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 5

UHRF1 is required for iTreg phenotypic stability and lung tissue engraftment following viral pneumonia.

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UHRF1 is required for iTreg phenotypic stability and lung tissue engraft...
DTx-treated, influenza A–infected Foxp3GFP-DTR recipient mice received retro-orbital adoptive transfer of 1 × 106 Foxp3-GFP+tdTomato+ Uhrf1fl/fl or Uhrf1+/+ iTregs at 5 DPI. iTregs were cultured with tamoxifen from day 0 to day 3, then harvested for adoptive transfer, as in Figure 1. Transferred Foxp3-GFP+tdTomato+ Uhrf1fl/fl or Uhrf1+/+ iTregs were sorted from the lungs of recipient Foxp3GFP-DTR mice 24 DPI for quantification and profiling via bulk RNA-Seq. (A) Frequency of Foxp3-GFP–tdTomato+ (ex-FOXP3) cells. (B) K-means clustering of 1,187 genes with FDR q < 0.05 comparing recovered Uhrf1+/+ and Uhrf1fl/fl iTregs with k = 2. (C) MA plot comparing gene expression of recovered Uhrf1+/+ and Uhrf1fl/fl iTregs. Genes of interest are annotated. (D) GSEA dot plot highlighting key statistics (FDR q value and normalized enrichment score [NES]) and enriched gene sets. Red dots denote gene sets with a positive enrichment score or enrichment at the top of the ranked list. Blue dots denote gene sets with a negative enrichment score or enrichment at the bottom of the ranked list. (E) Enrichment plots of hallmark gene sets generated through GSEA pre-ranked testing of the expressed genes in Uhrf1+/+ and Uhrf1fl/fl iTregs. All gene sets displayed significant enrichment with FDR q value < 0.25. (F) Selected Gene Ontology (GO) processes from 945 and 105 total enriched gene sets with FDR q < 0.25 in Uhrf1fl/fl and Uhrf1+/+ iTregs, respectively. Gene sets are annotated and ranked by –log10-transformed FDR q value. (G) Frequency of tdTomato+ iTregs recovered. (H) Total number of tdTomato+ iTregs recovered. ***P < 0.0005 according to Mann-Whitney U test (A–F, Uhrf1+/+ n = 9, Uhrf1fl/fl n = 7; G, Uhrf1+/+ n = 8, Uhrf1fl/fl n = 7; H, Uhrf1+/+ n = 8, Uhrf1fl/fl n = 6). Data generated from 2 independent experiments.