Influence Of REV Infection On The Tolls Receptors Pathways In Chicken Bone Marrow Derived Dendritic Cells

Reticuloendotheliosis virus(REV)is a C-type avian retrovirus that causes immunosuppression,short syndromes and so on.The immunosuppression caused by the virus can cause secondary infection or/and mixed infection of the host,aggravating the extent of poultry diseases,increasing the incidence and mortality of poultry diseases,and causing serious economic losses to the global poultry industry.Recent research reports,REV is potential threat to new bird species,so it is very important to study the pathogenic mechanism of REV,especially its immunosuppressive mechanism,and it also has public hygienics meanings.Dendritic cell(DC)is a kind of immune cell that currently known to have the strongest antigen presentation function,it is the bridge between innate and adaptive immunity,and it plays an important role in body's immune response.Immature dendritic cells have strong ability to antigenic phagocytosis,and immature dendritic cells become mature gradually after uptake of antigen;mature dendritic cells have strong ability to present,they can activate immune response strongly,thus the mature dendritic cells could activate T lymphocytes,B lymphocytes and natural killer cells.On the basis of the successful establishment of chicken bone marrow derived dendritic cells(ch BM-DC)infected REV model,the morphological changes of cells were observed by inverted microscope and scanning electron microscope,the changes of cell surface markers were detected by flow cytometry,the expression of different genes m RNA in BM-DC infected with REV was detected by RT-PCR technique.The expression of protein was detected by ELISA.We systematically studied the antiviral innate immunity mediated by Toll-like receptors(TLR)after REV infection.The main results are as follows:(1)Morphological observation showed that the cultured cells had DCs typical morphological characteristics,the chicken BM-DCs showed high purity by detecting cells surface markers CD11 c and MHC ?.These results suggested that large number of chicken BM-DCs with high purity were successfully induced.(2)We optimized the cells culture conditions by designing two factors and four level experiments,and culture cells under this condition,measuring the cell growth curve by CCK-8 method.The results showed that the cell grow well.(3)The virus infection was identified by PCR method.We found that REV specific conserved sequence LTR was detected in the chicken BM-DCs infected REV,but the LTR sequence was not amplified in the control group cells.The results suggested we establish the model of REV infection chicken BM-DCs successful.(4)We detected the morphological changes and the expression of cells surface markers in control group,LPS group and REV infection group.The results showed that the cell veils of REV infection group were shorter than the LPS-stimulated group,and the MHC ? expression decreased significantly,indicating that REV could inhibit maturation of chicken BM-DCs.(5)After chicken BM-DCs infection with REV,TLR3?TLR7?TLR21?TRIF?My D88?NF-?B m RNA expression were significantly upregulated(P<0.01)at 6 hour post infection(hpi)as compared to controls;TLR3 m RNA expression were significantly downregulated(P<0.05 or P<0.01)at 12,24,and 48 hpi as compared to controls;TLR7?TLR21 m RNA expression were significantly downregulated(P<0.05 or P<0.01)at 12 and 24 hpi,but significantly upregulated(P<0.05 or P<0.01)at 48 and 72 hpi as compared to controls;TRIF m RNA expression were significantly downregulated(P<0.01)at 12 and 24 hpi as compared to controls,and no statistical difference was found at other times(P>0.05);My D88 m RNA expression were significantly downregulated(P<0.05)at 12 hpi,but significantly upregulated(P<0.05)at 48 hpi as compared to controls,and no statistical difference was found at other times(P>0.05);NF-?B m RNA expression were significantly upregulated(P<0.05 or P<0.01)at 24 and 48 hpi as compared to controls.The results indicated that REV could strongly induce innate immune response mediated by TLRs at early stage of REV infection,but during late stage of infection,REV evaded recognition of TLRs by inhibiting the expression of TLRs and its associated factors.(6)After chicken BM-DCs infection with REV,IL-6 m RNA expression were significantly upregulated(P<0.01)at 6hpi,IL-6 protein expression were significantly upregulated(P<0.01)at 12 and 72 hpi as compared to controls;IFN-? m RNA and protein expression were significantly upregulated(P<0.05 or P<0.01)at 6 and 12 hpi,IFN-? protein expression were significantly downregulated(P<0.05 or P<0.01)at 24 and 48 hpi,but significantly upregulated(P<0.01)at 72 hpi as compared to controls;IL-8 m RNA and protein expression were significantly upregulated(P<0.05 or P<0.01)at 6,12,24 and 48 hpi as compared to controls,and no statistical difference was found at other times(P>0.05).The results indicated that REV could strongly induce antiviral and inflammatory responses of chicken BM-DCs at the early stage of REV infection,but during late stage of infection,REV inhibit interferon in host cells by evading TLR recognition,in order to promote their survival and proliferation within host cells.