The Effect Of Hemagglutinin Glycosylation Sites Of H5N1 Subtype Avian Influenza Virus On Innate Immune Response Of Dendritic Cells In Mice

H5N1 subtype avian influenza virus(AIV)can cause high morbidity and mortality in poultry,and brings huge economic losses to the world poultry industry.The disease is epidemic in many parts of the world.It can also infect humans,thus posed a huge threat to human health.At present,the glycosylation site of the HA protein in the H5N1 subtype AIV mutates frequently,and the glycosylation pattern is becoming more complicated,which makes the epidemic of the H5N1 subtype AIVs more diversified,and also increases the difficulty of vaccine control.Previous research in our laboratory showed that deletion of glycosylation sites at position 158 or 169 of the HA protein head can significantly increase the pathogenicity of the H5N1 subtype AIV to mammals.The outbreak of "cytokine storm" is the main reason for the high pathogenicity of H5N1 subtype AIV Dendritic cells(DCs),serving as professional antigen presenting cells,also play a vital role in regulating the host's innate immune response in recent years.It may play an important role in the AIV-induced "cytokine storm",however,the specific mechanism still unclear.In this study,we first successfully induced murine bone marrow-derived DCs in vitro Secondly,the effects of different glycosylation-modified H5N1 subtypes of HA proteins(rS-WT,rS?158,and rS?169)on the innate immune levels of DCs were evaluated.Finally,we further clarified that the changes of glycosylation sites affected the recognition of AIV by C-type lectin receptors on DCs.This study provides a theoretical basis for the analysis of the pathogenic mechanism of H5N1 highly pathogenic AIV in mammals.1.Induction and identification of murine bone marrow-derived dendritic cells in vitroThe precursor cells of murine bone marrow were first isolated and induced by the combination of cytokines GM-CSF and IL-4 until they were differentiated into DCs.Secondly,the morphology,purity,and maturation of DCs were identified by flow cytometry and confocal laser scanning microscopy.The results showed that the cultured cells had a typical dendritic morphology,the purity can reach more than 90%(CD11c+).Cells can be transformed into a mature state under the stimulation of LPS.The above data suggested that the induced cells in accorded with the typical characteristics of DCs and can be used for subsequent experiments.2.Effect of H5N1 subtype AIV with deletion of 158 or 169 glycosylation sites on the level of innate immunity in murine DCsFirstly,absolute qRT=PCR was used to determine the infective ability of three recombinant AIV(rS-WT,rS?158,and rS?169)to DCs.The results showed that three AIVs have limited ability to infect DCs.Secondly,compared with rS-WT,rSA158/rS?169 significantly increased the morphological index of DCs,promoted the expression of phenotypic markers(CD80,CD86,CD40,and MHCII),early activation markers(CD69),and migration markers(CCR7)on DCs;promoted the secretion of pro-inflammatory cytokines(IL-1?,IL-6,IL-12,and TNF-a).In the mixed lymphoid reactions(MLRs)assay between infected DCs(MOI = 0.1/0.5/1)and CD4+T cells(DCs:T=1:1/1:5),the rSA158/rS?169 group significantly promoted the proliferation of CD4+T cells compared with rS-WT group.The assays of FITC-Dextran phagocytosed by virus-infected DCs showed that the phagocytosis capacity of rS?158-infected DCs was significantly reduced compared to that of rS-WT group.Finally,after nasal drops infecting mice with each strain,rS?158/rS?169 significantly promoted the migration of DCs to the submucosal mucosa of the nasal cavity and further into cervical lymph nodes compared to that of rS-WT.The main subtype of involved DCs was CD103+.These results indicated that H5N1 subtype AIV with deletion of 158 or 169 glycosylation sites significantly enhanced the level of innate natural immunity in mouse bone marrow-derived DCs compared with wiki-type strains.3.Role of C-type lectin receptor mediated the innate immunity of murine DCs by deletion of 158 or 169 glycosylation sites of H5N1 subtype AIVFirstly,in the sialic acid receptor inhibition assay,blocking the a-2,3 and a-2,6 sialic acid receptors on the surface of DCs using sialicdase showed that the expression levels of NPs in each group did not decrease completely,indicating that AIVs are not completely dependent on the sialic acid receptor pathway to infect DCs.Secondly,the results of EDTA and mannan inhibitor assays showed that the expression of NP protein in rS?158/rSA169 group was significantly decreased,indicating that C-type lectin might play an important role in virus invasion.Thirdly,immunflourescence staining of infected DCs and observation by laser confocal microscopy revealed that,compared with rS-WT group,the ability of fluorescent labeled rS?158/rS?169 virus to adhere to cells was enhanced,and co-localization between SIGN-R1/MR C-type lectin receptor(CLRs)and rS?158/rS?169 virus was more easily formed.However,the co-localization between MGL receptor and all of strains was weaker.Neutralizing antibody of CLRs blocking assay in vitro showed that SIGN-R1 and MR but not MGL neutralizing antibodies blocked the up?regulation of NP protein expression and the expression of phenotypic marker CD80 in the rS?158/rS?169 group.In vivo,after intranasal challenge of three strains in mice,compared with the rS-WT group,the rS?158/rSA169 virus was able to recruit the DCs which highly express of SIGN-R1 and MR but not MGL receptors.These results indicated that the H5N1 subtypes AIVs with deletion of 158 or 169 glycosylation sites mainly through the SIGN-R1 and MR,but not MGL CLRs dependent pathways to infect DCs,and further caused high levels of innate immune response.