The Immunologic Effects And Mechanism Of Methionine Enkephalin(MENK) On Influenza Virus PR8 Infection

Object:The aim of this investigation was to construct influenza model with influenza A virus strain PR8,and to look at whether therapeutic and prophylactic MENK treatment could have anti-influenza virus activity in the experiment of RAW264.7 cells infected with PR8 virus in vitro and mice infected with PR8 virus in vivo,and to explore the immune modulatory mechanism of anti-influenza virus.The investigation may provide basic experimental data for MENK as anti-influenza agent or vaccine adjuvant,so as to provide a new strategy for the prevention and treatment of clinical influenza virus.Methods:1.Regulation of RAW264.7 cells against influenza virus PR8 infection in vitro.The influenza virus（H1N1;PR8）was cultured in chicken embryos,then detected by hemagglutination test（HA）.RAW264.7 cells were infected with a virus dose of 100TCID₅₀ followed by treatments of therapeutic and prophylactic MENK administration.The best effect concentration of MENK was detected by MTS method.Morphological changes were observed by light microscopy and Hoechst staining.Apoptosis of RAW264.7 cells and expression of Influenza NP and NF-κB p65 were determined by FCM.Immunofluorescence staining was used to locate the expression of Influenza NP,NF-κB p65 and MOR.The levels of Virus,TLR4,TLR7,NF-κB p65 and MOR were analyzed by q PCR.2.Regulation of innate immune response of anti-influenza virus PR8in vivo.Female C57BL/6 were assigned to five groups:normal saline（NS）group,A/PR/8/34 influenza virus（PR8）model control,Pre-MENK group,MENK treated group and ribavirin（Rib）treated group.Mice were infected via intranasal instillation（i.n.）with10LD₅₀ virus,and monitored body weight and survival time daily for 14 days.Lungs of mice infected with influenza virus were collected at day 4 p.i.for detected immune related indexes,including the general morphology of lung tissue and HE staining,pulmonary virus titer by hemagglutination test（HA）,Influenza-NP protein localization by lung tissue immunofluorescence,expression of IFN-α,IFN-β,TNF-α,IL-6,IL-1βand Virus mRNA by q PCR,and MOR,DOR,Toll receptor pathway related factors of TLR7,MyD88,TRAF6,NF-κB p65 detected by Western blot,q PCR and immunohistochemistry.3.Effects of MENK on memory T cells in vivo.Female C57BL/6 were assigned to four groups:normal saline（NS）group,A/PR/8/34 influenza virus（PR8）model control,Pre-MENK group,and MENK treated group.Mice were infected via intranasal instillation（i.n.）with 2LD₅₀ virus.Bronchoalveolar lavage fluid,lung tissue and lymph nodes were collected at day 10 p.i.,and ground to single cell suspension.CD4⁺T,CD8⁺T,CD4⁺IFN-γ,CD8⁺IFN-γ,CD4⁺T_EM and CD8⁺T_EM cells were detected by FCM.Bronchoalveolar lavage fluid,lung tissue and lymph node cells were stimulated with PMA/ionomycin,influenza NP（366-374）and influenza PA（224-233）to detect the effector CD4⁺T_EM,CD8⁺T_EM and NP/PA specific CD4⁺T_EM,CD8⁺T_EMM cells.Results:1.Regulation of RAW264.7 cells against influenza virus infection in vitro.The virus titer of PR8 was increased from 1:32 to 1:512 through chicken embryo culture,and100 TCID₅₀/50μl was 1:28.2.The concentration of MENK in the range of 20 to 10^-7mg/ml could promote cell proliferation,and had statistically significant at the concentrations of10mg/ml,1mg/ml,10^-1mg/ml and 10^-2mg/ml compared with the normal control group,and 10mg/ml was the best concentration.The prophylactic or therapeutic administration of MENK inhibited the proliferation of the virus and reduced the apoptosis in RAW264.7 infected with influenza virus on 24h,48h,72h p.i..Microscopic examination showed that the morphology of cells changed from circular to irregular,with vacuoles and granular substances in cytoplasm after PR8 infection.The cells in Pre-MENK group and MENK treated group showed a long fusiform shape with many pseudopods,occasional appeared vacuoles and granular substances.Hoechst staining showed that the nuclei of normal cells were dark blue,while a large number of apoptotic cells appeared in PR8 group and the nuclei were densely stained with bright blue.The cells infected with influenza virus in Pre-MENK group and MENK treated group were occasionally accompanied by spot-like staining of the nucleus.Both therapeutic and prophylactic administration of MENK down-regulated expression of Influenza NP protein and up-regulated NF-κB p65 level compared with PR8 group by FCM,q PCR and immunofluorescence staining（P<0.05）.The expression of TLR4 m RNA in RAW264.7cells was slightly higher than that in control group on 48h p.i.