Mycobacterium tuberculosis MPT64 stimulates the activation of murine macrophage modulated by IFN-γ

Q. Fan, M. Lu, Z.-Y. Xia, L. Bao

Laboratory of Infection and Immunity, School of Basic Medical Sciences, West China Center of Medical Science, Sichuan University, No. 17, 3rd Section, Ren Min Nan, Chengdu, Sichuan, China. baolang@scu.edu.cn

 

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• Infectious Diseases

BACKGROUND AND AIMS: MPT64, a major target of cell-mediated immunity in tuberculosis, is proposed to be a promising candidate for novel vaccines and diagnostic tests; it also involves in virulence mechanism of Mycobacterium tuberculosis. In this study, we investigated the effect of MPT64 on activation of RAW264.7 macrophage, and explored the possible mechanism.

MATERIALS AND METHODS: MPT64 protein was prepared by cloning, expression and purification from Escherichia (E.) coli. Cytokines (IL-1β, IL-6, IL-10 and TNF-α) expression in RAW264.7 macrophage induced by MPT64 was evaluated by xMAP technology. Cell counting kit (CCK8) assay was employed to detect the viability of MPT64-treated macrophage; moreover, flow cytometric analysis and Western Blot analysis were used to measure the effect of MPT64 on apoptosis in macrophage and cleaved caspase-3 expression respectively.

RESULTS: MPT64 significantly promoted the levels of IL-1β, IL-6, IL-10 and TNF-α; pretreatment of MPT64 followed by IFN-γ stimulation further up-regulated IL-6 production (p < 0.01, compared with MPT64-treated group). In CCK8 assay, significantly increased absorbance of MPT64-treated macrophage suggested that cell viability might be enhanced. Additionally, MPT64 modulated the apoptosis of RAW264.7 macrophage partly through caspase-3 pathway.

CONCLUSIONS: MPT64 activated RAW264.7 macrophage to secrete IL-1β, IL-6, IL-10 and TNF-α; it might down-regulate apoptosis of macrophage partially via caspase-3 pathway. Furthermore, the effect of MPT64 on RAW264.7 macrophage could be modulated by IFN-γ, and co-stimulation of MPT64 with IFN-γ was conducive to the host immune, which might be applied for vaccine design and provide a reference for the treatment of tuberculosis.

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