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.

Keywords: Immunology; Intermediary metabolism; Mitochondria; Oncology; Pulmonology; T cells.

Figure 1. AMPKα1/α2 are dispensable for Treg cell–mediated immune self-tolerance and Treg cell suppressive function at homeostasis.

(A) CD8⁺ conventional T (Tconv) cell absolute counts per milligram (mg) of Prkaa1/2^wt/wtFoxp3^YFP–Cre (control) and Prkaa1/2^fl/flFoxp3^YFP–Cre mouse spleen (n = 4 control, n = 4 Prkaa1/2^fl/flFoxp3^YFP–Cre), thymus (n = 4 control, n = 3 Prkaa1/2^fl/flFoxp3^YFP–Cre), and lung (n = 4 control, n = 4 Prkaa1/2^fl/flFoxp3^YFP–Cre). (B) Spleen mass of 8–12 week-old control (n = 5) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 5) mice. (C and D) Frequency of naive (CD62L^HiCD44^Lo; C) and effector (CD62^LoCD44^Hi; D) splenic CD8⁺ and CD4⁺ Tconv cells out of total CD8⁺ and CD4⁺ cells, respectively (n = 5 control, n = 5 Prkaa1/2^fl/flFoxp3^YFP–Cre). (E) CD4⁺Foxp3^YFP+ cell absolute counts per mg of control and Prkaa1/2^fl/flFoxp3^YFP–Cre mouse spleen (n = 4 control, n = 4 Prkaa1/2^fl/flFoxp3^YFP–Cre), thymus (n = 4 control, n = 3 Prkaa1/2^fl/flFoxp3^YFP–Cre), and lung (n = 4 control, n = 4 Prkaa1/2^fl/flFoxp3^YFP–Cre). (F) Frequency of Ki-67⁺CD4⁺Foxp3^YFP+ cells out of total CD4⁺Foxp3^YFP+ splenocytes (n = 5 control, n = 5 Prkaa1/2^fl/flFoxp3^YFP–Cre). (G) Frequency of central (CD62L^HiCD44^Lo) and effector (CD62^LoCD44^Hi) CD4⁺Foxp3^YFP+ cells of total CD4⁺Foxp3^YFP+ splenocytes (n = 5 control, n = 5 Prkaa1/2^fl/flFoxp3^YFP–Cre). (H and I) Foxp3^YFP (H) and FOXP3-PE-Cy7 (I) mean fluorescence intensity (MFI) of CD4⁺Foxp3^YFP+ splenocytes (n = 8 control, n = 8 Prkaa1/2^fl/flFoxp3^YFP–Cre). (J) Division index of CD4⁺Foxp3^YFP– splenic responder T (Tresp) cells cocultured with CD4⁺Foxp3^YFP+ splenocytes (n = 4 control, n=3 Prkaa1/2^fl/flFoxp3^YFP–Cre) for 72 hours. (K) K-means clustering of 78 significant differentially expressed genes (FDR q < 0.05) identified between splenic CD4⁺Foxp3^YFP+ cells sorted from control (n = 4) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 4) mice with k = 3 and scaled as Z-scores across rows. (L) Enrichment plot of the GSE15659_NONSUPPRESSIVE_TCELL_Versus_ACTIVATED_TREG_UP gene set generated through gene set enrichment analysis (GSEA) preranked testing of the expressed genes of Prkaa1/2^fl/flFoxp3^YFP–Cre and control splenic Treg cells identified by RNA-seq. **P or q < 0.01; ***P or q < 0.001; nd, no discovery, NS, not significant according to Mann-Whitney U test (B, F, H, and I) with 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (A, C–E, G, and J).

Figure 2. AMPKα1/α2 loss is sufficient to impair Treg cell suppressive function in the TME.

