感染猪布鲁菌S_2株死亡的巨噬细胞碎片启动抗布鲁菌感染的免疫应答

The fragments of dead macrophages infected by Brucellasuis S_2 strain activates the immune response against Brucella infection

目的研究猪布鲁菌S2株感染后死亡的巨噬细胞在启动抗布鲁菌感染免疫应答中的作用。方法以猪布鲁菌S2株体外感染小鼠腹腔巨噬细胞,感染1 h后,饥饿培养5 d致细胞死亡;细胞碎片与骨髓源性树突状细胞(DC)共培养24、48、72 h,ELISA检测细胞上清中白细胞介素12(IL-12)、肿瘤坏死因子α(TNF-α);以S2株体外感染羧基荧光黄二乙酸盐琥珀酰亚胺酯(CFSE)标记的小鼠巨噬细胞,饥饿避光培养,将细胞碎片与藻红蛋白标记的DC避光共培养1 h后,激光共聚焦显微镜检测DC摄取巨噬细胞碎片情况;以S2株感染后死亡的巨噬细胞碎片接种于BALB/c小鼠腹腔,1周后加强免疫1次;加强免疫3 d后,ELISA检测血清中IL-4、IL-2和γ干扰素(IFN-γ)水平。结果负载S2株感染后死亡巨噬细胞碎片的DC TNF-α的分泌水平明显高于未感染组,但不分泌IL-12;且S2株感染后死亡的巨噬细胞能被DC摄取到细胞内;接种S2株感染后死亡的巨噬细胞的小鼠血清中IL-2、IL-4和IFN-γ水平明显高于对照组。结论感染猪布鲁菌S2株死亡的巨噬细胞可激活DC、使其提呈抗原、并诱导机体产生抗感染免疫。

Objective To investigate the role of the dead macrophages infected by Brucella suis $2 strain in the initiation of immune response to Brucella. Methods The mouse peritoneal macrophages were infected with Brucella suis S2 strain in vitro. After one hour, the cells were cultured in serum-free RPMI1640 medium for 5 days until all of them were dead because of starvation. The dead cell fragments and the bone marrow-derived dendritic cells （BMDCs） were co-cultured for 24, 48 and 72 hours, and then interleukin 12 （IL-12）, tumor necrosis factor α （TNF-α） in the co-cultivation supernatant were detected by ELISA. The mouse macrophages marked by carboxyfluorescein diacetate succinimidyl ester （CFSE） were infected with Brucella suis S= strain in vitro, and then were cultured without serum in the dark; the dead macrophages fragments and BMDCs labeled with anti-CDllc-PE were co-cultured for 1 hour away from light, and then the changes that BMDCs swallowed the fragments of macrophages were observed by laser scanning confocal microscopy. BALB/c mice were inoculated with the fragments of dead macrophages infected by S2 strain through abdominal cavity. After one week, a second immunization was done. The serum levels of IL-4, IL-2 and IFN-γ were detected with ELISA at 3 days post-secondary immunization. Results The macrophages fragments infected by S2 strain could be swallowed by DCs. The level of TNF-α in BMDCs swallowing macrophages fragments infected by S2 strain was significantly higher than that in the control group, but the former did not secret IL-12. The levels of IL-2, IL-4 and IFN-γ in the sera from the mice inoculated with the macrophages fragments infected by S2 strain were dramatically higher than those in the control groups. Conclusion The dead macrophages infected by Brucella suis S2 strain can activate DCs to present antigen and induce the anti-Brucella immune response.