（P>0.05）.The level of TLR4 mRNA was up-regulated in Pre-MENK group and MENK treated group on 48h p.i.（P<0.05）.The expression of MOR mRNA in RAW264.7 cells had no significantly changes on 48h p.i.（P>0.05）.The MOR were significantly higher in Pre-MENK group and MENK treated group than those in normal control group and virus control group（P<0.01）.2.Regulation of innate immune response of anti-influenza virus in vivo.The therapeutic and prophylactic administration of MENK alleviated the weight loss and prolonged the survival of mice infected with PR8.The results demonstrated that prophylactic or therapeutic administration of MENK ameliorated lethal influenza infection,and prophylactic efficacy of MENK was much better than its therapeutic effect.The gross morphology showed that extensive hemorrhagic/inflammatory areas in mice infected with PR8 virus.In comparison,less lung lesion occurred in mice treated with MENK,while more restricted damage was observed in the Pre-MENK and Rib treatment groups.Histopathological examination of mice in the PR8 group revealed extensive parenchymal and peribronchiolar inflammation,epithelial necrosis of airways,diffused alveolar damage with inflammatory cells infiltration,edema,and hemorrhage.Compared with PR8 group,Pre-MENK and MENK treatment reduced the pathology,lesion rates accounted for 23.22%,44.59%respectively（P<0.01）.The results of Influenza NP showed that prophylactic and therapeutic MENK administration could effectively reduce the replication of influenza virus in mice with method of Hemagglutination test（HA）,qPCR and immunofluorescence,and the effect of prophylactic treatment was more obvious.qPCR results showed that MENK significantly reduced the expression of IFN-α,IFN-β,TNF-α,IL-6 and IL-1βmRNA and alleviated the lung injury caused by the imbalance of inflammatory mediators.The expression of MOR and DOR in lung tissue were increased by prophylactic and therapeutic MENK administration,but there were no significant changes of receptors expression between PR8 and Rib treated groups（P>0.05）.The expression of TLR7,MyD88,TRAF6 and NF-κB p65 in PR8 group were significantly increased with method of Western blot,qPCR and immunohistochemical staining,while prophylactic and therapeutic MENK administration both reduced the expression of TLR7,MyD88,TRAF6 and NF-κB p65（P<0.05）.3.Effects of MENK on memory T cells in vivo.FCM analysis showed that percents of CD8⁺T in BALF and lung tissue of Pre-MENK group were significantly higher than that in PR8 group（P<0.05）,the percent of CD8⁺T in lung tissue of MENK group was higher than that in PR8 group（P<0.05）,while CD4⁺T cells in Pre-MENK and MENK group despite had increase,but did not reach statistical significance（P>0.05）.The percents of CD4⁺IFN-γand CD8⁺IFN-γin bronchoalveolar lavage fluid and lung tissue of Pre-MENK group were significantly higher than that in PR8 group（P<0.05）,while various types of cells in MENK group despite had different degrees of increase,but did not reach statistical significance（P>0.05）.The percents of CD4⁺T_EM and CD8⁺T_EM cells in bronchoalveolar lavage fluid,lung tissues and CD4⁺T_CM and CD8⁺T_CM in lymph nodes were significantly increased in Pre-MENK group compared with those in PR8 group（P<0.01）,While the percents of CD4⁺T_EM and CD8⁺T_EM cells in lymph nodes did not change significantly（P>0.05）.Compare with PR8 group,the percents of CD4⁺T_CM,CD8⁺T_CM,CD4⁺T_EM and CD8⁺T_EM cells in bronchoalveolar lavage fluid,lung tissue and lymph node of MENK group were higher,but there were no significant differences（P>0.05）.The percents of effector CD4⁺T_EM,CD8⁺T_EM and NP/PA-specific CD4⁺T_EM,CD8⁺T_EM cells in bronchoalveolar lavage fluid and lung tissue of Pre-MENK group significantly increased compared with PR8 group（P<0.01）.While the percents of effector CD4⁺T_EM,CD8⁺T_EMM and NP/PA-specific CD4⁺T_EM,CD8⁺T_EM cells in MENK group showed an upward trend compared with PR8 group,but did not reach statistical significance（P>0.05）.Conclusions:1.MENK positively regulates the TLR4-NF-κB p65 signaling pathway by up-regulating opioid receptor,and enhances the antiviral effect of RAW264.7 cell.2.MENK can inhibit excessive innate immune response by down-regulating the TLR7-MyD88-NF-κB p65 signaling pathway.Also prophylactic or therapeutic administration of MENK can regulate the innate immune response to play a role in anti-influenza virus infection,and prophylactic efficacy of MENK is much better than its therapeutic effect.3.MENK can up-regulate immune response of virus-specific T cells and enhance immune memory against the conserved epitopes of influenza virus.