Study On The Effect Of Mycoplasma Ovipneumoniae Infection On Phagocytosis And Autophagy Function Of Macrophages

Background:Mycoplasma ovipneumoniae is one of the main pathogens causing infectious pleuropneumonia in sheep and goats(especially lambs).It is a cell-wall-free prokaryotic microorganism with a cell size between virus and bacteria.MO can cause chronic infection of the body,accompanied by co-infection of other pathogenic microorganisms in some cases.As an important member of the defense system,macrophages play a key role in both innate and adaptive immune.Macrophages can phagocytose,hydrolyze,clear and present the corresponding antigens by phagocytosis,autophagy,etc.,and activate the body's immune response to cope with pathogen infection.By studying the effects of MO on phagocytosis and autophagy of macrophages,it is expected to reveal the mechanism of chronic infection and synergistic infection caused by MO.In this experiment,sheep primary alveolar macrophages and mouse macrophage cell line RAW264.7 were used as research objects to establish a MO Y98 infection model to study the survival,phagocytosis and autophagy of infected macrophages.We found that:(1)DiO fluorescence staining showed that primary alveolar macrophages and RAW264.7 cells of sheep could engulf MO Y98.(2)The expression of BID and BAD in primary alveolar macrophages of sheep infected with MO Y98 decreased with the prolongation of infection time,while the expression of BCL-2 was up-regulated.The expression of Bad,Bak and Caspase-9 in RAW264.7 cells infected with MO Y98 was down-regulated significantly at 24h.The results showed that MO Y98 infection could inhibit the apoptosis of primary alveolar macrophages and RAW264.7 cells in sheep.(3)Proteomic analysis was used to detect the expression of phagocyte-related protein in primary alveolar macrophages of sheep infected with MO Y98 for 24h.It was found that the expression of early phagosome-associated protein was up-regulated,while that of late phage and mature lysosome was down-regulated.MO Y98 infection can up-regulated the expression of early phagosome-associated protein Eeal in RAW264.7 macrophages,but the expression of late phagosome-associated protein Lamp 1,Lamp 2 and Phagolysosome-related protein Atp6vla was significantly decreased.The results suggest that MO Y98 infection can promote the maturation of early phagosomes of sheep primary alveolar macrophages and RAW264.7 cells,but affect the maturation of late phagosomes and lysosome function.(4)MO Y98 could enhance the uptake of Escherichia coli by RAW264.7 cells after 24 h infected,but inhibit the clearance of intracellular Escherichia coli.(5)In the primary alveolar macrophage model of sheep infected with MO Y98,the expression of autophagy-related protein LC3 and Beclinl increased significantly at 6 h and 12h of infection,while the expression of autophagy-related protein decreased at 24 h and 36 h of infection.In the MO Y98 infected RAW264.7 cell model,the expression of autophagy-related factors was significantly increased at 24 h,while the expression of autophagy-related factors was significantly decreased at 48h of infection.The results showed that macrophage autophagy could be activated in the early stage of MO Y98 infection,and autophagy was inhibited with the prolongation of infection time.In conclusion,the results showed that macrophages could phagocytize MO Y98;MO Y98 infection could enhance the phagocytosis ability of macrophages to Eschenchia coll;MO Y98 infection could inhibit the expression of late phagosomes and lysosome-related proteins,and inhibit the clearance function of macrophages;MO Y98 infection could inhibit the apoptosis of macrophages;autophagy of macrophages is activated in the early stage of MO Y98 infection,but autophagy is inhibited with persistent infection.This study found the immune escape mode of MO,preliminarily revealed the possible causes of chronic infection and co-infection of the host caused by MO,and provided research ideas for the pathogenesis of MO.