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

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.