Molecular Mechanism Of Phagocytosis Of Salmonella In TLR4 Overexpression Sheep Monocyte-macrophages

Monocyte-macrophages are important immunocytes in animal body,which exert crucial functions during bacterial infection.After bacteria is phagocytized by immunocytes,all kinds of immune reactions are activated and start the clearance of invaded bacteria.Toll-like receptor(TLR)as a kind of pattern recognition receptor could trigger various immune reactions by multifarious ligands.Toll-like receptor 4(TLR4)is an important member of TLR family,which exists in various immunocytes.TLR4 can recognize component of cell wall of gram-negative bacterium including S.salmonella and E.coli and subsequently activates multiple signaling pathways and leads to phagocytosis,inflammatory response,oxidative stress and clearance of bacteria.Recognition and phagocytosis bacteria by animal monocyte-macrophages is the first defensive line of innate immunity and TLR4 is the most important receptor of gram-negative bacteria.Southern blot is considered as the most authoritative method to identify transgenic individual.In this experiment,digoxigenin-labeled probes and chemiluminescence detection are used in Southern blot.To achieve strong signal,low background and high sensitivity Southern blot,we optimize the procedure by extracting high quality genome DNA,controlling condition of digestion,purifying the probes and optimizing process of transferring and immunoassay.The mRNA level and protein level in transgenic sheep is higher.After the Tg sheep monocyte-macrophages are stimulated with LPS,the MAPK signaling and PI3K were rapidly activated.Meanwhile,the expression of scavenger receptors are significantly increased.Furthermore,after the cells are stimulated with S.typhimurium,TLR4 overexpression upregulated expression of scavenger receptors(including SR-A,CD36 and Lox-1)and effectively activated MAPK and PI3K signaling pathways.More,action polymerization and adhesive capacity are both enhanced in TLR4-overexpressing sheep within 30min.Knock-down or inhibition of TLR4 decreased phagocytosis and adhesive capacity of sheep monocyte-macrophages.To study which signaling pathways are involved in TLR4-depended bacterial phagocytosis,we inhibited p38,JNK,ERK and PI3K respectively.Finally,we find that both MAPK signaling and PI3K signaling are involved in TLR4-mediated phagocytosis.From the above,our findings indicate that bacterial infection triggers TLR4-dependent activation of MAPK and PI3K signaling and the subsequent activation of F-actin and scavenger receptors promotes the phagocytosis of bacteria in sheep.Meanwhile,we find that overexpression of TLR4 enhances phagocytosis in sheep monocytes-macrophages.This study enriches the understanding of the relationship between TLR4 and the mechanism of phagocytosis in sheep.Furthermore,we provide an important theoretical basis and a new exploration for resistance breeding.