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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. [ABSTRACT FROM AUTHOR]

Our previous studies have demonstrated that tetrandrine can induce the generation of regulatory T (Treg) cells in vitro and in vivo. But, the underlying mechanism of tetrandrine remains obscure. Naïve CD4+ T cells are isolated from the mesenteric lymph nodes of mice for the differentiation of Treg cells. Flow cytometry is used to detect the frequencies of Treg cells. Non‐targeted metabolomics analysis based on UHPLC‐QTOF/MS is performed to assess the intracellular metabolic profiles. ChIP‐PCR analysis is conducted to detect the level of H3K27ac at Foxp3 promoter and CNS regions. Tetrandrine treatment alters the metabolic profile of Treg cells, and pathway enrichment of differential metabolites mainly involves fatty acid oxidation (FAO). Tetrandrine promotes the mRNA expression of carnitine palmitoyl transferase‐1, and increases the level of acetyl coenzyme A (acetyl‐CoA) and the intracellular oxygen consumption rate. Either CPT1 inhibitor (etomoxir) or siRNA markedly diminishes the promotion of tetrandrine on Treg cell differentiation. Furthermore, tetrandrine enhances the acetylation of H3K27 in the promoter and CNS1 regions of Foxp3 through the acetyl‐CoA derived from FAO. In the mice with collagen‐induced arthritis, tetrandrine also induces Treg cell generation through FAO pathway. In addition, tetrandrine enhances the immunosuppressive function of Treg cells both in vitro and in vivo. The findings indicate that tetrandrine promotes Treg cell differentiation by enhancing FAO‐mediated Foxp3 acetylation, and the CPT1‐mediated FAO can serve as the target for the discovery of novel inducers of Treg cell generation. [ABSTRACT FROM AUTHOR]

Norisoboldine (NOR), an alkaloid isolated from Radix Lindera, was previously reported to promote the differentiation of regulatory T cells (Treg cells), an important subtype of lymphocytes capable of controlling autoimmune diseases. The present study was performed to explore the mechanism of NOR in the view of cellular metabolism. A global metabolomic analysis indicated that NOR preferentially altered the fatty acid oxidation (FAO) pathway and elevated the content of related metabolites during Treg cell differentiation. The detection of oxygen consumption rate (OCR) and mRNA expression of FAO‐related enzymes demonstrated that NOR promoted FAO in the early stage of Treg cell differentiation. Consistently, pharmacological or genetic inhibition of FAO markedly diminished the induction of NOR on Treg cell differentiation. Furthermore, NOR was shown to elevate the level of acetyl‐CoA derived from FAO and acetylation of lysine 27 on histone 3 (H3K27) at the Foxp3 promoter and CNS2 regions. A knockdown of CPT1, the rate‐limiting enzyme of FAO, weakened the promotion of NOR on the development, acetyl‐CoA level, and acetylation of H3K27 of Treg cells in vitro and in the mice with collagen‐induced arthritis, and attenuated the anti‐arthritic effect of NOR. These findings demonstrate that NOR induces the development of Treg cells through promoting FAO, therefore, facilitating gene transcription of Foxp3 via acetyl‐CoA‐mediated H3K27 acetylation modification, and FAO might serve as a novel target to induce Treg cell development. [ABSTRACT FROM AUTHOR]

Objective Norisoboldine (NOR), an isoquinoline alkaloid with very poor oral bioavailability, was previously proven to have a unique anti-arthritis activity in rats by inducing intestinal Treg cells. Herein, we explored its underlying mechanism in view of aryl hydrocarbon receptor (AhR). Methods The differentiation of regulatory T cells (Treg cells) and IL-17-producing T cells (Th17 cells) from naïve T cells was analyzed in vitro . The key role of AhR was ascertained using siRNA transfection. AhR agonistic effect was verified based on the activation of downstream signaling pathway and target genes. The correlation between AhR activation and Treg cell induction as well as pathological changes of joints was confirmed in collagen-induced arthritis (CIA) mouse model. Results NOR promoted intestinal Treg cell differentiation and function in an AhR-dependent manner. It acted as an AhR agonist, as evidenced by induction of CYP1A1 expression and activity, promotion of AhR/Hsp90 dissociation and AhR nuclear translocation, induction of XRE reporter activity, and facilitation of AhR/XRE binding. In CIA mice, NOR exerted substantial anti-arthritic effect through enhancing AhR activation in intestinal tissues and inducing intestinal Treg cell generation, which could be largely abolished by resveratrol (a antagonist of AhR). An adoptive transfer of Treg cells from NOR-treated mice could successfully alleviate arthritis symptoms in CIA mice. Conclusion Oral NOR induces the generation of intestinal Treg cells by the activation of AhR, and thereby exerts anti-arthritic effect. [ABSTRACT FROM AUTHOR]

[Display omitted] In this study, we used chemical modification to improve the pharmacological activity of norisoboldine (NOR). A new NOR-benzoic acid derivative, named DC-01, showed more potent induction of Treg cell differentiation than NOR. The in vitro effective concentration of DC-01 (1 μM) is about an order of magnitude lower than that of NOR (10 μM). DC-01 (28, 56 mg/kg) showed better amelioration of dextran sodium sulfate-induced colitis in mice than NOR (20, 40 mg/kg), and DC-01 (28 mg/kg) increased the number of Treg cells slightly better than NOR (20 mg/kg). In summary, DC-01 exerts more potent induction of Treg cell generation, which might be a candidate drug for the treatment of inflammation- and immune-related diseases. [ABSTRACT FROM AUTHOR]