Dataset Information

Transcriptomic profiling of three-dimensional cholangiocyte spheroids long term exposed to repetitive Clonorchis sinensis excretory-secretory products.

ABSTRACT:

Background

Biliary tract infection with the carcinogenic human liver fluke, Clonorchis sinensis, provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma. Complications are proportional to the intensity and duration of the infection. In addition to mechanical irritation of the biliary epithelia from worms, their excretory-secretory products (ESPs) cause chemical irritation, which leads to inflammation, proliferation, and free radical generation.

Methods

A three-dimensional in vitro cholangiocyte spheroid culture model was established, followed by ESP treatment. This allowed us to examine the intrinsic pathological mechanisms of clonorchiasis via the imitation of prolonged and repetitive in vivo infection.

Results

Microarray and RNA-Seq analysis revealed that ESP-treated cholangiocyte H69 spheroids displayed global changes in gene expression compared to untreated spheroids. In ESP-treated H69 spheroids, 185 and 63 probes were found to be significantly upregulated and downregulated, respectively, corresponding to 209 genes (p < 0.01, fold change > 2). RNA-Seq was performed for the validation of the microarray results, and the gene expression patterns in both transcriptome platforms were well matched for 209 significant genes. Gene ontology analysis demonstrated that differentially expressed genes were mainly classified into immune system processes, the extracellular region, and the extracellular matrix. Among the upregulated genes, four genes (XAF1, TRIM22, CXCL10, and BST2) were selected for confirmation using quantitative RT-PCR, resulting in 100% similar expression patterns in microarray and RNA-Seq.

Conclusions

These findings broaden our understanding of the pathological pathways of liver fluke-associated hepatobiliary disorders and suggest a novel therapeutic strategy for this infectious cancer.

SUBMITTER: Kim JW

PROVIDER: S-EPMC8056535 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundClonorchiasis, caused by the infection of Clonorchis sinensis (C. sinensis), is a kind of neglected tropical disease, but it is highly related to cholangiocarcinoma and hepatocellular carcinoma (HCC). It has been well known that the excretory/secretory products of C. sinensis (CsESPs) play key roles in clonorchiasis associated carcinoma. From genome and transcriptome of C. sinensis, we identified one component of CsESPs, severin (Csseverin), which had three putative gelsolin domains. Its homologues are supposed to play a vital role in apoptosis resistance of tumour cell.Methodology/principal findingsThere was significant similarity in tertiary structures between human gelsolin and Csseverin by bioinformatics analysis. We identified that Csseverin expressed at life stage of adult worm, metacercaria and egg by the method of quantitative real-time PCR and western blotting. Csseverin distributed in vitellarium and intrauterine eggs of adult worm and tegument of metacercaria by immunofluorence assay. We obtained recombinant Csseverin (rCsseverin) and confirmed that rCsseverin could bind with calciumion in circular dichroism spectrum analysis. It was demonstrated that rCsseverin was of the capability of actin binding by gel overlay assay and immunocytochemistry. Both Annexin V/PI assay and mitochondrial membrane potential assay of human hepatocarcinoma cell line PLC showed apoptosis resistance after incubation with different concentrations of rCsseverin. Morphological analysis, apoptosis-associated changes of mitochondrial membrane potential and Annexin V/PI apoptosis assay showed that co-incubation of PLC cells with rCsseverin in vitro led to an inhibition of apoptosis induced by serum-starved for 24 h.Conclusions/significanceCollectively, the molecular properties of Csseverin, a molecule of CsESPs, were characterized in our study. rCsseverin could cause obvious apoptotic inhibition in human HCC cell line. Csseverin might exacerbate the process of HCC patients combined with C. sinensis infection.

Project description:Liver fluke proteins, including excretory-secretory products (ESPs) and tegument proteins, are critical for the pathogenesis, nutrient metabolism, etiology and immune response of liver cancer. To understand the functions of various proteins in Clonorchis sinensis physiology and human clonorchiasis, the ESPs and tegument proteins of C. sinensis were identified. Supernatants containing ESPs from adult C. sinensis after culture for 6 h were harvested and concentrated. The tegument was detached using a freeze/thaw method and successively extracted using various extraction buffers. The outer surface proteins of C. sinensis were labeled with biotin, and the biotinylated proteins were purified. The ESP, tegument and labeled outer surface proteins were identified and analyzed by high-resolution LC-MS/MS. The identified proteins were compared with those of other flukes, and the protein functions associated with pathogenesis, carcinogenesis and potential vaccine antigens and drug targets were predicted and analyzed. A total of 175 proteins were identified after the 6-h culture of C. sinensis ESPs. A total of 352 tegument proteins were identified through sequential solubilization of the isolated teguments, and a subset of these proteins were localized to the surface membrane of the tegument by labeling with biotin. Thirty identified proteins, including annexins, actin and tetraspanins, were identified as potential immunomodulators and promising vaccine antigens. Interestingly, among the 352 tegument proteins, as many as 155 were enzymes, and most were oxidoreductases, hydrolases or transferases. A comparison of the outer surface proteins of C. sinensis with those of other flukes indicated that flukes have some common outer surface proteins, such as actin, tetraspanin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and annexin. Granulin, thioredoxin peroxiredoxin, carbonyl reductase 1 and cystatin were identified in the C. sinensis proteome and predicted to be related to liver disease and cancer. The analysis of the C. sinensis proteome could contribute to a more in-depth understanding of complex parasite-host relationships, improve the diagnosis of clonorchiasis and benefit research on the pathogenesis and development of novel interventions, drugs and vaccines to control C. sinensis infection.

