Study On Mechanism Of Lipid-associated Membrane Proteins And GroEL From Mycoplasma Gallisepticum Inducing DF-1 Cells To Release IL-1?

Mycoplasma gallisepticum(M.gallisepticum)is the pathogen of chronic respiratory disease(CRD),which can lead to a large number of damage in poultry industry.There are many lipid-associated membrane proteins(LAMPs)in the surface of Mycoplasma,LAMPs may cause the host innate immune response in a variety of Mycoplasma.Moreover,LAMPs can adhere to the corresponding receptors on the surface of host cell membrane,stimulating the cytokine release.Innate immune system is the first line of defense against the invasion of pathogenic microorganisms in vertebrates.TLR2 is one of the members of Toll-like Receptors(TLRs)family,which is the receptor of natural immune system to identify pathogen-associated molecular patterns(PAMP).TLR2 can identify pathogenic signals and enroll downstream signaling molecules,such as My D88 to activate NF-?B signaling pathway and cause the release of host cell factors.NF-?B is an important transcription factor of immune gene expression and is able to regulate inflammatory,the subunit of NF-?B is made up of p50 and p65,p65 subunit into nuclear and phosphorylation are the representative of NF-?B signaling pathway activated.LAMPs are a mixture and contain many membrane proteins with high immunogenicity.Which protein of LAMPs can activate the innate immune has not been reported.Gro EL has been confirmed immunogenicity in some bacteria.DF-1 cells were stimulated with a certain amount of M.gallisepticum's LAMPs or Gro EL,respectively.The expression of IL-1? was detected by Real-time PCR and ELISA.Results showed that M.gallisepticum's LAMPs and Gro EL could induce the release of IL-1? in DF-1 cells.Further,the nuclear translocation of p65 induced by LAMPs was detected by confocal laser microscope.Phosphorylation level of p65 induced by LAMPs or Gro EL was detected by Western blot.The results suggested that LAMPs and GroEL can induce cell p65 into nuclear,activing NF-?B signaling pathway and cause the release of IL-1?.To study the involvement of TLR2 and My D88 in IL-1? expressing induced by lipid-associated membrane proteins(LAMPs)of M.gallisepticum,the expression level of IL-1? was detected by RT-PCR and ELISA in the DF-1 cells stimulated with the LAMPs.One group cells were transfected with recombinant plasmid of p CMV-HA-TLR2 or p CMV-HA-My D88,respectively,for over expression of the TLR2 or My D88.Cell in other groups were pretreated with anti-TLR2 monclonal antibody(MAb)or transfected with interferencerecombinant plasmid of p CMV-HA-DN-My D88,respectively.Then the two groups were both treated with LAMPs.The results showed that the expression of IL-1? m RNA and IL-1? were found to be upregulated in the TLR2 or My D88 overexpression group,but it was deregulated the expression of IL-1? m RNA and IL-1? in the anti-TLR2 MAb pretreated group or transfected with interference plasmid group.In order to further recognize the role of Gro EL,the Gro EL was identified as a protein-binding protein using glutathione S-transferase(GST)pulldown and subsequent mass spectrometry analysis of DF-1cells.The results of Coimmunoprecipitation and GST pulldown suggested that Gro EL interacts with the DF-1 Annexin2 protein.