The Mechanism Of HMGB1 Protein In Inflammatory Response Induced By Chicken Newcastle Disease Virus

Newcastle disease(ND)is a highly pathogenic viral disease caused by Newcastle disease virus(NDV)infection.Because Newcastle disease has a high rate of infection and mortality,as well as it can spread around the world,it is a threat to the global poultry industry.NDV infection can activate the hosts' innate immune response,inducing a large number of inflammatory factors.And different virus strains of NDV induce different levels of cytokine expression.The respiratory tract and gastrointestinal mucosa,etc.will produce bleed and other exudative inflammatory pathology in the NDV infected poultries.The HMGB1 protein,which can be actively released into the supernatant of the cells,has a proinflammatory cytokine effect and is an inflammatory regulatory factor.There are fewer reports on the function of chicken HMGB1 proteins.The role of the chicken HMGB1 protein in the inflammatory response caused by Newcastle disease virus is worth to be studied.In order to better control the clinical response caused by the infection of Newcastle disease virus,reducing the death rate caused by the inflammation,we conducted the following studies.(1)In this study,chicken HMGB1 and human HMGB1 gene sequences were amplified from CEF cells and HeLa cells.And then the sequences of chicken HMGB1 and human HMGB1 were compared.It was found that both of them had high similarity in A box and B box,and the C-terminal has difference.The recombinant plasmid pEGFP-N1-chHMGB1 was constructed and transfect into HeLa cells.Then the result shows that the chicken HMGB1 were located in the nucleus.The distribution of HMGB1 proteins in the different organs of adult SFP chickens were analyzed by semi-quantitative RT-PCR.It was found that the HMGB1 proteins were widely distributed among the tissues of chicken tissues.The abundance in bursa of fabrics and intestinal abundance was high and the abundance in muscle and viscera was low.(2)In this study,we constructed the recombinant protein of chicken HMGB1 by prokaryotic expression vector of pET-28a-chHMGB1,and immunized the mice.After immunization for five times,the mice were sacrificed to prepare polyclonal antibody.At the same time we fuse the spleen cells from the immunized mice and sp2/0 cells.After four subclones,three monoclonal antibodies against recombinant chicken HMGB1 were obtained.Experiments show that the prepared polyclonal antibody and three monoclonal antibodies all have ELISA,Western Blot and IFA titers.The cell specific experiments found that the polyclonal antibody was specific to chicken HMGB1 protein,and three monoclonal antibodies were versatile and could identify chicken,human,mouse,monkey and pig HMGB1 proteins.The successful development of the monoclonal antibodies will provide the necessary biological reagents for further study of chicken HMGB1 protein function and its role in Newcastle disease virus infection.(3)In this study,the effects of the Newcastle disease virus on the intracellular HMGB1 protein were analyzed.We used NDV Herts/33 strain to infect DF1 cells and A549 cells,and then indirect immunofluorescence experiments showed that Newcastle disease virus infection could lead to the HMGB1 protein in the nucleus relocated in the cytoplasmic domain.Then,the Western Blot and fluorescence quantitative PCR showed that the expression of HMGB1 protein was not significantly induced in NDV infected DF1 and A549 cells.We also infected A549 cells with the Herts/33 strain and La Sota strain,and the level of HMGB1 protein in the supernatants was detected by ELISA.The results showed that the expression of HMGB1 proteins in NDV infected cells were not significantly induced,but NDV infection could induce the HMGB1 protein to migrate to the cytoplasmic region and then release into the extracellular environment.It was also found that the releasing of HMGB1 induced by this NDV infection was in a dose-dependent manner.(4)This study analyzed the effect of HMGB1 proteins on NDV replication in cells.We first transfect FLAG-chHMGB1 and FLAG-huHMGB1 plasmids into DF1 /A549 cells and then infected Herts/33 strain.The results showed that over expression of HMGB1 did not affect the replication of NDV in the cells.Then the exogenous HMGB1 proteins were used after Herts/33 infecton,and the results showed that addition of HMGB1 did not affect the replication of NDV in the cells.To fully validate this,we used two kinds of HMGB1 inhibitors-ethyl pyruvate and glycyrrhizic acid and the chHMGB1 neutralizing antibody.These two inhibitors or chHMGB1 neutralizing antibodies were used in the NDV infected cells,respectively.The results showed that the use of two inhibitors or chHMGB1 neutralizing antibodies did not inhibit the replication of Newcastle disease virus in cells.The above results indicate that the HMGB1 proteins does not affect the replication of Newcastle disease virus in cells.(5)Finally,this study analyzed the effect of HMGB1 proteins on the activation of NF-?B signaling pathway and the up-regulation of inflammatory factors induced by Newcastle disease virus.First,the results of real-time PCR and ELISA demonstrated that Herts/33 strain and La Sota strain could induce the up-regulation of IL-1?,IL-6,IL-8 and TNF-? mRNA levels,and secretion into the supernatant of the cells.Also the level of inflammatory factors secret into the supernatant was in a dose-dependent manner with Herts/33 infection.Then we proved that the NF-?B signaling pathway was activated in the NDV infected cells.The above experiments show that NDV can activate the NF-?B signaling pathway and up-regulate the downstream inflammatory cytokines and then secret into the supernatant.We added HMGB1 proteins after 12 hours of Newcastle disease virus infection.It was found that exogenous HMGB1 proteins could promote the activation of NF-?B signaling pathway by Newcastle disease virus and up-regulate the mRNA level of downstream inflammatory factor.We also used HMGB1 inhibitors and chHGMB1 neutralizing antibody after NDV infection.The results showed that HMGB1 inhibitors and chHGMB1 neutralizing antibody could inhibit the up-regulation of Newcastle disease virus-induced inflammatory cytokine mRNA level.We also studied the expression of the receptor protein in this process by interfering with TLR3,RAGE and TLR4 receptors after detecting the level of inflammatory cytokines and the results showed with the siRAGE and siTLR4 receptors can inhibit the up-regulation of cytokines in NDV-infected cells.The in vivo experiments of chicken HMGB1 neutralization antibodies show that the usage of chicken HMGB1 neutralizing antibodies can delay the death caused by infection of Newcastle disease virus by neutralizing the proinflammatory effect of the chicken HMGB1 proteins.All the results ultimately proved that NDV infection induced HMGB1 proteins migrate from the nucleus to the cytoplasmic region and then released into the extracellular space,the released HMGB1 proteins in the extracellular space dose not significantly impacte the replication of NDV in cells,but HMGB1 proteins were involved in the activation of NF-?B signaling pathway and the expression of downstream inflammatory factors in NDV infected cells.And in the process RAGE and TLR4 receptors were involved in signaling pathway.