Comparative Research On Polarization Of Murine Th17 Cells Induced By Virulent And Attenuated Mycobacterium Bovis Strains

M.bovis is the predominant pathogens of bovine tuberculosis,and thus brings enormous economic pressure to today's cattle-feeding industry.As a zoonotic disease,bovine tuberculosis also brings a great threat to human health.M.bovis infects the body through the aerosol which could cause chronic inflammatory reaction.BCG is a vaccine strain from M.bovis with good immunogenicity and BCG is the only vaccine against tuberculosis.IL-17 is mainly produced by Th17 cells and plays an important role in inflammatory response and antimicrobial infection.While,its role in the infection of M.bovis is not well understood.Previous studies in our laboratory have shown that M.bovis can increase the secretion of CD4~+T derived IL-17 through DCs,while,the ability of BCG to induce IL-17 expression was weaker.The aim of this study was to investigate the role of IL-17 and Th17 cells in M.bovis infection through the model of mouse.The main results are as follows:(1)TGF-?,IL-6,IL-1?,IL-23 are key cytokines in Th17 cell differentiation.Infection of dendritic cells with M.bovis and BCG of MOI(10:1),24 hours after infection we detected the mRNA expression of the key cytokines in the infected cells.We found that TGF-?,IL-6,IL-1?,and IL-23 were up-regulated significantly,and the induction effect of M.bovis was significantly stronger than that of BCG.(2)ROR-?t is a characteristic transcription factor of Th17 cells,and IL-17 is a characteristic cytokine of Th17 cells.We carried out experiments about co-culture of CD4~+T and DCs infected by the virulent and attenuated strains,then detected the mRNA expression of ROR-?t,IL-17,TGF-?,IL-6,IL-1?,IL-23.We found that ROR-?t,IL-17,TGF-?,IL-6,IL-1?,IL-23 were up-regulated significantly.And the induction effect of M.bovis was significantly stronger than that of BCG.M.bovis and BCG are able to induce the differentiation of Th17 by the up-regulation of TGF-?,IL-6,IL-1? and IL-23,and the induction of M.bovis was significantly higher than that of BCGThrough the above two experiments in vitro.(3)The strains that were cultured to logarithmic phase were injected into mice by tail vein of 2×105CFU.The mice were weighed and killed at 1,4,7,14,21,28,35,49 and 56 days,respectively.The body weight and pathological changes of the mice were detected and analyzed.The body weights of mice infected with different strains are loss and M.bovis decreased significantly.The infected strains could induce histopathological response in mices,M.bovis can cause pulmonary alveolar wall more thick,macrophage,lymphocyte infiltration,and even see the appearance of a typical tuberculosis in 21 days.BCG can also cause similar inflammatory reactions,but the symptoms are mild.The effects of M.bovis on the spleen were as follows: increased macrophage,reduced lymphocyte and the congestion of red pulp zone leads to edema,the symptoms of BCG group were lighter.The results showed that we infected mice with M.bovis and BCG through the tail vein successfully.(4)The Th17 and IL-17 in spleen and lungs of infected mice in different time were analyzed through flow cytometry and RT-qPCR.The results showed that both virulent and attenuated strains could increase the Th17 and IL-17 in spleen and lung,and the up regulation of IL-17 was earlier than Th17.In addition,the up regulation of IL-17 in the lung was earlier and lasted longer than the spleen.(5)We explored the distribution characteristics of IL-17 in spleen and lung of mice through IHC.The results showed that the IL-17 induced by the virulent and attenuated strains were mainly concentrated the red pulp area,the spleen trabecula and under the membranein of the spleen.The expression of IL-17 induced by M.bovis was higher than that of BCG.However,it is much lesser in the lung of mice,and mainly in the alveoli,pulmonary vessels and pulmonary trachea.These results lay a foundation for the future research on the role of Th17 and IL-17 in M.bovis infection.In summary,this study confirmed that M.bovis and BCG infected mice could induce the differentiation of Th17 and and the expression of IL-17,and the induction ability M.bovis was stronger than BCG.The results provide important evidence for further elucidation of the pathogenesis of bovine tuberculosis.