Effect Of NO,PGE₂ Production And Treg Differentiation By Dendritic Cells After Infection With Mycobacterium Bovis And Preliminary Study Of Novel TB Maker TRIM25

Tuberculosis(TB)is a chronic consumptive zoonotic disease which is caused by Mycobacterium tuberculosis complex(MBTC).Mycobacterium bovis(M.bovis)is the main pathogen causing bovine tuberculosis(BTB),and humans or other livestock are susceptible animals of it.BTB is not only a serious threat to human health and life,but also a great damage to the sustainable development of animal husbandry.There are about200 million TB patients dying in the end each year including 1 million people being infected with M.bovis.Dendritic cell(DC)is the most powerful kind of professional antigen presenting cells(APC),which is capable of initiating innate immune responses and activating T-cell responses.Nitric oxide(NO)and prostaglandin E₂(PGE₂)are important cytokines in the immune response,which can control the inflammatory response and prevent M.tb invasion.However,in order to avoid anti-infective mechanism of the body,M.tb induces the production of regulatory T cells(Treg)to limit the level of immune response and achieve immune escape.Previous studies in our laboratory have shown that M.bovis and BCG can cause a significant increase in NO and PGE₂ by infecting DCs,and a high expression of Treg transcription factor Foxp3 through the co-culture of DCs and CD4⁺T cells.Therefore,the aim of this study is to confirm the roles and interrelationship of NO,PGE₂ and Treg in M.bovis and BCG infection by in vivo and in vitro experiments through the models of cells and mouse.The main results are as follows:1.The production and simultaneous effect of NO and PGE₂The concentration of NO and PGE₂ and the expression of inducible enzymes i NOS,COX2 and m PGES1 increased gradually in a time-dependent manner.The ability of M.bovis to induce PGE₂ was significantly stronger than that of BCG,but the ability of M.bovis to induce NO was significantly weaker than that of BCG.Furthermore,NO and PGE₂ had some simultaneous effect.When the concentration of NO decreased,the conc entration of urea was increased,and the ability of M.bovis to induce urea was significantly stronger than that of BCG.The main receptors of PGE₂ were EP2 and EP4,and the ability of M.bovis to enhance EP2 and EP4 was significantly stronger than that of BCG.2.The concentration-dependent effect of NO and PGE₂ on DC differentiation,apoptosis and cytokinesWhen NO and PGE₂ are inhibited,the differentiation and maturation of DC could be reduced,and the inhibition of NO and PGE₂inhibited apoptosis and the expression of TNF-?,promoted the expression of IL-1?,but had no effect on the expression of IL-6.When NO inducer IFN-?1?L(100ng/m L)and exogenous PGE₂ 5?L(20ng/m L)are added,the maturation of DC could be promoted,and the apoptosis could be promoted.3.The effect of M.bovis and BCG on CD4⁺T cell differentiationM.bovis was more potent in stimulating Th2 and Treg,and the ability to induce PGE₂and NO was stronger than that of BCG,but was less potent in stimulating Th1.When the concentration of PGE₂ was significantly decreased,the ability of DCs to stimulate differentiation of CD4⁺T cell was decreased,which caused that the differentiation of Treg and the expression of Th1-related cytokine IFN-?and Th17-related cytokine IL-17A were significantly decreased.4.The immune response and Treg differentiation of M.bovis and BCG-infected miceCompared with BCG,the expression of IFN-?,IL-1?,IL-6,COX2 and i NOS were significantly increased in M.bovis group in most of the infection time,such as 1,7,14,21,28,42,49,56 days,but the expression of TNF-?and m PGES1 were significantly increased only in middle and late infection(day 14 to day 56),indicating that M.bovis infection could cause significant inflammatory response and active nitrogen reaction while inhibit the body to produce TNF-?and PGE₂ in early infection(day 1 to day 7).M.bovis could induce more obvious pathological changes,including red pulp zone congestion,increased macrophages and reduced lymphocytes in spleen lymph node.The distribution of Treg was mainly concentrated in the red pulp zone,the trabecular trabeculae and the membrane of the spleen,which is the main lesion site.In the whole process of the infection,the expression of Foxp3 and the differentiation of Treg were enhanced in M.bovis group,but the ability of BCG to stimulate Foxp3 expression and Treg differentiation was relatively weak.5.The effect of M.tb-infected cells and patients on tripartite motif 25(TRIM25)The expression of TRIM25 in the blood of the newly diagnosed tuberculosis patients was significantly higher than that of the healthy people.When TRIM25 was inhibited,the expression of cytokines IL-1?,IL-6,TNF-?,NO,PGE₂ and signaling factor p-p65 were significantly increased,but the expression of signaling factor p-p38 was significantly decreased,indicating that M.tb 1458 could induce the expression of TRIM25 to activate p38 pathway,inhibit NF-?B pathway and reduce the expression of IL-1?,IL-6,TNF-?,NO,PGE₂,which might be one of the mechanisms to reduce cellular immune response and achieve immune escape in M.tb.In summary,this study confirmed that M.bovis and BCG-infected cells and mice can induce NO and PGE₂ production and Treg differentiation,M.bovis induced higher PGE₂production and stronger Treg differentiation,but induced less NO production compared with BCG.NO,PGE₂and Treg differentiation are interrelated,high concentration of PGE₂ can inhibit NO production,and PGE₂ can promote Treg differentiation.When NO and PGE₂ are inhibited,the differentiation,maturation and apoptosis of DC could be reduced.M.tb 1458 induced high expression of TRIM25 to inhibit NF-?B pathway and reduce immune response.These results provided some evidences to further clarify the pathogenesis and immune escape mechanism of M.bovis and M.tb.