The Study Of The Role And Mechanism Of LTB4 In The Mac Rophage Against Mycobacterium Tuberculosis

Tuberculosis is still a worldwide public health problem.Mycobacterium tuberculosis(Mtb), the causative agent of tuberculosis(TB), is a facultative intracellular parasit e of macrophages.Macrophages serve as the major host cell niche for intracellular growth and persistence of Mtb.The macrophage plays an critical role in immune r esponse,constitute the first line of host defense against Mtb.In addition, macroph ages are responsible for activation of protective immune responses.both innate and acquired,thus playing a critical role in the ongoing cross-talk that is necessary to control or eliminate the infection.Mtb involve the engulfment of the bacilli by ma crophages and often their immediate killing by different macrophage bactericidal m echanisms, including the generation of reactive oxygen intermediates(ROI),cytoki nes,apoptosis and antigen presentation.The host immune response to Mtb is so c omplicate that many inflammatory mediators are involved.LTB4 is one of them an d Macrophages is the main source of LTB4.Leukotriene is Proinflammatory lipid medium by 5-lipoxygenase pathway metabolic products.Studies have shown that L TB4 has multiple functions,it can cause leukocyte chemotaxis,adhesion and degra nulation,cytokine secretion,and affect a variety of biological effects,also involve d and play an important role in many pathological process of autoimmune disease,such as asthma,rheumatoid arthritis.In recent years,the role of LTB4 in infectio us diseases receives more and more attention including bacteria, fungi and parasite s infection. But there are few reports about the effect of LTB4 on Mtb infection. This thesis intends to explore the following questions:Whether tuberculosis promot e macrophage express LTB4,To investigate the role and mechanism of LTB4 in th e Macrophage against Mycobacterium tuberculosis.Provide experimental evidences f or clinical adjuvant treatments of TB.Part 1: The influence of Mycobacterium tuberculosis and its components on macrophage LTB4 expressionObjective: To investigate mycobacterium tuberculosis and its components which ind uced the expression of LTB4.Methods: Using different doses of recombinant Ag85 B, ESAT-6 and strain H37 Ra and BCG incubation with macrophages about 4,24 h, then collect culture supernatant and detect LTB4 expression quantity by ELISA.Re sults: After incubation 4,24 h, Ag85 B and ESAT-6 can induce dose dependent the e xpression of LTB4, BCG promote the LTB4 formation of macrophage.But there is no statistical difference between H37 Ra and control group.The expression of LTB4 in 24 h is less than 4 h. Discussion: BCG and secretory protein of Mtb(ESAT-6,A g85B) promote LTB4 release from macrophage in the early stage of infection, and heat-inactivated H37 Ra has no effect on the expression of LTB4.Part 2: The role of LTB4 in the Macrophage against Mycobacterium tuberculosis Objective: To investigate the role of LTB4 in the macrophage against mycobacteriu m tuberculosis.Methods: Mtb were co-cultured with macrophage for different time. Phagocytosis rate was calculated by modified acid-fast staining method and FCM. ROS and apoptotic cells were detected by FCM. Macrophage were cocultured with Mtb for 4h and stained for acid phosphatase following the instruction of the kit.E xcept control and infection group,macrophages were pretreated with LTB4.Results:After four hours, different concentrations of exogenous LTB4(1-100 n M) can promote phagocytosis,the rate of phagocytosis increased with increasing of dosage. This eff ect can be blocked by U75302. LTB4 can promote production of reactive oxygen s pecies that released by macrophage, and improve the reactive oxygen species of inf ected cells.LTB4 promote apoptosis of infected cells.Discussion: Exogenous leukotri ene B4 enhanced the phagocytosis of THP-1 cells, LTB4 influence macrophage anti-tb immune function by producing reactive oxygen species and promote cell apopto sis.Part 3: The mechanism of LTB4 in the macrophage against Mycobacterium tuberculosis.Objective: To explore the mechanism of LTB4 in the macrophage against Mycobac terium tuberculosis.Methods: Macrophage were cocultured with bacteria for 4h or 24 h, and applied with different intervention.The level of LTA4 H m RNA and TNF-α m RNA were detected by q PCR,The expression of LTB4 and TNF-α were detected by ELISA.Results: Exogenous LTB4 has no influence in TNF-α expression of inf ect cells,Bestatin which is LTA4 H inhibitor can reduce the level of TNF-α m RNA after 4h. After 24 hours,the level of TNF-α m RNA was blocked by U75302.Discu ssion: Exogenous LTB4 does not affect the expression of TNF-α,TNF-α m RNA e xpression level can be partially blocked by LTB4 inhibitors.