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Clonorchis sinensis lysophospholipase A upregulates IL-25 expression in macrophages as a potential pathway to liver fibrosis.

ABSTRACT: Liver fibrosis is an excessive wound-healing reaction that requires the participation of inflammatory cells and hepatic stellate cells (HSCs). The pathogenesis of liver fibrosis caused by viruses and alcohol has been well characterized, but the molecular mechanisms underlying liver fibrosis induced by the liver fluke Clonorchis sinensis are poorly understood. Lysophospholipase A (LysoPLA), which deacylates lysophospholipids, plays a critical role in mediating the virulence and pathogenesis of parasites and fungi; however, the roles of C. sinensis lysophospholipase A (CsLysoPLA) in C. sinensis-induced liver fibrosis remain unknown.A mouse macrophage cell line (RAW264.7) was cultured and treated with CsLysoPLA. IL-25 and members of its associated signaling pathway were detected by performing quantitative real-time PCR, Western blotting and immunofluorescent staining. A human hepatic stellate cell line (LX-2) was cultured and exposed to IL-25. LX-2 cell activation markers were examined via quantitative real-time PCR, Western blotting and immunofluorescent staining. Migration was analyzed in transwell plates.Treating RAW264.7 cells with CsLysoPLA significantly induced IL-25 expression. Elevated PKA, B-Raf, and ERK1/2 mRNA levels and phosphorylated B-Raf and ERK1/2 were detected in CsLysoPLA-stimulated RAW264.7 cells. The PKA inhibitor H-89 weakened B-Raf and ERK1/2 phosphorylation whereas the AKT activator SC79 attenuated ERK1/2 phosphorylation in RAW264.7 cells. Both H-89 and SC79 inhibited CsLysoPLA-induced IL-25 upregulation. In addition, stimulation of LX-2 cells with IL-25 upregulated the expression of mesenchymal cell markers, including ?-smooth muscle actin (?-SMA) and collagen type I (Collagen-I), and promoted cell migration.CsLysoPLA activates HSCs by upregulating IL-25 in macrophages through the PKA-dependent B-Raf/ERK1/2 pathway and potentially promotes hepatic fibrosis during C. sinensis infection.

SUBMITTER: Zhou L

PROVIDER: S-EPMC5474055 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Clonorchis sinensis lysophospholipase A upregulates IL-25 expression in macrophages as a potential pathway to liver fibrosis.

Zhou Lina L Shi Mengchen M Zhao Lu L Lin Zhipeng Z Tang Zeli Z Sun Hengchang H Chen Tingjin T Lv Zhiyue Z Xu Jin J Huang Yan Y Yu Xinbing X

Parasites & vectors 20170617 1

<h4>Background</h4>Liver fibrosis is an excessive wound-healing reaction that requires the participation of inflammatory cells and hepatic stellate cells (HSCs). The pathogenesis of liver fibrosis caused by viruses and alcohol has been well characterized, but the molecular mechanisms underlying liver fibrosis induced by the liver fluke Clonorchis sinensis are poorly understood. Lysophospholipase A (LysoPLA), which deacylates lysophospholipids, plays a critical role in mediating the virulence and ...[more]

PMID: 28623940

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Project description:BackgroundChronic infection with Clonorchis sinensis can cause hepatobiliary fibrosis and even lead to hepatobiliary carcinoma. Epstein-Barr virus-induced gene 3 protein (EBI3) is a subunit of interleukin 35, which can regulate inflammatory response and the occurrence of fibrotic diseases. Previous studies have reported that the expression of EBI3 in the serum of patients with liver cirrhosis is reduced. The present study aims to investigate the biological effects of EBI3 on liver fibrosis caused by C. sinensis and the underlying molecular mechanisms.MethodsWe first established a mouse model of liver fibrosis induced by C. sinensis infection and then measured the serum expression of EBI3 during the inflammatory and fibrotic phase. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed to investigate the potential role of EBI3 in liver fibrosis by regulating the extracellular matrix structural constituent and collagen catabolic process. Recombinant protein EBI3 (rEBI3) was added to hepatic stellate cells (HSCs) in vitro with C. sinensis antigen to explore its function. Finally, the therapeutic effect of rEBI3 was verified by intravenous injection into C. sinensis-infected mice.ResultsThe results showed that the serum expression of EBI3 increased in the inflammatory response phase but decreased in the fibrotic phase. The excretory-secretory products of C. sinensis (Cs.ESP) were able to stimulate HSC activation, while rEBI3 reduced the activation of HSCs induced by Cs.ESP. Also, the protein expression of gp130 and downstream protein expressions of JAK1, p-JAK1, STAT3 and p-STAT3 in HSCs were increased after rEBI3 incubation. Finally, intravenously injected rEBI3 inhibited hepatic epithelial-mesenchymal transition in C. sinensis-infected mice by inhibiting HSC activation and reducing liver injury.ConclusionThis study confirms that rEBI3 can attenuate C. sinensis-induced liver fibrosis by inhibiting HSC activation and may be one of the potential treatments for liver fibrosis.

