Abstract: 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 hepatic NK 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.

Key words: Echinococcus multilocularis, Lymphocyte activation gene 3, Natural killer cell, Hepatic fibrosis, Mouse

摘要： 目的　探讨淋巴细胞活化基因3（lymphocyte activation gene 3，LAG3）缺陷（LAG3⁃/⁃）对多房棘球蚴感染小鼠自然杀伤（natural killer，NK）细胞功能及肝纤维化的影响。方法　取体质量为（20 ± 2） g的C57BL/6小鼠，分为LAG3⁃/⁃组和野生型（wild type，WT）组，分别经肝门静脉接种3 000个多房棘球蚴原头节。感染后12周，取小鼠肝脏制备肝脏组织切片，天狼星红染色观察肝脏病灶和纤维化程度。分离小鼠肝脏和脾脏淋巴细胞，流式细胞术检测小鼠肝脏和脾脏NK细胞比例，NK细胞表面CD25、CD44和CD69分子表达水平，以及γ干扰素（interferon γ，IFN⁃γ）、肿瘤坏死因子⁃α（tumor necrosis factor α，TNF⁃α）、白细胞介素（interleukin，IL）⁃4、IL⁃10、IL⁃17A 等相关细胞因子分泌水平。结果　天狼星红染色结果显示，与WT组相比，LAG3⁃/⁃组小鼠肝脏病灶周围炎性细胞带增宽、纤维化结缔组织增生，且呈现较多胶原纤维沉积。流式细胞术检测结果显示，LAG3⁃/⁃组小鼠肝脏（6.29% ± 1.06% vs. 11.91% ± 1.85%，P < 0.000 1）和脾脏NK细胞比例（4.44% ± 1.22% vs. 5.85% ± 1.10%，P > 0.05）均低于WT组；LAG3⁃/⁃组小鼠肝脏NK细胞表面CD44分子平均荧光强度显著高于WT组（t = −3.234，P < 0.01），而LAG3⁃/⁃组和WT组小鼠肝脏和脾脏NK细胞表面CD25和CD69分子平均荧光强度差异均无统计学意义（P均 > 0.05）。LAG3⁃/⁃组和WT组小鼠肝脏NK细胞分泌IFN⁃γ、TNF⁃α、IL⁃4、IL⁃10、IL⁃17A的比例差异均无统计学意义（t = −0.723、−0.659、−0.263、−0.455、0.091，P均 > 0.05）；LAG3⁃/⁃组小鼠脾脏NK细胞分泌IFN⁃γ的比例显著高于WT组（58.40% ± 1.64% vs. 50.40% ± 4.13%；t = −4.042，P < 0.01），但分泌TNF⁃α、IL⁃4、IL⁃10、IL⁃17A的比例差异均无统计学意义（t = −1.902、−1.333、−1.356、0.529，P均 > 0.05）。结论　在多房棘球蚴感染小鼠过程中，LAG3⁃/⁃促进脾脏NK细胞高分泌IFN⁃γ，可能参与逆转NK细胞免疫功能，从而导致肝脏纤维化加重。 　

关键词: 多房棘球蚴, 淋巴细胞活化基因3, 自然杀伤细胞, 肝纤维化, 小鼠