Global changes in gene expression related to Opisthorchis felineus liver fluke infection reveal temporal heterogeneity of a mammalian host response

Abstract

The food-borne trematode Opisthorchis felineus colonizes bile ducts of the liver of fish-eating mammals including humans. Among chronically infected individuals, this opisthorchiasis involves hepatobiliary problems, including chronic inflammation, periductal fibrosis, biliary intraepithelial neoplasia, and even cholangiocarcinoma. Despite numerous studies at the pathomorphological level, the systemic response and cellular pathogenesis of these disorders are not well studied. To conduct in-depth research and to gain insights into the mechanism by which O. felineus infection causes precancerous liver lesions, we (i) applied a next-generation-sequencing-based technology (high-throughput mRNA sequencing) to identify differentially expressed genes in the liver of golden hamsters infected with O. felineus at 1 and 3 months postinfection and (ii) verified the most pronounced changes in gene expression by western blotting and immunohistochemistry. A total of 2151 genes were found to be differentially expressed between uninfected and infected hamsters ("infection" factor), whereas 371 genes were differentially expressed when we analyzed "time × infection" interaction. Cluster analysis revealed that sets of activated genes of cellular pathways were different between acute (1 month postinfection) and chronic (3 months postinfection) opisthorchiasis. This enriched KEGG pathways were "Cell adhesion molecules", "Hippo signaling", "ECM-receptor interaction", "Cell cycle", "TGF-beta", and "P53 signaling". Moreover, epithelial-mesenchymal transition was the most enriched (q-value = 2.2E-07) MSigDB hallmark in the set of differentially expressed genes of all O. felineus-infected animals. Transcriptomic data were supported by the results of western blotting and immunohistochemistry revealing the upregulation of vimentin, N-cadherin, and α-smooth muscle actin postinfection. Our data expand knowledge about global changes in gene expression in the O. felineus-infected host liver and contribute to understanding the biliary neoplasia associated with the liver fluke infection.

Keywords: DEGs, differentially expressed genes; Differentially expressed gene; EMT, epithelial–mesenchymal transition; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes, O. felineus; Liver; Mesocricetus auratus; OF, Opisthorchis felineus; Opisthorchis felineus; hamster.

Fig. 1

Differentially expressed genes (DEGs) in the liver of Opisthorchis felineus–infected hamsters at 1 and 3 months postinfection. A. Principal component analysis identified two distinct clusters in the data separated along the first principal component. B. The most enriched KEGG pathways in the set of DEGs (interaction of factors: “infection × time”). C. A Euler diagram illustrating overlaps of the sets of genes matching the enriched KEGG pathways among the DEGs upregulated (>2-fold) in the OF-infected hamsters Mesocricetus auratus (“infection” factor). ECM: extracellular matrix.

Fig. 2

Clustering analysis of 376 DEGs (p_adj < 0.1; interaction of factors: “infection × time”) found in the liver of hamsters (Mesocricetus auratus) infected with Opisthorchis felineus (at 1 and 3 months p.i.).

Fig. 3

Histopathological analysis of a hamster liver at 3 months postinfection (p.i.) with Opisthorchis felineus metacercariae. A. The liver of an uninfected animal; B and C: biliary intraepithelial neoplasia at 3 months p.i. Biliary neoplasia was detectable, including the presence of nucleoli and loss of polarity with a transition from the normal duct epithelium to an atypical epithelium. E: epithelial cells; OF: O. felineus.

Fig. 4

Expression of epithelial–mesenchymal transition (EMT) genes in the liver of hamsters (Mesocricetus auratus) at 1 and 3 months postinfection (p.i.) with Opisthorchis felineus (OF). A. Immunohistochemical analysis of expression levels of MMP9, E-cadherin, N-cadherin, type 1 collagen, and α-smooth muscle actin in an OF-infected M. auratus liver at 1 and 3 months p.i. BD: bile duct, PF: periductal fibrosis, BV: blood vessel, Inf: inflammatory infiltrate, and E: epithelial cells. B. A heat map illustrating z-scored mRNA abundance of 16 EMT signature genes was constructed using the heatmap.2 (v.2.38) R-package. Z-scoring normalization was conducted using DESeq2 R-package). C. E-cadherin, N-cadherin, vimentin, and α-smooth muscle actin protein levels assessed by western blotting. α-SMA: α-smooth muscle actin. D. Protein levels were determined by densitometry of immunoblots (E), and the results were normalized to β-actin. The data are presented as mean ± SD; *p < 0.05. SD was calculated from the results of three independent experiments.

Fig. 5

Multiple pathways implicated in neoplasia formation. Chronic inflammation during Opisthorchis felineus infection leads to the activation of signaling cascades including p53, NF-κB, JAK/STAT, and TGF-β pathways. Fluke-derived substances and metabolites secreted into the host microenvironment (oxysterols and excretory-secretory [ES] products) may induce metabolic processes (including oxidative stress) that facilitate damage to the chromosomal DNA of cholangiocytes. In addition, physical damage to host tissues by parasites together with the wound healing process cause recurrent wound healing and cell proliferation. Such cofactors as dietary nitrosamines or changes in the microbiome may promote the pathology. Combined events in the parasite–host interaction (chronic inflammation, parasite-derived substances, and physical damage) and their effects on host chromosomes alter cell growth, proliferation, and survival. Created with BioRender.com.