[Effect of LAG3 deficiency on natural killer cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis].

OBJECTIVE: To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. METHODS: C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. RESULTS: Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = -3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = -0.723, P > 0.05), TNF-α (t = -0.659, P > 0.05), IL-4 (t = -0.263, P > 0.05), IL-10 (t = -0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = -4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = -1.902, P > 0.05), IL-4 (t = -1.333, P > 0.05), IL-10 (t = -1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). CONCLUSIONS: During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.

[Effect of Echinococcus multilocularis infection on Tim3 expression in spleen natural killer cells of mice].

OBJECTIVE: To investigate the effect of Echinococcus multilocularis infection on Tim3 expression and its co-expression with immune checkpoint molecules 2B4 and LAG3 in spleen natural killer (NK) cells of mice. METHODS: C57BL/6 mice, each weighing (20 ± 2) g, were randomly divided into a high-dose infection group (15 mice), a low-dose infection group (13 mice), and a control group (11 mice). Mice in the high- and low-dose infection groups were inoculated with 2 000 and 50 Echinococcus multilocularis protoscolices via the hepatic portal vein, while animals in the control group was injected with an equivalent amount of physiological saline via the hepatic portal vein. Mouse spleen cells were harvested 12 and 24 weeks post-infection, and Tim3 expression and its co-expression with 2B4 and LAG3 in NK cells were detected using flow cytometry. RESULTS: There were significant differences in the proportions of Tim3 expression (F = 13.559, P < 0.001) and Tim3 and 2B4 co-expression (F = 12.465, P < 0.001) in mouse spleen NK cells among groups 12 weeks post-infection with E. multilocularis, and the proportion of Tim3 expression was significantly higher in mouse spleen NK cells in the low-dose infection group [(23.84 ± 2.28)%] than in the high-dose infection group [(15.72 ± 3.67)%] and the control group [(16.14 ± 3.83)%] (both P values < 0.01), while the proportion of Tim3 and 2B4 co-expression was significantly higher in mouse spleen NK cells in the low-dose infection group [(22.20 ± 2.13)%] than in the high-dose infection group [(14.17 ± 3.81)%] and the control group [(15.20 ± 3.77)%] (both P values < 0.01). There were significant differences in the proportions of Tim3 expression (F = 5.243, P < 0.05) and Tim3 and 2B4 co-expression (F = 4.659, P < 0.05) in mouse spleen NK cells among groups 24 weeks post-infection with E. multilocularis infection, and the proportions of Tim3 expression and Tim3 and 2B4 co-expression were significantly lower in mouse spleen NK cells in the high-dose infection group [(20.55 ± 7.04)% and (20.98 ± 7.12)%] than in the control group [(31.38 ± 3.19)% and (31.25 ± 3.06)%] (both P values < 0.05), and there were no significantly difference between the proportions of Tim3 expression and Tim3 and 2B4 co-expression in splenic NK cells in the low-dose infection group [(26.80 ± 6.47)% and (26.48 ± 6.48)%] and the control group (both P > 0.05). There were no significant differences in the proportions of Tim3 and LAG3 co-expression in mouse spleen NK cells among groups 12 (F = 2.283, P > 0.05) and 24 weeks post-infection (F = 0.375, P > 0.05). In the low-dose infection group, there were no significant differences in the proportions of Tim3 expression or Tim3 and 2B4 co-expression in mouse spleen NK cells 12 (t = -1.137, P > 0.05) or 24 weeks post-infection (t = -1.658, P > 0.05), and the proportion of Tim3 and LAG3 co-expression increased in mouse spleen NK cells 24 weeks post-infection relative to 12 weeks post-infection (t = -5.261, P < 0.01). In the highdose infection group, there was no significant difference in the proportion of Tim3 expression in mouse spleen NK cells 12 and 24 weeks post-infection (t = -1.546, P > 0.05); however, the proportions of Tim3 co-expression with 2B4 and LAG3 increased in mouse splenic NK cells 24 weeks post-infection relative to 12 weeks post-infection (t = -2.425 and -4.745, both P values < 0.05). CONCLUSIONS: The Tim3 expression and Tim3 co-expression with LAG3 and 2B4 on spleen NK cells is affected by doses of E. multilocularis infection and disease stages, and present different phenotypes during the course of alveolar echinococcosis. NK cells tend to form an immunosuppressive phenotype with the progression of E. multilocularis infection, which facilitates immune escape and chronic parasitism of E. multilocularis.