title: Mycobacterium tuberculosis MPT64 stimulates the activation of murine macrophage modulated by IFN-γ.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: 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  
authors.forename: Q
authors.initials: Q
authors.lastname: Fan
authors.forename: M
authors.initials: M
authors.lastname: Lu
authors.forename: Z-Y
authors.initials: ZY
authors.lastname: Xia
authors.forename: L
authors.initials: L
authors.lastname: Bao
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D007371:Interferon-gamma / Q000276:immunology* / Q000378:metabolism; D016753:Interleukin-10 / Q000276:immunology / Q000378:metabolism; D053583:Interleukin-1beta / Q000276:immunology / Q000378:metabolism; D015850:Interleukin-6 / Q000276:immunology / Q000378:metabolism; D008262:Macrophage Activation; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; C556062:IL10 protein, mouse; D053583:Interleukin-1beta; D015850:Interleukin-6; C510537:MPT64 protein, Mycobacterium tuberculosis; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D007371:Interferon-gamma; C505409:Casp3 protein, mouse; D053148:Caspase 3