Mycobacteria directly induce cytoskeletal rearrangements for macrophage spreading and polarization through TLR2-dependent PI3K signaling.
ABSTRACT Macrophage migration and adhesion are important for the control of mycobacterial infection and are critically dependent on the reorganization of the cytoskeleton. Mycobacteria elicit rapid morphological changes, such as cell spreading, a process relevant to in vivo changes of macrophage shape during extravasation and migration. In this study, we investigated the BCG mycobacteria-induced signaling events leading to macrophage cytoskeletal rearrangements employing specific pharmacological inhibitors to suppress distinct kinase pathways known to be elicited by infection. Viable or lysed mycobacteria, as well as purified cell wall lipoprotein p19, TLR2 agonist, induced RAW264.7 cells to extend actin-rich pseudopods, which impart radial spreading within 3 h, leading later to persistent cell polarization. BCG induced rapid activation of phosphatidylinositol 3-kinase, PI3K, activation that was recruited to the activated TLR2 receptor. TLR2- neutralizing antibody inhibited macrophage spreading and PI3K activation induced by p19. Additionally, BCG induced spreading and polarization of bone marrow-derived macrophages from TLR2- expressing mice in contrast to their TLR2-knockout counterparts. Neither MEK1/ERK, p38 MAPK, nor NF-kappaB activation were important for the early cytoskeletal rearrangements observed, although suppression of these pathways is known to inhibit chemokine secretion by activated macrophages. Beta2-integrins blockade with a corresponding antibody inhibited macrophage spreading and polarization but had no effect on pseudopodia protrusions demonstrating the downstream position of integrin-mediated adhesion in PI3K- dependent signaling pathway leading to the motility phenotype. The obtained data demonstrate that the direct effect of mycobacteria on macrophage shape might be mediated through TLR2-dependent PI3K activation.
Article: Inhibitory effects of honokiol on LPS and PMA-induced cellular responses of macrophages and monocytes.[show abstract] [hide abstract]
ABSTRACT: The regulatory effects of honokiol on the cellular responses of macrophages and monocytes were evaluated. Specifically, we investigated the effects of honokiol with respect to lipopolysaccharide (LPS)-induced cytotoxicity, LPS- or phorbol-12-myristate-13-acetate (PMA)-mediated morphological changes, and relevant events (FITC-dextran-induced phagocytic uptake). Honokiol blocked the LPS-induced cytotoxicity of RAW264.7 cells in a dose-dependent manner. In addition, honokiol appeared to block the production of cytotoxic cytokines such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha, nitric oxide (NO), and reactive oxygen species (ROS). Moreover, honokiol strongly prevented the morphological changes in RAW 264.7 and U937 cells that were induced by LPS and PMA. The surface levels of marker proteins, which are up-regulated under the morphological changes of RAW264.7 and U937 cells, were also diminished. The data presented here strongly suggest that the honokiol modulates various cellular responses managed by macrophages and monocytes. [BMB reports 2009; 42(9): 574-579].BMB reports 09/2009; 42(9):574-9. · 1.72 Impact Factor
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ABSTRACT: The obligately intracellular bacterium Ehrlichia chaffeensis that resides in mononuclear phagocytes is the causative agent of human monocytotropic ehrlichiosis. Ehrlichia muris and Ixodes ovatus Ehrlichia (IOE) are agents of mouse models of ehrlichiosis. The mechanism by which Ehrlichia are transported from an infected host cell to a non-infected cell has not been demonstrated. Using fluorescence microscopy and transmission and scanning electron microscopy, we demonstrated that Ehrlichia was transported through the filopodia of macrophages during early stages of infection. If host cells were not present in the vicinity of an Ehrlichia-infected cell, the leading edge of the filopodium formed a fan-shaped structure filled with the pathogen. Formation of filopodia in the host macrophages was inhibited by cytochalasin D and ehrlichial transport were prevented due to the absence of filopodia formation. At late stages of infection the host cell membrane was ruptured, and the bacteria were released. Ehrlichia are transported through the host cell filopodium during initial stages of infection, but are released by host cell membrane rupture during later stages of infection.PLoS ONE 01/2010; 5(12):e15775. · 4.09 Impact Factor
Article: Evidence for oxidative stress and defective antioxidant response in guinea pigs with tuberculosis.[show abstract] [hide abstract]
ABSTRACT: The development of granulomatous inflammation with caseous necrosis is an important but poorly understood manifestation of tuberculosis in humans and some animal models. In this study we measured the byproducts of oxidative stress in granulomatous lesions as well as the systemic antioxidant capacity of BCG vaccinated and non-vaccinated guinea pigs experimentally infected with Mycobacterium tuberculosis. In non-vaccinated guinea pigs, oxidative stress was evident within 2 weeks of infection as measured by a decrease in the serum total antioxidant capacity and blood glutathione levels accompanied by an increase in malondialdehyde, a byproduct of lipid peroxidation, within lesions. Despite a decrease in total and reduced blood glutathione concentrations, there was an increase in lesion glutathione by immunohistochemistry in response to localized oxidative stress. In addition there was an increase in the expression of the host transcription factor nuclear erythroid 2 p45-related factor 2 (Nrf2), which regulates several protein and non-proteins antioxidants, including glutathione. Despite the increase in cytoplasmic expression of Nrf2, immunohistochemical staining revealed a defect in Nrf2 nuclear translocation within granulomatous lesions as well as a decrease in the expression of the Nrf2-regulated antioxidant protein NQO1. Treating M. tuberculosis-infected guinea pigs with the antioxidant drug N-acetyl cysteine (NAC) partially restored blood glutathione concentrations and the serum total antioxidant capacity. Treatment with NAC also decreased spleen bacterial counts, as well as decreased the lung and spleen lesion burden and the severity of lesion necrosis. These data suggest that the progressive oxidative stress during experimental tuberculosis in guinea pigs is due in part to a defect in host antioxidant defenses, which, we show here, can be partially restored with antioxidant treatment. These data suggest that the therapeutic strategies that reduce oxidant-mediated tissue damage may be beneficial as an adjunct therapy in the treatment and prevention of tuberculosis in humans.PLoS ONE 01/2011; 6(10):e26254. · 4.09 Impact Factor