吲哚胺2,3-双加氧酶1上调促进骨包虫组织浸润和免疫逃逸的机制研究OA

Objective To explore the molecular mechanisms of upregulated indoleamine 2,3-dioxygenase 1(IDO1)in promoting tissue infiltration and immune evasion of bone hydatid disease(BHD).Methods Immunofluorescence staining was used to detect the expression of surface markers CD206 and CD163 on M2 phenotype macrophages,as well as IDO1 expression in the surgically resected BHD cyst tissues and knee osteoarthritis(OA)tissues.Pearson correlation analysis was conducted to analyze the correlation between the number of CD206+CD163+macrophages and IDO1+cells per visual field in BHD tissues.The human monocyte leukemia cell line THP1 was cultured in vitro.THP1 cells were induced to polarize towards the M2 phenotype,and the hydatid cyst fluid native antigen B(EgB)was added during the induction process.For cell experiment Group 1,cells were divided into the Control group,the EgB-treated group.CCK-8 assay was used to detect the cell proliferation ability.For cell experiment Group 2,cells were divided into the M0-induced-Control group,the M1-induced-Control group,the M2-induced-Control group,the M0-induced-EgB-treated group,the M1-induced-EgB-treated group,and the M2-induced-EgB-treated group.RT-qPCR was used to detect the mRNA expression levels of CD68,F4/80,transforming growth factor-β1(TGF-β1),CD86,and CD163.Western blot was used to detect the protein expression levels of IDO1 and TGF-β1 in the M2-induced-Control group and the M2-induced-EgB-treated group.For cell experiment Group 3,cells were divided into the M2-induced-Control group,the M2-induced-EgB-treated group,the M2-induced-EgB+shRNA-IDO1 group,the M2-induced-EgB+shRNA-NT group,the M2-induced-EgB+OE-IDO1 group,and the M2-induced-EgB+vector group.RT-qPCR was used to detect the mRNA expression levels of CD68 and CD163 in cells.Western blot was used to detect the expression levels of IDO1,TGF-β1,programmed cell death protein 1(PD-1),phosphoinositol 3-kinase(PI3K),p-PI3K,protein kinase B(Akt),p-Akt,mammalian target of rapamycin(mTOR)and p-mTORin cells.Results Compared with OA tissues,the numbers of IDO1+cells and CD206+CD163+macrophages per visual field in BHD tissues were significantly increased(P<0.05).Pearson correlation analysis results showed that the number of IDO1+cells per visual field was positively correlated with the number of CD206+CD163+macrophages per visual field(r=0.933 1,P<0.01).Compared with the Control group,the proliferation ability of THP1 cells in the EgB-treated group was enhanced(P<0.01).Compared with the M0-induced-Control group,the mRNA expression levels of M0 phenotype polarization markers CD68 and F4/80 in the M0-induced-EgB-treated group were increased(P<0.01).Compared with the M2-induced-Control group,the mRNA expression levels of M2 phenotype polarization markers CD68 and CD163 in the M2-induced-EgB-treated group were increased(P<0.01).There was no statistically significant difference in the mRNA expression levels of TNF-α and CD86 between the M1-induced-Control group and the M1-induced-EgB-treated group(P>0.05).Compared with the M2-induced-Control group,the protein expression levels of IDO1,TGF-β1,and PD-1,as well as the ratios of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR in the M2-induced-EgB-treated group were increased(P<0.05).Compared with the M2-induced-EgB-treated group,the mRNA expression levels of CD68 and CD163,the protein expression levels of IDO1,TGF-β1,and PD-1,and the ratios of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR in the M2-induced-EgB+shRNA-IDO1 group were all decreased(P<0.05);while the mRNA expression levels of CD68 and CD163,the protein expression levels of IDO1,TGF-β1,and PD-1,and the ratios of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR in the M2-induced-EgB+OE-IDO1 group were all increased(P<0.05).Conclusion EgB can enhance the expression of IDO1 to stimulate the polarization of macrophages toward the M2 phenotype,and activate the TGF-β1,PD-1,PI3K/Akt/mTOR signaling pathways,thereby mediating the formation of immunosuppressive microenvironment during bone echinococcosis infection.