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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE307854 | GEO | 2026/03/25

REPOSITORIES: GEO

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Project description:Fibrotic interstitial lung disease (ILD) are lung disorders characterized by the accumulation of extracellular matrix, ultimately resulting in the destruction of the pulmonary scaffold. Continuous pro-fibrotic signaling perpetuates the remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. Studies that address this detrimental crosstalk between lung epithelial cells and fibroblasts are key to understanding ILD. With the aim of identifying functionally relevant targets that drive mesenchymal-epithelial crosstalk and their potential as new avenues to therapeutic strategies, we developed an organoid co-culture system based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells and lung fibroblasts from ILD patients as well as IMR-90 controls. While organoid formation capacity and organoid size was comparable in the presence of ILD or control lung fibroblasts, metabolic activity was significantly increased in ILD co-cultures. Alveolar organoids cultured with ILD fibroblasts further demonstrated reduced stem cell function supported by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. In order to identify key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used secretome mass spectrometry to identify key signals secreted by end stage ILD lung fibroblasts. Over 2000 proteins were detected in a single-shot experiment with 47 differentially upregulated proteins when comparing ILD and non-chronic lung disease control fibroblasts. The secretome profile was dominated by chemokines of the C-X-C motif family, including CXCL1, -3, and -8, all interfering with (epithelial) growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating 3D monocultures of iAT2s with IL11 we recapitulated the co-culture results obtained with primary ILD fibroblasts including changes in metabolic activity as well as organoid formation capacity and size. In summary, our analysis identified mesenchyme-derived mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD by using sophisticated alveolar organoid co-cultures indicating the importance of cytokine-driven aberrant epithelial differentiation and confirmed IL11 as a key player in ILD using an unbiased approach.

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

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