Fig. 3
Acetyl Co-A related transcriptional metabolic dysregulation in obese T-regs. Six-week old C57BL/6 mice were fed a high fat diet or normal chow for 14–16 weeks to generate obese (DIO) mice and normal (norm) controls, respectively (n= 5–6 mice/group). Single-cell suspensions of tissue-infiltrating leukocytes were generated, and the frequencies of Tregs among the recovered leukocytes (CD45+ cells) were found by flow cytometry (a) representative flow plots of lung and lymph nodes, (b) the levels of Foxp3 markers of activated, (c) “effector” Tregs PD-1 and (d) Ki67 were found by flow cytometry. (e) Expression of Treg-associated mRNAs by lung Tregs was also analyzed by qRT-PCR and fold change in DIO vs. norm controls are shown. (f) Pathways that result in increased metabolic activity (OxPhos) and acetyl Co-A metabolism (butanoate metabolism) are dysregulated in Tregs from obese vs. non-obese LLC (node size = -log(pVal), increased score/absolute value score = more transcriptionally dysregulated). (g) Expression of select metabolic genes by Tregs isolated from the lungs of obese and non-obese mice. Foxp3+/CD25+/CD4+ Tregs were recovered from the lung tissues of C57BL/6 mice fed either a high fat (obese) or control diet (non-obese) by FACS for mRNA isolation. Levels of the indicated transcripts were measured by RT-PCR. Shown are mean fold changes in gene expression in obese vs. non-obese Tregs. (h) Acetyl-CoA transcriptional signature shows separation between obese and non-obese Tregs. Sorted Treg and Tcon cells were slide-immobilized by Cytospin and subjected to proximity ligation assay to observe levels of acetylated FOXP3 protein (red) and nuclei counter stained with DAPI (blue). (i) Representative 20x images, with insets at 40X and (j) the mean proportion of Tregs with acetylated Foxp3 across 10–20X fields (~200 cells) are shown. Shown are mean values ±SEM or representative flow plots (panels b and c) from 2–7 independent experiments. In panel j Error bars depict the SEM. N=5 mice/group. Ac-Foxp3 = acetylated Foxp3