Project description:Clonorchis sinensis is the causative agent of the life-threatening disease endemic to China, Korea, and Vietnam. It is estimated that about 15 million people are infected with this fluke. C. sinensis provokes inflammation, epithelial hyperplasia, and periductal fibrosis in bile ducts, and may cause cholangiocarcinoma in chronically infected individuals. Accumulation of a large amount of biological information about the adult stage of this liver fluke in recent years has advanced our understanding of the pathological interplay between this parasite and its hosts. However, no developmental gene expression profiles of C. sinensis have been published. In this study, we generated gene expression profiles of three developmental stages of C. sinensis by analyzing expressed sequence tags (ESTs). Complementary DNA libraries were constructed from the adult, metacercaria, and egg developmental stages of C. sinensis. A total of 52,745 ESTs were generated and assembled into 12,830 C. sinensis assembled EST sequences, and then these assemblies were further categorized into groups according to biological functions and developmental stages. Most of the genes that were differentially expressed in the different stages were consistent with the biological and physical features of the particular developmental stage; high energy metabolism, motility and reproduction genes were differentially expressed in adults, minimal metabolism and final host adaptation genes were differentially expressed in metacercariae, and embryonic genes were differentially expressed in eggs. The higher expression of glucose transporters, proteases, and antioxidant enzymes in the adults accounts for active uptake of nutrients and defense against host immune attacks. The types of ion channels present in C. sinensis are consistent with its parasitic nature and phylogenetic placement in the tree of life. We anticipate that the transcriptomic information on essential regulators of development, bile chemotaxis, and physico-metabolic pathways in C. sinensis that presented in this study will guide further studies to identify novel drug targets and diagnostic antigens.

Project description:BackgroundLiver fibrosis is a hallmark of clonorchiasis suffered by millions people in Eastern Asian countries. Recent studies showed that the activation of TGF-β/Smad signaling pathway can potently regulate the hepatic fibrogenesis including Schistosoma spp. and Echinococcus multilocularis-caused liver fibrosis. However, little is known to date about the expression of transforming growth factor-β (TGF-β) and other molecules in TGF-β/Smad signaling pathway which may play an important role in hepatic fibrosis caused by C. sinensis.MethodsA total of 24 mice were individually infected orally with 45 metacercariae, both experimental mice and mocked-infected control mice were anesthetized at 4 week post-infection (wk p.i.), 8 wk p.i. and 16 wk p.i., respectively. For each time-point, the liver and serum from each animal were collected to analyze histological findings and various fibrotic parameters including TGF-β₁, TGF-β receptors and down-stream Smads activation, as well as fibrosis markers expression.ResultsThe results showed that collagen deposition indicated by hydroxyproline content and Masson's trichrome staining was increased gradually with the development of infection. The expression of collagen type α1 (Col1a) mRNA transcripts was steadily increased during the whole infection. The mRNA levels of Smad2, Smad3 as well as the protein of Smad3 in the liver of C. sinensis-infected mice were increased after 4 wk p.i. (P < 0.05, compared with normal control) whereas the TGF-β₁, TGF-β type I receptor (TGFβRI) and TGF-β type II receptor (TGFβRII) mRNA expression in C. sinensis-infected mice were higher than those of normal control mice after 8 wk p.i. (P < 0.05). However, the gene expression of Smad4 and Smad7 were peaked at 4 wk p.i. (P < 0.05), and thereafter dropped to the basal level at 8 wk p.i., and 16 wk p.i., respectively. The concentrations of TGF-β₁ in serum in the C. sinensis-infected mice at 8 wk p.i. and 16 wk p.i (P < 0.05) were significantly higher than those in the control mice.ConclusionsThe results of the present study indicated for the first time that the activation of TGF-β/Smad signaling pathway might contribute to the synthesis of collagen type I which leads to liver fibrosis caused by C. sinensis.

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Clonorchis sinensis lysophospholipase A upregulates IL-25 expression in macrophages as a potential pathway to liver fibrosis.

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Clonorchis sinensis lysophospholipase A upregulates IL-25 expression in macrophages as a potential pathway to liver fibrosis.

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