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OBJECTIVE@#To investigate the dynamic changes of macrophage numbers and apoptosis during Schistosoma japonicum infection, and to investigate the possible mechanisms of macrophage apoptosis induced by S. japonicum soluble egg antigen (SEA).@*METHODS@#C57BL/6 mice at ages of 6~8 weeks were randomly divided into 4 groups, including three experimental groups and a normal control group. Each mouse in the experimental groups was infected with (12 ± 1) cercariae of S. japonicum via the abdominal skin, and all mice in an experimental group were sacrificed 3, 5, 8 weeks post-infection, respectively, while mice in the control group were not infected with S. japonicum cercariae and sacrificed on the day of S. japonicum infection in the experimental group. Mouse liver specimens and peritoneal exudation cells were sampled in each group, and the dynamic changes of macrophage numbers and apoptosis were detected. Mouse peritoneal macrophages were isolated, purified and treated with S. japonicum SEA, PBS and ovalbumin (OVA) in vitro, and the macrophage apoptosis was detected using flow cytometry. The mRNA and protein expression of BCL-2 protein family members were determined in macrophages using real-time quantitative PCR (qP-CR) and Western blotting assays, and the activation of caspase 3 was determined using flow cytometry and Western blotting. In addition, macrophages were in vitro treated with S. japonicum SEA in presence of a caspase inhibitor, H2O2 or N-acetyl-L-cysteine, and the apoptosis of macrophages was detected using flow cytometry.@*RESULTS@#The total macrophage numbers continued to increase in mouse liver [(0.873 ± 0.106) × 106, (2.737 ± 0.460) × 106 and (3.107 ± 0.367) × 106 cells, respectively; F = 81.900, P < 0.01] and peritoneal specimens [(5.282 ± 1.136) × 105, (7.500 ± 1.200) × 105 and (12.800 ± 0.800) × 105 cells, respectively; F = 55.720, P < 0.01] 3, 5 and 8 weeks post-infection with S. japonicum, and the numbers of apoptotic macrophages also continued to increase in mouse liver [(0.092 ± 0.018) × 106, (0.186 ± 0.025) × 106 and (0.173 ± 0.0270) × 106 cells; F = 57.780, P < 0.01] and peritoneal specimens [(0.335 ± 0.022) × 105, (0.771 ± 0.099) × 105 and (1.094 ± 0.051) × 105 cells; F = 49.460, P < 0.01] 3, 5 and 8 weeks post-infection with S. japonicum. The apoptotic rate of SEA-treated macrophages [(24.330 ± 0.784)%] was significantly higher than that of PBS-[(18.500 ± 1.077)%] and OVA-treated macrophages [(18.900 ± 1.350)%] (both P values < 0.01). There were no significant differences in the mRNA or protein expression of Bcl-2 [Bcl - 2 mRNA expression: (1.662 ± 0.943) vs. (1.000 ± 0.000), t = 1.215, P > 0.05; BCL protein expression: (0.068 ± 0.004) vs. (0.070 ± 0.005), t = 0.699, P > 0.05], Bax [Bax mRNA expression: (0.711 ± 0.200) vs. (1.000 ± 0.000), t = 2.507, P > 0.05; BAX protein expression: (0.089 ± 0.005) vs. (0.097 ± 0.003), t = 2.232, P > 0.05] and Bak [Bak mRNA expression: (1.255 ± 0.049) vs. (1.00 ± 0.00), t = 0.897, P > 0.05; BAK protein expression: (0.439 ± 0.048) vs. (0.571 ± 0.091), t = 2.231, P > 0.05] between in SEA- and PBS-treated macrophages. S. japonicum SEA induced macrophage apoptosis in the presence of a caspase inhibitor (F = 0.411, P > 0.05); however, SEA failed to induce macrophage apoptosis in the presence of H2O2 or NAC (F = 11.880 and 9.897, both P values < 0.05).@*CONCLUSIONS@#S. japonicum SEA may induce macrophage apoptosis through promoting reactive oxygen species expression during S. japonicum infections in mice.
