结核分枝杆菌CFP10和ESAT6对巨噬细胞RAW264.7凋亡及AIM2/ASC/Caspase-8通路的影响

Effects of CFP10 and ESAT6 on cell apoptosis and AIM2/ASC/Caspase-8 pathway in RAW264.7 macrophages

摘要:

【背景】10 kD培养滤液蛋白(culture filtrate protein 10，CFP10)和6 kD早期分泌性抗原靶蛋白(early secretary antigenic target-6 kD，ESAT6)是结核分枝杆菌(Mycobacterium tuberculosis，Mtb)重要的毒力因子，能引起巨噬细胞的凋亡。【目的】探讨CFP10和ESAT6对巨噬细胞RAW264.7凋亡及AIM2/ASC/Caspase-8信号通路的影响。【方法】利用大肠杆菌表达并纯化获得了CFP10和ESAT6蛋白，重组蛋白处理巨噬细胞RAW264.7后，利用CCK8试剂盒检测细胞存活率，确定重组蛋白处理细胞浓度，利用Western blotting技术检测细胞凋亡相关蛋白及AIM2和ASC炎性小体的变化，利用流式细胞术检测细胞凋亡率。【结果】SDS-PAGE和Western blotting结果表明重组蛋白CFP10和ESAT6表达正确，不同浓度的CFP10和ESAT6处理RAW264.7后，对细胞的增殖能力具有明显的抑制作用，当CFP10和ESAT6单独处理且浓度为5 μg/mL时，细胞存活率较对照组有显著下降(P<0.001)，且随着蛋白浓度的增加细胞存活率显著下降(P<0.001)。Western blotting结果表明，CFP10和ESAT6蛋白单独处理巨噬细胞24 h后均能引起巨噬细胞发生凋亡。当终浓度为5 μg/mL的CFP10和ESAT6共处理巨噬细胞时，共处理组凋亡相关蛋白BAD、CHOP、Caspase-8和Caspase-3较ESAT6单独处理组有极显著差异(P<0.001)，说明CFP10和ESAT6共处理显著降低了ESAT6单独处理引起的巨噬细胞的凋亡，进一步研究发现ESAT6能激活AIM2、ASC炎性小体。【结论】结核分枝杆菌CFP10和ESAT6处理RAW264.7后均能引起巨噬细胞发生凋亡，当二者共处理时，CFP10会显著降低ESAT6单独处理引起的细胞的凋亡，进一步的研究表明，ESAT6可能通过激活AIM2/ASC/Caspase-8信号通路从而引起巨噬细胞发生凋亡。研究结果为进一步探究Mtb感染过程中CFP10和ESAT6蛋白对巨噬细胞凋亡的调控作用及其分子机制奠定了基础。

Abstract:

[Background] CFP10 and ESAT6 are important virulence factors of Mycobacterium tuberculosis (Mtb), and can cause the apoptosis of macrophages. [Objective] To investigate the effects of CFP10 and ESAT6 on cell apoptosis and AIM2/ASC/Caspase-8 pathway in RAW264.7 macrophages. [Methods] E. coli expression system was used to express and purify recombinant CFP10 and ESAT6 proteins of Mtb. Then, the RAW264.7 cells were treated with the recombinant proteins with different concentrations. The cell viability of RAW264.7 cells after incubation with CFP10 and ESAT6 at different concentrations for 24 h was measured by CCK-8 assay, expression of apoptosis related proteins as well as AIM2 and ASC inflammasomes were determined by Western blotting analysis, and the cell apoptosis was detected by flow cytometry. [Results] The results of SDS-PAGE and Western blotting showed that we have successfully purified the recombinant proteins of CFP10 and ESAT6 from E. coli. After treatment of RAW264.7 cells with different concentrations of CFP10 and ESAT6, the cells proliferation was significantly inhibited. When CFP10 and ESAT6 were treated separately at the concentration of 5 μg/ml, the cell viability rate decreased significantly as compared with the control group (P<0.001), and the cell viability rate decreased significantly in a dose dependent manner (P<0.001). Western blotting results further showed that both of CFP10 and ESAT6 with the concentration of 5 μg/ml could lead to RAW264.7 cells apoptosis after 24 h treatment. When RAW264.7 cells were treated with CFP10 and ESAT6 together at a final concentration of 5 μg/ml respectively, the expression of apoptosis-related proteins of BAD, CHOP, Caspase-8 and Caspase-3 decreased significantly as compared to ESAT6 treatment group (P<0.001), an indication that the co-treatment of RAW264.7 cells with CFP10 and ESAT6 significantly reduced the apoptosis of macrophages caused by ESAT6 treatment alone. Furthermore, the results of Western blotting showed that ESAT6 treatment could activate the expression of AIM2 and ASC inflammasomes. [Conclusion] CFP10 and ESAT6 of Mtb alone at the concentration of 5 μg/ml could cause apoptosis of RAW264.7. The co-treatment of RAW264.7 cells with CFP10 and ESAT6 could reduce the apoptosis of macrophages caused by ESAT6 treatment alone. Further studies show that ESAT6 may cause RAW264.7 cell apoptosis by activating AIM2/ASC/Caspase-8 signaling pathway. The results of this study will lay a foundation for fully understanding the immunoregulatory roles of CFP10 and ESAT6 on cell apoptosis and its molecular mechanisms in macrophage defense against Mtb infection.