(A) Growth of B16 melanoma tumors in Prkaa1/2^wt/wtFoxp3^YFP–Cre (control, n = 6) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 5) mice. (B) Tumor mass of control (n = 14) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 10) mice at day 15 after engraftment. (C) Ratio of live CD8⁺ cell counts to live CD4⁺Foxp3^YFP+ (Treg) cell counts in B16 melanoma tumors of control (n = 19) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 19) mice at day 15 after engraftment. (D) Absolute counts of CD8⁺ Tconv cells and Treg cells per mg of tumor from control (n = 14, CD8⁺ Tconv cells; n = 6, Treg cells) and Prkaa1/2^fl/flFoxp3^YFP–Cre mice (n = 15, CD8⁺ Tconv cells; n = 6, Treg cells). (E) K-means clustering of differentially expressed genes (FDR q < 0.05) identified between Treg cells sorted from B16 melanoma tumors of control (n = 5) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 3) mice at day 15 after engraftment with k = 3 and scaled as z-scores across rows. (F) Average z-scores for the 3 clusters shown in (E). (G) Ppargc1a expression (n = 5 control, n = 3 Prkaa1/2^fl/flFoxp3^YFP–Cre). (H) Selection of top gene ontology (GO) processes (FDR q < 0.05). (I) K-means clustering of differentially expressed genes (FDR q < 0.05) identified between Treg cells sorted from B16 melanoma tumors of control (n = 6) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 6) mice at day 12 after engraftment with k = 2 and scaled as z-scores across rows. (J and K) GSEA preranked test enrichment plots (P < 0.05, FDR q < 0.25) of the REACTOME_DNA_METHYLATION (J) and REACTOME_HDACS_DEACETYLATE_HISTONES (K) from tumor-infiltrating Prkaa1/2^fl/flFoxp3^YFP–Cre and control Treg cells on day 12 after engraftment. ***P < 0.001 according to 2-way ANOVA with 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (A). **P < 0.01. according to Mann Whitney U test (B and C). 1 outlier was identified and excluded from (B) and 2 from (C) using the ROUT method (Q = 0.5%).

Figure 3. The metabolic landscape of the influenza virus–injured lung resembles the TME in its metabolite abundance; however, they differ in the abundance of key carbon sources.

(A) Principal component (PC) analysis of the peak intensities of metabolites identified via liquid chromatography tandem mass spectrometry (LC-MS) from B16 melanoma tumor (n = 3) and influenza virus–infected lung (flu, n = 7) interstitial fluid (IF) and paired plasma (n = 4 tumor, n = 6 flu) from the same animals. (B) Heatmap of the 70 most differentially represented metabolites in plasma, tumor IF, and flu IF according to 1-way ANOVA (P < 0.1). (C–F) Abundance of key significant differentially represented metabolites: 2-hydroxyglutarate (C), lactic acid (D), glucose (E), and glutamine (F). (G) Results from overrepresentation analysis of the significant (P < 0.1) differentially represented metabolites between tumor IF and plasma. (H) Results from overrepresentation analysis of the significant (P < 0.1) differentially represented metabolites between flu IF and plasma. (I) Overlap in significantly (P < 0.1) enriched metabolite sets between tumor IF versus plasma comparison and flu IF versus plasma comparison according to overrepresentation analysis of flu IF versus plasma and tumor IF versus plasma.

Figure 4. AMPKα1/α2 are necessary for optimal Treg cell function in the lung during influenza pneumonia.

(A) Enrichment plot of the REACTOME_INFLUENZA_INFECTION gene set (P < 0.05, FDR q < 0.25) generated through GSEA preranked testing of the expressed genes of Prkaa1/2^wt/wtFoxp3^YFP–Cre (control) and Prkaa1/2^fl/flFoxp3^YFP–Cre CD4⁺Foxp3^YFP+ splenocytes identified by RNA-seq shown in Figure 1K. (B) Survival of control (n = 23) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 25) mice following intratracheal inoculation of 12.5 plaque forming units (PFUs) of influenza A/WSN/33 H1N1 (influenza) virus. (C and D) Weight (C), and arterial oxyhemoglobin saturation (D) over time of control (n = 6) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 8) mice following intratracheal inoculation of 12.5 PFUs of influenza virus. (E–H) Absolute counts of CD45⁺ cells (E), CD8⁺ cells (F), CD4⁺Foxp3^YFP+ cells (G), and CD4⁺ cells (H) per pair of lungs in control (n = 6) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 9) mice at day 10 after influenza virus inoculation. (I) Volcano plot of abundance of metabolites detected in control (n = 4) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 4) Treg cells sorted from lungs at day 10 after influenza virus inoculation. (J) Heatmap of top 50 differentially represented metabolites between control (n = 4) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 4) Treg cells sorted from lungs at day 10 after influenza virus inoculation. (K–M) Peak intensities measured for lactic acid (K), pyruvic acid (L), and glutathione GSH (M) in Treg cells from the lungs of control (n = 4) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 4) mice at day 10 after influenza virus inoculation. (N) Results of overrepresentation analysis from the significant (P < 0.1, log₂(FC) ≥ 1.5 or ≤ –1.5) differentially represented metabolites identified in I. Survival curve (B) P was determined using log-rank (Mantel-Cox) test. *q < 0.05 according to 2-way ANOVA with 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (C–D). *P < 0.05, NS not significant according to Mann-Whitney U test (E–H).

