Department of Liver Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, 510080, People's Republic of China.
Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, 510080, People's Republic of China.
J Exp Clin Cancer Res. 2019 Jul 11;38(1):303. doi: 10.1186/s13046-019-1271-3.
Gut microbiota and the tumor microenvironment are thought to be critical factors that modulate the processes of liver diseases, including hepatocellular carcinoma (HCC). Interleukin-25 (IL-25) promotes type 2 immunity via alternative activation of macrophages, and is closely associated with inflammation-related diseases, even malignancies. However, it is not clear which role IL-25 plays in the development of HCC, and whether gut microbiota are involved.
IL-25 was detected by ELISA, Western blotting (WB), and immunohistochemistry. Chemokines were measured by RT-qPCR and WB. After co-culture with IL-25-stimulated macrophages, the cell growth, migration, invasion and EMT marker of HCC cell lines (MHCC97L and HepG2) were evaluated by Brdu proliferation, Transwell assays and WB. An antibody neutralization assay of chemokine CXCL10 was performed to confirm its role in HCC development. Furthermore, the effects of IL-25 in HCC were investigated in vivo. Dysbiosis of gut microflora was induced by antibiotics (vancomycin, cefoperazone or combination of ampicillin, neomycin, metronidazole, and vancomycin). We used feces suspension to treat colonic epithelial NCM460 cells, and detected IL-25 and tuft cell marker DCLK1 using WB and immunofluorescence staining.
We found that the level of IL-25 was significantly elevated in HCC patients, and was negatively correlated with survival rate after hepatectomy. However, IL-25 did not directly promote the development of HCC cells. Then, we observed the significant positive correlation between IL-25 level and M2 percentage (CD206/CD68) in HCC tumors. In vitro and in vivo, IL-25 induced alternative activation of macrophages promoted HCC cell migration, invasion and tumorigenesis, increased the expression of vimentin, Snail and phospho-ERK, and decreased the expression of E-cadherin in HCC cells. After IL-25 treatment, chemokine CXCL10 was increased in macrophages. Neutralizing CXCL10 in macrophage-conditioned medium reversed the IL-25-mediated effect on HCC cells. Vancomycin-induced dysbiosis promoted the growth of orthotopic HCC homograft. Surprisedly, we found the hyperplasia of colonic epithelial tuft cells, from which more IL-25 was secreted .
IL-25 promotes the progression of HCC through inducing alternative activation and CXCL10 secretion of macrophages in tumor microenvironment, and IL-25 secretion may partly result from hyperplastic epithelial tuft cells in colon, induced by gut microbiota dysbiosis.
肠道微生物群和肿瘤微环境被认为是调节肝脏疾病（包括肝细胞癌[HCC]）过程的关键因素。白细胞介素 25（IL-25）通过巨噬细胞的替代性激活促进 2 型免疫，并且与炎症相关疾病甚至恶性肿瘤密切相关。然而，IL-25 在 HCC 发展中的作用以及肠道微生物群是否参与其中尚不清楚。
通过 ELISA、Western blot（WB）和免疫组织化学检测 IL-25。通过 RT-qPCR 和 WB 测量趋化因子。在与 IL-25 刺激的巨噬细胞共培养后，通过 Brdu 增殖、Transwell 测定和 WB 评估 HCC 细胞系（MHCC97L 和 HepG2）的细胞生长、迁移、侵袭和 EMT 标志物。通过趋化因子 CXCL10 的抗体中和测定来确认其在 HCC 发展中的作用。此外，还在体内研究了 IL-25 在 HCC 中的作用。抗生素（万古霉素、头孢哌酮或氨苄西林、新霉素、甲硝唑和万古霉素的组合）诱导肠道微生物群失调。我们使用粪便悬浮液处理结肠上皮 NCM460 细胞，并用 WB 和免疫荧光染色检测 IL-25 和绒毛细胞标志物 DCLK1。
我们发现 HCC 患者的 IL-25 水平显着升高，并且与肝切除术后的生存率呈负相关。然而，IL-25 并没有直接促进 HCC 细胞的发展。然后，我们观察到 IL-25 水平与 HCC 肿瘤中 M2 百分比（CD206/CD68）之间存在显着正相关。在体外和体内，IL-25 诱导的巨噬细胞替代性激活促进了 HCC 细胞的迁移、侵袭和肿瘤发生，增加了 HCC 细胞中波形蛋白、Snail 和磷酸化 ERK 的表达，降低了 E-钙粘蛋白的表达。在 IL-25 处理后，趋化因子 CXCL10 在巨噬细胞中增加。在巨噬细胞条件培养基中中和 CXCL10 逆转了 IL-25 对 HCC 细胞的介导作用。万古霉素诱导的微生物群失调促进了原位 HCC 同种移植物的生长。令人惊讶的是，我们发现结肠上皮绒毛细胞增生，从中分泌出更多的 IL-25。
IL-25 通过诱导肿瘤微环境中的巨噬细胞替代性激活和 CXCL10 分泌来促进 HCC 的进展，而 IL-25 的分泌可能部分来自于肠道微生物群失调引起的结肠上皮绒毛细胞增生。
