Polarization effect of Echinococcus granulosus antigen B on the mouse macrophage RAW264.7

doi:10.12140/j.issn.1000-7423.2023.01.004

Abstract

Abstract:

Objective To investigate the regulatory effect of Echinococcus granulosus antigen B (AgB) on macrophage polarization. Methods After cultivated for 24 h, the RAW264.7 macrophages cells were designated to 6 groups: M1, M2, AgB, AgB+M1, AgB+M2 and blank control (M0), 3 wells each group. After all the cells attached to the well wall for 3 h, the AgB、AgB+M1、AgB+M2 group was respectively added with natural AgB extracted from sheep hydatid cyst fluid (1 000 ng/ml, final concentration); 1 h post-stimulation, the M1 and AgB+M1 group was respectively added with lipopolysaccharide (LPS, final concentration 100 ng/ml), and IFN-γ (20 ng/ml, final concentration) to stimulate differentiation for 20 h; M2 and AgB+M2 group was added with interleukin 4 (IL-4) and IL-13 (final concentration 20 ng/ml) to stimulate differentiation for 20 h; the control group was cultured in parallel without changing medium. The morphology of macrophage cells were observed microscopically. Total RNA of the macrophages in all groups was extracted for performing RT-PCR to detect the relative transcription levels of the surface markers on stimulated macrophages, including arginase 1 (Arg-1) and tumor necrosis factor α (TNF-α). The relative expression levels of Arg-1 and inducible nitric oxide synthase (iNOS) were analyzed by Western blotting. The change of IL-10 and TNF-α expression in the culture supernatant of stimulated macrophages were detected by ELISA. Results After stimulation and differentiation, most cells in the M1 group and AgB+M1 group were irregularly shaped and had antennae. The cells of M2 group and AgB+M2 group were mostly round or oval, and very few were irregular. The cells of M0 group and AgB group were partly round and oval, and partly irregular. RT-PCR showed that the relative transcription levels of Arg-1 mRNA in the M2 group and the AgB+M2 group were 189.49 ± 68.43 and 435.83 ± 123.57, respectively (t = 246.30, P < 0.01). They were higher than those in the M0 group (1.00 ± 0.00), M1 group (1.87 ± 1.29), AgB group (2.37 ± 2.06), AgB+M1 group (3.96 ± 1.92) (t = 188.50, 187.60, 187.10, 185.50, P < 0.01; t = 434.80, 434.00, 433.50, 431.90, all P < 0.01). The relative transcription levels of TNF-α mRNA in the M1 group and the AgB+M1 group were 8.34 ± 2.92 and 8.10 ± 1.54, respectively (t = 0.24, P > 0.05). They were higher than that of the M0 group (1.00 ± 0.00), M2 group (1.37 ± 0.64), AgB group (2.86 ± 0.44) and AgB+M2 group (1.62 ± 0.27) (t = 7.34, 6.97, 5.48, 6.71, P < 0.01; t = 7.10, 6.74, 5.24, 6.48, P < 0.01). Western blotting showed that the relative expression level of Arg-1 protein in the M2 group was 1.18 ± 0.35, which was higher than that in the M1 group (0.33 ± 0.18) and the AgB+M1 group (0.58 ± 0.10) (t = 0.67，0.61, P < 0.01). There was no significant difference on the Arg-1 protein relative expression level between the AgB group (1.05 ± 0.17) and the AgB+M2 group (0.97 ± 0.27) (t =0.20, 0.13, P > 0.05). There was statistical significance in AgB+M2 group compared with M1 group and AgB+M1 group (t = 0.52, 0.48, P < 0.05). The iNOS relative expression levels of M1 group and AgB+M1 group were 0.95 ± 0.21 and 0.88 ± 0.02 (t = 0.07, P > 0.05), respectively. They were higher than those in M0 group (0.03 ± 0.00), M2 group (0), AgB group (0) and AgB+M2 group (0) (t = 0.92, 0.95, 0.95, 0.95, P < 0.01; t = 0.85, 0.88, 0.88, 0.88, P < 0.01). The ELISA results showed that the expression level of IL-10 cytokine in the cell supernatant was 166.67 ± 56.67 in the AgB+M1 group and 213.33 ± 16.67 in the AgB+M2 group, respectively. They were higher than those in the M0 group (0.00 ± 0.00), M1 group (43.33 ± 36.67), M2 group (50.00 ± 43.00) and AgB group (47.50 ± 25.00) (t = 166.70, 123.30, 116.70, 119.20, all P < 0.05. t = 213.30, 170.00, 163.30, 165.80, P < 0.01). The expression levels of TNF-α cytokines in M1 group and AgB+M1 group were 833.13 ± 3.09 and 745.63 ± 118.00, respectively (t = 87.50, P > 0.05). They were higher than those in M0 group (217.50 ± 32.26), M2 group (224.69 ± 17.68), AgB group (308.44 ± 4.42), AgB+M2 group (251.25 ± 1.33) (t = 615.60, 608.40, 524.70, 581.90, P < 0.01; t = 528.10, 520.90, 437.20, 494.40, P < 0.01). Conclusion AgB can up-regulate the expression of Arg-1 in macrophages and polarize it towards M2 type, which may be an important regulatory molecule in the host-parasite immune responses, involving in the immune regulation of macrophages.

Key words: Echinococcus granulosus, Secreted antigen, Antigen B, Polarization of macrophages

CLC Number:

R383.33

JIAO Hongjie, QI Wenjing, GUO Gang, BAO Jianling, WU Chuanchuan, SONG Chuanlong, LI Jun, ZHANG Wenbao, YAN Mei. Polarization effect of Echinococcus granulosus antigen B on the mouse macrophage RAW264.7[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(1): 23-28.

Figures/Tables 2

References 19

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