弓形虫宿主依赖因子Cbl-b负性调控TLRs/MyD88介导的天然免疫的机制研究

Toxoplasma gondii is an important zoonotic protozoan, which has to exploit host proteins for successful infection. Such host proteins are called host dependency factors (HDFs). Our previous study used lentiviral CRISPR-Cas9-sgRNA libraries to edit the genome of host cells to get gene knockout cell library, then the cells were infected with Toxoplasma gondii to screen the HDFs required for Toxoplasma gondii infection. One of the screened HDFs is Casitas B-lineage lymphoma-b (Cbl-b) protein. Cbl-b interacts with C-type lectin receptors (CLRs) to ubiquitinate CLR for degradation, and consequently to inhibit host innate immunity against fungi infection. As host toll-like receptors (TLRs)/ MyD88 pathway plays a very important role in host innate immunity against Toxoplasma gondii infection, and it is reported that ring-finger domain of Cbl-b binds with MyD88 for degradation in LPS activated murine macrophages. We proposed that Cbl-b interacted with MyD88 when host cells were infected by Toxoplasma gondii, and then ubiquitinate MyD88 for degradation, as a result, negatively regulate host's anti-Toxoplasma gondii innate immunity. Cbl-b gene knockout cells or mice will be used to verify whether Cbl-b is a HDF required for Toxoplasma gondii infection. After host cells being infected with Toxoplasma gondii, the binding of Cbl-b with MyD88 to ubiquitinate MyD88 for degradation will also be tested, and Cbl-b's negative regulation on TLRs/MyD88 dependant anti-Toxoplasma gondii innate immunity through reducing the production of anti-infection cytokines will be evaluated. HDFs screening and functional analysis is of important innovation significance for the research of Toxoplasma gondii infection and immunity, and for anti-T. gondii infection therapeutic agents designing.

弓形虫为重要人兽共患病原虫，需依赖宿主蛋白完成感染，这些蛋白即宿主依赖因子（HDFs）。我们利用人sgRNA慢病毒库构建基因敲除细胞库并用弓形虫感染筛选其HDFs，发现泛素连接酶E3（Cbl-b）为潜在HDF。Cbl-b可与C型凝集素受体（CLRs）结合并将其泛素化降解，抑制细胞针对真菌的天然免疫。弓形虫感染时宿主TLRs/MyD88天然免疫发挥重要作用,据报道LPS刺激小鼠巨噬细胞时Cbl-b可使MyD88降解。我们提出：弓形虫感染致使Cbl-b与MyD88结合，并泛素化降解MyD88，从而抑制TLRs/MyD88介导的抗弓形虫天然免疫。本项目拟应用基因敲除细胞与小鼠验证Cbl-b为弓形虫HDF,阐明Cbl-b通过与MyD88结合、使其泛素化降解，减少抗感染细胞因子分泌从而负性调控TLRs/MyD88天然免疫的机制。HDF筛选鉴定及功能分析对弓形虫感染免疫研究及药物靶标筛选具有重要意义。

弓形虫需依赖宿主因子完成感染和增殖，即宿主依赖因子（HDFs）。本研究利用针对人的CRISPR库构建基因敲除细胞库并感染弓形虫筛选其HDFs。结果共筛到1183个基因和17条成熟miRNA为潜在HDFs。研究发现，Rac1的弓形虫HDF, 可被弓形虫感染所激活；弓形虫入侵时Rac1被招募到纳虫泡膜（PVM），完全入侵后随着时间延长Rac1从PVM上消退直到消失；随着入侵时间延长Rac1磷酸化水平也升高；Rac1特异性抑制剂可阻碍宿主F-Actin聚合，且显著抑制弓形虫的入侵和增殖。Cbl-b也是弓形虫的HDF，敲低Cbl-b可显著抑制弓形虫增殖，弓形虫感染可导致Cbl-b高表达及MyD88降解，Cbl-b可与MyD88发生相互作用，敲除Cbl-b可增强宿主抗弓形虫免疫应答。这些结果说明Cbl-b可负性调控TLRs/MyD88介导的抗弓形虫天然免疫。该研究可为弓形虫感染机制研究和药物靶标筛选提供理论基础。TLR7是固有免疫受体分子。TLR7在B细胞介导的免疫应答中的作用尚不详。本研究采用日本血吸虫感染小鼠。感染小鼠脾脏的B细胞占比和绝对数量均增加。感染小鼠更多的脾脏免疫细胞表达TLR7，并且B细胞是最主要的细胞群。此外，与感染WT小鼠相比，感染日本血吸虫的TLR7 KO小鼠血清中针对SEA特异性抗体水平较低。在相同剂量SEA刺激作用下，未感染WT小鼠B细胞的活化水平高于TLR7 KO小鼠B细胞。并且，TLR7对B细胞的活化作用依赖于NF-κB p65的磷酸化。总之我们发现TLR7可调节脾脏B细胞免疫应答。该研究有助于全面解析B细胞在血吸虫病患者中的作用及机制。目前γδT细胞在疟原虫感染小鼠肺脏中的作用尚不清。本研究采用疟原虫感染小鼠后发现，疟原虫感染后肺脏γδT细胞的占比和绝对数均增加。感染后，更多的γδT细胞表达CD80、CD11b和PD-1，更少的γδT细胞表达CD34、CD62L和CD127。感染后IL-4+、IL-5+、IL-6+、IL-21+、IL-1α+、IL-17+ 的γδT细胞百分率升高，而表达IFN-γ的γδT细胞百分比降低。与野生感染小鼠相比，γδTCR敲除小鼠感染疟原虫后其肺脏T细胞比例和绝对数均显著下降，分泌IFN-γ的能力增强。总之我们发现γδT细胞在疟原虫感染过程中参与T细胞免疫应答。本研究有助于了解γδT细胞在疟疾患者中的作用。

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