Role Of Macrophage Autophagy In M.Ovipneumoniae Infection Resistance

Contagious ovine pleuropneumonia caused by Mycoplasma ovipneumoniae(MO)is a serious disease of sheep,which treaten the development of sheep industry.This disease is widely distributed,especially in the western developing areas of animal husbandry of China,which resulting in huge economic losses.Autophagy is a conserved cellular physiological mechanism of eukaryotic cells and it fight against stress and maintain a stable cell environment.And it phagocytoses and degrades the cellular aging organelles and long-lived proteins,those are fused with lysosome.The degradation products are recycled to maintain the function of cellular organelles and cellular homeostasis.In addition,autophagy play an important function to defense bacteria infections.In recent years,a number of studies have shown that MO can adhere to sheep lung cells and damage host cells.However,it is not clear that whether macrophage autophage can be mediatedd by MO,nor the mechanism of MO infection macrophage autophage.In this research,the macrophages of mice lines(RAW264.7)as study object.From the morphological and molecular level to illustrate whether MO could mediate the occurrence of autophagy or not.And RNAi technology was used to detect the affect of MO in autophagic macrophage.Results as follow(1)Red MO dyed DiI were found around the nucleus,which indicating MO could be phagocytosed by RAW264.7.(2)LC3 and Atg16L fluorescent spots were found around the nucleus by Immunofluorescence,which indicating that the autophagosome appeared after the MO infection.In addition,fluorescence microscopy results showed that LC3 protein accumulated after 6 hour post infection detected by,which indicate appearance of autophagosome.The occurrence of autolysosome is found after 12h and 24h post infection.(3)Double-membrane's autophagosome and single-membrane's autolysosome was detected at 12hours post infection by detecting with immunoelectron microscopy.(4)qPCR results showed that mRNA expression of autophagy-related genes,such as LC3,p62,Atg5,Atg7 were upregulated when infected by MO.While,Western blot results revealed a similar pattern compared with qPCR results.(5)The CCU experimental suggested that macrophage autophagy involved in the clerance of MO.(6)After RNAi silencing autophagy key factors p62 and Atg7,autophagy was inhibit but MO was proliferation,it indicating that autophagy inhibited the proliferation of MO.As these results show:MO infection could activate RAW264.7 cells autophagy and the key autophgic factors upregulation,the highest level of autophagy occurs in 12h.MO can not only adhere to the cell surface,but also into the cell.RNAi technology was used to silence the key factors of p62 and Atg7,which could inhibit macrophage autophagy.While increasing the numbers of MO in the cell.It indicated macrophage autophagy inhibtes the macrophage clerance of MO.