UHRF1-Mediated Maintenance DNA Methylation is Required for Induced Regulatory T Cell Function Following Influenza Induced Lung Injury in Young Adult Mice [BiSulfite-seq]

Regulatory T cells (Tregs) promote resolution of inflammation and repair of epithelial damage following lung injury, thus representing a possible cellular therapeutic for ARDS. Foxp3+ natural regulatory T cells (nTregs) require specific DNA methylation patterns maintained by the epigenetic regulator, Ubiquitin-like with PHD and RING finger domains 1, (UHRF1), to function, but the DNA methylation requirements of in vitro induced Tregs (iTregs) remain unknown. Here we tested whether the loss of Uhrf1 would augment the pro-recovery function of iTregs during viral pneumonia. We found that the loss of maintenance DNA methylation in iTregs results in reduced engraftment and a delayed repair response after experimental influenza pneumonia. Transcriptional and DNA methylation profiling of sorted Uhrf1-deficient iTregs post infection provide insight into the mechanisms underlying their impaired ability to promote repair, with Uhrf1-defiicent iTregs demonstrating greater instability via gain of alternate effector T cell lineage-defining transcription factors. Strategies to promote stability iTregs could be leveraged to further augment their pro-recovery function during pneumonia and ARDS. Overall design: CD4+FoxP3- T cells were isolated from the spleens and lymph nodes of Uhrf1fl/fl and Uhrf1+/+Foxp3GFP-CreERT2Rosa26SorCAG-tdTomato mice and cultured in iTreg skewing media plus tamoxifen for 5 days (denoted as the "early" cohort). Cells were transitioned to resting media on day 5 and cultured for an additional 7 days. A separate cohort of cells was cultured in iTreg skewing media for 5 days but not supplemented with tamoxifen until after the transition to resting media (denoted as the "delayed" cohort). To profile genomic CpG DNA methylation and confirm that maintenance DNA methylation was lost upon UHRF1 deletion, DNA libraries utilizing modified reduced representation bisulfite sequencing were generated from sorted Foxp3GFP+Tomato+ cells on day 12 of culture from both cohorts.