Maintenance DNA methylation is required for induced Treg reparative function following viral pneumonia in mice
Anthony M. Joudi, Jonathan K. Gurkan, Qianli Liu, Elizabeth M. Steinert, Manuel A. Torres Acosta, Kathryn A. Helmin, Luisa Morales-Nebreda, Nurbek Mambetsariev, Carla Patricia Reyes Flores, Hiam Abdala-Valencia, Samuel E. Weinberg, Benjamin D. Singer
Anthony M. Joudi, Jonathan K. Gurkan, Qianli Liu, Elizabeth M. Steinert, Manuel A. Torres Acosta, Kathryn A. Helmin, Luisa Morales-Nebreda, Nurbek Mambetsariev, Carla Patricia Reyes Flores, Hiam Abdala-Valencia, Samuel E. Weinberg, Benjamin D. Singer
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Research Article Immunology Inflammation Pulmonology

Maintenance DNA methylation is required for induced Treg reparative function following viral pneumonia in mice

Abstract

FOXP3+ natural regulatory T cells (nTregs) promote resolution of inflammation and repair of epithelial damage following viral pneumonia–induced lung injury, thus representing a cellular therapy for patients with severe viral pneumonia and the acute respiratory distress syndrome. Whether in vitro–induced Tregs (iTregs), which can be rapidly generated in substantial numbers from conventional T cells, also promote lung recovery is unknown. nTregs require specific DNA methylation patterns maintained by the epigenetic regulator ubiquitin-like with PHD and RING finger domains 1 (UHRF1). Here, we tested whether iTregs promote recovery following viral pneumonia and whether iTregs require UHRF1 for their pro-recovery function. We found that adoptive transfer of iTregs to mice with influenza virus pneumonia promotes lung recovery and that loss of UHRF1-mediated maintenance DNA methylation in iTregs leads to reduced engraftment and a delayed repair response. Transcriptional and DNA methylation profiling of adoptively transferred UHRF1-deficient iTregs that had trafficked to influenza-injured lungs demonstrated transcriptional instability with gain of transcription factors that define effector T cell lineage. Strategies to promote the stability of iTregs could be leveraged to further augment their pro-recovery function during viral pneumonia and other causes of severe lung injury.

Authors

Anthony M. Joudi, Jonathan K. Gurkan, Qianli Liu, Elizabeth M. Steinert, Manuel A. Torres Acosta, Kathryn A. Helmin, Luisa Morales-Nebreda, Nurbek Mambetsariev, Carla Patricia Reyes Flores, Hiam Abdala-Valencia, Samuel E. Weinberg, Benjamin D. Singer

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Figure 6

Loss of UHRF1 results in delayed transcriptional changes over the course of viral pneumonia.

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Loss of UHRF1 results in delayed transcriptional changes over the course...
Foxp3GFP-DTR mice were treated with DTx every 48 hours beginning 2 days before inoculation with 6.5 PFU of influenza A/WSN/33 H1N1 virus and then received retro-orbital adoptive transfer of 1 × 106 Foxp3-GFP+tdTomato+ Uhrf1fl/fl or Uhrf1+/+ iTregs at 5 DPI as in Figure 1. iTregs were treated with tamoxifen from day 0 to day 3 of culture, then harvested for adoptive transfer on culture day 5. Adoptively transferred Foxp3-GFP+tdTomato+ Uhrf1fl/fl or Uhrf1+/+ iTregs were sorted from the lungs of recipient Foxp3GFP-DTR mice 11 or 24 DPI and were compared via RNA transcriptomic and DNA methylation analysis. (A) PCA of 2,117 DEGs identified from ANOVA-like testing with FDR q < 0.05. Independent biological replicates are shown. (B) GSEA dot plot highlighting key statistics (FDR q value and NES) and enriched gene sets for Uhrf1+/+ iTregs at 11 DPI. Blue dots denote gene sets with a negative enrichment score or enrichment at the bottom of the ranked list. (C) Selection of representative enriched GO processes from 40 total enriched gene sets with an FDR q < 0.25 in Uhrf1+/+ iTregs at 11 DPI. Gene sets are annotated and ranked by –log10-transformed FDR q value. (D) K-means clustering of 34 differentially methylated regions with a difference of ≥10% between Uhrf1+/+ and Uhrf1fl/fl iTregs. (A–D, 24 DPI Uhrf1+/+ iTreg recipients n = 9, Uhrf1fl/fl iTreg recipients n = 7, 11 DPI Uhrf1+/+ and Uhrf1fl/fl iTreg recipients n = 8).