Molecular Mechanism Of Brucella OMP25and L7/L12Induce Macrophage Apoptosis

Brucellosis (referred to as brucellosis) is an internationally recognized natural foci of disease in animal populations naturally carry and spread. Cattle, sheep, pig are the most commons, dogs and more than60kinds of livestock, poultry and wild animals have varying degrees of susceptibility to brucellosis. More than170countries and regions, people and livestock brucellosis in over200countries and regions around the world. Every year abort3billion costed by brucellosis in Worldwide.In China, Xinjiang and Qinghai, only the annual brucellosis caused nearly100million.Macrophages are the first line of defense of the innate immune. And plays a very important role in the survival of Brucella. First through degradation of Brucella and to present antigens to initiate immune responses in early macrophage,when this pathway is blocked, infected macrophages can pass to initiate apoptosis to Brucella to lost the protection of the environment, to facilitate the body to clear. In fact, Brucella can replication in host cells from apoptosis. Thus, the induction of macrophage apoptosis to clear Brucella infection is an important significance.Studies have found that the protein of Gram-negative bacteria can regulation of macrophage function and secretion cytokine.Omp25can regulate the generation of TNF-α, and to limit or clear the bacteria within the macrophages; L7/L12is an ribosomal protein of Brucella, can promote γ interferon (IFN-gamma) transcription and expression.IFN-γ upregulation is directly related to macrophage apoptosis. Therefore, in vitro studies Omp25and L7/L12for the target protein, effected on macrophage apoptosis.In this study, omp25and L7/L12gene were cloned. The omp25into the prokaryotic expression vector pGEX-6p-1, L7/L12into the prokaryotic expression vector pET28a-the SUMO Maded Omp25and L7/L12polyclonal antibody. Omp25and L7/L12insert transfer vector pFastBacHTB, multiple cloning sites to construct the baculovirus recombinant transfer vector transfection of insect cells (of sf9) protein expression.The expression of proteins in vitro the role of macrophages, the phagocytic activity of macrophages, secretion of cytokines, inducing apoptosis detection. Results:1) Omp25and L7/L12effect on the phagocytic activity of macrophagesMacrophages load Omp25and L7/L12, the difference in phagocytic activity compared to resting state was significantly (P<0.01), compared with the negative control, the difference was significant (P<0.01), Omp25and LPS synergy, giant phagocytic activity was significantly increased, compared the phagocytic activity of Omp25with the macrophage load, the difference was significant (P<0.01); the L7/L12synergy with LPS, the macrophage phagocytic activity was significantly improved, and macrophage load The L7/L12after phagocytic activity compared to the significant difference (P<0.01).2) Omp25and L7/L12effect on macrophage secretion of cytokinesMacrophage load Omp25from6h to detect IL-1β, IL-6, IL-8, IL-10secretion to12h of IL-6secretion reaches its maximum, to48h of IL-1β, IL-8of IL-10secretion of the maximum, compared with the expression of wild poisonous protein differences significant with (P<0.01); with LPS synergistic role of macrophages, secretion of cytokines and LPS group compared to the significant difference (P<0.01), compared with negative control, the difference was significant (P <0.01), polyclonal antibody blocked the cytokine secretion in decline, compared with non-blocking group, the difference was significant (P<0.01).Macrophages load L7/L12, from6h detected IL-1β, IL-6, IL-8, IL-10secretion,12h of IL-6secretion reaches its maximum, to48h of IL-1β, IL-8, IL-10secretion reaches its maximum, compared with the field strains were significantly (P<0.01); collaborative role of macrophages with LPS secretion of cytokines and LPS group compared to the significant difference (P<0.01), compared to field strains, significant differences (P<0.01), polyclonal antibody after blocking, the cells factor secretion in decline, compared with non-blocking group, the difference was significant (P<0.01).3) Omp25and L7/L12macrophage apoptosisMacrophage load Omp25stimulate macrophages to produce a small amount of caspase-3after stimulation with LPS synergy can produce more caspase-3; macrophages load Omp25is not typical of apoptotic bodies, but omp25synergy with LPS to produce the typical apoptotic bodies; Omp25after the load macrophage staining found that the macrophage cell membrane occurred after the load Omp25minor damage to the cells blue fluorescence, and stronger than the control cells. Omp25and LPS co-stimulatory cell membrane changes, a strong blue fluorescence.The omp25role of macrophages promote the secretion of macrophages caused by LPS stimulation of TNF-α to increase. The omp25promote activation by LPS-induced NF-κB nuclear translocation,NF-κB is activated; polyclonal antibody to block omp25, NF-κB is not activated.Macrophage load cell L7/L12can stimulate macrophages to produce small amounts of caspase-3, after stimulation with LPS synergy can produce a small amount of caspase-3, macrophage load L7/L12could not have typical apoptotic body to produce L7/L12with LPS synergy can produce the typical apoptotic bodies; load the L7/L12after the macrophage staining found macrophage load L7/L12, membrane slight damage in cells blue color fluorescence. L7/L12and LPS co-stimulatory cell membrane changes, a strong blue fluorescence.The the L7/L12role of macrophages by LPS stimulation can promote macrophage secretion of TNF-α increase. L7/L12can promote activation by LPS-induced NF-κB nuclear translocation, L7/L12and P38MAPK signal transduction inhibitor SB203580role of NF-κB is not activated; when polyclonal antibodies against L7/L12blocked, NF-κB is not activated.In this study, obtained through the above test:1) Omp25and L7/L12can activation of macrophages, increase the phagocytic activity of macrophages enhanced the bactericidal capacity, and promote macrophage pathogen clearance;2) Omp25and L7/L12enhanced macrophage secretion of IL-1β, IL-6, IL-8and promote apoptosis in macrophages in vitro.3) In this study, Omp25and L7/L12induced macrophage secretion of caspase-3, increase the secretion of TNF-α, activation of NF-κB to promote the LPS-induced macrophage apoptosis.