The Immune Response Of NKT Cells In Mice Infected With Listeria Monocytogenes L-form

Objective: Natural killer T cells (NKT cells) are kind of immune cells that can mediateinnate and acquired immune response, which can be activated by recognizingglycolipids antigen presented by CDld molecules beard in APC and plays an importantrole in resisting intracellular bacteria infection. A variety of factors in vitro and in vivocan make the bacteria cell wall deficiency, and then mutate into L-form leading tocomposition contains more lipid exposed. Accordingly, we speculate that whetherL-form be more effective in activating NKT cell compared with the wild type bacteria?A lot of studies have reported about the nonspecific response and specific response ofactivated NKT cells to the wild type, but little is known about the response of activatedNKT to L-form. Listeria monocytogenes had been used in this study, which was afacultative cell entophyte, and can be induced into L-form by penicillin. Wild type andL-form animal infection models were established respectively by infecting mice(C57BL/6) with caudal vein infection. The difference of pathogenicity between the wildtype and L-form listeria can be observed by detecting the colony-forming units (CFU)in the liver and spleen of infected mice. The activation rate and the absolute number ofNKT cell in the liver and spleen of the mice which were infected differently at differenttime points have been detected by using flow cytometry. Whether the activation to NKTcells by L-form and wild type are different would be discussed. The contents of IL-4and IFN-γ in blood serum of different models have been detected and the differences ofcytokine secretion in the mice infected by L-form listeria and the IL-4have beencompared by enzyme-linked immunosorbent.Methods:1. Induced listeria from wild type into L-form by penicillin, and then adjustedthe final concentration of them to2×105CFU/ml;2. Confirmed the scope of applicationof different liver lymphocyte separation methods by comparing percentage and absolutenumber of lymphocyte, NKT and NK cell obtained by those above methods.3. Theinfected animal model have been established by caudal vein infection of C57BL/6micewith0.2ml PBS, wild type or L-form listeria,4mice in each group;4. The liver andspleen were isolated sterilely on day3after infection, and the CFU of wild type andL-form in the two organs were determined;5. The lymphocyte of liver and spleen wereseparated on day1and day3after infection, respectively, percentage and absolutenumber of CD3+NK1.1+NKT, CD3+NK1.1+CD4+NKT, CD3-NK1.1+NK cells in thetwo organs in each group were detected by flow cytometry;6. Peripheral blood was obtained by removing the eyeball of mice in each group, the contents of IFN-γ and IL-4in mice serum were detected by ELISA assay.Results:1. L-form listeria colonies showing a characteristic ‘fried egg’ like appearanceon Listeria L-form medium agar media induced by penicillin, Some bacteria changedfrom Gram-positive into Gram-negative and became pleomorphic; cell wall stainingshows that bacteria in vivo stained thickly caused by cell wall-deficient.2. The availability of lymphocyte were affected by the pretreatment of the liver, the totalabsolute number of lymphocytes, percentage and absolute number of T cells obtained byunblooding method were the highest; the con-percentage of lymphocytes obtained byblooding with removing eyeball combined with systemic circulation systemiccirculation was the highest; the percentage of CD3+NK1.1+NKT cells and CD3-NK1.1+NK cells obtained by blooding method with removing eyeball were the highest.Lymphocytes access was affected by different Percoll separation method. Thepercentage of lymphocytes, T cells, CD3+NK1.1+NKT cells and CD3-NK1.1+NK cellsobtained by discontinuous density gradient (33%+70%) were higher than33%group.3. The CFU numbers in the liver and the spleen of the L-form–treated group weresignificantly lower than the wild type-treated group on day1and day3after infection.4. By comparing the proportion and the absolute number of CD3+NK1.1+NKT、CD3+NK1.1+CD4+NKT、CD3-NK1.1+NK cells in liver lymphocytes on day1and day3after infection, we found that the above-mentioned numerical value of L-form-treatedgroup were higher than the bacterial-treated group; all values of the above-mentionedinfected group were higher than the uninfected group, excepted the proportion ofCD3+NK1.1+CD4+NKT cells on day1after infection; the proportion and absolutenumber of CD3+NK1.1+NKT、CD3-NK1.1+NK cells on day3declined slightlycompared to day1. The absolute number of CD3+NK1.1+CD4+NKT cells on day1andday3after infection with bacteria and L-form listeria were increased, and day3waslower than day1; but the proportion of CD3+NK1.1+CD4+NKT cells increased on day3of L-form-treated group, while declined on day1and increased on day3ofbacteria-treated group.5. We found the proportion and the absolute number of CD3+NK1.1+NKT andCD3-NK1.1+NK cells in spleen of the L-form-treated group were higher than thebacteria-treated group by comparing all the mice, and both of the infected groups werehigher than the uninfected group; the proportion and absolute number of these two kindsof cells on day3in spleen were higher than day1, which was different from the liver. Compared with the uninfected cells, the proportion of CD3+NK1.1+CD4+NKT cells inspleen of two infected group increased on day1after infection, while decreased on day3, but the absolute numbers on day1and day3were both higher than the uninfectedgroup. Campared with the bacteria-treated group, the proportion of CD3+NK1.1+CD4+NKT cells of L-form-treated group was higher on day1, but lower on day3, while thecell absolute numbers were both higher on day1and day3.6. The secretion conditions of IFN-γ and IL-4of the peripheral blood of the infectedmice were detected by using ELISA assay, the results showed that the IFN-γ levelsincreased constantly with the infection time increased, and L-form-treated group washigher than the bacteria-treated group; With the infection time increased, the IL-4levelof serum first increased and then decreased, and L-form-treated group lower than thebacteria-treated group.Conclusions:1. Mononuclearcell suspension of liver could be obtained by bloodletting with removingeyeball combined with heart perfusion, a large proportion of liver lymphocyte and itssubsets could be obtained by discontinuous density gradient centrifugation.2. Listeria monocytogenes L-form is more likely to be cleared than the wild type.3. Proliferation of CD3+NK1.1+NKT, CD3+NK1.1+CD4+NKT and CD3-NK1.1+NKcells could be stimulated more effectively by L-form compared with the wide type, thismight be one of the reasons leading to weak pathogenicity of L-form.4. The peak of role played by the early stages of infection of the NKT and NK cell inthe liver, which was an innate immune organ, came earlier compared with the spleen inthe non-specific immunity process of anti-listeria infection.5. The activated CD3+NK1.1+NKT cells in the liver played a major role byCD3+NK1.1+CD4+NKT subsets, while giving priority to the CD3+NK1.1+CD4-NKTsubsets in the spleen.6. L-form listeria could induce immune system to generate more IFN-γ to play a role ofanti-inflammatory compared with the wild type.