UHRF1-Mediated Maintenance DNA Methylation is Required for Induced Regulatory T Cell Function Following Influenza Induced Lung Injury in Young Adult Mice [invitro RNA-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. CD4+Foxp3+ cells (nTregs) and CD4+FoxP3- conventional T cells were isolated from the spleens and lymph nodes of Uhrf1fl/fl and Uhrf1+/+Foxp3GFP-CreERT2Rosa26SorCAG-tdTomato mice. nTreg and CD4+FoxP3- conventional T cells were cultured with (IL-2 and αCD3/αCD28) or iTreg skewing media (TGF-β, IL-2, αCD3/αCD28), respectively, plus tamoxifen for 5 days (denoted as the "early" cohort). Cells were transitioned to resting media on day 5 (IL-2 only) and cultured for an additional 7 days. A separate cohort of cells was cultured with IL-2 and αCD3/αCD28 or iTreg skewing media (TGF-β, IL-2, αCD3/αCD28) for 5 days but not supplemented with tamoxifen until after the transition to resting media (denoted as the "delayed" cohort). Foxp3GFP+Tomato+ cells from both cohorts were flow cytometry sorted on days 5 and 12 of culture. RNAseq was leveraged to profile the gene expression of nTregs and iTregs that lost Uhrf1 concurrent with or after Foxp3 induction.