| Avian reticuloendotheliosis(reticuloendotheliosis,RE),caused by avian reticuloendotheiosis virus(reticuloendotheiosis virus,REV),is a viral and immunosuppressive tumor disease of poultry.REV affects the poultry industry development seriously.TLRs a gonist are powerful immune adjuvant.In order to explore the expression of the polarized phenotype of macrophages after REV infection and the role of TLRs in this process,this study started with the interaction between REV and immune cells,and selected HD11 chicken macrophage cell line as the experimental subject.Subjects were divided into REV infection group(referred to as group REV),uninfected REV control group(referred to as group C),Poly(I:C)(HMW)stimulation group [referred to as Poly(I:C)group] and Poly(I:C)(HMW)pre-treatment and then infected with REV group [abbreviated as Poly(I:C)+REV group] four groups,using cell culture technology and real-time fluorescent quantitative polymerase chain reaction method,indirect enzyme-linked immunosorbent method,Western blotting,CCK-8 and Griess method,etc.we detected the changes in viral load and cell viability in HD11 cells at 6,12,24,48,and 72 h after the action of REV and the changes of macrophage polarization marker genes M1(i NOS),M2(IL-10),innate immune Toll-like receptors TLR3 and TLR7,antiviral factor IFN-β respectively.The main research results are as follows:(1)After HD11 cells were stimulated by 0,30,50,100 ng/m L Poly(I:C)(HMW)for 6h,CCK-8 method was used to detect the light absorption values at 450 nm at 6,12,24,48,and 72 h after REV infection.The results showed that at 12-72 hpi,compared with 0 ng/ml,the absorbance of HD11 cells at 450 nm was significantly decreased when the concentration of Poly(I:C)(HMW)was 100 ng/ml(P<0.001),but there was no significant difference when the concentration of Poly(I:C)(HMW)was 50 ng/ml(P>0.05).The results showed that when the concentration of Poly(I:C)(HMW)was 50 ng/ml,the absorbance value of cells at 450 nm was less affected,so the Poly(I:C)(HMW)of 50 ng/ml concentration was used as the optimal concentration of Poly(I:C)(HMW)in this study.(2)The cell viability of HD11 cells in REV,C,Poly(I:C)and Poly(I:C)+REV group were detected by CCK-8 method.The results showed that the cell viability of REV,Poly(I:C)Group and Poly(I:C)+REV group was lower than that of group C at 6-72 h.Among them,the cell viability of REV group was extremely significant at 6 h,48 h and 72 h(P<0.01 or P<0.001)lower than group C,indicating that REV inhibits the activity of HD11 cells.(3)The virus was detected at each time point after REV infection of HD11 cells,indicating that REV replicated and proliferated in HD11 cells;and at 24-48 hpi,the viral load increased significantly(P<0.05),and The virus reached its peak at 48 hpi;in Poly(I:C)+REV group,we found that the viral load in HD11 cells was significantly reduced and maintained at a relatively low level;there was no REV detected in the Poly(I:C)group and C group.It shows that REV replicates in HD11 cells,and the TLR3 agonist Poly(I:C)(HMW)has a certain inhibitory effect on REV replication.(4)Through the quantitative detection of HD11 cell polarization marker factors i NOS and IL-10 in REV group,C group,Poly(I:C)group and Poly(I:C)+REV group,the results found :1)The IL-10 m RNA expression level of HD11 cells in group REVwas higher than that in group C at all time points tested,and reached its peak at 24 hpi,indicating that after REV infects HD11 cells,M2 type polarization was present in the early stage;compared with group C,the IL-10 m RNA expression of HD11 cells in REV group showed a trend of first decline and then increase,and IL-10 m RNA expression was significantly lower than that of group C at 12 hpi(P<0.05);Compared with group REV,HD11 cells in Poly(I:C)+REV group showed signi ficant(P<0.05)or extremely significant(P<0.0001)reduction in IL-10 m RNA expression at 12,24 and 48 hpi and the IL-10 protein content in the HD11 cell culture supernatant of the Poly(I:C)+REV group was significantly(P<0.01)lower than and significantly(P<0.05)higher than that in the C group at 24 and 48 hpi,respectively.The above results show that after REV infection with Poly(I:C)pre-stimulated HD11 cells,the macrophages tend to be M2-type polarization levels significantly reduced.2)The expression of i NOS m RNA in HD11 cells of REV group was significantly higher than that of C group at 24 and 48 hpi(P<0.01);the expression of i NOS m RNA in Poly(I:C)group was higher than that in C group at all time points,and reached