An Immunoregulatory Role Of Wnt/β-catenin Signaling In Macrophage In Response To Mycobacterium Tuberculosis Infection

Tuberculosis (TB) is an important infection disease of human and animals caused by Mycobacterium tuberculosis (Mtb), which remains a major public health problem in most parts of the world. Alveolar macrophages are main targets of Mtb infection, which are able to provide critical intracellular niches for Mtb establishing infection in host. The inflammatory responses, apoptosis and necrosis of host-pathogen interaction of macrophages and Mtb are crucial for the development and outcome of Mtb infection, which remain elusive. The process of Mtb infection of macrophages is regulated by many signals. Increasing evidence has demonstrated an ability of Wnt/β-catenin signaling to modulate the processes of many infectious diseases, However, the role of Wnt/β-catenin signaling in regulation of immune response, macrophage apoptosis and necrosis in response to Mtb infection remains unclear. Therefore, to explore it allows us to better understand the immunoregulatory role of Wnt/p-catenin signaling in the target cells of mycobacteria.In order to probe the molecular mechanism of an immunoregulatory role of Wnt/β-catenin signaling in macrophages in response to Mycobacterium tuberculosis infection, murine alveolar macrophage RAW264.7was transfected with Dkkl or cultured with Wnt3a CM followed by Mycobacterium bovis BCG infection; Wnt/p-catenin signaling were experimentally validated using luciferase reporte vectors; The inflammatory cytokins and apoptotic characteristics and necrotic characteristics were ascertained by flow cytometry method,electron microscope,laser confocal technology. ELISA, qRT-PCR and Western blotting assays. The results are shown as below:1. Wnt/β-catenin signaling significantly was able to suppress the BCG-induced production of IL-6and TNF-α and increase IL-10in both naive macrophages and BCG-infected cells with a down-regulation of TLR2and myD88andTRAF6and NFκB. This finding supported the notion of a negative regulatory role of Wnt/β-catenin signaling in host inflammatory responses against mycobacterial infection in macrophages.2. Wnt/β-catenin signaling could significantly reduce mitochondrial potential in both naive RAW264.7cells and the BCG-infected cells, as compared with the control cells. Molecular analysis by Western blotting assay and caspase inhibiting assay by Z-VAD further revealed that the Wnt/β-catenin induced apoptosis was accompanied with up-regulation of pro-apoptotic proteins Bax and cleaved caspase-3, and down-regulation of anti-apoptotic proteins Mcl-1macrophages, suggesting that the Wnt/p-catenin signaling promoted BCG-infected RAW264.7cell apoptosis was in part through a caspase-dependent mechanism. Cell cycle analysis further demonstrated an increasing G0/G1phase cell fraction and decreasing fractions of S phases and G2/M phase, These data suggested that Wnt3a was capable of regulating macrophages cell cycle progression.3. BCG was capable of inducing a dose-dependent and time-dependent necrosis in macrophages. The activation of the Wnt/p-catenin pathway could subsequently promote GSH content, suppress ROS and PARP-1and AIF of RAW264.7infected with BCG, accordingly suppress cell to necrosis. Based on above findings, we therefore proposed a below model of mechanism whereby the regulatory role of Wnt/β-catenin signaling in the apoptosis and necrosis of macrophages upon mycobacterial infection. In this model, upon invasion of mycobacteria, the pathogen is first recognized by TLRs of the cells, which in turn triggers the TLR signaling cascade to initiate inflammatory response. During the infection, the activated TLR signaling components and their down-stream target genes, including the NFκB and TNF-a, which in turn activate Wnt/β-catenin signaling that plays a negative feedback inhibitory role in modulation of inflammatory responses. Meanwhile, the activation of the Wnt/β-catenin pathway subsequently promotes a switch of necrosis to apoptosis in macrophages, in part by a mechanism of down-regulating expression of ROS and PARP-1and anti-apoptosis factors, such as Mcl-1, and up-regulating expression of pro-apoptotic factors BAX and caspase3.