AhR activation promotes Treg cell generation by enhancing Lkb1‐mediated fatty acid oxidation via the Skp2/K63‐ubiquitination pathway

Abstract Several aryl hydrocarbon receptor (AhR) agonists have been reported to promote the generation of regulatory T cells (Treg cells), and the action mechanisms need to be identified. In this study, we addressed the underlying mechanism of AhR activation to induce the generation of Treg cells in the view of cellular metabolism. Naïve CD4+ T cells were purified with mouse CD4+ CD62L+ T Cells Isolation Kits. The proportions of Treg cells were detected by flow cytometry. The value of oxygen consumption rate (OCR) of CD4+ T cells was detected by the Seahorse XFe 96 analyzer. The activation of fatty acid oxidation (FAO)‐related metabolic pathways was detected by Western blotting. Intracellular localization of Lkb1 was detected by immunofluorescence. The Strad‐Mo25‐Lkb1 complex formation and K63 chain ubiquitination modification of Lkb1 were detected by co‐immunoprecipitation. The binding of AhR to the Skp2 promoter was detected by constructing luciferase reporter gene. AhR or carnitine palmitoyltransferases 1 was knockdown in dextran sulphate sodium (DSS)‐induced colitis or collagen‐induced arthritis (CIA) mice by infecting mice with adeno‐associated virus via the tail vein injection. Compared to the control group, exogenous and endogenous AhR agonists 3,3′‐diindolylmethane (DIM) and 2‐(1 ′ H‐indole‐3′‐carbonyl)‐thiazole‐4‐carboxylic acid methyl ester (ITE) were shown to preferentially upregulate the mRNA expression of FAO‐related enzymes and the value of OCR. Consistently, pharmacological or genetic inhibition of FAO markedly diminished the induction of DIM and ITE on the differentiation of Treg cells. DIM and ITE functioned mainly through activating the liver kinase B1 (Lkb1)‐AMPK pathway via promotion of Lkb1‐Strad‐Mo25 complex formation and Lkb1 K63 ubiquitination. DIM and ITE were also shown to upregulate the mRNA expression of Skp2, a ubiquitination‐related enzyme, and facilitate the binding of AhR to the xenobiotic responsive element of Skp2 promoter region by luciferase reporter gene assay. Furthermore, the contribution of Skp2/K63 ubiquitination/Lkb1/FAO axis was verified in (DSS)‐induced colitis or CIA mice. In summary, these findings indicate that AhR activation promotes Treg cell generation by enhancing Lkb1‐mediated FAO via the Skp2/K63‐ubiquitination pathway, and AhR agonists may be used as inducers of Treg cells to prevent and treat autoimmune diseases.