Lipopolysaccharide Promote Anti-tuberculosis Immunity In Mouse Model

Background: Mycobacterium tuberculosis(M.tb) is caused by tuberculosis pathogens. Studies have shown [1], the body after infection M.tb, M.tb can be hidden within the host macrophages by inhibiting macrophage autophagy anti M.tb, escape immune destruction, the formation of so-called "immune escape" cause latent infection. There have been a variety of studies have shown clear intracellular bacterial infection mainly rely on innate immune cells and induce protective immunity.Generally believed that macrophages and lymphocytes play an important role in TB infection immunity. Our previous study from horizontal cells found: LPS via TLR4 expression levels increase, and thus contributing to increased ROS production in macrophages M.tb oxygen-dependent killing, IFN-γ can promote macrophage pathogens autophagy. Therefore, this study from animal models to further study the level of non-M.tb sources TLR4 agonist LPS in mice monocyte- macrophage regulation of anti-TB immune function.Objective: To study on the effects of LPS on the anti-tuberculosis immunity in mouse infected with M.tb.Methods: 1. Take M.tb(H37Ra) in Su Tong’s liquid medium, incubator a month after transfection species in 7H11 solid medium culture incubator at 37 ° C within a month, using a sterile inoculation loop charge M.tb strains homogenizer in 1 ml, was added Tween-80 repeated grinding, take the bacterial suspension, with Tween-80 by10000 rpm × 2 min and washed three times, acid-fast staining and microscopic observation of bacteria dispersed. Using a spectrophotometer to detect bacterial concentration and diluted with saline to a concentration of 2 × 108 CFU / m L suspension of M.tb, placed 4 ° C refrigerator spare.2. Adjusted the concentration of living M.tb to 2 × 108 CFU / ml, bacillus were injected into the tail vein of C57 BL / 6 mice. After that, sacrificed mice at 7th day,14 th day, 21 st day, and took the lung, spleen tissue of mice for living bacillus culture,or organ weight index detection, Determine the success of M.tb infected mice.3. According to the reagent manual of LPS, the dose which can induce fever in mice for LPS is 500 ng / kg. So, the LPS dose to 250 ng / kg, 500 ng / kg, 750 ng / kg,then injected intravenously into C57 BL / 6 mice. Sacrificed the mice after 10 hours of intravenous injection to confirm the appropriate dose of LPS treatment in mice by method of HE staining.4. After LPS treatment for 7 days, 14 days, and 21 days, peripheral blood of model mice was collected by extirpating eyeballs. Level of TLR4 and ROS expression in monocytes of periphery blood were detected by flow cytometry.5.After LPS treatment for 7 days, 14 days, and 21 days,take the periphery of mouse anti-coagulation, intracellular staining, flow cytometry, and IFN-γ expression of IL-4T cell sub-population proportion.6. After treatment with LPS for 7 days, 14 days, and 21 days, tissues of lung and spleen were taken out from sacrificed mice, and the tissue damages were confirmed by method of histology.7. Grinded Tissues of lung and spleen from sacrificed model mice were cultured on7H11 dishes to find the living bacillus.Results:1. After treatment with different concentration of LPS, the histopathological results showed that the maximum safe dose of a LPS which can be used in mice was250 ng / kg.2. The infection model can be made after the mice were infected with 2 × 108 CFU /ml attenuated Mycobacterium tuberculosis strain H37 Ra by tail vein injection.3. Mice infected with m.tb, in LPS to 7 day, expression level of TLR4 in peripheral blood mononuclear cells(80.00±37.97) %, were significantly higher than thosewithout drug group(15.66±10.14) %, the difference was statistically significant(p <0.01).That LPS can strengthen mice is m. b inhibition of mononuclear macrophage surface TLR4 receptor expression rate.4. Mice infected with m.tb, in LPS to 14 days after ROS production in the peripheral blood mononuclear cells(104.76±18.65) is significantly higher than not to drug group(55.70±3.43)(p < 0.01).That LPS can strengthen mice by M.tb inhibits monocyte surface production of ROS, which expression quantity decreased gradually after 7days.5. After treated with LPS for 14 days, the percentage of peripheral blood IFN-γ+ T cells was(11.52±1.43)%, which was higher than(3.84±1.11)% in group of control.After treated with LPS for 7 days, the percentage of IL-4+ T cells was(2.67±0.14)%in test group, which was lower than(4.78±1.14)% in control group(P<0.01).6. The results of living culture and histopathological staning showed that LPS improved the status of lung and spleen injury in M.tb infected mice.Conclusions:1. LPS can upregulate the immune function of anti-tuberculosis in H37 Ra infected mice model.2. LPS can improve the tissue injury of H37 Ra infected mice model at low concentration.