Herth, F. | bioRxiv Science

bioRxiv2023

Epigenetic deregulation of IFN and WNT pathways in AT2 cells impairs alveolar regeneration (in COPD)

Chronic lung diseases, including chronic obstructive pulmonary disease (COPD), affect over 500 million people and are a leading cause of death worldwide. A common feature of both chronic and acute lung diseases is altered respiratory barrier integrity and impaired lung regeneration. We hypothesized that alveolar type 2 (AT2) cells, as alveolar epithelial progenitors, will carry molecular alterations that compromise alveolar regeneration in COPD. Sorted AT2 cells from ex-smokers with and without COPD at different disease stages were subjected to RNA sequencing and whole-genome bisulfite sequencing to generate unbiased transcriptome and DNA methylation maps of alveolar progenitors in the lung. Our analysis revealed genome-wide epigenetic changes in AT2 cells during COPD that were associated with global gene expression changes. Integrative data analysis uncovered a strong anti-correlation between gene expression and promoter methylation, suggesting that dysregulation of COPD-associated pathways in AT2 cells may be regulated by DNA methylation. Interferon (IFN) signaling was the top-upregulated pathway associated with the concomitant loss of promoter DNA methylation. Epigenetic regulation of the IFN pathway was validated in both global and targeted DNA demethylation assays in A549 cells. Notably, targeted DNA demethylation of IRF9 triggered upregulation of IFN signaling, mimicking the effects observed in COPD AT2 cells in the profiling data. Our findings suggest that COPD-triggered epigenetic alterations in AT2 cells may impair internal regeneration programs in human lung parenchyma.

cell biology↗

bioRxiv2020

Versatile workflow for cell type resolved transcriptional and epigenetic profiles from cryopreserved human lung.

The complexity of the lung microenvironment together with changes in cellular composition during disease progression make it exceptionally hard to understand the molecular mechanisms leading to the development of chronic lung diseases. Although recent advances in cell type resolved and single-cell sequencing approaches hold great promise for studying complex diseases, their implementation greatly relies on local access to fresh tissue, as traditional methods to process and store tissue do not allow viable cell isolation. To overcome these hurdles, we developed a novel, versatile workflow that allows long-term storage of human lung tissue with high cell viability, permits thorough sample quality check before cell isolation, and is compatible with next generation sequencing-based profiling, including single-cell approaches. We demonstrate that cryopreservation is suitable for isolation of multiple cell types from different lung locations and is applicable to both healthy and diseased tissue, including COPD and tumor samples. Basal cells isolated from cryopreserved airways retain the ability to differentiate, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing (RNA-seq) and Illumina EPIC Array, we show that genome-wide gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for -omics profiling of human lung cells. In addition, we obtained high-quality single-cell RNA sequencing data of cells isolated from cryopreserved human lung, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our cryopreservation workflow is well-suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to human tissue.

cell biology↗