The activation of PPARγ enhances Treg responses through up-regulating CD36/CPT1-mediated fatty acid oxidation and subsequent N-glycan branching of TβRII/IL-2Rα

Background: Peroxisome Proliferator-activated Receptor gamma (PPARγ) is an enhancer of Treg responses, but the mechanisms remain elusive. This study aimed to solve this problem in view of cellular metabolism.

Methods: Three recognized PPARγ agonists (synthetic agonist: rosiglitazone; endogenous ligand: 15d-PGJ2; natural product: morin) were used as the tools to activate PPARγ. The fatty acid oxidation (FAO) was evaluated through the detection of fatty acid uptake, oxygen consumption rate, mitochondrial mass, mitochondrial membrane potential and acetyl-CoA level. The involvement of UDP-GlcNAc/N-linked glycosylation axis and the exact role of PPARγ in the action of PPARγ agonists were determined by flow cytometry, Q-PCR, western blotting, a commercial kit for Enzyme activity and CRISPR/Cas9-mediated knockout.

Results: Rosiglitazone, 15d-PGJ2 and morin all increased the frequency of CD4⁺Foxp3⁺ Treg cells generated from naïve CD4⁺ T cells, boosted the transcription of Foxp3, IL-10, CTLA4 and TIGIT, and facilitated the function of Treg cells. They significantly promoted FAO in differentiating Treg cells by up-regulating the levels of CD36 and CPT1 but not other enzymes involved in FAO such as ACADL, ACADM, HADHA or HADHB, and siCD36 or siCPT1 dampened PPARγ agonists-promoted Treg responses. Moreover, PPARγ agonists enhanced UDP-GlcNAc biosynthesis and subsequent N-linked glycosylation, but did not affect the expressions of N-glycan branching enzymes Mgat1, 2, 4 and 5. Notably, the Enzyme activity of phosphofructokinase (PFK) was inhibited by PPARγ agonists and the effect was limited by siCD36 or siCPT1, implying PFK to be a link between PPARγ agonists-promoted FAO and UDP-GlcNAc biosynthesis aside from acetyl-CoA. Furthermore, PPARγ agonists facilitated the cell surface abundance of TβRII and IL-2Rα via N-linked glycosylation, thereby activating TGF-β/Smads and IL-2/STAT5 signaling, and the connection between N-linked glycosylation and Treg responses was revealed by tunicamycin. However, the increased surface abundance of CD36 was demonstrated to be mainly owing to PPARγ agonists-up-regulated overall expression. Finally, PPARγ Antagonist GW9662 or CRISPR/Cas9-mediated knockout of PPARγ constrained the effects of rosiglitazone, 15d-PGJ2 and morin, confirming the exact role of PPARγ.

Conclusions: The activation of PPARγ enhances Treg responses through up-regulating CD36/CPT1-mediated fatty acid oxidation and subsequent N-glycan branching of TβRII/IL-2Rα, which is beneficial for inflammatory and autoimmune diseases. Video Abstract.