Study On The Release Of IL-1β In EBL Cells Induced By Mycoplasma Bovis-Derived LAMPs

Mb AD is short for diseases caused by Mycoplasma bovis(M.bovis), such as pneumonia, arthritis, mastitis, keratitis and so on. Mb AD is one of the main diseases that lead to calf deaths in our country. But the pathogenicity and pathogenic mechanism of M.bovis is still unknown.In vertebrates, innate immune response is the first line of defense against microbial invasion. Innate immune cells include epithelial cells, neutrophils, dendritic cells and macrophages. As an important member of the host innate immune cells, epithelial cell plays its immunological function by direct removal of pathogenic microorganisms and the release of corresponding cytokines and chemokines to recruit other immune cells to the site of infection. Lung is the target organ of M.bovis infection, immune response of lung epithelial cells against M.bovis and its important virulence proteins can help us understand the host defense strategy and the corresponding escape strategy of M.bovis. Embryonic bovine lung(EBL) cell is a primary cell established from embryonic bovine lung which is pregnant for about 7 months. The cell is an important cell model for the study of bovine viral, bacterial diseases.Mycoplasma membrane surface exists plenty of lipid-associated membrane proteins(LAMPs). LAMPs is the foundation of mediating the adhesion and invasion of Mycoplasma to host. At the same time, LAMPs can bind to the corresponding receptor on host cell membrane surface, stimulating the cytokine release. In this study, we used EBL cell as cell model, studying the molecular mechanism of the release of IL-1β in host induced by M.bovis LAMPs.1. The cloning of key moleculars in innate immune signaling pathwaysKey molecules including TLR1,TLR2,My D88,IRAK4,and p65 in NF-κB signaling pathway and upstream Toll-like receptor signaling pathway were cloned, then their recombinant plasmids were constructed and named as p CMV- HA – TLR1; p CMV- HA- TLR2; p CMV- HA- My D88; p CMV-HA- IRAK4; p EGFP- p65 and so on, providing tools for studying the molecular mechanism of the release of IL-1β in EBL cells induced by M.bovis LAMPs.2. Study on the molecular mechanism of the release of IL-1β in EBL cells induced by M.bovis LAMPs.As cell model, EBL cells were stimulated with a certain amount of heat-inactivated M.bovis or live M.bovis, respectively. The relative expression of IL-1β was examined by SYBR Green I Real-time PCR. Results showed that both live M.bovis and heat-inactivated M.bovis could induce the release of IL-1β in EBL cells. On the other hand, LAMPs could also induce the release of IL-1β in EBL cells, moreover, the expression of IL-1β is time and dose-dependent. Further, the nuclear translocation of p65 induced by LAMPs was detected by confocal laser microscope and Western Blotting. The nuclear translocation of p65 leads to the activation of NF-κB signaling pathway and downstream release of IL-1β, and transmembrane receptor TLR2 plays an important role in this process.