关键词: 多房棘球蚴病, NK1.1, CD4⁺ T细胞, CD8⁺ T细胞

Abstract:

Objective To observe the changes in NK1.1 expression of liver T cells in the advanced stage of mice infected with Echinococcus multilocularis, and analyze the effect of NK1.1 expression on liver T cells’ function. Methods C57BL/6N mice were assigned into the infection group and the control group randomly. Mice in the infection group were injected with 2 000 E. multilocularis protoscoleces through hepatic portal vein, while mice in the control group injected with the same volume of normal saline. The livers were collected at the advanced stage of infection (36 weeks post-infection). The pathological changes of the liver tissues were observed by hematoxylin-eosin (HE) staining, and the degree of live fibrosis was observed by Masson staining and Sirius red staining. The lymphocytes were collected and the flow cytometry was performed to detect the proportions of CD4⁺ T and CD8⁺ T cells. The expression changes of NK1.1, granzyme B (GrB), perforin, interferon-γ (IFN-γ) and interleukin-17 (IL-17) in different T cells were also detected. SPSS 25.0 software was used for statistical analysis, and an independent sample t-test was used to compare the mean between the two groups. Results HE staining showed that lesions with germinal layer structure were formed in the livers of mice in the infection group 36 weeks post-infection, with infiltration of inflammatory cells in surrounding tissues, and results of Masson staining and Sirius red staining showed the significant hepatic fibrosis. Flow cytometry results showed the proportions of CD4⁺ T and CD8⁺ T cells in the liver of mice in the infection group 36 weeks post-infection were (32.0 ± 6.0)% and (42.0 ± 13.9)%, respectively, which were (36.6 ± 9.0)% and (50.2 ± 5.2)% in the control group, with no significant differences (t = 1.068, 1.446, both P > 0.05). The proportion of NK1.1⁺CD4⁺ T cells in the infection group was (30.0 ± 14.5)%, which was lower than NK1.1⁺CD4⁺ T cells in the control group [(69.0 ± 10.7)%] (t = 5.573, P < 0.01) and NK1.1^-CD4⁺ T cells in the infection group [(51.6 ± 10.8)%] (t = 2.920, P < 0.05). The proportion of NK1.1⁺CD8⁺ T cells in the infection group was (22.0 ± 12.9)%, which was lower than NK1.1⁺CD8⁺ T cells in the control group [(43.6 ± 5.9)%] (t = 3.999, P < 0.01) and NK1.1^-CD8⁺ T cells in the infection group [(64.0 ± 13.6)%] (t = 5.496，P < 0.01). The proportions of NK1.1⁺CD4⁺ T cells that secreted GrB, IFN-γ and IL-17 in the infection group were (4.8 ± 2.8)%, (6.8 ± 6.0)% and (6.9 ± 5.2)%, respectively, which were lower than those in the control group [(12.6 ± 4.8)%, (23.6 ± 7.3)% and (22.1 ± 7.1)%] (t = 3.462, 4.171, 4.124, all P < 0.01). The proportions of NK1.1⁺CD8⁺ T cells that secreted GrB, IFN-γ and IL-17 in the infection group were (1.5 ± 0.9)%, (5.3 ± 2.7)% and (3.7 ± 2.3)%, respectively, which were lower than those in the control group [(6.0 ± 2.7)%, (18.1 ± 9.5)% and (16.9 ± 9.3)%] (t = 3.861, 3.196, 3.361, P < 0.01, 0.05, 0.01). The proportions of NK1.1⁺CD4⁺ T and NK1.1⁺CD8⁺ T cells that secreted perforin in the infection group were (3.0 ± 1.3)% and (2.7 ± 1.2)%, respectively, which were (2.3 ± 1.2)% and (2.1 ± 1.6)% in the control group, with no significant differences (t = 1.027, 0.717, both P > 0.05). In the infection group, the proportion of NK1.1^-CD4⁺ T cells secreted GrB was (6.7 ± 2.1)%, which was higher than NK1.1⁺CD4⁺ T cells (t = 2.629, P < 0.05); the proportion of NK1.1^-CD8⁺ T cells secreted IFN-γ was (13.7 ± 9.2)%, which was higher than NK1.1⁺CD8⁺ T cells (t = 2.609, P < 0.05); the proportion of NK1.1^-CD8⁺ T cells secreted IL-17 was (1.0 ± 0.4)%, which was lower than NK1.1⁺CD8⁺ T cells (t = 2.740, P < 0.05). Conclusion The number of NK1.1⁺CD4⁺ T and NK1.1⁺CD8⁺ T cells decreased in the liver of mice infected with E. multilocularis in the advanced stage, and the secretion of GrB, IFN-γ and IL-17 were weakened, which indicated the existence of immune depletion of NK1.1⁺ T cells.

Key words: Alveolar echinococcoisis, NK1.1, CD4⁺ T cell, CD8⁺ T cell