| Objectives: Toxoplasma gondii is an important opportunistic protozoa,which cause a cosmopolitan zoonotic toxoplasmosis in both human and mammals.The infection is usually asymptomatic in immunocompetent individuals,but when an infected person becomes immunosuppressed,the organism will disseminate rapidly,and then lead to fatal disorders.NKG2 D is an activating receptor expressed by NK cells and subsets of T cells.Once recognising and binding various ligands expressed on the cell as the result of stress or tissue injury,the activation will participate in the genesis of several inflammatory diseases.Soluble MULT1(sMULT1)of mice can promote the expression of NKG2 D on the surface of all NK cells and enhance the function of NK cells.However,the role of sMULT1 in the immune regulation of T.gondii infection has remained poor understanding.This study aimed to investigate the effect of NKG2 D activation on anti-T.gondii immunity by overexpression sMULT1 in mice.Methods: Eight-week-old female BALB/c mice were infected intraperitoneally with fifty tachyzoites of T.gondii RH strains for one week,and the nature of NK cells and lymphocytes were analyzed by flow cytometry.s MULT-expressing plasmid p EGFP-N1-sMULT1(1.5 ml,40 μg/mouse)was injected by tail vein for three times with an interval of two days starting at one day before infection of T.gondii.The survival time of infected mice,protozoa burden and liver pathology were investigated.In addition,the mice were sacrificed one week after infection and spleen mononuclear cells were isolated.The percentage of CD4+T cells,CD8+T cells,NK cells and NKT cells in spleen cells was analyzed by flow cytometry,the expression of CX5,CD69,KLRG1,CD27 and the expression of intracellular IL-4,IL-10,IFN-γ or the transcription factors Foxp3 and Ki67 were also detected.Results: The proportion of CD8+T cells in the spleen of T.gondii infection was significantly increased after one week of infection,and the expression of NKG2 D,CD69,and CD27 on CD8+T cells was up-regulated.However,the proportion of CD4+T cells was not significantly changed,but the expression of CD69 was increased.Although the proportion of NK and NKT cells in the spleen of mice decreased after infection for one week,flow cytometry analysis showed that the NKG2 D,CD69,and CD27 expression of NK cells were significantly increased.In addition,the expression of Foxp3 in CD4+T and CD8+T cells in the spleen after infection was not significantly changed,although the proportion of Ki67 in the CD4+Foxp3+ and CD8+Foxp3+ cell populations was found to be increased by flow cytometry.We employed the plasmid DNA expressing sMULT1 and found that overexpression of sMULT1 increased the proportion of CD8+T and CD4+T cells in the spleen of mice infected with T.gondii and did not affect the ratio of splenic NK and NKT cells,and the expression of NKG2 D in CD8+T cells,CD4+T cells and NKT cells,but it promoted the expression of NKG2 D on the surface of NK cells.In addition,we observed the effect of overexpression of sMULT1 on the activation and proliferation of major immune cell subsets in mice infected with T.gondii.We found that sMULT1 overexpression did not affect the expression of CD69,CD27,and the inhibitory receptor KLRG1 on NK and NKT cells.We also found that IFN-γ+NK cells was increased in spleen mice infected with T.gondii after s MULT injection,and the expression of IL-4 was also increased,but there was no significant effect on IL-10 secretion in all cell subsets.Overexpression of sMULT1 improved the survival of infected mice to some extent.We detected the load of 529 bp gene in the liver and peripheral blood by real time-PCR and the results showed that overexpression of sMULT1 significantly reduced the T.gondii burden in the peripheral blood and the liver of mice.Consistent with the results of the Real time-PCR,the size of the liver was reduced and HE staining revealed that the infiltration of inflammatory cells in the liver tissue was also significantly less,and the degree of degeneration and necrosis was significantly reduced.Conclusion: sMULT1 recombinant plasmid employment can activate the receptor NKG2 D,promote the activation and proliferative potential of NK and CD8+T cells,and promote the expression of IFN-γ,this enhanced Type I immunity accompanied with supressed Foxp3+T cells.Reduced protozoa burden in the liver and peripheral blood of the mice are consequently resulted from the overexpression of sMULT1,and the liver pathology are moderately improved.However,the increased secretion of IL-4 by NK cells demonstrates that anti-inflammatory Typt Ⅱ immunity may also induced at the early stage of infection,the down-regulation is occurred to counrterweight the excessive inflammation leading to a fatal pathogenicity. |
