Effects of TLR4-PI3K-Rac1 pathway on cytoskeleton rearrangement and phagocytosis of macrophage

Abstract

Objective: To investigate the effects of Toll-like receptor 4 (TLR4)-phosphatidylinositol 3-kinase (PI3K) -Ras-related C3 botulinum toxin 1 (Rac1) signaling pathway on macrophage cytoskeleton rearrangement and phagocytosis. Methods: Mouse macrophage cell line RAW264.7 was divided into blank group, negative control group and TLR4-RNA interference (RNAi) group. The lentivirus carrying TLR4 short hairpin RNA (shRNA) and nonsense control sequence were respectively transfected into TLR4-RNAi group and negative control group. The cells in blank group were not transfected. The silencing efficiency of TLR4 was detected by Western blot. Real-time fluorescence quantitative PCR was used to detect the expression of PI3K and Rac1 mRNA in each group. The expressions of PI3K, p-Rac1 and Rac1 protein were detected by Western blot. Cytoskeleton was observed by laser scanning confocal microscopy. Mean fluorescence intensity (MFI) and the percentage of cells phagocytosing flurescein inothiocyanate-labeled Eseherichina coli (FITC-E.coli) (Phagocytosed cell %) were detected by flow cytometry. Results: The RAW264.7 cells can be successfully transfected by TLR4-shRNA lentivirus, and the transfection efficiency ranged from 80% to 90%. The silencing efficiency of TLR4 gene was (63±4)%. After silencing the TLR4 gene, the relative expression of TLR4 mRNA and protein (0.20±0.03, 0.37±0.04), PI3K mRNA and protein (0.64±0.06, 0.75±0.06), Rac1 mRNA, protein and p-Rac1 protein (0.75±0.04, 0.76±0.01, 0.74±0.05) in TLR4-RNAi group were significantly lower than those in negative control group and blank group (all P<0.01). The change of cytoskeleton: after silencing the TLR4 gene, the celluar pseudopods were short and stiff, with the impaired capacity of phagocytosing FITC-E.coli. Cells in blank group and negative control group extended good pseudopodia, and the capacity of phagocytosing FITC-E.coli was normal. The MFI and Phagocytosed cells % of TLR4-RNAi group[(7 453±564), (70.20±2.27)%]were significantly lower than those in the blank group and the negative control group (all P<0.01). Positive correlations were existed between mRNA, protein expression of TLR4, PI3K, Rac1 and MFI, Phagocytosed cells% (all P<0.05) in all groups. Conclusion: TLR4-PI3K-Rac1 pathway involves in the cytoskeleton rearrangement and impairs the phagocytosis of macrophages.