Sujets)
Animaux , Souris , Apoptose , Caspases , Peroxyde d'hydrogène , Macrophages , Souris de lignée C57BL , ARN messager , Schistosoma japonicum , Schistosomiase artérioveineuse , Protéine BaxRésumé
OBJECTIVE@#To investigate the serum microRNA (miRNA) expression and examine the impact of miRNA expression profiles on T helper type 17 (Th17)/regulatory T cells (Treg) imbalance among patients with cystic echinococcosis, so as to provide insights into the illustration of the mechanisms underlying chronic Echinococcus granulosus infections, and long-term pathogenesis.@*METHODS@#Total RNA was extracted from the sera of cystic echinococcosis patients and healthy controls, and subjected to high-throughput sequencing with the Illumina sequencing platform. Known miRNAs were annotated and new miRNAs were predicted using the miRBase database and the miRDeep2 tool, and differentially expressed miRNAs were identified. The target genes of differentially expressed miRNAs were predicted using the software miRanda and TargetScan, and the intersection was selected for Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Among the differentially expressed miRNAs with the 20 highest fold changes, miRNAs that targeted genes relating to key transcription factors RORC and FOXP3 that determine the production of Th17 and Treg cells or their important regulatory pathways (PI3K-Akt and mTOR pathways) were matched.@*RESULTS@#A total of 53 differentially expressed miRNAs were screened in sera of cystic echinococcosis patients and healthy controls, including 47 up-regulated miRNAs and 6 down-regulated miRNAs. GO enrichment analysis showed that these differentially expressed miRNA were involved DNA transcription and translation, cell components, cell morphology, neurodevelopment and metabolic decomposition, and KEGG pathway analysis showed that the differentially expressed miRNA were mainly involved in MAPK, PI3K-Akt and mTOR signaling pathways. Among the differentially expressed miRNAs with the 20 highest fold changes, there were 3 miRNAs that had a potential for target regulation of RORC, and 15 miRNAs that had a potential to target the PI3K-Akt and mTOR signaling pathways.@*CONCLUSIONS@#Significant changes are found in serum miRNA expression profiles among patients with E. granulosus infections, and differentially expressed miRNAs may lead to Th17/Treg imbalance through targeting the key transcription factors of Th17/Treg or PI3K-Akt and mTOR pathways, which facilitates the long-term parasitism of E. granulosus in hosts and causes a chronic disease.
Sujets)
Humains , Échinococcose/génétique , Analyse de profil d'expression de gènes , microARN/métabolisme , Phosphatidylinositol 3-kinases/génétique , Protéines proto-oncogènes c-akt/génétique , Lymphocytes T régulateurs , Sérine-thréonine kinases TOR/génétique , Cellules Th17 , Facteurs de transcription/génétiqueRésumé
Objective To investigate the effect of gender on hepatic pathology and antibody-mediated immunity in Schistosoma japonicum-infected C57BL/6 mice. Methods Female and male C57BL/6 mice were infected with S. japonicum, and the hepatic pathological changes were observed using HE and picrosirius red staining in mice 8 weeks post-infection. The serum specific IgG antibody levels against the soluble adult worm antigen (SWA) and soluble egg antigen (SEA) were measured in mice using enzyme-linked immunosorbent assay (ELISA), and the percentages of follicular helper T (Tfh) cells and regulatory T (Treg) cells were detected in mouse spleen and lymph nodes using flow cytometry. Results HE staining showed no significant difference in the mean area of a single hepatic egg granuloma between female and male mice 8 weeks post-infection with S. japonicum [(28.050 ± 3.576) × 104 μm2 vs. (26.740 ± 4.093) × 104 μm2; t = 0.241, P = 0.821], and picrosirius red staining revealed no statistical differences between female and male mice in terms of the mean proportion of picrosirius red stained hepatic tissues [(7.667 ± 1.856)% vs. (7.667 ± 1.764)%; t = 0, P = 1] or the mean optical density [(0.023 ± 