关键词: 多房棘球蚴病, 多房棘球蚴, 脾脏, 巨噬细胞, 极化

Abstract:

Objective To investigate the changes of splenic macrophages subsets and their polarization phenotype in mice infected with different numbers of Echinococcus multilocularis protoscoleces. Methods Sixty female C57BL/6 mice were randomly assigned to mild, moderate, and heavy infection groups and control group with 15 mice in each group. The mice in mild, moderate and heavy infection groups were inoculated with 50, 500, and 2 000 protoscoleces via hepatic portal vein puncture, respectively, while the control group was injected with the same volume of saline. At 2, 12, and 24 weeks after infection, the spleens from five mice randomly selected from each group were collected, followed by calculating the spleen coefficients after weighted, and isolating splenic lymphocytes. The proportions and numbers of different macrophages subsets were determined by flow cytometry. The total RNA of splenic lymphocytes was extracted, and the relative level of mRNA transcription of M1 and M2 polarization related genes in splenic macrophages were examined with real-time quantitative PCR. The M1 type polarization related genes were those involving interleukin-1β (IL-1β), interferon-γ (IFN-γ), chemokine (C-X-C motif) ligand 11 (CXCL11), chemokine (C-C motif) receptor 7 (CCR7) and CD86, and the M2 type polarization related genes were involving mannose receptor C-type 1 (MRC1), resistin like alpha (Retnla) and arginase 1 (ARG1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference. SPSS 26.0 software was used for statistical analysis. The comparison between multiple groups at different time points was analyzed by one-way ANOVA, and the pairwise comparative analysis at the same time point was tested by LSD method. Results Two weeks after infection, the spleen weight, spleen coefficient, the number of splenic lymphocytes, the proportion of splenic macrophages, the number of splenic macrophages and Ly-6C^high splenic macrophage subset proportion in heavy infection group were (157.2 ± 22.8) mg,(0.8 ± 0.1)%,(10.3 ± 2.9) × 10⁷, (9.2 ± 6.4)%,(48.9 ± 32.7) × 10⁵ and (75.8 ± 4.6)%, respectively, which were all significantly higher than that in the control group [(75.0 ± 18.3) mg, (0.4 ± 0.1)%, (3.1 ± 1.3) × 10⁷, (4.3 ± 0.7)%, (7.7 ± 4.1) × 10⁵, (52.1 ± 8.4)%], mild infection group [(89.2 ± 7.4) mg, (0.4 ± 0.0)%, (5.4 ± 2.3) × 10⁷, (3.0 ± 0.9)%, (9.3 ± 6.9) × 10⁵, (50.1 ± 8.8)%], and in moderate infection group [(102.6 ± 15.2) mg, (0.5 ± 0.1)%, (7.0 ± 2.1) × 10⁷, (3.5 ± 0.3)%, (13.7 ± 3.9) × 10⁵, (60.3 ± 8.7)%] (F = 22.744, 23.542, 9.318, 3.935, 6.617, 11.197, P < 0.05 or P < 0.01). Ly-6C^low macrophage subset proportion (20.3 ± 4.2)% was significantly milder than that in the control group (39.0 ± 7.1)%, mild infection group (41.2 ± 8.6)% and in moderate infection group (34.2 ± 7.1)%(F = 9.157, P < 0.01). At 12 weeks after infection, the spleen weight, spleen coefficient, the proportion and number of macrophages and the proportion of the Ly-6C^high macrophage subset in the spleen were significantly higher in heavy infection group than that in the control group, mild and moderate infection groups (F = 12.730, 14.173, 20.380, 7.943, 25.838, P < 0.01), the proportion of Ly-6C^low macrophages was significantly lower than that in the control group, mild and moderate infection groups (F = 27.668, P < 0.01). At 24 weeks after infection, the spleen weight, spleen coefficient, the number of lymphocytes, the proportion and number of macrophages in the spleen were also significantly higher in heavy infection group (F = 8.664, 7.318, 13.047, 3.315, 6.007, P < 0.05 or P < 0.01). The proportions of Ly-6C^high and Ly-6C^low macrophages were not significantly different from those of the control group, mild and moderate infection groups (F = 3.177 and 2.709, P > 0.05). With longer time of infection, the proportion of Ly-6C^high macrophages was gradually decreased (F = 30.649, P < 0.01), and the proportion of Ly-6C^low macrophages was gradually increased (F = 32.407, P < 0.01). Real-time quantitative PCR showed that at 24 weeks after infection, the relative level of mRNA transcription of CXCL11 in heavy infection group (28.2 ± 36.3) was higher than that in control group (1.9 ± 2.7) (t = 2.243, P < 0.05), and the relative level of mRNA transcription of CD86 (0.2 ± 0.1) was significantly lower than that in the control group (0.5 ± 0.3) (t = -2.255, P < 0.05). The relative transcription level of mRNA of MRC1, Retnla, and ARG1 (5.2 ± 2.9, 201.8 ± 176.4, 51.2 ± 69.6) were significantly higher than those in control group (1.8 ± 1.5, 0.8 ± 0.8, 1.2 ± 0.8, respectively) (t = 2.313, 3.470 and 2.185, P < 0.05) Conclusion Two weeks after infection, large numbers of Ly-6C positive monocyte derived macrophages were recruited into spleen in the heavy infection group and the Ly-6C^high macrophage subset gradually transformed shifted to the Ly-6C^low subset during infection. At 12 weeks and 24 weeks after infection, the macrophages in the spleen were dominated by the M2 type, inducing immune tolerance, favorable to chronic infection of Echinococcus multilocularis metacestode.

Key words: Alveolar echinococcoisis, Echinococcus multilocularis, Spleen, Macrophage, Polarization