关键词: 日本血吸虫, B细胞, RNA测序, 转录组

Abstract:

Objective To analyze the transcriptomic characteristics of splenic B cells in mice infected with Schistosoma japonicum, screen and validate differentially expressed genes (DEGs), and provide a basis for elucidating the regulatory mechanisms of B cells during S. japonicum infection. Methods Balb/C mice were randomly divided into the control group (NC group), the 4-week post-infection group (Sj_4W group), and the 6-week post-infection group (Sj_6W group). Mice in the Sj_4W and Sj_6W groups were infected percutaneously using the patch method with (25 ± 1) S. japonicum cercariae. Mice were sacrificed at week 4 and 6 post-infection and spleen tissues were collected, respectively. Splenic B cells were isolated and enriched by magnetic bead sorting. Total RNA was extracted from splenic B cells, and transcriptome libraries were constructed. RNA sequencing was performed on the Illumina platform. Differential expression analysis was conducted using the limma and edgeR packages to screen for DEGs. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis and gene ontology (GO) functional enrichment classification were performed on the DEGs. Three highly expressed genes were selected for validation by reverse transcription quantitative Real-time PCR (RT-qPCR). Results Cluster analysis revealed that a total of 6 443 DEGs were identified between the Sj_6W and NC groups, including 4 421 up-regulated genes and 2 022 down-regulated genes. A total of 6 757 DEGs were identified between the Sj_6W and Sj_4W groups, including 4 754 up-regulated genes and 2 003 down-regulated genes. GO functional enrichment analysis showed that downregulated genes were primarily involved in lymphocyte differentiation, immune response regulatory signaling pathways, and lymphocyte proliferation, whereas upregulated genes were mainly enriched in lymphocyte-mediated immunity, production of immune response molecular mediators, B cell-mediated immunity, and humoral immunity. KEGG pathway enrichment analysis revealed that downregulated genes were mainly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, Th1 and Th2 cell differentiation, and Th17 cell differentiation, whereas upregulated genes were mainly enriched in cytokine-cytokine receptor interaction, extracellular matrix-receptor interaction, and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. RT-qPCR validation confirmed that the relative mRNA expression levels of interleukin-27 (IL-27), matrix metalloproteinase-8 (MMP-8), and eosinophil protein X (EPX) in splenic B cells of the Sj_6W group were 2.14 ± 1.18, 21.86 ± 14.06, and 324.40 ± 252.40, respectively, which were significantly higher than those in the Sj_4W group (0.31 ± 0.18, 0.03 ± 0.02, and 0.44 ± 0.72; Mann-Whitney U test, P < 0.01) and also higher than those in the NC group (1.04 ± 0.29, 1.20 ± 0.72, and 1.17 ± 0.69; Mann-Whitney U test, P < 0.05). Conclusion Following S. japonicum infection, splenic B cells in mice underwent transcriptional reprogramming, with upregulated expression of IL-27, EPX, and MMP-8, accompanied by alterations in pathways related to lymphocyte differentiation and proliferation, Th1/Th2 and Th17 cell differentiation, production of immune response molecular mediators, B cell-mediated immunity and humoral immune regulation, cytokine-cytokine receptor interaction, and extracellular matrix-receptor interaction.

Key words: Schistosoma japonicum, B cells, RNA-sequencing, Transcriptome