0.003) vs. (0.027 ± 0.007); t = 0.447, P = 0.678]. ELISA detected no significant differences in the serum IgG antibody levels against SWA [(2.098 ± 0.037) vs. (1.970 ± 0.071); t = 1.595, P = 0.162] or SEA [(3.738 ± 0.039) vs. (3.708 ± 0.043); t = 0.512, P = 0.623] between female and male mice 8 weeks post-infection with S. japonicum. Flow cytometry detected significantly greater percentages of Tfh cells in the spleen [female mice, (8.645 ± 1.356)% vs. (1.730 ± 0.181)%, t = 5.055, P = 0.002; male mice, (8.470 ± 1.161)% vs. (1.583 ± 0.218)%, t = 5.829, P = 0.001] and lymph nodes [female mice, (3.218 ± 0.153)% vs. (1.095 ± 0.116)%, t = 11.040, P < 0.001; male mice, (3.673 ± 0.347)% vs. (0.935 ± 0.075)%, t = 8.994, P = 0.001) of both female and male mice 8 weeks post-infection with S. japonicum than in uninfected mice; however, no significant differences were seen between female and male mice 8 weeks post-infection with S. japonicum in terms of the percentages of Tfh cells in the spleen [(8.645 ± 1.356)% vs. (8.470 ± 1.161)%; t = 0.098, P = 0.925] or lymph nodes [(3.218 ± 0.153)% vs. (3.673 ± 0.347)%; t = 1.332, P = 0.241]. There was no significant difference in the proportion of Treg cells in the spleen of male mice between infected and uninfected mice [(10.060 ± 0.361)% vs. (10.130 ± 0.142)%; t = 0.174, P = 0.867], while a higher proportion of Treg cells was seen in the spleen of female mice 8 weeks post-infection with S. japonicum than in uninfected mice [(10.530 ± 0.242)% vs. (9.450 ± 0.263)%; t = 3.021, P = 0.023]. There was no significant difference in the proportion of Treg cells in the spleen between female and male mice infected with S. japonicum [(10.530 ± 0.242)% vs. (10.060 ± 0.361)%; t =1.077, P = 0.323]. In addition, the proportions of Treg cells were significantly greater in the lymph node of S. japonicum -infected female [(17.150 ± 0.805)% vs. (13.100 ± 0.265)%; t = 4.781, P = 0.003] and male mice [(18.550 ± 0.732)% vs. (12.630 ± 0.566)%; t = 6.402, P = 0.001] than in uninfected mice; however, no significant difference was seen between female and male mice 8 weeks post-infection [(17.150 ± 0.805)% vs. (18.550 ± 0.732)%; t = 1.287, P = 0.246]. Conclusion There are no gender-specific hepatic pathological changes or antibody-mediated immunity in C57BL/6 mice post-infection with S. japonicum.
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Objective To investigate the immunological functions of heat shock protein 40 kDa of Schistosoma japonicum (SjHSP40). Methods The homology of the SjHSP40 protein sequence was analyzed and the B and T cell epitopes of SjHSP40 were predicted using bioinformatics tools. The full-length SjHSP40 gene was amplified using a PCR assay, and cloned into the prokaryotic expression vector pGEX-6P-1, which was transformed into Escherichia coli BL-21. The protein expression was induced with isopropyl β-D-thiogalactoside (IPDG), and then, the recombinant protein was purified with glutathione-sepharose 4B resin to yield the fusion protein GST-SjHSP40, which was checked with SDS-PAGE and Western blotting. Following immunization with GST-SjHSP40, the serum levels of anti-SjHSP40 IgG antibody and IgG1 and IgG2a subtypes were detected in BALB/c mice using ELISA. In addition, the effect of SjHSP40 on CD4+ T-cell subset differentiation was examined using flow cytometry. Results SjHSP40 contained 7 potential B cell epitopes and multiple T cell epitopes (CTL epitopes and Th epitopes). The prokaryotic expression plasmid pGEX-6p-1-SjSHP40 was successfully constructed, and the fusion protein GST-SjHSP40 was obtained following IPDG induction and protein purification. Significantly higher serum levels of anti-SjHSP40 IgG, IgG1 and IgG2a antibodies were detected in mice immunized with GST-SjHSP40 than in other groups; however, SjHSP40 showed no remarkable effects on CD4+ T-cell subset differentiation. Conclusions SjHSP40 may induce specific humoral immune responses in mice; however, it does not affect the balance of Th immune responses. It is suggested that SjHSP40 may be a potential vaccine candidate.
