关键词: 多房棘球蚴, 固有淋巴细胞, 肝纤维化, 免疫调控

Abstract:

Objective To investigate the progression of hepatic fibrosis and the dynamic changes in the proportions of innate lymphoid cells (ILCs) and their subtypes in mice infected with Echinococcus multilocularis at various stages, and to examine the association between E. multilocularis-induced hepatic fibrosis and ILCs, so as to provide the theoretical basis for unraveling the immunoregulatory mechanisms underlying E. multilocularis-induced hepatic fibrosis. Methods BALB/c mice were randomly divided into an infection group and a control group (18 mice in each group). Each mouse in the infection group was injected with 1 500 E. multilocularis protoscoleces, and mice were euthanized 30, 90, and 180 days post-infection. Changes in the structure of liver tissues, inflammation and collagen deposition were observed using hematoxylin and eosin (HE) staining and Masson’s trichrome staining, and the expression of fibrosis-related proteins was quantified using immunofluorescence staining, including α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β1, Collagen Ⅰ, and Collagen Ⅲ. The proportion of ILCs in liver immune cells and the proportions of ILC subtypes (ILC1, ILC2, ILC3) in total ILCs were estimated using flow cytometry, and the relative IL-33 expression was quantified using quantitative Real-time PCR (qPCR) assay. The associations between ILC subtypes and fibrosis-related indicators were examined using Pearson correlation analysis. Results Vesicle-like lesions were seen in mouse livers 30 days post-infection with E. multilocularis, which gradually enlarged over time, and the lesions invaded surrounding liver tissues, leading to inflammation and even fibrotic lesions. HE staining showed aggravation of inflammation over time post-infection, and fiber spacing gradually formed 180 days post-infection. Masson’s trichrome staining showed higher proportions of collagen areas 90 days [(37.26 ± 7.83)% vs. (1.57 ± 1.17)%; t = 7.80, P < 0.01] and 180 days post-infection [(55.36 ± 8.21)% vs. (0.83 ± 0.73)%; t = 11.47, P < 0.01] than in controls. Immunofluorescence staining revealed higher ratios of the fluorescence intensity of α-SMA (1.08% vs. 0.51%; t = 4.62, P < 0.01), TGF-β1 (0.94% vs. 0.12%; t = 4.62, P < 0.01), Collagen Ⅰ (3.33% vs. 0.20%; t = 4.62, P < 0.01), and Collagen Ⅲ to 4', 6-diamidino-2-phenylindole (DAPI) (1.55% vs. 0.62%; t = 5.54, P < 0.01) in the infection group than in the control group, and flow cytometry showed a higher proportion of ILC in mouse liver leukocytes expressing CD45.2⁺ cells in the infection group than in the control group 30 days post-infection [(2.54 ± 0.43)% vs. (2.29 ± 0.31)%; t = 0.91, P > 0.05], a lower proportion of ILCs in the infection group than in the control group 90 days post-infection [(1.16 ± 0.51)% vs. (1.88 ± 0.31)%; t = 2.69, P < 0.05], and a higher proportion of ILCs in the infection group than in the control group 180 days post-infection [(0.56 ± 0.13)% vs. (0.31 ± 0.07)%; t = 3.02, P < 0.05]. The proportions of ILC1 in ILCs were lower in mice in the infection group than in the control group 90 [(61.22 ± 4.38)% vs. (75.96 ± 4.81)%; t = 5.067, P < 0.01] and 180 days post-infection [(11.11 ± 3.61)% vs. (47.50 ± 4.95)%; t = 10.29, P < 0.01], and negatively correlated with the ratio of collagen areas as revealed by Masson’s trichrome staining (r < 0, P < 0.05). The proportion of ILC2 in ILCs were higher in mice in the infection group than in the control group 180 days post-infection [(78.50 ± 4.10)% vs. (33.23 ± 4.57)%; t = 12.78, P < 0.01], and positively correlated with the ratio of collagen areas as revealed by Masson’s trichrome staining (r = 0.72, P < 0.05). There were no significant differences between the infection and control groups in terms of the proportion of ILC3 in ILCs 30 [(5.58 ± 2.37)% vs. (6.08 ± 0.83)%; t = 0.40, P > 0.05], 90 [(2.90 ± 0.84)% vs. (2.66 ± 0.68)%; t = 0.49, P > 0.05] and 180 days post-infection [(4.07 ± 2.94)% vs. (5.24 ± 3.74)%; t = 0.43, P > 0.05], and no significant associations were detected between the proportion of ILC3 in ILCs and fibrosis-related parameters. qPCR assay quantified no significant difference in the IL-33 mRNA expression in mouse liver tissues between the infection (1.37 ± 0.28) and control groups (1.03 ± 0.17) 30 days post-infection (t = 1.06, P > 0.05), higher IL-33 mRNA expression in the infection group (2.88 ± 0.49) than in the control group (0.74 ± 0.14) 90 days post-infection (t = 3.62, P < 0.05), and no significant difference in the IL-33 mRNA expression between the infection (1.55 ± 0.31) and control groups (1.06 ± 0.26) 30 days post-infection (t = 1.10, P > 0.05). Conclusion E. multilocularis infection may induce hepatic fibrosis in mice and alter the proportions of ILCs and their subtypes in hepatic immune cells. Notably, ILC2 expression is significantly upregulated at late stage of E. multilocularis infection, which may contribute to regulating hepatic stellate cell activation and promoting fibrosis.

Key words: Echinococcus multilocularis, Innate lymphoid cell, Hepatic fibrosis, Immune modulation