The role of GLS1-mediated glutaminolysis/2-HG/H3K4me3 and GSH/ROS signals in Th17 responses counteracted by PPARγ agonists.
The role of GLS1-mediated glutaminolysis/2-HG/H3K4me3 and GSH/ROS signals in Th17 responses counteracted by PPARγ agonists.
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作者：
Miao Y;Zheng Y;Geng Y;Yang L;Cao N;Dai Y;Wei Z
背景资料：过氧化物酶体增殖物激活受体γ（过氧化物酶体增殖物激活受体γ）具有对抗Th17应答的能力，但完整的机制仍不清楚。在此，我们的目的是阐明这一过程中的细胞代谢，特别是β-内酰胺酶。方法：MTT法、CCK-8法、Annexin V-FITC/PI染色法和台盼蓝拒染法检测细胞毒性。应用流式细胞术和Q-PCR测定来确定Th17应答。使用商业试剂盒检测代谢物水平，并使用蛋白质印迹法检测限速酶表达，以说明代谢活性。使用ChIP测定来检查H3K4me3修饰。建立了葡聚糖硫酸钠（DSS）诱导的结肠炎和屋尘螨（HDM）/脂多糖（LPS）诱导的哮喘小鼠模型，以证实体外研究的机制。结果如下：通过检测细胞内乳酸和α-KG以及BCECF-AM的荧光比率表明，在Th17-偏斜条件下，PPAR γ激动剂罗格列酮和吡格列酮阻断了α-氨基醇解，但不阻断糖酵解。PPAR γ激动剂阻止谷氨酰胺的利用，从而直接限制了Th17应答，即使Foxp3缺乏。其机制被归因于通过降低限速酶GLS 1的表达来限制谷氨酰胺向谷氨酸的转化，GLS 1过表达证实了这一点。补充α-KG和2-HG而非琥珀酸可减弱PPAR γ激动剂的作用，而siIDH1/2可抑制α-KG促进的Th17应答。抑制KDM5而非KDM4/6可抑制PPAR γ激动剂对IL-17 A表达的抑制作用，而2-HG和GLS 1过表达可恢复PPAR γ激动剂下调的IL-17基因启动子和CNS2区的H3K4me3水平。然而，2-HG不能阻止PPAR γ激动剂对ROR γ t mRNA表达的限制，而是由于GLS 1后的GSH/ROS信号。GW9662或PPAR γ基因敲除证实了PPAR γ的确切作用，GLS 1过表达进一步证实了PPAR γ抑制Th17应答的机制。结论：PPAR γ激动剂可通过抑制GLS 1介导的γ-谷氨酰胺分解/2-HG/H3K4me3和GSH/ROS信号而抑制Th17细胞应答，这有利于Th17细胞相关的免疫失调。
Background: Peroxisome proliferator-activated receptor gamma (PPARγ) has the ability to counter Th17 responses, but the full mechanisms remain elusive. Herein, we aimed to elucidate this process in view of cellular metabolism, especially glutaminolysis. Methods: MTT, CCK-8, Annexin V-FITC/PI staining or trypan blue exclusion assays were used to analyze cytotoxicity. Flow cytometry and Q-PCR assays were applied to determine Th17 responses. The detection of metabolite levels using commercial kits and rate-limiting enzyme expression using western blotting assays was performed to illustrate the metabolic activity. ChIP assays were used to examine H3K4me3 modifications. Mouse models of dextran sulfate sodium (DSS)-induced colitis and house dust mite (HDM)/lipopolysaccharide (LPS)-induced asthma were established to confirm the mechanisms studied in vitro. Results: The PPARγ agonists rosiglitazone and pioglitazone blocked glutaminolysis but not glycolysis under Th17-skewing conditions, as indicated by the detection of intracellular lactate and α-KG and the fluorescence ratios of BCECF-AM. The PPARγ agonists prevented the utilization of glutamine and thus directly limited Th17 responses even when Foxp3 was deficient. The mechanisms were ascribed to restricted conversion of glutamine to glutamate by reducing the expression of the rate-limiting enzyme GLS1, which was confirmed by GLS1 overexpression. Replenishment of α-KG and 2-HG but not succinate weakened the effects of PPARγ agonists, and α-KG-promoted Th17 responses were dampened by siIDH1/2. Inhibition of KDM5 but not KDM4/6 restrained the inhibitory effect of PPARγ agonists on IL-17A expression, and the H3K4me3 level in the promoter and CNS2 region of the il-17 gene locus down-regulated by PPARγ agonists was rescued by 2-HG and GLS1 overexpression. However, the limitation of PPARγ agonists on the mRNA expression of RORγt was unable to be stopped by 2-HG but was attributed to GSH/ROS signals subsequent to GLS1. The exact role of PPARγ was proved by GW9662 or PPARγ knockout, and the mechanisms for PPARγ-inhibited Th17 responses were further confirmed by GLS1 overexpression in vivo. Conclusion: PPARγ agonists repressed Th17 responses by counteracting GLS1-mediated glutaminolysis/2-HG/H3K4me3 and GSH/ROS signals, which is beneficial for Th17 cell-related immune dysregulation.