STAT6介导的巨噬细胞极化对布鲁氏菌胞内存活的影响

旨在探讨STAT6介导的巨噬细胞极化对布鲁氏菌胞内生存的影响。本研究采用布鲁氏菌光滑株S2308（S2308）和粗糙型疫苗株RB51（RB51）侵染巨噬细胞。利用qRT-PCR检测M1型巨噬细胞标志因子p65、NOS2和IL-1β，M2型巨噬细胞标志因子STAT6、ARG1、IL-10的mRNA表达水平；流式细胞术检测M1型标记分子CD86和M2型标记分子CD206的表达；Western blot检测p-STAT6蛋白及抑制剂AS对蛋白的抑制作用；ELISA检测M1型细胞因子TNF-α、IL-12和M2型细胞因子IL-4、IL-10的表达量；最后对胞内菌落进行CFU计数。qRT-PCR结果显示，在感染8、12 h时可显著诱导M1型因子mRNA转录表达，72 h时低表达，而M2型因子在72 h时高表达；流式细胞术结果显示，S2308感染12 h可显著诱导CD86的表达，感染72 h可显著诱导CD206的表达，但RB51对二者无影响；Western blot结果显示，S2308菌株在感染72 h时激活STAT6信号通路，而RB51几乎不激活该通路，抑制剂AS在2 μmol·L^-1浓度时抑制效果最佳；ELISA结果显示，AS抑制剂可显著抑制IL-4、IL-10的释放，并促进TNF-α、IL-12的释放；CFU计数结果显示，S2308组的胞内菌呈先降低后显著上升趋势，加入AS抑制剂后可显著抑制布鲁氏菌胞内复制。布鲁氏菌S2308在感染后期能够通过STAT6诱导M1型巨噬细胞向M2型转化，并促进Th2型细胞因子的释放，从而有利于布鲁氏菌的胞内生存。而RB51几乎不激活该通路，不影响胞内生存。

Effect of STAT6 Mediated Macrophage Polarization on Intracellular Survival of Brucella

This study aimed to investigate the effect of STAT6 mediated macrophage polarization on intracellular survival of Brucella.Brucella smooth strain S2308(S2308)and rough vaccine strain RB51(RB51)were used to infect macrophages.M1-type macrophage marker factors p65,NOS2 and IL-1βwere detected by qRT-PCR.mRNA expression levels of M2-type macrophage marker factors STAT6,ARG1 and IL-10 were detected by qRT-PCR.The expression of M1-type marker CD86 and M2-type marker CD206 was detected by flow cytometry.The inhibitory effect of p-STAT6 protein and inhibitor AS on the protein was detected by Western Blot.The expression levels of M1-type cytokines TNF-αand IL-12 and M2-type cytokines IL-4 and IL-10 were detected by ELISA.Finally,CFU count of intracellular colonies was carried out.The qRT-PCR results showed that the mRNA expression of M1 type factor was significantly induced at 8 h and 12 h after infection,the expression of M2 type factor was low at 72 h,and the expression of M2 type factor was high at 72 h.Flow cytometry showed that S2308 could significantly induce CD86 expression at 12 h after infection,and CD206 expression at 72 h after infection,but RB51 had no effect on both.Western Blot results showed that strain S2308 activated STAT6 signaling pathway 72 h after infection,while RB51 hardly activated STAT6 signaling pathway.The inhibitory effect of inhibitor AS was the best at 2μmol·L^-1 concentration.ELISA results showed that AS inhibitors could significantly inhibit the release of IL-4 and IL-10,and promote the release of TNF-αand IL-12.CFU count results showed that intracellular bacteria in S2308 group were firstly decreased and then significantly increased,and S2308+IL-4 group was significantly higher than S2308 group,S2308+AS group was significantly lower than S2308 group,and there was no significant difference in RB51 group.Brucella S2308 can induce the transformation of M1 type macrophages to M2 type macrophages through STAT6 in the late stage of infection,and promote the release of Th2 type cytokines,which is conducive to the intracellular survival of Brucella.RB51 does not activate this pathway and does not affect intracellular survival.