(C) PLOS One
This story was originally published by PLOS One and is unaltered.
 . . . . . . . . . .



TLR3 activation by Clonorchis sinensis infection alleviates the fluke-induced liver fibrosis [1]

['Yuru Wang', 'State Key Laboratory For Zoonotic Diseases', 'Key Laboratory For Zoonosis Research Of The Ministry Of Education', 'Institute Of Zoonosis', 'College Of Veterinary Medicine', 'Jilin University', 'Changchun', 'Pengtao Gong', 'Xuancheng Zhang', 'Xiaocen Wang']

Date: 2023-05 

Abstract Clonorchis sinensis is a zoonotic parasite associated with liver fibrosis and cholangiocarcinoma development. The role of toll-like receptors (TLRs) in C. sinensis infection has not yet been fully elucidated. Here, the TLR3 signaling pathway, cytokine expression and liver fibrosis were examined in C. sinensis-infected wildtype (WT) and TLR3-/- mice. Polyinosinic-polycytidylic acid (Poly (I:C)) was used to treat C. sinensis infections. The results showed that TLR3 deficiency caused severe clonorchiasis with increased parasite burden, exacerbated proinflammatory cytokine expression and liver lesions, promoted the TGF-β1/Smad2/3 pathway and myofibroblast activation, exacerbated liver fibrosis (compared to WT mice). Poly (I:C) intervention increased the body weight, decreased mouse mortality and parasite burden, reduced liver inflammation, and alleviated C. sinensis-induced liver fibrosis. Furthermore, C. sinensis extracellular vesicles (CsEVs) promote the production of IL-6, TNF in WT biliary epithelial cells (BECs) via p38/ERK pathway, compared with control group, while TLR3 deletion induced much higher levels of IL-6 and TNF in TLR3-/- BECs than that in WT BECs. Taken together, TLR3 inhibit IL-6 and TNF production via p38/ERK signaling pathway, a phenomenon that resulted in the alleviation of C. sinensis-induced liver fibrosis. Poly (I:C) is a potential treatment for clonorchiasis.

Citation: Wang Y, Gong P, Zhang X, Wang X, Zhang X, Zhang N, et al. (2023) TLR3 activation by Clonorchis sinensis infection alleviates the fluke-induced liver fibrosis. PLoS Negl Trop Dis 17(5): e0011325. https://doi.org/10.1371/journal.pntd.0011325 Editor: Krystyna Cwiklinski, University of Liverpool, UNITED KINGDOM Received: January 9, 2023; Accepted: April 21, 2023; Published: May 11, 2023 Copyright: © 2023 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: We received support for this work through the National Key R&D Program of China (https://www.most.gov.cn), grant number "2017YFD0501200 / 2017YFD0501305" to JL and XZ. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Summary Clonorchis sinensis is a zoonotic parasite associated with liver fibrosis and cholangiocarcinoma development. Further understanding of the pathogenesis of C. sinensis especially liver fibrosis would help us develop a novel strategy for controlling clonorchiasis. Toll-like receptors (TLRs) serve as the host’s first line of defense against pathogens, however the role of TLRs in C. sinensis infection has not yet been fully elucidated. To elucidate the potential mechanism of TLR3 in C. sinensis-induced liver fibrosis, liver fibrosis modeling assays and cellular level assays in vivo and in vitro were performed using TLR3-deficient or normal C57/BL6 mice in our research. A new role for TLR3 in controlling C. sinensis-induced liver fibrosis was identified. TLR3 deficiency resulted in severe clonorchiasis increased parasite burden, exacerbated proinflammatory cytokine expression and liver lesions, promoted the TGF-β1/Smad2/3 pathway and myofibroblast activation, aggravated liver fibrosis. Poly (I:C) intervention alleviated liver inflammation and liver fibrosis in mice infected with C. sinensis and is a promising drug for clonorchiasis treatment. Our results will help in understanding the molecular mechanisms governing the host’s immune responses to C. sinensis infections and provide new information about clonorchiasis treatment.

