Department of Pharmacology of Chinese Materia Medica, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China.
Theranostics. 2021 Mar 4;11(9):4531-4548. doi: 10.7150/thno.54803. eCollection 2021.
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. 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 . 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 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 . 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.
过氧化物酶体增殖物激活受体 γ（PPARγ）具有拮抗 Th17 反应的能力，但完整的机制仍不清楚。在此，我们旨在从细胞代谢的角度，特别是谷氨酰胺分解代谢的角度阐明这一过程。MTT、CCK-8、Annexin V-FITC/PI 染色或台盼蓝排斥试验用于分析细胞毒性。流式细胞术和 Q-PCR 试验用于确定 Th17 反应。使用商业试剂盒检测代谢物水平，并使用 Western blot 试验检测限速酶表达，以说明代谢活性。ChIP 试验用于检测 H3K4me3 修饰。建立葡聚糖硫酸钠（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，以证实所研究的机制。PPARγ 激动剂罗格列酮和吡格列酮在 Th17 偏倚条件下阻断了谷氨酰胺分解代谢，但不阻断糖酵解，这可以通过检测细胞内乳酸和 α-KG 以及 BCECF-AM 的荧光比值来证实。PPARγ 激动剂阻止了谷氨酰胺的利用，从而即使在 Foxp3 缺乏的情况下也直接限制了 Th17 反应。这些机制归因于通过降低限速酶 GLS1 的表达来限制谷氨酰胺转化为谷氨酸，这通过 GLS1 的过表达得到了证实。补充 α-KG 和 2-HG 但不是琥珀酸削弱了 PPARγ 激动剂的作用，并且 siIDH1/2 减弱了 α-KG 促进的 Th17 反应。抑制 KDM5 但不是 KDM4/6 抑制了 PPARγ 激动剂对 IL-17A 表达的抑制作用，PPARγ 激动剂下调的基因座启动子和 CNS2 区域的 H3K4me3 水平被 2-HG 和 GLS1 的过表达所挽救。然而，PPARγ 激动剂对 RORγt mRNA 表达的限制作用不能被 2-HG 阻断，而是归因于 GLS1 后的 GSH/ROS 信号。GW9662 或 PPARγ 敲除证实了 PPARγ 的确切作用，并且通过 GLS1 过表达进一步证实了 PPARγ 抑制 Th17 反应的机制。PPARγ 激动剂通过拮抗 GLS1 介导的谷氨酰胺分解代谢/2-HG/H3K4me3 和 GSH/ROS 信号来抑制 Th17 反应，这有利于 Th17 细胞相关的免疫失调。
