T细胞对非适应性宿主抗血吸虫病的作用

Schistosomiasis is a major infectious disease in which the immunopathological mechanisms still need to be elucidated. Previous studies have revealed the critical roles of T cells in the occurrence of schistosomiasis in susceptible hosts such as humans and mice. However very little is known about the T cell role in resistance of non-permissive hosts such as the vole termed Microtus Fortis and SD rat to schistosomiasis. To unmask whether and how T cells are involved in such an infectious disorder, it is essential to perform genetic engineering and develop genetic models. In this study, we will use the CRISPR/Cas9 genome editing technology to inactivate in vivo the Lck gene which is required for T cell development in thymus, to obtain T cell deficient animals. The applicant has succeeded in generating the Lck deleted SD rats and also found that transplant of thymus tissue of Microtus Fortis into the renal capsule of NOG recipient mice allowed for development of xenografted T cells, which resulted in a protective function against schistosomiasis in the host mice. In this study, we will further obtain the Lck disrupted T cell deficient Microtus Fortis, and verify the resistance to schistosomiasis of the genetically engineered animals. In the T cell deficient animals and T cell sufficient controls, the parasitic worm development will be compared and so the histopathological changes in the host livers. Using mice as control, we will sort T cells from the non-permissive hosts and analyze the T helper cell differentiation by real-time quantitative PCR. This project is set to determine the role of T cells in resistance of non-permissive hosts to schistosomiasis, which may provide a new perspective for prevention and treatment of schistosomiasis.

血吸虫病是严重危害人类健康的感染性疾病，研究其免疫机制十分必要。科学家们已揭示了易感宿主如人和小鼠T细胞在血吸虫病中的重要作用，但对非适应性宿主如东方田鼠和SD大鼠抗血吸虫病是否依赖T细胞还缺乏研究。明确这一问题需要在遗传模型上取得突破。本项目利用CRISPR/Cas9技术敲除东方田鼠和SD大鼠T细胞发育所必需的Lck基因，获得T细胞缺陷遗传模型。申请人已完成SD大鼠Lck基因敲除，并在前期实验中成功将东方田鼠胸腺移植到NOG小鼠肾被膜，后者能发育东方田鼠T细胞且获得对血吸虫病的保护作用。本项目将获得T细胞缺陷东方田鼠，验证其与SD大鼠对血吸虫病抗性是否依赖T细胞，比较T细胞缺陷动物与野生型对照感染血吸虫后虫体发育、宿主肝脏病理变化。进一步分选感染后不同宿主的脾脏T细胞，用荧光定量PCR分析其辅助T细胞分化的差异。本项目旨在阐明T细胞对非适宜宿主抗血吸虫病的作用，为血吸虫病防治提供新思路。

血吸虫病是严重危害人类健康的感染性疾病，每年引起全球范围内约二百万人感染。人和小鼠是易感宿主而东方田鼠和SD大鼠是天然的非适应性宿主。科学家们已揭示了易感宿主T细胞在血吸虫病中的重要作用，但对非适应性宿主如东方田鼠和SD大鼠抗血吸虫病是否依赖T细胞还缺乏研究。申请人在早期实验中首先成功将东方田鼠胸腺移植到NOG小鼠肾被膜，后者能发育东方田鼠T细胞，且血吸虫感染后的肝脏病理损害较未移植的NOG对照组明显减轻，说明原本是易感宿主的移植小鼠获得了对血吸虫病的保护。本项目通过利用CRISPR/Cas9基因组编辑技术以敲除T细胞发育所必需的Lck基因，由于一直未能获得基因敲除东方田鼠模型，因此后续实验聚焦于T细胞缺陷SD大鼠模型。在感染血吸虫条件下，研究从寄生虫发育及宿主病理、免疫调节这两个大的方面对T细胞缺陷动物与野生型对照进行对比，所检测的指标包括虫体发育（虫长、虫宽）、肝门肠系膜成虫回收率、宿主肝脏病理变化、宿主外周血及脾脏中免疫表型变化等，证实了SD大鼠血吸虫病抗性依赖于T细胞的作用。同时用荧光定量PCR检测易感宿主小鼠和非适应性宿主SD大鼠在感染前后辅助性T细胞转录因子表达的差异，结果发现大鼠和小鼠中检测的五种转录因子表达变化具有相同的趋势，其中Tbx21和Bcl6的表达被诱导轻微上调，另三种转录因子表达没有显著变化。本项目还发现细胞（趋化）因子LIX、CCL20、TIMP1和VEFG在T细胞缺失和T细胞正常的SD大鼠分别感染血吸虫后呈现截然不同的表达趋势，提示在下一步研究中可以将这些细胞因子条件性敲除或者特异性过表达，以进一步揭示它们为抗血吸虫病的贡献作用。