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牙龈卟啉单胞菌通过CCR6+ Treg促进口腔鳞癌免疫抑制微环境形成的机制研究

Prophyromonas gingivalis Promotes the Formation of Immunosuppressive Microenvironment in Oral Squamous Cell Carcinoma by CCR6+ Regulatory T Cells: A Study of the Mechanisms Invovled

    摘要
  • 摘要:
    目的 本研究旨在探讨在口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）中牙龈卟啉单胞菌（Porphyromonas gingivalis, P. gingivalis）通过募集趋化因子受体6（chemokine receptor 6, CCR6）阳性（CCR6+）调节性T细胞（regulatory T cells, Treg）到肿瘤微环境（tumor microenvironment, TME）中促进OSCC的恶性进展的机制。
    方法 使用TCGA数据库分析趋化因子配体20（chemokine ligand 20, CCL20）-CCR6-Treg之间的相关性，OSCC患者CCR6高表达组中Treg富集指数、白细胞介素（interleukin, IL）-10和肿瘤坏死因子β1（tumor necrosis factor β1, TGF-β1）的表达。C57BL/6小鼠随机分为2组，每组6只，对照组颊部注射1次小鼠头颈鳞癌细胞系SCC7，实验组颊部注射1次SCC7细胞和P. gingivalis混合液，均为100 μL。2周后处死小鼠，免疫组化检测CCR6和叉头盒蛋白3（forkheadbox protein 3, FOXP3）在OSCC中的表达，流式细胞术明确P. gingivalis对OSCC恶性生物学行为及对CCR6+ Treg细胞和免疫微环境的影响。
    结果 通过生信分析确定CCL20-CCR6-Treg之间具有相关性（r=0.373，P<0.0001），OSCC中CCR6高表达患者中Treg富集评分较高，IL-10表达升高。动物实验表明P. gingivalis能够促进小鼠OSCC的瘤体体积（mm3）（对照组：0.294±0.105；实验组：0.526±0.101；P<0.01）和质量（mg）（对照组：206.200±53.950；实验组：376.000±119.200；P<0.01）的增加；免疫组化验证CCR6与FOXP3间存在相关关系（r=0.659, P<0.05），且P. gingivalis促进CCR6与FOXP3的表达；流式分析表明在OSCC中P. gingivalis通过募集更多CCR6+ Treg（%）（对照组：13.780±1.506；实验组：18.260±2.257；P<0.01），降低CD8+ T细胞比例（%）（对照组：27.120±1.647；实验组：21.060±3.148；P<0.01），进而促进免疫抑制微环境形成。
    结论 P. gingivalis通过肿瘤细胞募集CCR6+ Treg细胞形成肿瘤免疫抑制性微环境，促进OSCC的恶性进展。

     

    Abstract:
    Objective To investigate the mechanisms by which Porphyromonas gingivalis (P. gingivalis) promotes the malignant progression of oral squamous cell carcinoma (OSCC) through the recruitment of chemokine receptor 6-positive (CCR6+) regulatory T cells (Treg) in the tumor microenvironment (TME).
    Methods The Cancer Genome Atlas (TCGA) database was used to analyze the correlation between chemokine ligand 20 (CCL20), CCR6, and Treg. The Treg enrichment index and the expression levels of interleukin (IL)-10 and tumor necrosis factor β1 (TGF-β1) were assessed in the high CCR6 expression group of OSCC patients. C57BL/6 mice were randomly assigned to a control group and an experimental group (n = 6 in each group). The control group received a single injection of 100 μL SCC7, a mice head and neck squamous carcinoma cell line, while the experimental group received a single injection of 100 μL mixture of SCC7 cells and P. gingivalis in the cheek. After two weeks, the mice were sacrificed, and immunohistochemistry was performed to assess the expression levels of CCR6 and forkhead box protein 3 (FOXP3) in OSCC. Flow cytometry was performed to analyze the effects of P. gingivalis on OSCC malignant biological behavior, CCR6+ Treg cells, and the immune microenvironment.
    Results Bioinformatics analysis revealed a correlation between CCL20, CCR6, and Treg (r = 0.373, P < 0.000 1). OSCC patients with high CCR6 expression showed higher Treg enrichment scores and increased IL-10 expression. Animal experiments showed that P. gingivalis promoted the increase in the tumor volume (mm3) (0.294 ± 0.105 in the control group and 0.526 ± 0.101 in the experimental group, P < 0.01) and mass (mg) (206.200 ± 53.950 in the control group and 376.000 ± 119.200 in the experimental group, P < 0.01) in mice with OSCC. Immunohistochemistry confirmed a correlation between CCR6 and FOXP3 (r = 0.659, P < 0.05), and P. gingivalis promoted the expression of CCR6 and FOXP3. Flow cytometry analysis showed that P. gingivalis increased the proportion of CCR6+ Treg (%) (13.780 ± 1.506 in the control group and 18.260 ± 2.257 in the experimental group, P < 0.01) and decreased the proportion of CD8+ T cells (%) (27.120 ± 1.647 in the control group and 21.060 ± 3.148 in the experimental group, P < 0.01) in OSCC, thereby promoting the formation of a immunosuppressive microenvironment.
    Conclusion P. gingivalis promotes the malignant progression of OSCC by recruiting CCR6+ Treg cells to form an immunosuppressive TME.

     

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