Transcriptomic profiling of Kupffer cells exposed to Clonorchis sinensis extracellular vesicles: Unraveling the role of CSF3 in hepatic fibrosis

Clonorchiasis, caused by <i>Clonorchis sinensis</i>, is a significant public health issue in China and East Asia. However, understanding its pathological mechanisms underlying this disease remains limited. Here, we isolated and characterized <i>C. sinensis</i> extracellular vesicles (CsEVs) and evaluated their uptake by Kupffer cells (KCs) <i>in vitro</i> in immortalized KC (ImKC) line and <i>in vivo</i> in a model of <i>C. sinensis</i> infection, investigating genes with altered gene expression after treatment with CsEVs and during progression to liver fibrosis. CsEVs were successfully taken up by KCs to regulate gene expression. RNA-sequencing analysis identified 694 differentially expressed genes (DEGs), including upregulation of those encoding <i>colony-stimulating factor</i> 3 (<i>CSF</i>3), <i>IL1b</i>, and others. Further validation showed increased expressions of these genes in mice infected with <i>C. sinensis</i>. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that these DEGs were involved in pathways such as TNF signaling, and NF-κB signaling. During liver fibrosis progression, <i>CSF</i>3 expression positively correlated with alpha smooth muscle actin (α-SMA) levels in the liver, both of which were higher compared with negative controls. <i>CSF</i>3 inhibition caused a significant decrease in α-SMA expression. This study was the first to report differential gene expression changes in ImKCs stimulated by CsEVs, with <i>CSF</i>3, the most significantly upregulated gene, having a potential role in liver fibrosis. These findings provide important data for the pathology of clonorchiasis and could identify new targets for treatment.