Project description:Clonorchiasis, which is caused by Clonorchis sinensis, is an important foodborne disease worldwide. The excretory-secretory products (ESPs) of C. sinensis play important roles in host-parasite interactions by acting as causative agents. In the present study, the ESPs and sera positive for C. sinensis were collected to identify proteins specific to the sera of C. sinensis (i.e., proteins that do not cross-react with Fasciola hepatica and Schistosoma japonicum) at different infection periods. Briefly, white Japanese rabbits were artificially infected with C. sinensis, and their sera were collected at 7 days post-infection (dpi), 14 dpi, 35 dpi, and 77 dpi. To identify the specific proteins in C. sinensis, a co-immunoprecipitation (Co-IP) assay was conducted using shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) to pull down the sera roots of C. sinensis, F. hepatica, and S. japonicum. For the annotated proteins, 32, 18, 39, and 35 proteins specific to C. sinensis were pulled down by the infected sera at 7, 14, 35, and 77 dpi, respectively. Three proteins, Dynein light chain-1, Dynein light chain-2 and Myoferlin were detected in all infection periods. Of these proteins, myoferlin is known to be overexpressed in several human cancers and could be a promising biomarker and therapeutic target for cancer cases. Accordingly, this protein was selected for further studies. To achieve a better expression, myoferlin was truncated into two parts, Myof1 and Myof2 (1,500 bp and 810 bp), based on the antigenic epitopes provided by bioinformatics. The estimated molecular weight of the recombinant proteins was 57.3 ku (Myof1) and 31.3 ku (Myof2). Further, both Myof1 and Myof2 could be probed by the sera from rabbits infected with C. sinensis. No cross-reaction occurred with the positive sera of S. japonica, F. hepatica, and negative controls. Such findings indicate that myoferlin may be an important diagnostic antigen present in the ESPs. Overall, the present study provides new insights into proteomic changes between ESPs and hosts in different infection periods by LC-MS/MS. Moreover, myoferlin, as a biomarker, may be used to develop an objective method for future diagnosis of clonorchiasis.

Project description:BACKGROUND:Clonorchiasis is endemic in East and Southeast Asian countries. For a preventive strategy against infectious diseases, vaccination is the most effective. Here, we evaluated the molecular characteristics and immune responses of CsAg17 protein from Clonorchis sinensis, and investigated its protective effects against C. sinensis challenge. METHODS:A cDNA clone encoding CsAg17 protein and containing a secretory signal peptide at the N-terminus was retrieved from the C. sinensis transcriptome bank. Recombinant CsAg17 B-cell epitope protein and cDNA vaccines were produced and their immune responses were evaluated in FVB mice. The proportional changes of CD3+/CD4+ and CD3+/CD8+ T cells were detected by flow cytometry, and immune effectors were measured by ELISA. RESULTS:The CsAg17 mRNA was transcribed at a higher level in C. sinensis adults than in metacercariae. The CsAg17 protein was distributed in the sperms, oral and ventral suckers, and mesenchymal tissues of C. sinensis adults. In mice challenged with C. sinensis metacercariae, vaccination with CsAg17 protein and cDNA resulted in a reduction to 64% and 69% in worm burden, respectively. Both CsAg17 protein and cDNA vaccines increased the proportion of CD3+/CD4+ and CD3+/CD8+ T cells and stimulated the production of Th1 type cytokines such as interleukin (IL)-2, IL-12, and interferon-?, while maintaining minimum levels of Th2 cytokines. The levels of IgG specific to CsAg17 protein steeply increased in the two vaccinated groups from 2 weeks after immunization. The liver tissue retained good morphology in the mice vaccinated with CsAg17 protein or cDNA, whereas severe inflammation and large serous cysts were observed in the liver of the unvaccinated mice. CONCLUSIONS:Vaccination with CsAg17 protein and cDNA reduced the pathological changes in the bile duct and liver, and ameliorated the worm burden via cellular and humoral immune responses. Thus, they may serve as good vaccine candidates against C. sinensis infections.