| Background: Toxoplasma gondii can actively invade almost any mammalian cell type,including phagocytes.Early events in phagocytes,such as dendritic cells,are not only key to establishing parasitic infection,but in turn play a key role in initiating host immunity.It is now recognized that in addition to changes in some immune markers and mediators,metabolic changes also occur after macrophage activation.These metabolic changes are important to support a developing immune response,but may affect the intracellular pathogen of Toxoplasma gondii.However,Chinese 1 genotype Toxoplasma gondii infection of macrophages,especially how infection changes their metabolism,has not been extensively studied.Objective: The aim of this study was to investigate the changes of main metabolic pathways and polarization shift of macrophages in mice infected with Chinese 1 Toxoplasma gondii,and to provide a new perspective for immune metabolism of Chinese 1 Toxoplasma gondii infected cells.Methods:（1）Uninfected RAW264.7 cells were used as the control group,and macrophages were stimulated with TgCtWh3 and TgCtWh6 for 24 h,respectively.The changes of protein expression in macrophages were detected by non-standard proteomics.We conducted systematic bioinformatics analysis of all identified proteins（annotation of protein function）.Functional classification,functional enrichment and cluster analysis based on functional enrichment were carried out for all differentially expressed proteins.（2）Uninfected RAW264.7 cells were used as control group,and macrophages were stimulated with TgCtWh3,TgCtWh6 and LPS（100ng/ml）for 24 h,respectively.LCMS mass spectrometry was used to detect the differential expression of glucose metabolites in the cells.（3）Uninfected RAW264.7 cells were used as control group,and macrophages were stimulated with TgCtWh3,TgCtWh6 and LPS（100ng/ml）for 24 h,respectively.The m RNA and protein expression levels of i NOS（TNF-α）and M2（TGF-β）related genes in macrophages were detected by fluorescence quantitative PCR（RT-PCR）and Western blotting.Results:（1）The protein expression changes of RAW264.7 cells in the control group and Chinese 1 infected cells in the experimental group were measured using a non-standard quantitative proteome.A total of 6,136 proteins were identified,of which 4,432 contained quantitative information.We found that with 1.5 times as the differential expression change threshold and t-test p-value<0.05 as the significance threshold,The expression of TgCtWh3 protein mass increased by 177 proteins and decreased by 484 proteins compared to the control group.Compared with the control group,element expression of 476 proteins in TgCtWh6 was significantly increased,while element expression of 632 proteins was significantly decreased.Compared with TgCtWh6 group,the expression of elements in 254 proteins in TgCtWh3 group was significantly increased,while the expression of elements in 266 proteins was significantly decreased.（2）In GO classification,we focus on Biological processes and the differential protein functions are mainly concentrated in metabolic processes（such as carboxylic acid metabolism,ketoic acid metabolism,cell lipid metabolism,etc.）.KEGG pathway obtained through enrichment analysis showed that the pathways involved in the differential proteins included carbon metabolism,amino acid metabolism,sulfur metabolism,etc.To further clarify the overall difference of differential proteins in macrophages infected by TgCtWh3 and TgCtWh6,we conducted KEGG pathway enrichment analysis for differential proteins in macrophages infected by TgCtWh3 and TgCtWh6 and the control group,respectively.The results showed that the KEGG pathway induced by the enrichment analysis of differential proteins in TgCtWh3 infected macrophages and the control group mainly included pyruvate metabolism,arginine and proline metabolism,amino acid metabolism and so on.The KEGG pathway was obtained by the enrichment analysis of differential proteins in TgCtWh6 infected macrophages and the control group.The changes in metabolic pathways caused by KEGG pathway mainly included glycolysis and sphinolipid metabolism.（3）Macrophages infected with TgCtWh3 and TgCtWh6 tend to change key enzymes in glycolysis,tricarboxylic acid cycle and arginine metabolic pathway,and the increased or decreased expression of metabolic enzyme proteins may lead to the increase or decrease of some intermediates.（4）Changes in glycolysis and TCA cycling-related metabolites were observed in TgCtWh3,TgCtWh6 infection group and LPS stimulation group.（5）m RNA expression of M1 and M2-related genes was detected by real-time fluorescence quantitative PCR: i NOS and TNF-α of M1-related genes were highly expressed in TgCtWh3 and TgCtWh6 infected macrophages,but the expression level in TgCtWh3 group was higher than that in TgCtWh6 group,the difference was statistically significant（p<0.05）.Arg-1 of M2-related genes was highly expressed in TgCtWh6 infected macrophages,and the expression level of TgCtWh6 group was higher than that of TgCtWh3,and the difference was statistically significant（p<0.0001）.It was highly expressed in TgCtWh3 and TgCtWh6 groups.（6）The expression of M1 and M2-related proteins was detected by Western-blot: the expression of i NOS and TNF-α in TgCtWh3 group was higher than that in TgCtWh6 group（p <0.05）.The difference was statistically significant（p<0.05）.The expression levels of ARG-1 and TGF-β of M2-related proteins in TgCtWh6 group were higher than those in TgCtWh3 group,and the difference was statistically significant（p <0.001）.（7）Cell surface marker molecules were detected by flow cytometry: the proportion of CD11C-labeled M1 macrophages in TgCtWh3 group was higher than that in TgCtWh6 group.The proportion of CD206-labeled M2 macrophages in TgCtWh6 group was higher than that in TgCtWh3 group.Conclusion: Macrophages infected with TgCtWh3 and TgCtWh6 have profound effects on metabolism,and their altered metabolic patterns may be closely related to macrophages’ response to pathogen infection.The multiomics approach used here provides a large number of metabolic and proteomic indicators of other pathways affected,including arginine metabolism,and provides insights into the potential interactions between host and parasite biochemistry. |
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