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.
Xiaoyan Su,Ming Zhao,Xinyu Fu,Xiaodan Ma,Wei Xu,Songhua Hu
Polysaccharides from Rubus chingii Hu fruits (RFPs) were extracted, purified, characterized and investigated for immunomodulatory activity. Results indicated that RFPs were heteropolysaccharides with molecular weight of 51.7 kDa and contained carbohydrate contents of 96.83%. RFPs with characteristic absorptive peaks of polysaccharide structure and α-configuration were composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and fucose with a molecular ratio of 0.08:0.17:0.16:0.48:5.73:1.00:1.79:0.03. RFPs promoted splenocyte proliferation, NO release, cytokine production and intracellular Ca 2+ concentration. The mechanism underlying splenocyte activation by RFPs was elucidated by RNA-Seq, in which 2214 differentially expressed genes (DEGs), 2030 GO terms and 92 KEGG pathways were identified. RFPs were further proved to induce a balanced Th1/Th2/Th17 response pattern through MAPK, NF-κB, Jak-STAT and Calcium signaling pathways in splenocytes. These findings provided a fundamental basis for the further application of RFPs as immunomodulators in food and medicine.
Background Extensive research has revealed the favorable effects of celastrol (CEL) against various diseases, but the role of CEL in autoimmune uveitis remains unexplored. Methods We first assessed the prophylactical and therapeutical effects of CEL on autoimmune uveitis via rat experimental autoimmune uveitis model. After network pharmacology, functional enrichment and molecular docking analyses, we predicted the potential target of CEL and validated its effect on EAU by clinical and histopathological scores, Evans blue staining, immunofluorescence assay and western blotting. Then we evaluated the role of CEL in the gut environment by 16S rRNA sequencing and untargeted metabolomic analysis. Results We confirmed that CEL treatment suppressed the pathological TH17 response, inhibited the migration of inflammatory cells, and preserved the integrity of BRB via targeting STAT3-IL17 pathway. Furthermore, CEL was found to reduce the relative abundance of opportunistic pathogenic bacteria including Clostridium_sensu_stricto_1 , Parasutterella and GCA-900066575 , and enrich the relative abundance of beneficial Oscillospirales and Ruminococcus_torques_group in EAU rats by fecal 16S rRNA sequencing. Meanwhile, CEL treatment reshaped the gut metabolites in the EAU rats by increasing the relative concentrations of cholic acid, progesterone and guggulsterone, and decreasing the relative levels of isoproterenol, creatinine and phenylacetylglutamine. Conclusions CEL exerts its ameliorative effects on the experimental autoimmune uveitis through the dual mechanisms of targeting STAT3 and reprofiling the gut microenvironment.
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