IL-17D调控CD93^(+)CTL在肺脏肿瘤微环境中的杀伤功能及桔梗的促进作用

IL-17D improves cytotoxic function of CD93^(+)CTL in lung tumor microenvironment and promotion of Platycodon grandiflorum

目的:探讨IL-17D调控CD93^(+)CTL在肺脏肿瘤微环境(TME)中的杀伤功能及桔梗(PG)的促进作用。方法:采用PG或IL-17D处理小鼠肺脏B16黑色素瘤模型和对照小鼠,观察肺脏肿瘤克隆形成;应用单细胞测序、免疫荧光染色、流式细胞术等方法检测肿瘤小鼠肺脏IL-17D表达变化;采用单细胞测序、流式细胞术等方法分别检测各组小鼠肺脏CD93^(+)CTL含量、细胞表型和功能因子改变(包括CD107a、穿孔素、颗粒酶B、趋化因子CCL2、CXCL9及趋化因子受体CCR2、CXCR3等);利用EL4细胞系,采用Western blot、RT-PCR检测桔梗皂苷D对IL-17D及其转录调控因子NRF2表达的作用。结果:小鼠肺脏CD93^(+)CTL较CD93−CTL高表达穿孔素、CD107a等细胞毒性效应因子和趋化因子受体CXCR3、CCR2等,而颗粒酶B分泌能力无显著差异;小鼠肺脏肿瘤模型中,肺脏IL-17D表达及其CD93^(+)CTL含量明显下降(P<0.001);肿瘤小鼠经IL-17D回补实验,或经PG处理10 d后,肺脏IL-17D和CD93^(+)CTL比例和数量明显增多(P<0.05),肿瘤克隆明显减少。同时,肿瘤局部CD93^(+)CTL募集和功能相关细胞因子CCL2、CXCL9、IL-17D等表达明显上调。PG对IL-17D的上调作用在EL4细胞系体外实验中也得到了验证,桔梗皂苷D可显著上调EL4细胞IL-17D及其转录调控因子NRF2表达。结论:中药PG及其提取物可上调肺脏IL-17D表达,增强肺脏CD93^(+)CTL浸润和杀伤功能,拮抗肿瘤在肺脏的恶性进展,是PG改善肺脏TME的重要药理机制。

Objective:To investigate mechanisms of IL-17D on cytotoxic function of CD93^(+)CTL in lung tumor microenvironment(TME)and promotion of Platycodon grandiflorum(PG).Methods:Lung of B16 melanoma model mice and control mice were treated with PG or IL-17D,and lung tumor clone formation was observed.IL-17D expression change in lung was detected by single cell sequencing,immunofluorescence staining and flow cytometry.Single cell sequencing and flow cytometry were used to detect CD93^(+)CTL content,cytotoxic phenotype and functional factors changes,including CD107a,perforin,granzyme B,chemokine CCL2,CXCL9 and their ligand CCR2 and CXCR3.Western blot and RT-PCR were used to detect effect of Platycodonopsis saponin D on IL-17D and its transcriptional regulator NRF2 expressions in EL4 cells.Results:Pulmonary CD93^(+)CTL highly expressed cytotoxic effectors such as perforin and CD107a and chemokine receptors CXCR3 and CCR2 than CD93−CTL,while there was no significant difference in secretion of granzyme B.In mouse lung tumor model,pulmonary IL-17D and CD93^(+)CTL were significantly decreased(P<0.001);in tumor-bearing mice after IL-17D backfill assay,or after 10 days of treatment with PG,proportion and absolute number of IL-17D and CD93^(+)CTL in lungs were significantly increased(P<0.05),and tumor clones were significantly reduced;meanwhile,tumor-local expressions of cytokines CCL2,CXCL9,which are related to recruitment and function of CD93^(+)CTL,and IL-17D were significantly upregulated.Up-regulation of IL-17D and its transcriptional regulator NRF2 by PG was verified in vitro experiments on EL4 cell line by PD.Conclusion:Traditional Chinese medicine PG and its extracts can up-regulate expression of IL-17D in lungs,improve infiltration and cytotoxic function of CD93^(+)CTL and antagonize malignant progression of lung tumors,this is an important pharmacological mechanism of PG in improving immune TME of tumors in lung.