the peak at 48 h pi with significant difference(P<0.01,P<0.001),but there was no significant difference at other time points(P>0.05);Compared with REV group,the expression of i NOS m RNA in Poly(I:C)+REV group increased at6-24 hpi,and reached its peak at 24 hpi with significant difference(P<0.0001);The results showed that Poly(I:C)(HMW)prestimulation HD11 cell infected with REV tended to M1 type.3)At the protein level,the i NOS protein content in the HD11 cell culture supernatant of the REV group was lower than that of the C group at different levels except for 48 h;the i NOS in the HD11 cell culture supernatant of the Poly(I:C)group was significantly(P<0.05)or extremely significantly(P<0.001)higher than that of group C at 12-48 hpi;the content of i NOS protein in HD11 cell culture supernatant of Poly(I:C)+REV group was Significantly(P<0.05)higher than that of group C and REV group at 12-72 hpi,and the other time points tested were no significantly difference(P>0.05);the above results show that Poly(I:C)(HMW)stimulated HD11 cells and re-infected with REV,HD11 cells tend to M1-type.(5)In this study,TLR3,TLR7,IFN-β m RNA expression changes in the REV,C,Poly(I:C)and Poly(I:C)+REV group of HD11 cells were tested at 6,12,24,48,and 72 hpi.The results are as follows:1)The expression of TLR3 m RNA in REV group was higher than that in group C at 6-72 hpi,and there were significant(P<0.05)and extremely significant(P<0.0001)statistical differences at6 and 12 hpi;the TLR3 m RNA expression of HD11 cells in the Poly(I:C)group and Poly(I:C)+REV group was higher than that in the C group at all time points tested,and reached a very significant(P<0.0001)peak at 48 hpi;while the TLR3 m RNA expression in HD11 cells of the REV group was significantly(P<0.05)or extremely significantly(P<0.001)lower than Poly(I:C)+REV group and Poly(I:C)group at 6-48 hpi;the expression of TLR7 m RNA in the REV,Poly(I:C),Poly(I:C)group was no significantly difference than that of the C group at all time points(P>0.05).The results showed that the effects of REV infection on the polarization phenotype of HD11 cells were mainly mediated by TLR3;Poly(I:C)(HMW)successfully activated TLR3 and its mediated signaling pathways.2)In terms of protein level,the TLR3 protein expression of HD11 cells in group REV showed a downward trend at 12-48 hpi compared with group C,and was significantly lower than group C at 48 hpi(P<0.05),although it was higher than C Group at 72 hpi,but no statistical significance(P>0.05);the expression of TLR3 protein in Poly(I:C)+REV group was significantly higher than group REV at 24,48 and 72 h(P<0.01 or P<0.05).The results showed that REV infection inhibited the expression of TLR3 protein in HD11 cells.Poly(I:C)(HMW)stimulated HD11 cells a t each time point under examination.The expression of TLR3 protein in HD11 cells increased significantly(P<0.05).Poly(I:C)(HMW)After pre-stimulating HD11 cells and reinfected REV,the expression of TLR3 protein in HD11 cells was increased.(6)The IFN-β m RNA expression of HD11 cells in group REV showed a downward trend at each time point of inspection;IFN-β m RNA expression of cells in the Poly(I:C)group was higher than that of group C at all time points.IFN-β m RNA expression in Poly(I:C)+REV group was higher than that in C group at 6-48 hpi;meanwhile,IFN-β protein content in HD11 cell culture supernatant of Poly(I:C)group was very significant(P<0.0001)or significantly(P<0.05)higher than group C at 12-72 hpi;the content of IFN-β protein in HD11 cell culture supernatant in Poly(I:C)+REV group is extremely significant or significant at 12-48 hpi(P<0.0001 or P<0.001 or P<0.05)higher than group C.The changes of IFN-β protein content is basically same as that of TLR3,indicating that IFN-β is closely related to TLR3.Based on the above experimental results,it is not difficult to find that after REV infects HD11 cells,it mainly inhibits the expression of TLR3,IFN-β and other innate immune factors,which makes the polarization of macrophages tend to M2 type.The large-scale production of M2 type IL-10 factor is beneficial to REV replication;after the application of Poly(I:C)(HMW),the expression of TLR3 increased significantly;and after Poly(I:C)(HMW)stimulated HD11 cells then reinfected with REV,TLR3 was expressed in large quantities,effectively identifying REV invasion,mediating HD11 cells transform to M1 type,activating IFN-β m RNA expression to activate the host’s natural immune defense mechanism and preventing REV replication in HD11 cells. |
