College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui Province, 230036, PR China.
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, National Center for Tropical Diseases Research, WHO Collaborating Center for Tropical Diseases, Key Laboratory of Parasite and Vector Biology, National Health and Family Planning Commission, Shanghai 200025, PR China.
Trop Biomed. 2021 Mar 1;38(1):172-179. doi: 10.47665/tb.38.1.029.
The objective of this study was to investigate the mechanism of Toll-like receptor (TLR4)- mediated dendritic cell (DC) immune against Cryptosporidium parvum infection. C. parvum sporozoites were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester. Murine bone marrow-derived DCs were isolated, and divided into TLR4 antibody blocking (TAB; infected with 2 × 10 labeled sporozoites and 0.5 μg TLR4 blocking antibody), TLR4 antibody unblocking (TAU; infected with 2 × 10 labeled sporozoites), and blank control (BC; with 1.5 mL Roswell Park Memorial Institute 1640 medium) groups. The adhesion of Cryptosporidium sporozoites to DCs and CD11c levels were examined by fluorescence microscopy and flow cytometry. Male KM mice were orally injected with C. parvum. The proliferation of T lymphocytes in spleen, expression of cytokines in peripheral blood, and TLR4 distribution features in different organs were further determined by immunohistochemistry. A significantly higher expression of CD11c and higher C. parvum sporozoite adhesion were found in the TAU group compared with other groups. The expression of CD4CD8 /CD8CD4 in the spleen were obviously differences between the TAB and TAU groups. The expression of TLR4, interleukin IL-4, IL-12, IL-18 and IFN-γ improved in the TAU group compared with TAB group. Higher expression of TLR4 was detected in the lymph nodes of mice in the TAU group, with pathological changes in the small intestine. Hence, TLR4 could mediate DCs to recognize C. parvum, inducing Th1 immune reaction to control C. parvum infection.
本研究旨在探讨 Toll 样受体（TLR4）介导的树突状细胞（DC）对微小隐孢子虫感染的免疫机制。微小隐孢子虫卵囊用 5,6-羧基荧光素二乙酸琥珀酰亚胺酯进行标记。分离培养鼠骨髓来源的树突状细胞，分为 TLR4 抗体阻断（TAB；感染 2×10 个标记的卵囊和 0.5 μg TLR4 阻断抗体）、TLR4 抗体非阻断（TAU；感染 2×10 个标记的卵囊）和空白对照组（BC；用 1.5 mL 罗氏培养基 1640）。荧光显微镜和流式细胞术检测隐孢子虫卵囊与 DC 的黏附及 CD11c 水平。雄性 KM 小鼠经口感染微小隐孢子虫。免疫组化法进一步检测脾 T 淋巴细胞增殖、外周血细胞因子表达及各脏器 TLR4 分布特征。TAU 组 CD11c 表达明显高于其他组，卵囊黏附率也较高。TAB 和 TAU 组小鼠脾 CD4CD8 /CD8CD4 表达差异明显。TAU 组 TLR4 及细胞因子 IL-4、IL-12、IL-18 和 IFN-γ 表达均高于 TAB 组。TAU 组小鼠淋巴结 TLR4 表达升高，小肠出现病理改变。提示 TLR4 介导 DC 识别微小隐孢子虫，诱导 Th1 免疫反应以控制微小隐孢子虫感染。
