AMPK is necessary for Treg functional adaptation to microenvironmental stress during malignancy and viral pneumonia
Manuel A. Torres Acosta, Jonathan K. Gurkan, Qianli Liu, Nurbek Mambetsariev, Carla Reyes Flores, Kathryn A. Helmin, Anthony M. Joudi, Luisa Morales-Nebreda, Kathleen Cheng, Hiam Abdala-Valencia, Samuel E. Weinberg, Benjamin D. Singer
Manuel A. Torres Acosta, Jonathan K. Gurkan, Qianli Liu, Nurbek Mambetsariev, Carla Reyes Flores, Kathryn A. Helmin, Anthony M. Joudi, Luisa Morales-Nebreda, Kathleen Cheng, Hiam Abdala-Valencia, Samuel E. Weinberg, Benjamin D. Singer
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Research Article Immunology Oncology Pulmonology

AMPK is necessary for Treg functional adaptation to microenvironmental stress during malignancy and viral pneumonia

Abstract

CD4+FOXP3+ Treg cells maintain self tolerance, suppress the immune response to cancer, and protect against tissue injury during acute inflammation. Treg cells require mitochondrial metabolism to function, but how Treg cells adapt their metabolic programs to optimize their function during an immune response occurring in a metabolically stressed microenvironment remains unclear. Here, we tested whether Treg cells require the energy homeostasis–maintaining enzyme AMPK to adapt to metabolically aberrant microenvironments caused by malignancy or lung injury, finding that AMPK is dispensable for Treg cell immune-homeostatic function but is necessary for full Treg cell function in B16 melanoma tumors and during influenza virus pneumonia. AMPK-deficient Treg cells had lower mitochondrial mass and exhibited an impaired ability to maximize aerobic respiration. Mechanistically, we found that AMPK regulates DNA methyltransferase 1 to promote transcriptional programs associated with mitochondrial function in the tumor microenvironment. During viral pneumonia, we found that AMPK sustains metabolic homeostasis and mitochondrial activity. Induction of DNA hypomethylation was sufficient to rescue mitochondrial mass in AMPK-deficient Treg cells, linking AMPK function to mitochondrial metabolism via DNA methylation. These results define AMPK as a determinant of Treg cell adaptation to metabolic stress and offer potential therapeutic targets in cancer and tissue injury.

Authors

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

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Figure 6

AMPKα1 interacts with DNMT1 to demethylate the promoter of mitochondrial genes in tumor-infiltrating Treg cells.

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AMPKα1 interacts with DNMT1 to demethylate the promoter of mitochondrial...
(A–C) CpG methylation of all gene promoters (A), gene promoters of cluster 1 genes identified by k-means clustering of the RNA-seq shown in Figure 2E (B), and the Ppargc1a promoter (C) in tumor-infiltrating CD4+Foxp3YFP+ cells (n = 4 Prkaa1/2wt/wtFoxp3YFP–Cre or control, n = 2 Prkaa1/2fl/flFoxp3YFP–Cre) and splenic CD4+Foxp3YFP+ cells at homeostasis (n = 3 control, n = 3 Prkaa1/2fl/flFoxp3YFP–Cre) (D) DNMT1 protein expression of splenic CD4+Foxp3YFP+ (Treg) cells at homeostasis (n = 7 control, n = 7 Prkaa1/2fl/flFoxp3YFP–Cre). 3 independent experiments are shown. DNMT1 peak intensity area was normalized to the corresponding sample’s β-actin peak intensity area. (E) Dnmt1 gene expression of splenic CD4+Foxp3YFP+ cells at homeostasis (n = 4 control, n = 4 Prkaa1/2fl/flFoxp3YFP–Cre) as measured by RNA-seq shown in Figure 1. (F) Anti-AMPKα1 and isotype control immunoprecipitates from ex vivo induced (i)Treg cell lysates blotted for DNMT1 protein. Independent biological replicates are shown. (G) Representative microscopy images of AMPKα-sufficient iTreg cells showing AMPKα1 and DNMT1 subcellular localization. Scale bars: 5 μm. (H) MitoTracker Deep Red (MitoTracker DR) mean fluorescence intensity (MFI) of AMPKα-sufficient (control) and -deficient splenic CD4+Foxp3YFP+ cells treated with either vehicle (n = 8 control, n = 10 Prkaa1/2fl/flFoxp3YFP–Cre), 50 nM decitabine (DAC, n = 7 control, n = 7 Prkaa1/2fl/flFoxp3YFP–Cre), or 100 nM DAC (n = 7 control, n = 7 Prkaa1/2fl/flFoxp3YFP–Cre). *P or q < 0.05, NS, not significant according to Mann-Whitney U test (D and E) with 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (H).