Abstract:

Objective To investigate the dynamic changes of the polarization of myeloid-derived suppressor cells (MDSC) to M2 macrophages stimulated by Echinococcus granulosus protoscolex-derived exosomes, and to explore the biological significance of their synergistic immunosuppressive effect. Methods The exosomes were collected by ultracentrifugation of supernatant of E. granulosus protoscoleces in vitro culture. The morphology of exosomes was observed by transmission electron microscope, and the expression of exosomal protein CD9 and enolase were detected by Western blotting. Subsequently, MDSC stimulated from myeloid cells of 3 healthy BALB/c mice femoral bone marrow were added at 1 × 10⁶ MDSC per well of a 6-well plate, and divided into experimental group, positive control group and negative control group. 20 μl exosomes (5 μg), 20 μl IL-4 (40 ng) and 20 μl RPMI 1640 medium were added respectively for co-culture. The proportions of monocytic DMSC (M-MDSC) and the expression of M2 macrophages molecular markers F480 and CD206 in co-cultured cells at 24 h, 48 h, and 72 h, were analyzed by Flow cytometry, respectively. Statistical analysis was performed by statistical software GraphPad Prism 8.0.2. Results E. granulosus protoscolex-derived exosomes are of circular membrane structure with a diameter of 60-90 nm by transmission electron microscopy. The expression of specific protein CD9 and enolase in exosomes was detected by Western blotting. The proportions of M-MDSC in MDSC in the negative control, experimental and positive control group were (78.13 ± 0.81)%, (77.14 ± 0.78)%, and (75.74 ± 0.50)%, respectively after 24 h of stimulation by exosomes. The differences were not significant (P > 0.05). The proportions of M-MDSC in the negative control, experimental and positive control group were (80.47 ± 0.85)%, (78.27 ± 0.69)%, and (68.01 ± 1.33)%, respectively, after 48 h of stimulation, showed no significant difference between experimental group and negative control group (P > 0.05), however significant difference was observed between the positive control and the negative control group (P < 0.01). After 72 h, the proportions of M-MDSC in the experimental and positive control groups in MDSC were (50.03 ± 0.59)% and (46.14 ± 0.87)%, which are lower than negative control group (74.94 ± 1.53)%, and the differences were statistically significant (P < 0.001). After stimulation with exosomes for 24 h, 48 h, and 72 h, the proportions of M-MDSC expressing M2 macrophages molecular markers in experimental and positive control group were (11.83 ± 0.06)%, (12.48 ± 0.12)%; (15.11 ± 0.21)%, (15.42 ± 0.27)%; and (29.12 ± 1.34)%, (31.83 ± 1.27)%, respectively, which were higher than those in the negative control group(8.52 ± 1.11)%, (8.79 ± 0.35)%, and (15.49 ± 0.26)%, and all differences were statistically significant (P < 0.05 or P < 0.01). Conclusion E. granulosus protoscolex-derived exosomes could regulate the polarization of M-MDSC to M2 macrophages, and the polarization continued to advance with the stimulation time.

Key words: Echinococcus granulosus, Exosomes, Myeloid-derived suppressor cells, M2 macrophages