Cytokine Release Molecular Mechanism Of Macrophages By Mycoplasma Ovipneumoniae And LAMPs-Treated

Mycoplasma ovipneumoniae(M.ovi),a highly contagious disease,causes mycoplasmal pneumonia of sheep and goats(MPSG).The recognition of mycoplasma by innate immune cells using the receptors in the cytoplasm and cytoplasm and the activation of the cells may be the main cause of some important complications of MPSG.Toll-like receptors(TLRs)are known as the main receptors,which play an important role in the recognition of PAMPs and DAMPs.These receptors are involved in the recognition of mycoplasma pneumoniae to induce an appropriate immune response in the host to clear infection,or to induce chronic inflammation.1.This experiment using M.ovi and lipid-related membrane proteins(LAMPs)stimulated C57BL/6J,Tlr2-/-and Nlrp3-/-three kinds of mice abdominal macrophages.The expression,activation and secretion of TLR2 and NLRP3 receptors,related NF-?B,MAPK and caspase-1 signaling pathways,and cytokines TNF-a and IL-1? were detected by fluorescence quantitative PCR,flow cytometry,Western blot and ELISA,respectively.The experimental results showed that(1)M.ovi and LAMPs could increase the expression levels of TLR2 and intracellular NLRP3 receptor genes and proteins on the surface of mouse peritoneal macrophages.(2)M.ovi and LAMPs activate NF-?B,MAPK and caspase-1 signaling pathways through mouse peritoneal macrophage by TLR2 and NLRP3 receptors.(3)M ovi and LAMPs can induce the increase of cytokines TNF-a and IL-1? secretion through mouse peritoneal macrophage by TLR2 and NLRP3 receptors.2.Based on the successful isolation of sheep alveolar macrophages,M ovi and its LAMPs were stimulated in this experiment,and the gene and protein expressions of TLR2 and NLRP3 receptors were detected by fluorescence quantitative PCR and flow cytometry.Expression levels of NF-?B and MAPK signaling pathways and cytokines TNF-a and 1L-1? were detected by Western blot and ELISA.At the same time,the receptors and the signal pathways were inhibited by the corresponding receptor functional inhibitory antibody and signal protein inhibitor,and the corresponding detection was performed.The experimental results showed that(1)M.ovi and LAMPs could increase the expression levels of TLR2 and intracellular NLRP3 receptor genes and proteins on the surface of alveolar macrophages of sheep.(2)M ovi and LAMPs activate NF-?B and MAPK signaling pathways in alveolar macrophages of sheep.(3)M.ovi and LAMPs increase the production of cytokines TNF-a and IL-1? in sheep alveolar macrophages.(4)Functional inhibitory antibody TLR2 blocks the NF-?B and MAPK signaling pathways in sheep alveolar macrophages activated by M ovi and its LAMPs,and reduces the secretion of cytokines TNF-a and IL-1?.(5)After M.ovi and its LAMPs stimulated sheep alveolar macrophages,NF-?B and MAPK functional inhibitors decreased the secretion of cytokines TNF-a and IL-1?.