LPS Activated Macrophage Autophagy And Cytokine Production Of Antituberculosis

Background: Tuberculosis (TB) is one of the diseases that are seriously harmful tohuman health. Mycobacterium tuberculosis (M.tb) is the causative agent for TB. M.tbcan hide in the host macrophages and evade the immune system’s recognizing andkilling, through interfering with the formation and maturation of phagolysosomes. Inrecent years, the role of autophagy in anti-TB immune response is getting widespreadconcerns. More and more studies showed that lack or inhibition of autophagy signalsmay be the key of that M.tb can escape from immune recognition and killing bymacrophages. The present data showed that LPS-induced autophagy could overcomethe obstacles of the formation and maturation of phagolysosomes, and promote M.tbcolocalization with autophagy, thus help to eliminate M.tb by autophagy lysosomalpathway in macrophages. Studies have found, that IFN-γ and the Th1/Th2cytokinespolarization to Th1, and so, can promote the autophagy to pathogens in humanmacrophages. To further investigate the effects of LPS on the production ofantituberculosis autophagy and cytokine in macrophages has important value forstudy on the possibility of LPS as anti-TB drugs or immune adjuvants.Objective: To investigate effects of LPS on the production of antituberculosisautophagy and cytokine in macrophages.Methods:1. An attenuated strain of M.tb, H37Ra was cultured in Sauton’s liquidmedium for one month and then cultured on Lowenstein-Jenden solid medium for onemonth at37°C. The M.tb was collected in1mL homogenizer for repeatedly grindingwith normal saline, and the M.tb bacterial suspension was washed3times bycentrifuge at10000r/min for2min, following identifying by acid-fast staining andmicroscopic observation. The bacterial concentration was measured byspectrophotometer, and M.tb suspension was prepared with normal saline at concentration of5×106/ml, preserved at4°C for further application.2. The effects of LPS (100ng/ml) on the autophagy of THP-1macrophage that infectedwith M.tb was identified by TEM.3. Effect of LPS (100ng/ml) on the IL-12p35and IL-12p40gene expression andprotein levels of THP-1cells before and after M.tb infection were detected byQuantitative PCR and ELISA, respectively. Western blot was used to detectautophagy-related protein LC3-II protein expression levels in THP-1(A) cells.4. Effect of LPS (100ng/ml) on IL-12expression was detected by quantitative PCRand ELISA in THP-1macrophages, while autophagy was blocked with trimethylpurine3-MA (3-Methyladenine), a specific inhibitor.5. peripheral blood taken from5cases of healthy people and5cases of tuberculosispatients, was stimulated with PMA and Ionomycin for2hours, and then cultured withMonensin for4hours. The cells were collected, dyed with a fluorescent labeledantibody CD3APC, CD8PECY5.5, and then with fluorescent labeled monoclonalanti-cytokine IFN-γ and IL-4after cell membrane was penetrated. The rates of T cellssecreting IFN-γ and IL-4were detected by flow cytometry.6. PBMCs of5cases of healthy people and5cases of tuberculosis patients wereisolated from their anticoagulation peripheral blood, and then resuspended withRPMI-1640containing5%NBS(1.5×106cells ml), seeded in24-well culture plate.LPS (100ng/ml) was added to the wells of TB group. the proportion of the T cellsubsets secreting IFN-γ and IL-4was detected by flow cytometry after24h.Results:1. Observed by TEM, the level of autophagy in THP-1macrophages (THP-1(A)) infected with M.tb increased after stimulated by100ng/ml LPS ()forPMA-induced differentiation of case development.2. The results of Western blot showed that LPS upregulated the expression of LC3-IIprotein in THP-1(A) macrophages infected with M.tb.3. The results of Quantitative PCR showed that LPS upregulated the expression IL-12p35and IL-12p40in THP-1(A) macrophages infected with M.tb significantly,but pretreatment with3-MA could inhibite this effection(p<0.05).4. The concentration of IL-12p70protein in THP-1(A) cell group was (28.63±5.78),which had no significant difference with (25.03±4.32) in non-infected group (p>0.05); Compared with control, the concentration of IL-12p70protein in LPSstimulated group increased significantly (p <0.05), and autophagy specific inhibitor3-MA could block this effect(p <0.05).5. The positive percentage of T cells expressing IFN-γ and IL-4were (11.45±4.10)%and (0.92±0.98)%in healthy human peripheral blood, compared with (5.11±1.17)%and (1.66±0.34)%in group of TB patients, there were statistical significance (p<0.05).6. For the peripheral blood T cells of TB patients, stimulation of100ng/ml LPS canincreased the level of IFN-γ expression (p<0.05), but it has no obvious effect on thelevel of IL-4expression(p>0.05).Conclusions:1. LPS can increase the expression of Th1-type cytokines, whichinhibited in TB patients;2. LPS can promote the levels of antituberculosis autophagy and cytokines inmacrophages;3. LPS may stimulate autophagy, and upregulate expression of IL-12in macrophages.