Abstract:

Objective To investigate the effects of Echinococcus multilocularis infection on the expression of CD47 and signal regulatory protein α (SIRPα) in macrophages and the function of macrophages, and to provide evidence for exploring the pathogenesis of alveolar echinococcosis (AE). Methods Liver tissue samples were collected from 30 AE patients. Tissues were categorized into close liver tissue (CLT) located 0.5 cm from the lesion and distal liver tissue (DLT) located more than 2 cm from the lesion. Paraffin sections were used for immunohistochemistry and frozen sections were used for immunofluorescence to analyze the expression levels of CD47 and SIRPα in CLT and DLT. Mouse monocyte-macrophage leukemia cells (RAW264.7) were co-cultured with E. multilocularis protoscoleces at a ratio of 500 ∶ 1. Cells were collected before the addition of protoscoleces and after co-culture for 24 and 48 hours, respectively. The expression changes of CD47 and SIRPα in macrophages were detected by flow cytometry, cellular immunofluorescence and quantitative real-time PCR (qPCR). RAW264.7 cells were divided into control group, infection group, inhibitor group and inhibitor-infection group (1 × 10⁵ cells per group). The infection group and inhibitor-infection group were supplemented with protoscoleces (200 per group), while the inhibitor group and inhibitor-infection group were treated with a CD47/SIRPα binding inhibitor (NCGC00138783TFA, 10 µmol/L). Enhanced green fluorescent protein (EGFP)-labeled Escherichia coli were added to each group (1 × 10⁶ per group) after co-culture for 48 hourse. Following fluorescence observation, the phagocytic capacity of macrophages was assessed by flow cytometry. Additionally, the mRNA relative transcription levels of macrophage polarization markers and cytokines were detected by qPCR. Independent samples t-test or paired t-test was used for comparisons between two groups, one-way ANOVA was used for multiple groups, and Tukey’s HSD method was used for multiple comparisons. Results Immunohistochemical analysis revealed that in AE patients, the proportions of CD47 and SIRPα positive cells in CLT were (61.99 ± 3.61)% and (54.06 ± 1.85)%, respectively, which were higher than (57.08 ± 3.38)% and (40.77 ± 1.49)% in DLT (t = 9.434, 58.840; both P < 0.01). Immunofluorescence analysis revealed that the Pearson correlation coefficient between CD47 and SIRPα in CLT was 0.66 ± 0.02, which was higher than that in DLT (0.45 ± 0.01) (t = 7.624, P < 0.01). The results of flow cytometry showed that the median fluorescence intensity of CD47 in macrophages after co-culture for 24 and 48 hours were 8 259.00 ± 66.01 and 9 445.00 ± 41.58, respectively, which were higher than the pre-co-culture value of 5 603.00 ± 193.40 (HSD = 0.691, 0.735; both P < 0.01). The median fluorescence intensity of SIRPα in macrophages after co-culture for 24 and 48 hours were 3 123.00 ± 184.60 and 2 931.00 ± 54.08, respectively, which were higher than the pre-co-culture value of 2 508.00 ± 43.15 (HSD = 0.491, 0.235; both P < 0.01). qPCR analysis revealed that the relative transcription levels of CD47 in macrophages after co-culture for 24 and 48 hours were 1.80 ± 0.02 and 1.64 ± 0.01, respectively, which were higher than the pre-co-culture level of 0.99 ± 0.01 (HSD = 0.098 and 0.125; both P < 0.01). The relative transcription levels of SIRPα in macrophages were 1.00 ± 0.02 before the addition of protoscoleces, 0.52 ± 0.05 and 1.27 ± 0.03 after co-culture for 24 and 48 hours, respectively. The level after 24 hours was lower than that before the addition of protoscoleces (HSD = 0.015, P < 0.01), while the level after 48 hours was higher than that before the addition of protoscoleces (HSD = 0.105, P < 0.01). Immunofluorescence analysis revealed that the Pearson correlation coefficients between CD47 and SIRPα after co-culture for 24 and 48 hours were 0.920 ± 0.001 and 0.990 ± 0.001, respectively, which were higher than the pre-co-culture value of 0.770 ± 0.001 (HSD = 0.091, 0.135; both P < 0.01). Fluorescence analysis revealed that the mean fluorescence intensity of EGFP in the inhibitor-infection group was 8 923.0 ± 49.3, which was higher than that in the infection group (7 537.0 ± 29.3) (HSD = 0.205, P < 0.01). Flow cytometry analysis revealed that the mean fluorescence intensity of EGFP in the inhibitor-infection group was 21.54 ± 0.03, which was higher than that in the infection group (18.55 ± 0.51) (HSD = 0.327, P < 0.01). qPCR analysis revealed that the mRNA relative transcription levels of inducible nitric oxide synthase (iNOS) and CD86, the markers of M1 macrophage, were 20.87 ± 0.40 and 40.64 ± 0.75 in the inhibitor-infection group, respectively, which were higher than 5.38 ± 0.11 and 3.79 ± 0.05 in the infection group (HSD = 0.194, 0.261; both P < 0.01). The mRNA relative transcription levels of arginase 1 (Arg1) and CD206, the markers of M2 macrophage, were 48.76 ± 2.22 and 6.33 ± 0.06 in the inhibitor-infection group, respectively, which were lower than 83.28 ± 0.58 and 12.33 ± 0.12 in the infection group (HSD = 0.283, 0.164; both P < 0.01). In the inhibitor-infection group, the mRNA relative transcription levels of interleukin 6 (IL-6), IL-1β and tumor necrosis factor α (TNF-α), the M1-type cytokines, were 3 896.00 ± 176.70, 271.30 ± 8.39 and 4.90 ± 0.10, respectively, which were higher than 2 869.00 ± 89.55, 154.90 ± 2.61 and 3.04 ± 0.03 in the infection group (HSD = 0.712, 0.625, 0.693; P < 0.05, 0.01, 0.01). The mRNA relative transcription levels of IL-10 and transforming growth factor β (TGF-β), the M2-type cytokines, in the inhibitor-infection group were 127.40 ± 4.92 and 1.34 ± 0.03, respectively, which were lower than 380.30 ± 8.55 and 1.61 ± 0.02 in the infection group (HSD = 0.324, 0.163; both P < 0.01). Conclusion The expression of CD47 and SIRPα in macrophages were increased after E. multilocularis infection, while the phagocytic function of macrophages was reduced. Specific inhibition of the CD47/SIRPα interaction could improve the phagocytic function and promote macrophage polarization towards the M1 phenotype.

Key words: Echinococcus multilocularis, Immune checkpoint, Phagocytosis, Macrophage polarization