| Newcastle disease virus(NDV)is an important pathogen that seriously endangers poultry industry.Since it was first reported in 1926,NDV has caused four pandemics worldwide,each of which is caused by different genotypes.Virulent NDV strains of different genotypes are distinctive in the pathogenesis,with the remarkable contrast between genotype ? and genotype ?.Compared with genotype ?,genotype ? NDV exhibits a strong invasiveness to the immune organs,characterized with extensive infiltration of inflammatory cells and severe tissue necrosis.Macrophages are an important component of the immune organs,which restrict virus infection by engulfing invaded virus,apoptotic cells and producing chemokines and cytokines.However,the role of macrophages in the pathogenesis of NDV and the related mechanism remain unclear.Therefore,in this study,the infectivity of NDV in macrophages was evaluated in chickens and chicken peripheral blood mononuclear cells(PBMCs).Subsequently,the role of macrophage in the pathogenicity of NDV was further evaluated through macrophage depletion in chickens.Finally,the chicken macrophage cell line HD11 was used to characterize the main cell death induced by NDV,and the mechanism of cell death was explored.Our results indicated that macrophages play an important role in the host denfense against NDV,and the virus may undermine the host antiviral response through inducing pyroptosis of macrophages,promoting virus replication and pathology.This study presents new information regarding the pathogenesis of NDV.1.Tropism of NDV to macrophagesTo determine the tropism of NDV to macrophages,we firstly isolated macrophages from chicken PBMCs and determined the purity of macrophages by flow cytometry(FACS).The results indicated a high purity of macrophages in the adherent cells.Secondly,the adherent and suspension cells isolated from PBMCs were infected with genotype ? JS5/05 strain and genotype ? Herts/33 strain.Virus copies in the cells were measured by real-time quantitative polymerase chain reaction(qRT-PCR).The in vitro results showed that NDV can replicate in macrophages(adherent cells)and lymphocytes(suspension cells),with a hihger tropism to macrophages.In addition,the tropism of NDV to macrophages was analyzed by multiple immunofluorescence(mIHC)staining.The results showed that there was an extensive co-localization of the viral antigen with KUL01+ cells,and the replication level of JS5/05 in the spleen was significantly higher than that of Herts/33.Finally,histopathological changes and the integrity of ellipsoid in the spleen were asessed.Neither JS5/05 nor Herts/33 had obvious destructive effect on the reticular fiber structure in the ellipsoid,but JS5/05 induced more severe necrosis in the ellipsoid.Therefore,in vitro and in vivo data confirmed that macrophages are important target cells for NDV infection.2.The role of macrophage in the pathogenicity of NDVThe role of macrophages in the pathogenesis of NDV was investigated through in vivo macrophage depletion.Firstly,optimization of the conditions of macrophage depletion showed that intravenous inoculation of 250 ?L of clodronate liposomes resulted in a significant reduction of macrophages(KUL01+),as measured by flow cytometry and immunofluorescent assays.Secondly,NDV infection was carried out after macrophage depletion,and virus replication,histopathology and apoptosis in the spleen were determined.The results showed that after the depletion of macrophages,viral load in the spleen increased,however,there were no significant changes in viral loads in other tissues including the liver,lung,thymus and intestine.The distribution of the NDV nucleoprotein(NP)in the spleen significantly increased in clodronate liposomes-treated chickens.In addition,histopathology assessment revealed that lymphocyte depletion and tissue necrosis in the spleen of clodronate liposomestreated chickens were exacerbated after JS5/05 infection compared with the PBS liposomes-treated chickens.The Tunel assay showed that the amount of apoptotic cells in the spleen of JS5/05-infected chickens significantly increased after clodronate liposomes treatment.The results showed that macrophage depletion led to the enhancement of virus replication,tissue injury and apoptosis in the spleen,indicating a critical role of macrophages in restricting NDV infection and alleviating tissue injury.3.Pyroptosis of macrophages induced by NDVBased on the antiviral role of macrophages and the high tropism of NDV to macrophages,it was speculated that NDV may undermine the host antiviral response by triggering cell death of macrophages,promoting virus replication and pathology.Therefore,we used chicken macrophage cell line HD11 as an in vitro model and cell death induced by NDV was systematically characterized.The results verified that NDV can induce pyroptosis in macrophages.Secondly,the expression levels of key pyroptosis-related genes were determiend using qRT-PCR.The results showed that NDV infection significantly up-regulated the expression levels of caspase 1,caspase 3 and NLRP3 genes in HD11 cells,but there was no significant change in IL-1? gene expression.Then,some key proteins in two major pathways regulating pyroptosis were blocked by treatment with inhibitors.The results demonstrated that the pan-caspase inhibitor(caspase 1,3,8)ZVAD-FMK and caspase 3 specific inhibitor Ac-DEVD-CHO significantly impaired pyroptosis caused by JS5/05 infection,but caspase 1 specific inhibitor VX-765?NLRP3 inhibitor MCC-950 and gasdermin D(GSDMD)inhibitor disulfiram had no impact on virus-induced pyroptosis.Furthermore,caspase 3 activity in virusinfected cells was measured,the results showed that NDV infection significantly enhanced caspase 3 activity in HD11 cells,and a large number of caspase 3 activated cells were PI-positive,suggesting that caspase 3 activation plays an important role in NDV-induced macrophage pyroptosis.The results showed that NDV can induce pyroptosis of macrophage by activating caspase 3,while the NLRP3-caspase 1-GSDMD pathway is not involved in virus-induced pyroptosis. |
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