关键词: 多房棘球蚴, 自然杀伤T细胞, 肝脏

Abstract:

Objective To examine the effects of early Echinococcus multilocularis infection on the quantity, subsets, and functions of natural killer T (NKT) cells in mouse hepatic tissues. Methdos C57BL/6N mice were randomly divided into a control group and an infection group. Mice in the infection group were inoculated with 2 000 E. multilocularis protoscoleces via the hepatic portal vein, while animals in the control group received an equal volume of physiological saline. Mouse liver tissues were collected 4 weeks post-infection. The pathological changes of liver tissues were observed by hematoxylin-eosin (HE) staining, and alterations in hepatic NKT cells were examined using immunofluorescence staining. Lymphocyte suspensions were prepared from mouse liver tissues. The percentages of NKT cells and their different subsets were quantified in mouse liver tissues in both control and infection groups using flow cytometry, and the expression of inhibitory receptors [natural killer group 2 member A （NKG2A） and programmed cell death protein 1 (PD-1)], an activating receptor NKG2D, chemokine receptors [C-X-C motif chemokine receptor 3 (CXCR3) and CXCR6], interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and transforming growth factor-β1 (TGF-β1) was detected in NKT cells and their respective subsets. Results HE staining revealed lesions exhibiting a germinal layer structure in mouse liver tissues in the infection group 4 weeks post-infection with E. multilocularis, surrounded by large number of inflammatory cells. Immunofluorescence staining showed that the average count of NKT cells was (1.20 ± 0.53) cells per field in the liver tissues of the infected group, which was significantly lower than that in the control group (3.73 ± 0.50) cells (t = 6.01, P < 0.01). Flow cytometry detected that the proportions of NKT cells and CD69⁺NKT cells were (11.83 ± 1.19)% and (76.93 ± 3.06)% in mouse liver tissues in the infection group, which were lower than those [(23.20 ± 0.20)% and (88.47 ± 1.53)%, respectively] in the control group (t = 16.28 and 5.84; both P < 0.01). The proportions of hepatic NKT cells secreting IFN-γ and IL-10 were (17.50 ± 5.04)% and (4.54 ± 0.20)% in the infection group, which were lower than those [(61.90 ± 2.25)% and (10.24 ± 1.37)%, respectively] in the control group (t = 13.93, 7.15, P < 0.01, 0.05). The proportion of hepatic NKT cells secreting TGF-β1 was higher in the infected group [9.04% (8.01%, 9.62%)] than in the control group [3.68% (3.48%, 3.68%)] (t = 8.60, P < 0.05), and there was no significant difference in the proportion of hepatic NKT cells producing TNF-α between the infection group [(20.83 ± 5.75)%] and the control group [(19.20 ± 5.91)%] (t = 0.34, P > 0.05). The proportions of NKG2A⁺NKT [(13.90 ± 3.18)% vs. (22.37 ± 2.61)%; t = 3.57, P < 0.05], CXCR3⁺NKT [(26.77 ± 6.00)% vs. (55.40 ± 13.64)%; t = 3.33, P < 0.05] and CXCR6⁺NKT cells [(57.20 ± 7.63)% vs. (86.73 ± 2.72)%; t = 6.31, P < 0.01] were significantly lower in mouse liver tissues in the infection group than in the control group. The percentage of PD-1⁺NKT cells [(10.04 ± 1.01)% vs. (6.56 ± 0.10)%; t = 5.92, P < 0.05] was significantly higher in mouse liver tissues in the infection group than in the control group, and there was no significant difference in the proportion of NKG2D⁺NKT cells in mouse liver tissues between the infection group and the control group [1.58% (1.56%, 2.14%) vs. 1.25% (1.15%, 1.31%); Z = ‒1.96, P > 0.05]. The proportions of CD4⁺NKT cells [(42.67 ± 4.15)% vs. (60.97 ± 1.80)%; t = 7.01, P < 0.01] and CD69⁺CD4⁺NKT cells [(44.80 ± 2.27)% vs. (62.30 ± 3.16)%; t = 7.79, PP < 0.01] were significantly lower in mouse liver tissues in the infection group than in the control group, and the proportions of CD4⁻CD8⁻ (double-negative, DN) NKT cells [(45.63 ± 3.61)% vs. (32.57 ± 2.06)%; t = 5.44, P < 0.01] and CD69⁺DN NKT cells [(44.53 ± 2.23)% vs. (32.17 ± 1.34)%; t = 8.23, P < 0.01] were higher in mouse liver tissues in the infection group than in the control group. The proportions of CD4⁺NKT cells secreting IL-10 [(9.97 ± 4.38)% vs. (52.00 ± 4.20)%; t = 11.99, P < 0.01], and DN NKT cells producing IFN-γ [(15.87 ± 2.57)% vs. (59.80 ± 4.70)%; t = 14.20, P < 0.01] and TNF-α [(6.61 ± 2.69)% vs. (30.57 ± 2.02)%; t = 12.33, P < 0.01] were lower in mouse hepatic tissues in the infection group than in the control group, and the proportions of CD8⁺NKT cells secreting IL-10 [(55.90 ± 13.68)% vs. (10.10 ± 3.24)%; t = 5.64, P < 0.01] and TGF-β1 [(45.87 ± 5.95)% vs. (19.69 ± 10.25)%; t = 3.83, P < 0.05] were significantly higher in mouse liver tissues in the infection group than in the control group. The proportions of NKG2A⁺CD4⁺NKT cells [(37.87 ± 4.80)% vs. (52.80 ± 5.17)%; t = 3.66, P < 0.05] and CXCR3⁺CD4⁺NKT cells [(40.40 ± 9.48)% vs. (57.00 ± 3.90)%; t = 2.81, P < 0.05] were significantly lower in the infection group than in the control group, and the proportion of NKG2A⁺DN NKT cells was higher in the infection group [(55.63 ± 3.46)%] than in the control group [(41.43 ± 2.31)%] (t = 5.91, P < 0.01). Conclusion During the early stage of E. multilocularis infection, NKT cells exhibite an overall state of functional suppression or exhaustion in mouse liver tissues. The NKT cell subsets demonstrate distinct expression characteristics: CD4⁺NKT cells show low expression, and CD8⁺NKT cells function by secreting IL-10 and TGF-β1, while the highly activated DN NKT cells have an impaired cytokine-secreting capacity due to high expression of NKG2A.

Key words: Echinococcus multilocularis, Natural killer T cell, Liver