Figure 5. AMPKα is necessary for maximal Treg cell mitochondrial function.

(A) Representative oxygen consumption rate (OCR) over time of CD4⁺Foxp3^YFP+ splenocytes from Prkaa1/2^wt/wtFoxp3^YFP–Cre (control, n = 3) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 2) mice following treatment of oligomycin (2.5 μM), carbonyl cyanide m-chlorophenylhydrazone (CCCP; 10 μM), and antimycin A/piercidin (A/P; 2 μM each), as measured by a metabolic flux assay. (B–C) Basal (B) and maximal (C) OCR of CD4⁺Foxp3^YFP+ splenocytes from control (n = 6) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 6) mice, some of which are shown in A. (D and E) MitoTracker Deep Red (MitoTracker DR) mean fluorescence intensity (MFI) of CD4⁺Foxp3^YFP+ splenocytes at homeostasis (D; n = 7 control, n = 8 Prkaa1/2^fl/flFoxp3^YFP–Cre mice) and lung CD4⁺Foxp3^YFP+ cells at day 10 after influenza virus inoculation (E; same cohort as in Figure 4, E–H, and Supplemental Figure 7, n = 6 control, n = 9 Prkaa1/2^fl/flFoxp3^YFP–Cre mice). (F and G) Basal (F) and maximal (G) extracellular acidification rate (ECAR) of CD4⁺Foxp3^YFP+ splenocytes from control (n = 6) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 7) mice. (H) LC3B-PE MFI of CD4⁺Foxp3^YFP+ splenocytes from control (n = 8) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 8) mice. (I) Mean LAMP-1-PE-Cy7 and MitoView Green colocalization in CD4⁺Foxp3^YFP+ splenocytes from control (n = 4) and Prkaa1/2^fl/flFoxp3^YFP–Cre (n = 4) mice. *P < 0.05, **P < 0.01, NS, not significant according to Mann-Whitney U test.

Figure 6. AMPKα1 interacts with DNMT1 to demethylate the promoter of mitochondrial genes in tumor-infiltrating Treg cells.

(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⁺Foxp3^YFP+ cells (n = 4 Prkaa1/2^wt/wtFoxp3^YFP–Cre or control, n = 2 Prkaa1/2^fl/flFoxp3^YFP–Cre) and splenic CD4⁺Foxp3^YFP+ cells at homeostasis (n = 3 control, n = 3 Prkaa1/2^fl/flFoxp3^YFP–Cre) (D) DNMT1 protein expression of splenic CD4⁺Foxp3^YFP+ (Treg) cells at homeostasis (n = 7 control, n = 7 Prkaa1/2^fl/flFoxp3^YFP–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⁺Foxp3^YFP+ cells at homeostasis (n = 4 control, n = 4 Prkaa1/2^fl/flFoxp3^YFP–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⁺Foxp3^YFP+ cells treated with either vehicle (n = 8 control, n = 10 Prkaa1/2^fl/flFoxp3^YFP–Cre), 50 nM decitabine (DAC, n = 7 control, n = 7 Prkaa1/2^fl/flFoxp3^YFP–Cre), or 100 nM DAC (n = 7 control, n = 7 Prkaa1/2^fl/flFoxp3^YFP–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).