激活TLR5和NLRC4通路对小鼠先天免疫细胞的影响

Impact of TLR5 and NLRC4 activation on innate immune cells in mice

目的:探讨用重组鞭毛素蛋白同时或者分别激活C57BL/6小鼠模型TLR5和NLRC4通路对小鼠先天免疫细胞的影响。方法:首先,表达和纯化重组鞭毛素蛋白即全长鞭毛素蛋白FliC(同时激活TLR5和NLRC4两条通路);FliCΔ90-97(不能激活TLR5通路);FliC-L3A(不能激活NLRC4通路);FliCΔ90-97:L3A(两条通路都不激活)。将小鼠分为5组,分别为:PBS组、FliC组、FliC-L3A组、FliCΔ90-97和FliCΔ90-97:L3A组。分别用PBS和10μg重组鞭毛素蛋白腹腔注射C57BL/6小鼠,每组3只。12 h后收集腹腔灌洗液,流式抗体染色检测腹腔灌洗液中的中性粒细胞和NK细胞的比例。同时,取小鼠脾脏制成脾细胞悬液,流式抗体染色检测DC表面共刺激分子CD80和CD86的表达情况及脾细胞中调节性T细胞(Treg)的比例。结果:激活TLR5(FliC组和FliC-L3A组)和NLRC4通路(FliCΔ90-97组)小鼠的腹腔灌洗液中中性粒细胞和NK细胞的比例都显著高于PBS组和FliCΔ90-97:L3A组(P<0.01)。激活TLR5通路的FliC组和FliC-L3A组的DC表面CD80和CD86表达水平显著高于PBS组、FliCΔ90-97和FliCΔ90-97:L3A组(P<0.01)。FliC-L3A组调节性T细胞的比例高于其他组(P<0.05)。结论:激活TLR5和NLRC4通路可以趋化中性粒细胞、NK细胞,两条通路激活对中性粒、NK细胞有相同的趋化能力。鞭毛素蛋白只有激活TLR5通路才可以上调DCs表面共刺激分子CD80和CD86,促进DCs的成熟。

Objective：To investigate the impact of recombinant flagellin targeting TLR5 and NLRC4 simultaneously or respectively on innate immune cells in mice. Methods： Induction,expression,purification and identification of recombiant FliC,which were FliC（activating both TLR5 and NLRC4）;FliCΔ90-97（unable to activate TLR5）,FliC-L3A（unable to activate NLRC4）,FliCΔ90-97：L3A（unable to activate both TLR5 and NLRC4）. The mice were divided into five groups,namely group FliC,FliC-L3A,FliCΔ90-97,FliCΔ90-97：L3A and PBS,which were injected with 100μl PBS or 10μg recombinant flagellin intraperitoneally,three mice in each group. 12 h later,the mice were executed using dislocation of cervical vertebra and the splenic and peritoneal cells were isolated. The spleen was grinded into single-cell suspension. The proportion of neutrophils,NK cells,DCs and the expression level of CD80 and CD86 on DCs were evaluated with flow cytometry. Results：Group FliC,group FliC-L3A and group FliCΔ90-97 shared the similar proportion of neutrophils in peritoneal cavity （ P〈0. 05 ） , and all of which were significantly higher than group PBS and group FliCΔ90-97 （ P〈0. 01）,and NK cells also showed the similar trend. Compared with group FliCΔ90-97 and FliCΔ90-97：L3A,the mean fluorescence intensities（MFIs） of CD80 and CD86 in group FliC and FliC-L3A increased significantly（P〈0. 01）. The proportion of Treg in spleen was highest among all groups. Conclusion：Activation of TLR5 and NLRC4 had similar chemotaxis of neutrophils and NK cells. The ex-pression of CD80 and CD86 on DCs were upregulated after stimulation by flagellin and TLR5-dependent. Activation of TLR5,but not NLRC4,increased the proportion of Treg in spleen.