关键词: 日本血吸虫病, 肝脏损伤, 巨噬细胞, 胞葬, A类清道夫受体

Abstract: Objective　To investigate the development, role, and regulatory mechanism of macrophage efferocytosis in the liver of hosts infected with Schistosoma japonicum. Methods　The expression of efferocytosis⁃related gene like efferocytosis receptors, efferocytosis⁃related bridging molecules,"eat me" signal and "don't eat me" signal was detected in the livers of patients and mice infected with S. japonicum in the Gene Expression Omnibus (GEO) database. Ten wild⁃type (WT) male mice (6 ~ 8 weeks old, weighing 20 ~ 25 g) were randomly divided into a Schistosoma japonicum infection (SJ) group and a normal control (NC) group, with 5 mice in each group. The efferocytosis of neutrophils and T cells by liver macrophages was detected in mice from SJ group and NC group using flow cytometry and immunofluorescence assay, respectively. The expression of efferocytosis⁃related Mer receptor tyrosine kinase (MerTK) and Axl receptor tyrosine kinase (Axl) proteins was determined in mouse liver tissues using Western blotting assay, and the proportion of MerTK＋ macrophages and the average fluorescence intensity of macrophage MerTK were detected in mouse livers using flow cytometry. Changes in liver granulomas and fibrosis were observed in mice infected with S. japonicum following injection of efferocytosis inhibitors, and S. japonicum⁃infected mice without injection of efferocytosis inhibitors served as controls. Bone marrow⁃derived macrophages (BMDMs) were isolated from macrophage scavenger receptor class A (SR⁃A) conditional knockout (CKO) and wild⁃type (WT) mice, and changes in apoptotic neutrophils were detected in SR⁃A CKO mouse macrophages in vitro using flow cytometry. Then, BMDMs was divided into the WT mono⁃culture group, the WT and apoptotic neutrophils co⁃culture group, the SR⁃A CKO mono⁃culture group, and the SR⁃A CKO and neutrophils co⁃culture group, and the expression of MerTK and Axl was quantified in vitro using Western blotting and real⁃time quantitative PCR (RT⁃qPCR) assays during neutrophil efferocytosis. The efferocytosis of neutrophils and T cells by mouse liver macrophages was detected in the SR⁃A CKO SJ group and the WT SJ group using flow cytometry and immunofluorescence assay, and the expression of MerTK and Axl proteins was determined in mouse liver tissues in both groups using Western blotting. In addition, the proportion of MerTK＋ macrophages and the average fluorescence intensity of macrophage MerTK were detected in mouse livers in both groups using flow cytometry. Results　Data from the GEO database showed that the expression of some efferocytosis receptors and efferocytosis⁃related bridging molecules, and "eat me" and "don't eat me" signals all appeared a tendency towards a rise in livers of patients and mice in the SJ group relative to the NC group, suggesting that S. japonicum infection⁃induced liver diseases may be associated with efferocytosis. Flow cytometry detected higher proportions of Ly6G＋cells [(13.13 ± 0.45)% vs. (6.48 ± 0.25)%; t = 22.30, P < 0.05] and CD3＋ cells [(7.60 ± 0.33)% vs. (3.30 ± 0.42)%; t = 13.98, P < 0.05] in mouse liver macrophages in the SJ group than in the NJ group, and the expression of MerTK protein [(2.30 ± 0.14) vs. (1.14 ± 0.46); t = 4.19, P < 0.05], the mean fluorescence intensity of macrophages [(160.67 ± 15.28) vs. (94.50 ± 19.61); t = 4.81, P < 0.05], and the proportion of MerTK＋ macrophages [(20.78 ± 4.17)% vs. (6.85 ± 0.39)%; t = 6.57, P < 0.05] were significantly higher in mouse liver tissues in the SJ group than in the NC group. The Axl expression was lower in mouse liver tissues in the SJ group than in the NC group [(1.25 ± 0.08) vs. (1.93 ± 0.37); t = 2.79, P < 0.05], and the area of granulomas around single eggs [(9.18 ± 1.81) × 104 μm2  vs.  (5.24 ± 1.35) × 104 μm2; t = 3.03, P < 0.05] and proportion of collagen fibers [(25.27 ± 3.99)% vs. (15.14 ± 4.02)%; t = 3.10, P < 0.05] were significantly greater in livers of S. japonicum⁃infected mice with injection of efferocytosis inhibitors than in mice without injection of efferocytosis inhibitors. The in vitro efferocytosis efficiency of BMDMs [(32.83 ± 3.17)% vs. (45.43 ± 2.34)%; t = 5.54, P < 0.05], and the proportions of Ly6G＋ [(9.37 ± 0.48)% vs. (13.13 ± 0.72)%; t = 7.50, P < 0.05] and CD3＋ cells [(4.95 ± 0.17)% vs. (7.64 ± 0.50)%; t = 8.87, P < 0.05] in liver macrophages post⁃infection with S. japonicum were significantly lower in SR⁃A CKO mice than in WT mice, and the expression of MerTK protein [(0.65 ± 0.25) vs. (1.96 ± 0.69); t = 3.10, P < 0.05], the average fluorescence intensity of macrophages [(138.33 ± 8.39) vs. (160.67 ± 15.28); t = 3.03, P < 0.05] and the proportion of MerTK＋ macrophages [(13.17 ± 5.01)% vs. (22.63 ± 2.06)%; t = 3.56, P < 0.05] were significantly lower in mouse liver tissues in the SR⁃A CKO SJ group than in the WT group. Western blotting detected no significant difference in the Axl protein expression in mouse liver tissues between the SR⁃A CKO SJ group and the WT SJ group [(0.48 ± 0.07) vs. (0.68 ± 0.30); t = 1.09, P > 0.05]. There were significant differences in the relative MerTK mRNA and protein expression during efferocytosis of BMDMs among the WT mono⁃culture group, the WT and apoptotic neutrophils co⁃culture group, the SR⁃A CKO mono⁃culture group, and the SR⁃A CKO and neutrophils co⁃culture group (F = 9.41 and 40.68, both P values < 0.05). In addition, there was a significant difference in the relative Axl mRNA expression during efferocytosis of BMDMs among the four groups (F = 13.62, P < 0.05); however, no significant difference was seen in the relative Axl protein expression (F = 1.27, P > 0.05). Conclusions　Efferocytosis of liver macrophages is seen in mice infected with S. japonicum and inhibits liver fibrosis. SR⁃A may up⁃regulate the efficiency of macrophage efferocytosis through regulating the expression of efferocytosis receptors.

Key words: Schistosomiasis japonica, Hepatic damage, Macrophage, Efferocytosis, Scavenger receptor class A