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

Clonorchiasis, caused by Clonorchis sinensis, 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 C. sinensis extracellular vesicles (CsEVs) and evaluated their uptake by Kupffer cells (KCs) in vitro in immortalized KC (ImKC) line and in vivo in a model of C. sinensis 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 colony-stimulating factor 3 (CSF3), IL1b, and others. Further validation showed increased expressions of these genes in mice infected with C. sinensis. 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, CSF3 expression positively correlated with alpha smooth muscle actin (α-SMA) levels in the liver, both of which were higher compared with negative controls. CSF3 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 CSF3, 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.