Journal: Mediators of Inflammation

Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

doi: 10.1155/2018/3685948

Figure Lengend Snippet: Mtb H37Rv-infected A549 cells reduced TLR signaling activity and cytokine production of U937 cells in response to mycobacterial infection. (a) Illustration of cell culture models used for infection in this study. A549 cells were cultured on the apical surface of transwells at an air-liquid interface state for 24 hours; then the transwell insert was transferred to a well containing PMA-stimulated U937 cells for infection. The coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (b) Representative blots of immunoblotting assay for indicated components of TLR signaling cascade (left panel) and fold of changes of proteins of interest in U937 cells semiquantitatively determined by densitometric assay using ImageJ software Fiji (right panel). H37Rv-infected A549 cells showed an ability to reduce TLR signaling activity in U937 cells in response to mycobacterial infection. Concentrations of TNF- α (c), IL-10 (d), and IL-6 (e) in culture media determined by ELISA. H37Rv-infected A549 cells led a reduction of cytokine production in U937 cells in response to Mycobacteria infection. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗∗ p < 0.01; compared to infection of U937 cell alone, ΔΔ p < 0.01. NI: noninfected control; AI: infection was performed on A549 cell alone; UI: infection was performed on U937 alone; CI: infection was performed on both A549 cells and U937 cells.

Article Snippet: To generate a cell contact-independent model, the epithelial A549 cells were seeded on the upper chamber of a transwell (24 mm diameter transwell with 0.4 μ m pore polyester membrane insert, 3450 Clear, Corning Life Sciences, Corning, NY, USA) at a density of 5 × 10 5 cells/insert in culture medium and cultured overnight; the medium of the upper chamber was subsequently removed and the cells were cultured for an additional 24 hours to establish an air-liquid interface (ALI).

Techniques: Infection, Activity Assay, Cell Culture, Western Blot, Software, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control