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

Objective: Natural killer T (NKT) cells are a group of cells that share somecharacteristics with T cells and natural killer (NK) cells, which can recognizeglycolipids presented by CD1d molecules expressed on antigen presenting cells throughcell recognition pathway. A large number of studies have shown that NKT cells play animportant role in anti-infection immunity. Listeria monocytogenes (LM) are a kind offacultative intracellular bacteria, it has become an important model for the research ofintracellular bacteria infection mechanism. There is a variety of factors in vitro and invivo, inducing bacteria to have a cell wall deficiency so as to become into L-form,leading to more lipid composition exposed. Studies have confirmed that the L-form ofListeria monocytogenes can more effectively activate NK1.1+NKT cells. NKT cells canexpress DX5in mice of different types. This research respectively establish animalinfection model infected by wide type bacteria and L-form, use flow cytometry to testNKT cells’ activation rate and absolute number at different time in different models, andinvestigate whether there are difference between the activation of L-forms and thebacteria to NK1.1+NKT cells and DX5+NKT cells.Method:1. Induce L-form LM by applying the method of oxacillin, and adjust thefinal concentration of wide type bacteria and L-form LM to be1×106CFU/ml;2. InfectC57BL/6mice through tail vein with normal sodium, wide type bacteria and L-forms ofLM to establish animal model of the infection，each group has6mice;3. Separate theliver and spleen in mice respectively0,1,3days after infection, then use flowcytometer to test NK1.1+NKT, DX5+NKT, CD4+NKT and CD4-NKT cells percentageand absolute number in every group, which belong to liver and spleen lymphocytes andsecrete IFN-γ. Result:1.Successfully obtained L-form Listeria monocytogenes after induction withthe oxacillin disk diffusion method, we can see fried-egg-like colony in the L-formmedium. Some bacteria changed into Gram-negative bacteria and became pleomorphic.2. In the liver, the proportion and absolute number of DX5+NKT cells rose with theinfection time increased both in the group of wide type and L-forms. But the first dayafter infection, the absolute number of L-form group was higher than the wide typegroup. The proportion and absolute number of NK1.1+NKT cells increased on the firstday after infection, and on the third day，it was lower than the first day. However, bothon the first day and the third day that number of L-form group was higher.3. In the spleen, the proportion and absolute number of DX5+NKT and NK1.1+NKTcells in the group of bacteria rose as the infection time increases. The proportion of cellsin the group of L-forms was higher than the group of wide type on the first and thirdday. But the absolute number of cells in the group of wide type was higher than thegroup of L-forms on the first day. It was just the opposite on the third day.4. In the liver, after the infection of L-forms, the proportion and absolute number ofDX5+NKT、CD4+DX5+NKT、CD4-DX5+NKT cells which secrete IFN-γ kept rising,and the absolute number of L-form group was higher than the wide type group on thefirst day after infection. The proportion of CD4+DX5+NKT cells which secret IFN-γafter the infection of L-forms kept decreasing, while the proportion of CD4-DX5+NKTwhich secret IFN-γ after the infection of L-forms kept increasing. The absolute numberkept increasing in the wide type group. On the first day after infection the absolutenumber of L-form group was higher than the wide type group. However, on the thirdday the absolute number of wide type group was higher than the L-form group.5. In the liver, the proportion of NK1.1+NKT cells which secret IFN-γ in wide typegroup increased on the first day after infection and decreased on the third day, and theproportion of L-form group was higher than the wide type group on the first day and thethird day after infection. On the third day the proportion of wide type was higher thanthe L-form group. The proportion of CD4+NK1.1+NKT which secret IFN-γ keptincreasing on the first and the third day after infection. The proportion of L-form group was higher than the wide type group on the third day. The absolute number ofCD4+NK1.1+NKT cells kept rising after infection.6. In the spleen, the proportion of DX5+NKT cells which secret IFN-γ increased on thefirst day and decreased on the third day after the infection of L-forms. The proportion ofL-form group was higher than the wide type group on the first day after infection. Theabsolute number of DX5+NKT cells which secret IFN-γ kept raising in both L-formgroup and wide type group. And the absolute number of L-form group was higher thanthe wide type both on the first and the third day. The proportion of CD4+DX5+NKTcells which secret IFN-γ increased on the first day and decreased on the third day afterthe infection of the wide type bacteia. The proportion of L-form group was higher thanthe wide type group on the third day after infection. The proportion of CD4-DX5+NKTcells which secret IFN-γ decreased on the first day and increased on the third day. Andthe proportion of wide type group was higher than the L-form group on the third dayafter infection. The absolute number of CD4+DX5+NKT cells which secret IFN-γ keptrising after the infection of L-forms. And the absolute number of L-form group washigher than the wide type group on the first and the third day after infection. Theabsolute number of CD4-DX5+NKT cells which secret IFN-γ kept increasing after theinfection of L-forms and wide type. On the first day the absolute number of L-formgroup was higher than the wide type group, however, on the third day the absolutenumber of wide type group was higher than the L-form group.7. In the spleen, NK1.1+NKT cells which secret IFN-γ kept increasing after infection,and the proportion of NK1.1+NKT cells which secret IFN-γ in the L-form group washigher than the wide type group on the first and the third day after infection. Theproportion and absolute number of CD4+NK1.1+NKT cells which secret IFN-γ keptincreasing after the infection of L-forms and wide type. The proportion and the absolutenumber of L-form group was higher than the wide type group on the first and the thirdday. The proportion of CD4-NK1.1+NKT cells which secret IFN-γ increased on thefirst day and decreased on the third day after infection of the wide type bacteria andL-forms. The proportion of wide type group was higher than the L-form group on thethird day after infection. Conclusion:1. The ability of Listeria monocytogenes L-form activated NKT cells is stronger thanthe wide type.2. In the liver, CD4+DX5+NKT cells and CD4-NK1.1+NKT cells play the major roleafter Listeria monocytogenes infection3. In the spleen, CD4-DX5+NKT cells and CD4+NK1.1+NKT cells play the major roleafter Listeria monocytogenes infection...