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Objective To explore the possible mechanisms by which Schistosoma japonicum heat shock protein 60(SjH-SP60)enhances CD4+CD25+regulatory T cell(Treg)immunosuppressive function.Methods An in vitro method was used to investigate the effect of SjHSP60 on Treg immunosuppressive activity.Co-cultures in transwells and in vitro suppression assay were performed to investigate how SjHSP60 enhanced the immunosuppressive function of Tregs.Intracellular cytokine staining combined with flow cytometry was used to detect Treg-expressing IL-10 and TGF-β,and flow cytometry was also used to analyze the expressions of Foxp3 and CTLA-4 in Tregs.Results SjHSP60 enhanced the immunosuppressive function of Tregs.Soluble cytokines IL-10 and TGF-β mediated inhibitory activity of SjHSP60-triggered Tregs.SjHSP60 induced significant increases in both IL-10 and TGF-β expressions of Tregs.Further investigation showed significant increased Foxp3 and CTLA-4 in SjHSP60-trggered Tregs.Conclusion SjHSP60 enhances Treg immunosuppressive function by promoting the expressions of IL-10 and TGF-β,possibly due to SjHSP60-mediated induction of Foxp3 and CTLA-4 in Tregs.
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Objective To assess the influence of glycolytic pathway on the proportion and numbers of regulatory T cells dur-ing Schistosoma japonicum infection. Methods A S. japonicum-infected mouse model was established,and C57/BL6 male mice infected with S.japonicum were subjected to intraperitoneal injections of with the glycolytic inhibitor 2-Deoxy-D-glucose (2DG)or PBS for 6 times,and then the cells from spleen or mesenteric lymph nodes(LNs)were isolated and analyzed by flow cytometry(FCM)to detect the percentage of Glut1+CD4+T cells and Treg cells. Results The proportions of Glut1+CD4+T cells in the spleen(43.58%±2.50% vs.21.15%±0.96%;t=8.834,P<0.01)and mesenteric LNs(38.97%±1.97% vs.28.40%± 2.11%;t=3.662,P<0.05)were higher in the normal mice than those in the infected mice,and the percentages of Treg cells in the spleen(6.83% ± 0.21% vs. 13.30% ± 0.35%;t = 15.65,P < 0.01)and LNs(8.26% ± 0.15% vs. 14.37% ± 0.44%;t =13.14,P<0.01)were lower in the normal mice than those in the infected mice.In addition,the proportions of Treg cells in the spleen(15.50%±0.76% vs.13.07%±0.15%;t=3.130,P<0.05)and LNs(17.00% ± 0.41% vs.13.83% ± 0.18%;t=6.947, P<0.01)were higher in the infected mice injected intraperitoneally with 2DG than those in the infected mice injected intraperi-toneally with PBS. Conclusion Glycolytic pathway inhibits Treg differentiation in the spleen and mesenteric LNs of S.japoni-cum-infected mice.
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Objective To investigate the role of TIGIT signal in Th1/Th2 balance in the process of Schistosoma japonicum in-fection.Methods Male C57BL/6 mice were infected with cercariae of S.japonicum,and normal uninfected mice served as the controls.The percentages of TIGIT+cells,Ki67+CD3+CD4+TIGIT+cells,Ki67+CD3+CD4+TIGIT-cells,IFN-γ+CD3+CD4+TIGIT+cells,IFN-γ+CD3+CD4+TIGIT- cells,IL-4+CD3+CD4+TIGIT+cells and IL-4+CD3+CD4+TIGIT- cells were evaluated in mouse spleen by flow cytometry.Results The proportion of TIGIT+CD4+T cells was higher in the spleen of S.japonicum-infected mice than in the normal uninfected mice(29.30%±0.70% vs.3.09%±0.50%;t=8.834,P<0.01).However,no significant differ-ence in the percentages of TIGIT+CD8+T cells between the infection group and normal controls(3.61% ± 0.26% vs. 3.58% ± 0.16%;t=0.108,P>0.05),and no significant difference was detected in the percentages of TIGIT+cells in non-T cells be-tween the infection group and controls(1.86%±0.19% vs.1.37%±0.17%;t=1.931,P>0.05).In addition,the proportion of Ki67 in the TIGIT+cells was higher than that in the TIGIT-cells(17.03%±0.64% vs.6.59%±0.37%;t=14.09,P<0.01).The Th2/Th1 ratio was higher in the TIGIT+CD4+T cells than in the TIGIT-CD4+T cells(39.28%±3.75% vs.11.79%±1.64%;t=6.721,P<0.01).Conclusion The TIGIT signaling may be involved in the development of Th2 responses in the mice infected with S.japonicum.