The Regulation Of IL-10 In The PCV2 Infection And Replication

Interleukin-10(IL-10)is an important anti-inflammatory factor.Its core mechanism of anti-inflammatory regulation is to regulate the balance of inflammation in the body,but it can be used by viruses and many other pathogenic microorganisms to escape the body's immune recognition and removal.Porcine circovirus type 2(PCV2)is the major cause of porcine circovirus disease,which is characterized by multi-system failure syndrome of weaned piglets.The typical symptoms of swine disease are immunosuppression and lymphocyte deletion disease.PCV2 infects porcine peripheral blood and tissues with high expression of IL-10,as an important regulatory factor in the body,il10 gene may play an important regulatory role in the process of PCV2 infection.However,the regulation of il10 gene and the effect of il10 on lymphocytes(T cells,B cells,and NK cells)are still largely unknow.In this study,wild-type C57BL/6 mice and il10-/-type C57BL/6 mice were used as model animals.PCV2-infected mice were established by inoculation of PCV2.Real-time PCR was used to detect the replication of PCV2 in mice.The expression of Cap protein in mice was detected by western blotting.Tissue lesions of PCV2 infected mice were identified by tissue section.The PCV2 infection model of il10-/-type C57BL/6 mice was reproduced,and the distribution and absolute number of lymphocytes(T cells,B cells and NK cells)in mice were detected by flow cytometry,by comparing the ratio and number of cells in the Mock group to determine the regulation of il10 gene deletion on PCV2 infection and replication.The following experimental results were obtained.1.A PCV2-infected wild-type mouse model was successfully established by inoculating PCV2 in mice.The PCV2 ORF2 fragment was identified in the peripheral blood,mandibular lymph nodes,lungs and liver of mice after inoculation with PCV2.The viral load in the lungs of infected mice was detected by Real-time PCR.After 2 and 4 weeks of PCV2 infection,the viral DNA level was significantly higher than that after 1 week infection,but the viral DNA level after 4 weeks infection was reduced to half compared with 2 weeks.Cap protein in lung,liver and mandibular lymph nodes was detected by western blotting.The tissue sections of the mice were observed under microscope.After 1,2 and 4 weeks of PCV2 infection,the lung tissue alveolar septa widened,alveolar septum with varying degrees of lymphocyte infiltration,showing the characteristics of interstitial pneumonia.The lymphocytes in the lymph nodes of mice were decreased at 1 week after PCV2 infection.After 2 weeks,the lymphocytes were further reduced,but the number of lymphocytes in the lymph nodes was restored 4 weeks after infection.2.The model of PCV2 infection il10-/-mouse was successfully established.PCV2 virus copy number was continuously increased in PCV2-infected wild-type mice,and lung tissue viral load in il10-/-mice reached the highest level at 2 weeks after infection(**P < 0.01),and decreased sharply(&&P < 0.01)at 4 weeks after infection,and was significantly lower than that in the first week(*P < 0.05).Western blotting showed that Cap protein was expressed in lung,liver and mandibular lymph nodes of PCV2-infected wild-type mice and il10-/-mice.The histopathological changes of lungs in il10-/-mice were similar to those in wild-type mice after PCV2 infection.However,compared with wild mice,infiltration of inflammatory cells was more significant in lung tissue 2 weeks after PCV2 inoculation,and lung tissue lesions were significantly reduced at 4 weeks after PCV2 inoculation.3.The mononuclear cells of the lungs and spleen of wild type mice and il10-/-mice were taken after PCV2 infection,and the results of flow cytometry were as follows: 1)PCV2 infection reduced the spleen CD45+ cells.The deletion of il10 gene could prevent the decrease of CD45+ cells in the spleen of 4 weeks after infection,reduce the number of CD45+ cells in lung tissue and increase the number of CD45+ cells in lung tissue at 4 weeks after infection;2)CD3+CD4+ T cells and CD3+CD8+ T cells were significantly increased at 2 weeks after PCV2 infection.The deletion of il10 gene mainly affected the number of CD3+CD4+ T cells in the spleen after 4 weeks of infection,but had no significant effect on CD3+CD8+ T during the whole infection;3)PCV2 infection increased the number and proportion of CD3+CD4+ T cells in the lungs,but had no significant effect on the number of CD3+CD8+ T cells.The number and proportion of CD3+CD4+ T cells in lung tissue were significantly higher than those in wild type mice at 2 weeks after infection,and the number/proportion of CD3+CD8+ T cells in lung tissue was significantly higher than that in mock or wild-type mice infection group;4)PCV2 infection did not affect the number of spleen NK,il10 gene deletion make the number of spleen NK cells increased significantly at 2 weeks and 4 weeks after infection,while il10 gene deletion 2 weeks after infection,the proportion of NK cells in lung tissue decreased significantly,4 weeks after infection and significantly increased;5)PCV2 infection reduced the number and proportion of B lymphocytes in the spleen of wild-type mice.The deletion of il10 gene could not change the trend of B lymphocytes.PCV2 infection increased the number of B lymphocytes in the lungs of wild-type mice,and the deletion of il10 gene failed to change this trend of B lymphocytes.Based on the above results,this study can be used to establish a mouse model of PCV2 infection.By successfully establishing the il10-/--type mouse model of PCV2 infection,it was clear that IL-10 played an important role in the pathogenesis of PCV2 infection and the formation of chronic lung lesions and lymphopenia.To determine the loss of IL-10 can reduce the amount of virus in the body of PCV2 infection and reduce the tissue lesion.The results clarified the role of IL-10 in the regulation of T,B and NK cells in vivo after PCV2 infection,and provided new theoretical data to further elucidate the mechanism of immunosuppression and lymphopenia in PCV2 infected pigs.