Alteration Of T Cell Subtypes In Spleen And Antibodies Of Serum In Mice Infected By Angiostrongylus Cantontensis

Angiostrongylus cantontensis (A. cantontensis) is the pathogen ofangiostrongyliasis (a new infection disease), which mainly invades the central nervoussystem and results in eosinophilia encephalitis or meningitis (EosinophilicMeningoencephalitis, EM). A. cantontensis is mainly prevalence in some islandcountries of the Pacific Ocean, India Ocean, Southeast Asia, China, Japan and so on.So far, more than3000cases have already been reported all over the world.Understanding the immune responses of the host after A. cantontensis infection isimportant for us to mater the host’s resistance to the parasite, pathogenic mechanismsand immunological diagnosis. However, the basic immunological research about A.cantontensis is rare, so that our knowledges on the mechanisms and characteristics ofimmune response in host are not enough. And it has also limited to activities ofprevention and control of A. cantontensis. Spleen is the largest peripheral immuneorgan in mammalian and the site of dealing with the antigens from blood. It’s also aplace settled by mature lymphocytes and other immunocytes. The changes of spleniclymphocytes after the infection may reveal the characteristics of the immune responseto parasite. So the BALB/c mice as animal models, and the alteration of T cellsubtypes in spleen and antibodies of serum in mice infected by A. cantontensis areobserved.Objective: To observe the alteration of T cell subtypes and the cytokines in spleen, and thedynamic changes of the specific IgG and IgE antibodies of serum in mice infected byA. cantontensis.Methods:1. Animal model and infection: Thirty of BALB/c mice were randomly dividedinto normal control group, experimental group1and experimental group2, eachgroup have ten mice. They were infected with the third stage larva of A. cantontensisfrom snail Achatina fulica. The experimental group1was infected20larvae permouse, and experimental group2infected40larvae.2. Sampling: The serums were collected from the caudal vein or eyeball atpre-infection and different time of post-infection. The spleens were harvested on the19th and25th day post-infection. Splenocytes were separated and counted, and thenumber of splenocytes was adjusted to2×106/ml per sample.3. Flow Cytometry: The splenocytes were incubated with PMA and Ionomycinin37℃,5%CO2incubator for1hour, and then incubation with BFA for4hours.The cultured cells were fixed by4%formaldehyde, and added buffer (includingSaponin) at4℃overnight. The cells were added into the tube, and then bound withmAbs labeled fluorescein. The subtypes of T cells in splenocytes were detected byFlow Cytometry.4. Detcetion of cytokines in cultured supernatants of splenocytes: The splenocytswere incubated with PMA and Ionomycin in the cell culture plate for72hours in37℃,5%CO2incubator. The concentrations of IL-4and IL-17in cultured supernatants ofsplenocytes were assessed by ELISA.5. Detection of specific antibodies in serums: ELISA plates were coated by theantigens of adult A. cantontensis. The specific IgG and IgE in serum of mice weredetected by ELISA.Results:1. In comparison with normal control group, the percentage of CD4~+T lymphocyte in splenocytes of the infected group was increased significantly. Thepercentage of CD8~+T cells was increased too. But there were not significantdifference between the infected group and the control group.2. The percentage of CD4~+IL-4~+T cells in splenocytes of the infected group wasmuch higher than that of the control group, however the percentages of CD4~+IL-17~+and CD4~+IFN-γ~+T cell were much lower.3. In comparison with control group, the concentration of IL-4in the culturedsupernatants of splenocytes in the infected group increased significantly, while theconcentration of IL-17decreased significantly.4. The levels of some immune factors includding CD4~+, CD8~+, CD4~+IL-4~+Tcells and IL-4in experimental group2were higher than experimental group1, whilethe levels of CD4~+IFN-γ~+T cells and IL-17in experimental group2were lower thanexperimental group1, but there was not significant difference between twoexperimental groups.5. The levels of specific IgG antibodies in serum rose a bit at the5th daypost-infection. And as the time gone, in the fluctuation, the levels of the antibodiesgradually reach a peak at the20th day post-infection.6. The levels of specific IgE antibodies in serums post-infection were higherthan pre-infection, and the IgE antibodies reached a higher level during the10th to15th days post-infection.Conclusion:1. The T lymphocyte in spleen of mice infected by A. cantontensis appearsproliferation phenomenon, which CD4~+T lymphocyte proliferation were moresignificant than CD8~+T cells.2. After A. cantontensis infection, the percentage of CD4~+IL-4~+T lymphocyte insplenocytes of the infected mice were increased significantly, while the percentagesof CD4~+IL-17~+and CD4~+IFN-γ~+T lymphocyte were decreased significantly.3. Under effect of immunological irritants, the levels of IL-4in the culturedsupernatants of splenocytes were increased significantly in the infected mice, while that of IL-17were decreased obviously.4. The factors including the number of larva infected and days after infectioncould influence the proportions of T cell subtypes and the levels of cytokine inspleen.5. It is evident that the CD4~+T lymphocytes of spleen in BALB/c mice infectedwith A. cantontensis proliferate markedly and polarize to Th2phenotypes.6. In the humoral immunity of the infected mice, the levels of specified IgG andIgE antibodies in serums were all increased, but the change of IgG antibodies wasmore significant than that of IgE antibodies.