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 2

UHRF1 is dispensable for iTreg FOXP3 expression and suppressive capacity but is required for transcriptional and epigenetic stability in vitro.

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UHRF1 is dispensable for iTreg FOXP3 expression and suppressive capacity...
(A–D) Natural and induced Tregs derived from Uhrf1+/+ and Uhrf1fl/fl mice were exposed to tamoxifen to delete UHRF1 from day 0 to day 5 (“early”) or day 6 to day 12 (“delayed”) of culture, then harvested on days 5 and 12 for flow cytometry and RNA-Seq analysis. (A) Schematic. (B) Frequency of Foxp3-GFP+tdTomato+ iTregs and Foxp3-GFP–tdTomato+ (ex-FOXP3) cells. (C) Percent suppression of CD4+CTV+Foxp3-GFP– splenic responder T cells cocultured for 72 hours at indicated ratios of experimental Tregs. (D) PCA of 6,978 DEGs, identified from ANOVA-like testing with FDR q < 0.05. (E–I) Induced Tregs derived from Uhrf1+/+ and Uhrf1fl/fl mice were exposed to tamoxifen to delete UHRF1 from day 6 to day 12 (“delayed”) of culture, then harvested on day 12 for RNA-Seq and DNA methylation analysis. (E) K-means clustering of 127 genes with an FDR q < 0.05 with k = 2. (F) MA plot comparing gene expression between groups. Genes of interest are annotated. (G) Enrichment plots of gene sets (P < 0.05, FDR q < 0.25) generated through GSEA pre-ranked testing of the expressed genes. (H) PCA of 81,179 differentially methylated cytosines identified from ANOVA-like testing with FDR q < 0.05. Ellipses represent normal contour lines with 1 SD probability. (I) Cumulative distribution function plot of differentially methylated cytosines expressed as β scores, with 0 representing unmethylated and 1 representing fully methylated; a shift in the cumulative distribution function up and to the left represents relative hypomethylation. (B, n = 3 per group; C, n = 3 per group; D–I, n = 2 for Uhrf1fl/fl, n = 3 for Uhrf1+/+). B and C representative of 3 independent biological replicates. *q < 0.05 according to 2-way ANOVA with 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5 (C).