IFN-γ-induced Autophagy Regulates Lactation, Cell Proliferation And Anti-bacterial Infection Of Mammary Epithelial Cells Of Lactating Cows

Nutrition directly affects immune defence and resistance to pathogens, a great deal of studies have confirmed that diet can alter the release of inflammatory cytokines, and develops a new direction for the etiology. In our country, limited arable land resulted in high quality forage far less to meet the requirements of the rapid growth of the dairy industry, so the high-concentrate agriculture by-product straw diet are used more and more for breeding industry. O ur previous study found that IFN-γ is present at high concentrations in lactating cows feeding with corn straw diets, with consequences appears noninfectious inflammatory state and became new risk factors for various diseases. However, the effects of IFN-γ on cow mammary gland disease remains unclear. Therefore, the present study is mainly aimed to evaluate the effects and underling molecular mechanisms of IFN-γ on cell proliferation, milk fat and milk protein synthesis and susceptibility to pathogens for bovine mammary gland epithelia l cel s(BMECs).Increase in autophagy was clearly observed in the mammary tissue of cows expressing high levels of IFN-γ and it’s receptors. To investigated whether IFN-γ induce autophagy in BMECs, western blotting, transmission electron microscopy and other methods were used and the results showed IFN-γ treatment of primary BMECs from the normal mammary tissue of lactating cows increased the expression of IFN-γ receptors and clearly induced autophagy within 24 h, as confirmed by the presence of autophagosomes and autophagolysosomes, the translocation of LC3 and degration of p62.To further search the underling molecular mechanisms of IFN-γ on autophagy, mass spectrometry, western blotting and other methods were used and the results indicate that IFN-γ promotes arginine depletion, activates the e IF2 a kinase general control nonderepressible-2(GCN2), and leads to an increase in the autophagic flux. Further, RNA interference directed against GCN2 inhibited IFN-γ–induced autophagy. Therefore, we identify a nove l mechanism by which IFN-γ activates autophagy in BMECs.To understand role of autophagy on the function of BMECs, MTT and other methods were used and the results indicate that long-term activation of autophagy accelerate BMECs cell cycle progression and cell growth and promote the ability to form colony in soft agar suggesting enhanced BMECs malignant transformation. Further studies shown that epithelial- mesenchymal transformation(EMT) is not involved in this process, however, autophagy promote c-Abl trarnslocation into nucleus of the cell and increase in activation of HDAC2, which regulation of the cycle-related proteins resulting in accelerate cell cycle progression.The most important functions for BMECs is lactation, in the present study we found that autophagy was clearly induced in primary BMECs in vitro within 24 h, and consequences significantly decrease in milk synthesis were observed following IFN-γ treatment. Interestingly, inhibition of autophagy can improve milk protein and milk fat synthesis. Conversely, upregulation of autophagy decreased milk synthesis. Further, arginine supplementation assists in recovering milk synthesis that had been reduced by IFN-γ-induced autophagy.We make a further investigation about the role of autophagy for BMECs a gainst staphylococcus aureus. The results indicated that BMECs, primed for 24 hwith IFN-γ exhibited an increase in internalized bacteria per cell compared to unprimed cells, and blockage of ITGA4 decreases IFN-stimulated internalization of Staphylococcus aureus. Electron microscopy confirmed BMECs contained double membrane vacuoles surrounding S. aureus, indicative of autophagosome formation, and plate counting method showed that inhibition of autophagy significantly increased intracellular bacteria, suggesting that autophagy stimulates clearance of S. aureus in BMECsIn summary, we have demonstrated that IFN-γ is mainly through the promotion of arginine depletion and activation of GCN2/e IF2 a signaling pathways to regulate autophagy. Activation of autophagy inhibition of milk fat milk protein synthesis and long-term activation of autophagy induced BMECs malignant transformation. In addition, autophagy mediated IFN-γ promoting intracellular clearance of staphylococcus aureus in BMECs. This study reveals the IFN-γ-induced autophagy and underlying molecular mechanism in BMECs, not only opened up new potential areas of regulation of milk fat and milk protein synthesis and the immune defense, but also provide new train of thought and experimental clues for the prevention and treatment of breast-related diseases.