Regulation Of MTOR-mediated Autophagy On Macrophage Polarization In Ketotic Dairy Cows

Ketosis is a nutritional and metabolic disease,which is caused by the negative energy balance(NEB)in perinatal cows.Excessive lipid mobilization increases the content of free fatty acids(FFA)in blood of ketosis cows,inhibited the immune function of dairy cows,and then led to inflammatory diseases.Macrophages are immune cells of the body that plays a central role in maintaining immune homeostasis.Macrophages can be polarized into pro-inflammatory M1 phenotype or anti-inflammatory M2 phenotype under different stimulation types.Mammalian target of rapamycin(mTOR)mediated autophagy is a major waste degradation process in response to stress conditions,and has a negative regulatory relationship with inflammatory response and immune function.However,the mechanism of mTOR mediated autophagy in inflammatory injury and immune response of ketosis cow macrophages is still unclear.The aim of this study was to investigate the effects of mTOR mediated autophagy in the activation of NF-κB(Nuclear factor kappa B)signaling and macrophage polarization in ketosis cows.Collect blood samples from healthy cows and cows with clinical ketosis(CK),extract blood monocytes and induce them into macrophages,detect the expression of autophagy and changes of macrophage polarization in macrophages.The results showed that the number of autophagosomes and autolysosomes in CK cows were decreased,indicating that autophagy in macrophages of ketosis cows was damaged.Compared with healthy cows,the protein expression of M1 marker i NOS was increased while the protein expression of Arg 1 was decreased,indicating that ketosis cows have serious immunosuppression.In this study,the bovine macrophage cell line was cultured in vitro,and different concentrations of FFA with or without LPS and IFN-γ were used to stimulate bovine macrophage cells for research.The results show that treated with 0,0.3,0.6 or 1.2 m M FFA with or without LPS and IFN-γ for 24 h,the expression levels of autophagy related proteins p-mTOR and p62 were increased in 0.3,0.6,1.2 m M FFA with or without LPS and IFN-γ in macrophages.The expression level of LC3 phosphatidylethanolamine conjugate(LC3-II)was significantly decreased.Transfection of recombinant adenovirus MRFP-GFP-LC3 for 48 h,treated with 1.2m M FFA with or without LPS and IFN-γ for 24 hours,The number of autophagosomes labeled with yellow puncta and autolysosomes labeled with red puncta were decreased.After treated with 0.3,0.6,1.2 m M FFA with or without LPS and IFN-γ,the protein abundance of p-P65 and the m RNA abundance of proinflammatory factor TNF-α,IL-1β and IL-6 were significantly increased.In addition,treated with 0.3,0.6,1.2 m M FFA with or without LPS and IFN-γ,the mean fluorescence intensity of CD86 and the protein expression of i NOS were increased,while the mean fluorescence intensity of CD206 and the protein expression of Arg1 were decreased.The study shows that FFA induces autophagic dysfunction of bovine macrophages,which in turn leads to inflammatory reaction and induces macrophages toward M1 phenotype.In order to investigate the regulatory mechanism of mTOR mediated autophagy on the immune function of bovine macrophages,the activator of mTOR,MHY1485 and LPS,IFN-γ or IL-4,IL-10 were added to the bovine macrophage.The results showed that the protein expression levels of p-mTOR and p62 were increased and the LC3-II protein level was decreased in MHY1485 with LPS and IFN-γ group.Furthermore,the protein abundance of i NOS was greater in the MHY1485 with LPS and IFN-γ group,but the protein abundance of Arg1 was lower in the MHY1485 with IL-4 and IL-10 group.The mean fluorescence intensity of CD86 was greater in the MHY1485 with LPS and IFN-γ group,but the mean fluorescence intensity of CD206 was lower in the MHY1485 with IL-4 and IL-10 group.The above results suggest that mTOR mediated autophagy has a regulatory effect on the polarization of bovine macrophages.In order to investigate the relationship between mTOR mediated autophagy,inflammatory reaction and macrophage polarization.Rapamycin(RAPA),an inhibitor of mTOR,with or without LPS and IFN-γ were used to treat bovine macrophage cell line.The results showed that RAPA pretreatment alleviated the increase of p-mTOR and p62 protein levels induced by FFA,and aggravated the decrease of LC3-II protein expression level and the number of autophagosomes labeled with yellow puncta and autolysosomes labeled with red puncta induced by FFA;By reducing the expression level of p-P65 protein and proinflammatory factor TNF-α,IL-1β,IL-6 m RNA expression level reduced FFA induced inflammatory response;In addition,the decrease of i NOS protein expression level and the mean fluorescence intensity of CD86 alleviated the immune dysfunction induced by FFA.These results indicate that high concentration of FFA in the blood of ketosis cows can damage autophagy,activate inflammatory pathways,and induce macrophages to polarize toward M1.In summary,ketosis in dairy cows results in increased polarization of macrophages towards M1 and impaired autophagic flux.Exogenous FFA activates NF-κ The B inflammatory signaling pathway induces macrophage polarization towards the M1 phenotype and inhibits autophagy activity by activating the mTOR signaling pathway in cow macrophages.MHY1485 inhibits autophagy by activating the mTOR signaling pathway,while RAPA has the opposite effect on the mTOR signaling pathway.Therefore,enhancing mTOR mediated autophagy may provide ideas for combating immune suppression caused by metabolic challenges in ketosis cows.