The Protective Effect of the Soluble Egg Antigen of Schistosoma japonicum in A Mouse Skin Transplantation Model

Abstract

Background: Organ transplantation is currently an effective method for treating organ failure. Long-term use of immunosuppressive drugs has huge side effects, which severely restricts the long-term survival of patients. Schistosoma can affect the host's immune system by synthesizing, secreting, or excreting a variety of immunomodulatory molecules, but its role in transplantation was not well defined. In order to explore whether Schistosoma-related products can suppress rejection and induce long-term survival of the transplant, we used soluble egg antigen (SEA) of Schistosoma japonicum in mouse skin transplantation models.

Materials and methods: Each mouse was intraperitoneally injected with 100 μg of SEA three times a week for four consecutive weeks before allogenic skin transplant. Skin transplants were performed on day 0 to observe graft survival. Pathological examination of skin grafts was conducted 7 days post transplantation. The skin grafts were subjected to mRNA sequencing. Bioinformatics analysis was conducted and the expression of hub genes was verified by qPCR. Flow cytometry analysis was performed to evaluate the immune status and validate the results from bioinformatic analysis.

Results: The mean survival time (MST) of mouse skin grafts in the SEA-treated group was 11.67 ± 0.69 days, while that of the control group was 8.00 ± 0.36 days. Pathological analysis showed that Sj SEA treatment led to reduced inflammatory infiltration within skin grafts 7 days after allogenic skin transplantation. Bioinformatics analysis identified 86 DEGs between the Sj SEA treatment group and the control group, including 39 upregulated genes and 47 downregulated genes. Further analysis revealed that Sj SEA mediated regulation on cellular response to interferon-γ, activation of IL-17 signaling and chemokine signaling pathways, as well as cytokine-cytokine receptor interaction. Flow cytometry analysis showed that SEA treatment led to higher percentages of CD4⁺IL-4⁺ T cells and CD4⁺Foxp3⁺ T cells and decreased CD4⁺IFN-γ⁺ T cells in skin transplantation.

Conclusion: Sj SEA treatment suppressed rejection and prolonged skin graft survival by regulating immune responses. Sj SEA treatment might be a potential new therapeutic strategy to facilitate anti-rejection therapy and even to induce tolerance.

Keywords: Schistosoma japonicum; cytokine–cytokine receptor interaction; gene expression; skin graft; soluble egg antigen.

Figure 1

SEA pretreatment can prolong the survival time of mouse skin grafts. (A) In the control group (n=5) has no special treatment, and in the treatment group (n = 6), each mouse is given SEA 100 μg pretreatment 28 days later (3 times a week, 4 weeks in a row), and the outer ears of BALB/c donor mice were transplanted to the back of C57BL/6 recipient mice. (B) Compared with the control group (8.00 ± 0.36), the treatment group (11.67 ± 0.69) significantly prolonged the survival time of skin grafts (p < 0.01). (C, D) On the 9th day after transplantation, there was no obvious inflammation, ulcer, and necrosis in the skin grafts in the SEA treatment group. Compared with the control group, the graft rejection score was significantly lower than that of the control group (p < 0.01). (E) The pathological changes of the skin grafts were observed on the 7th day after transplantation. Compared with the control group, the low-power and high-power microscopes showed that the neutrophil cell aggregates decreased.

Figure 2

Volcano map and hierarchical clustering heat map of DEGs. (A) (|log2Foldchange|>1, p adj < 0.05) DEG volcano distribution map; red represents upregulated transcripts; blue represents downregulated transcripts. (B) Hierarchical clustering heat map of DEGs (n = 3) in each group.

Figure 3

GO and KEGG analysis of the role of DEGs and screening enrichment pathways. (A) From the aspects of biological processes, cellular components, and molecular functions, select the top 10 most significant terms and draw a histogram for display. The abscissa in the figure is the description of GO Term, and the ordinate is the significance level of GO Term enrichment. The higher the value, the more significant. Orange represents BP, green represents CC, and blue represents MF. (B) Select the 20 most significant KEGG pathways to draw a scatter diagram for display. The abscissa in the figure is the ratio of the number of differential genes annotated to the KEGG pathway to the total number of differential genes, the ordinate is the description of the KEGG pathway, the size of the dot represents the number of genes annotated to the KEGG pathway, and the color from red to purple represents enrichment of the saliency size.

Figure 4

GSEA in Sj SEA-treated skin graft. We screened and analyzed 22 upregulated pathways and 22 downregulated pathways based on |NES| > 1 and NOM p-value < 0.05. The upregulated enrichment pathways include adherens junction, glycolysis and gluconeogenesis, and inositol phosphate metabolism, among others. The downregulated pathways include oxidative phosphorylation, allograft rejection, Parkinson disease, etc.

Figure 5

PPI analysis and screening of the hub gene and key signaling pathways in DEGs. (A) STRING database analyzes DEGs to get 48 nodes and 106 edges displayed on Cytoscape. Red represents upregulated genes, blue represents downregulated genes, and orange represents hub genes. (B) Further analysis with cytoHubba obtained the top 15 most significant genes as hub genes. (C) MCODE analysis obtains 12 genes and 52 edges of module 1, MCODE 1 score: 9.455. (D) The hub gene of the skin graft obtained by RNA sequencing was verified by qPCR. The significance level was tested by unpaired t test (n = 3 for each group). The data are shown as the mean ± SEM value. At least 3 independent experiments have also obtained similar results. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6

T-cell subsets in the spleen of mice in each group were analyzed by flow cytometry on day 28 after SEA treatment and on day 7 after skin transplantation. (A) Compared with the control group, on the 28th day after SEA treatment, the percentages of CD4⁺IL-4⁺ T and Treg cells in the spleen of mice in the experimental group were significantly upregulated, while CD4⁺ IFN-γ⁺ and CD4⁺ IL-17⁺ T-cell ratios were not significantly different. *p  < 0.05, **p  < 0.01, n = 6. (B) On the 7th day after transplantation, the percentages of CD4⁺ IL-4⁺ T and Treg cells in the spleen of mice in the experimental group were still significantly upregulated compared with the control group, and there was no significant difference in the ratio of CD4⁺ IL-17⁺ T cells, but compared with the control group, the percentage of CD4⁺ IFN-γ⁺ T cells was significantly decreased. *p  < 0.05, **p  < 0.01, n  = 6.