Study On γδT Cell-mediated Immune Mechanism Of Mammary Gland Against Staphylococcus Aureus Infection

Mastitis is an inflammatory disease of the breast caused by the infection of bacteria,fungus and virus in the breast,including clinical mastitis and subclinical mastitis,which restricts the development of dairy cattle and goats breeding industry in China and even the world.The lactation quantity and quality of the affected individuals are seriously reduced,which may even have a great impact on the health of the lamb and causes hundreds of millions of dollars in economic losses every year.As an important part of the body’s immune barrier system,the mammary gland contains abundantγδT cells.γδT cells are an important part of the body’s natural immune system.When activated,γδT cells secrete IL-17 or IFN-γto fight infection.However,there is no research evidence on howγδT cells are involved in the immunity of the mammary gland against pathogen infection and the regulatory mechanism of the activation of the mammary glandγδT cells.And the pathogenic mechanism of mastitis and the anti-infection immune response mechanism of the mammary gland is still not completely clear,therefore,there is no specific treatment for mastitis.Further research is urgently needed.In this study,milk from dairy goats with mastitis was collected and the cytokines associated with IL-17~+γδT cells induction and neutrophil migration were detected by real-time PCR.In vitro cultured dairy goat mammary epithelial cells were stimulated by staphylococcus aureus and IL-17,and the cytokines related to theγδT cells chemotaxis and neutrophil migration were quantitatively detected by quantitative PCR;With S.aureus mastitis mice as the model,the role ofγδT cells in mammary gland anti-S.aureus infection was studied by flow cytometry and real-time PCR,respectively,to explore the ways in which delta T cells participate in the immunity of mammary gland anti-pathogen infection.The results are as follows:1. The m RNA levels of IL-17,IL-23,IL-1βand TGF-βin milk of dairy goats with mastitis were significantly increased(P<0.05),and the relative m RNA expression levels of the chemokine(CCL2)and chemokine receptor(CCR2)ofγδT cells in milk were also significantly increased(P<0.01).2. After S.aureus stimulation,the expression ofγδT cells chemokine(CCL2)secreted by mammary epithelial cells increased significantly(P<0.001).S.aureus and IL-17 can significantly promote the high m RNA levels of IL-8,CXCL6 and CCL4(P<0.01).3. The detection results of IL-17~+γδT cells and related cytokines in the mice mammary gland showed that the proportion of IL-17~+γδT cells in the number of lymphocytes was significantly increased(P<0.001).The m RNA levels of Il1b,Il6 and Il17 increased significantly(P<0.05). There was no significant difference between the m RNA levels of Il23 and Tgfb(P>0.05).4. The detection results ofγδT cells and neutrophil chemokines in mice mammary gland showed that the m RNA levels of Ccr2 and Ccl2 increased significantly(P<0.05).The m RNA levels of neutrophil chemokines Ccl4 and Cxcl6 were significantly increased(P<0.05).Cxcr4m RNA levels associated with neutrophil activation were significantly increased(P<0.05).5. The m RNA levels of M2-type macrophage-related molecules Ym-1,Cd206,arginase-1,fizz-1 and IL10 in the mammary glands of infected mice were significantly increased(P<0.05).6. The m RNA levels of M2-type macrophage-related molecules Cd206,arginase-1 and IL10 were significantly increased in the milk of mastitis dairy goats(P<0.05).Accordingly,the following conclusions are drawn:1.The mammary glands of milk goats with mastitis form a local microenvironment suitable for IL-17~+γδT cells activation.2.S.aureus and IL-17 can stimulate the secretion ofγδT cells and neutrophil chemokines by mammary epithelial cells in vitro.3.In the natural immune response phase,IL-17 mediates mastitis from IL-17~+γδT cells in the mammary gland.4.Flow cytometry showed a significant increase in the percentage of IL-17~+γδT cells in the mammary gland,but no significant change in the percentage ofγδT cells in the spleen.5.M2-type macrophages secrete IL-10 to negatively regulate the immune response and avoid immunopathological damage.