Effects of lactate on metabolism and differentiation of CD4+T cells.

BACKGROUND: Lactate accumulation caused by abnormal tumor metabolism can induce the formation of an inhibitory immune microenvironment through a variety of pathways, which is characterized by regulatory T cells (Treg) infiltration and effector T cells (Teff) depletion. Studies have found that the key reason why Treg cells can survive in harsh environments lies in their flexible metabolic mode, which can use lactate in tumor microenvironment (TME) as an alternative energy substance to maintain their inhibitory activity. In addition, lactate could also promote the differentiation of CD4+T cells into Treg, but the mechanism was not completely clear. The purpose of this study was to investigate the possible mechanism by which lactate is utilized by CD4+T cells to influence Th17/Treg ratio. METHODS: Basal cytokines (anti-CD3, anti-CD28, TGF-ß) and 10 mM lactate was added into Naïve CD4+T cells basal medium for 3 days. After TCR stimulation, Naïve CD4+T converted to CD4+T. Flow cytometry was used to detect the proportion of Treg cells; ELISA was used to detect the activity of LDHA, LDHB and NADH and the amount of α -Ketoglutaric Acid (α-KG) and 2-Hydroxyglutaric Acid (2HG) after lactate entered the cells; Western Blot and RT-PCR were used to detect the protein and gene expression of Foxp3, RORγt, LDHA and LDHB. In the validation experiment, lactate uptake inhibitor AZD3965, LDHA inhibitor GSK2837808A and NADH conversion inhibitor Rotenone were added respectively to observe the differentiation ratio of Treg cells and confirm the key points of metabolism; the degradation of Treg cell transcription factor Foxp3 was interfered with ubiquitination inhibitors to observe whether it co-ubiquitinated with HIF-1α; the expression and activity of LDHA, LDHB and NADH in mitochondria and cytoplasm were detected to confirm cell localization. RESULTS: When basal cytokines (anti-CD3, anti-CD28, TGF-ß) stimulated, lactate was added to the culture medium, and CD4+T cells absorbed a large amount of lactate not only through MCT1 (monocarboxylic acid transporter), but also increased the expression of lactate dehydrogenase and accelerated the intracellular metabolism of lactate. LDHB in cytoplasm mainly catalyzed the dehydrogenation of lactate to pyruvate, accompanied by the transformation reaction between NAD+ and NADH. The latter further entered the mitochondria and participates in the tricarboxylic acid cycle metabolism. In addition, lactate could significantly increase the level of LDHA in mitochondria and promote the transformation of α-KG to 2HG, accompanied by the transformation of NADH to NAD+. These metabolic changes eventually led to an increase in the intracellular 2HG/α-KG ratio. Abnormal 2HG increased the proportion of Treg by inhibiting ATP5B-mediated phosphorylation of mTOR and the synthesis of HIF-1α, causing it not be enough to ubiquitinate and degrade with Foxp3. CONCLUSIONS: Lactate plays an important role in regulating the differentiation of Treg cells, inducing the expression and function of LDHA and promoting the transformation of α-KG to 2HG may be an important mechanism.

Multi-material basis and multi-mechanisms of the Dahuang Zhechong pill for regulating Treg/Th1 balance in hepatocellular carcinoma.

BACKGROUND: Dahuang Zhechong pill (DHZCP) improves the inhibitory immune status of mice with hepatocellular carcinoma (HCC) by regulating Treg/Th1 balance. HYPOTHESIS/PURPOSE: To study the multi-material basis and multi-mechanisms of DHZCP against HCC by regulating Treg/Th1 balance in vitro and in vivo. METHODS: UPLC-MS/MS was used to detect the dynamic changes in 29 characteristic components of different polar parts of DHZCP. H&E and TUNEL were used to check pathological condition in HCC mice. The number of CD4+T, CD8+T, Treg, Th1, and Th1-like Treg cells was counted by flow cytometry. TGF-ß, IL-10, IFN-γ, and TNF-α content were detected by ELISA. α-Ketoglutarate and glutamine levels were detected by Trace1310/TSQ8000 GC-MS/MS. p-Smad2, and p-Smad3 protein levels were detected by WB, mRNA expression of Smad2, alanine-serine-cysteine transporter-2, glutaminase, and glutamate dehydrogenase were detected by RT-PCR. Simca-p multivariate data analysis software was used to evaluate the relationship between the different polar parts of DHZCP and the proportion of Treg cells. RESULTS: Water-soluble (PW) and ethyl acetate (PE) polar parts of DHZCP affected the HCC immune system by inhibiting the differentiation of Tregs, reversing the balance of Treg/Th1, and significantly reduced the tumor volume and weight. However, petroleum ether and n-butanol polar parts had no above actions. The changes in emodin, chrysophanol, aloe vera emodin, emodin-8-O-ß-D-glycoside, gallic acid, naringenin, baicalein, wogonin, norwogonin, apigenin, chrysin, glycyrrhizin, formononetin, and palmitic acid were closely related to the changes of Treg cells, which is the main material basis of DHZCP inhibition of Treg differentiation. Additionally, PW mainly inhibit the differentiation of Treg cells by affecting the metabolism of hepatoma cells, improving tumor microenvironment acidity, and glutamine depletion. However, PE inhibited the differentiation of Treg cells mainly by regulating the TGF-ß/Smad pathway. CONCLUSION: In this study, accurate analysis of multi-component was combined with pharmacodynamic evaluations to identify the pharmacodynamic substances of DHZCP in regulating Treg/Th1 balance, and clarified the multi-target mechanism of DHZCP to improve tumor immunity. The study style offers a novel approach for pharmacological research on TCM.