肿瘤微环境中乳酸对巨噬细胞表型极化和功能的影响

Lactic acid regulates phenotype polarization and function of macrophages in tumor microenvironment

目的研究肿瘤微环境中乳酸对巨噬细胞表型极化和功能的影响。方法在Balb/c小鼠乳腺处接种乳腺癌细胞4T1,研磨肿瘤组织后测其乳酸浓度。以不同浓度乳酸处理RAW264.7巨噬细胞,用流式细胞术、RT-PCR和Western blot检测M1、M2型巨噬细胞的标志分子和NFκB P50/P65。蛋白芯片检测RAW细胞分泌的细胞因子。流式细胞术检测RAW细胞吞噬功能和抗原递呈功能。结果小鼠乳腺癌组织的乳酸浓度高于正常乳腺组织(P<0.05)。15 mmol/L浓度的乳酸可显著上调RAW细胞M2型标志分子的表达(P<0.05),下调M1标志分子的表达(P<0.05),使RAW细胞分泌的M1型细胞因子明显减少(P<0.05),同时使磷酸化NFκB P65降低(P<0.05)。乳酸对RAW细胞的吞噬功能没有明显的影响,但减弱其抗原递呈功能(P<0.05)。结论在乳酸的作用下,RAW巨噬细胞表型向M2型转变,抗原递呈功能减弱,NFκB参与调控乳酸对巨噬细胞的作用。提示肿瘤微环境中的乳酸对抗肿瘤免疫有较大影响。

Objective To investigate that lactic acid regulates polarization of phenotype and function of macrophages in tumor microenvironment. Methods Balb/c mice were inoculated by 4T1 breast tumor cells with the mammary gland injection.The tumor tissues were harvested at different time points and detected the lactic acid concentration. The expression of M1 and M2 markers in RAW264.7 cells treated with different concentration of lactic acid were detected by RT-PCR ,FACS and Western Blot. The mouse cytokine array chip was used to evaluate the release of cytokines from RAW cells treated with 15mmol/L lactic acid. Phagocytosis and antigen presenting function was evaluated by using FACS. Results lactic acid concentration of mouse breast tumor was higher than that of normal breast tissue（P〈0.05）. M2 markers of RAW cells were up-regulated and M1 markers were down-regulated by 15mmol/L lactic acid（P〈0.05）.M1 cytokines secretion of RAW cells treated with 15mmol/L lactic acid was down-regulated（P〈0.05）. Phosphorylated NFκB P65 was down-regulated by 15mmol/L lactic acid（P〈0.05）.The phagocytosis was not affected by lactic acid, but the expression of the antigen presenting and co-stimulatory molecules were down-regulated in RAW cells treated with 15mmol/L lactic acid（P〈0.05）. Conclusion lactic acid induces the M2 phenotype shift and inhibits the antigen presenting function of RAW cells. NFκB P50/P65 are involved in the phenotype polarization of macrophages.This study suggested that lactic acid plays a indispensable role in macrophages polarization in tumor microenvironment.