2 ablation. Bulk RNA sequencing was performed to identify PAD2-regulated genes. Differential expression was subsequently confirmed, and basal and TNFα-induced gene expression were measured, using quantitative PCR, flow cytometry, and/or ELISA. Results: PAD2 transcript levels in lung fibroblasts increased with age, a trend not observed in fibroblasts from other organs. While PAD2 deficiency had a limited impact on the overall transcriptome of lung fibroblasts, it consistently reduced the expression of Podoplanin (PDPN) and TNFRSF1b across fibroblasts from all mouse organs and human lung fibroblasts. Lung fibroblasts exhibited the highest IL-6 expression among all sources of fibroblast. Notably, PAD2 deficiency, but not PAD4 deficiency, attenuated TNFα-induced IL-6 expression in fibroblasts from lung and synovium but not other organs. Conclusion: PAD2 is expressed in lung fibroblasts in an age-dependent manner and cell-autonomously regulates TNFα-induced gene expression of fibroblasts based on their organ origin, particularly lung and synovium. These findings suggest a novel pathogenic role for PAD2 in rheumatoid arthritis (RA) and offer a plausible explanation for the observed lung-synovium connection.