Introduction Clonorchis sinensis is a zoonotic parasitic that is also known as the Chinese liver fluke. It inhabits the biliary system of humans and other piscivorous animals and can cause clonorchiasis [1]. Definitive hosts are infected by consuming under cooked or raw freshwater fish that contain metacercariae. Approximately 15 million people are infected by this liver flukes in East Asia, including over 13 million in China [2]. Infection is usually asymptomatic, although it may cause bile duct inflammation, hyperplasia, and fibrosis in chronically infected [1]. As it can cause cholangiocarcinoma (CCA) in chronic patients, C. sinensis has been classified as a class I carcinogen [3,4]. The recommended treatment for clonorchiasis is praziquantel; however, side effects such as mild or transient headaches, dizziness, nausea, abdominal discomfort, and drug resistance are unavoidable [1]. An effective vaccine for C. sinensis is currently unavailable [1,5]. Further understanding of the pathogenesis of C. sinensis especially liver fibrosis and CCA would help us develop a novel strategy for controlling clonorchiasis. Toll-like receptors (TLRs) are the most potent initiators of the inflammatory response and serve as the host’s first line of defense against pathogens [6]. The TLR4-TGF-β/Smad signaling pathway regulates myofibroblast activation induced by C. sinensis [7]. The TLR2-regulated MAPK pathway and the release of reactive oxygen species regulate the C. sinensis-induced inflammatory response [8]. TLR9 recognizes CsEVs, promotes IL-6 and TNF release, and regulates inflammatory responses in clonorchiosis [9]. Evidence suggests that TLR3 is involved in the progression of liver fibrosis. Studies have confirmed that TLR3 is an important target protein that regulates Hepatic stellate cells (HSCs) activation and promotes liver regeneration. Polyinosinic-polycytidylic acid (Poly (I:C) (an agonist of TLR3) can significantly inhibit the activation of HSCs and block liver regeneration [10]. Moreover, TLR3 activation promotes the secretion of IL-10 and PGE2 by bone marrow mesenchymal stem cells (BMMSCs) to improve the therapeutic function of BMMSCs in alcoholic liver disease [11]. However, whether C. sinensis can activate the TLR3 pathway and the role of TLR3 in liver fibrosis caused by C. sinensis has not been extensively explored. To investigate the role of TLR3 in C. sinensis-induced liver fibrosis, we established a mouse model of C. sinensis-induced liver fibrosis using wildtype (WT) and TLR3-/- mice and examined various parameters associated with clonorchiosis, including weight, mortality, parasite burden, liver inflammation and biliary injury, cytokine expression, myofibroblast activation, TGF-β1 expression, phosphorylation level of Smad2/3, and intrahepatic collagen deposition. We also intervened C. sinensis-infected mice with poly (I:C) and evaluated the above indicators to understand the therapeutic effects of the TLR3 agonist on clonorchiasis caused by C. sinensis. To determine whether TLR3 and its downstream pathways regulate proinflammatory cytokine expression, mouse biliary epithelial cell (BECs) were stimulated with C. sinensis extracellular vesicles (CsEVs), and phosphorylation of p38, ERK, and p65, as well as cytokine expression, were examined. In addition, the mouse model of CsEVs injection was established to investigate the roles of CsEVs in regulating the activation of TLR3 and the contribution to biliary injuries.

Materials and methods Ethics statement All experimental procedures involving animals were conducted in compliance with Chinese legal standards, and the experiments were approved by the Animal Welfare and Research Ethics Committee of Jilin University. (IACUC permit number: 20160612). Animals TLR3-/- and WT female C57BL/6 mice (aged 6–8 weeks) were housed under constant temperature and pathogen-free animal conditions for 12 h on a dark/light cycle with sterile water and normal mouse chow. Collection of C. sinensis and CsEVs The C. sinensis excretory/secretory proteins (CsESPs) were immediately used to isolate CsEVs [9]. The C. sinensis adults were isolated from bile duct of metacercariae infected WT C57BL/6 mice: The mice were euthanized after 35 days post infection (dpi) and disinfected with 75% alcohol for 15 min. Then, the mice were transferred to a sterile platform, and aseptic procedures were performed throughout the trial. Carefully isolate the liver, soak it in sterile PBS, and open the bile duct. Gently pressing the liver using ophthalmic forceps and picking out the adults. The isolate adults were incubated at density of five parasites per mL of serum-free RPMI-1640 at 37°C for 6 h. The incubated supernatant was collected and centrifuged at 280×g and 2 000×g, continuously. Then the supernatant was filtered through a syringe filter (0.22-μm, Millipore, Massachusetts, USA). Then a slightly modified differential centrifugation method was used to collect CsEVs from the CsESPs [12]. Briefly, CsESPs were centrifuged at 10 000 ×g for 1 h, the supernatant was collected then passed through a filter (0.22 μm). The filtered liquid was centrifuged at 100 000 ×g for 1 h, and the sediment was collected. The precipitate was dissolved in sterile phosphate-buffered saline (PBS) and the CsEVs concentration was determined using BCA. Then, CsEVs on copper grids were negatively stained using carbon and 3% phosphotungsticacid and observed under a transmission electron microscope (TEM) (HITACHI, Tokyo, Japan). And a fraction of CsEVs were treated with RNase A (Solarbio, Beijing, China) to remove dsRNAs in CsEVs-free ESPs (rCsEVs). Isolation of mouse BECs The biliary tree was obtained by removing liver membranes and hepatocytes through perfusion using HEPS solution (Biosharp, Anhui, China) (with 0.05mg/mL collagenase IV(Sigma-Aldrich, Missouri, US)) as conventional methods [13,14]. Then, the isolated biliary tree was divided into small pieces and further digested in 0.05mg/mL collagenase IV solution for 30 min under shaking conditions, and were centrifuged at 800 ×g for 10 min. The precipitate was retained, digested by 0.25% trypsin-digested (VivaCell, Shanghai, China) at 37° C for 10min. After centrifugation (800 ×g for 10 min), trypsin was discarded and RPMI-1640 medium with 5% serum was added to stop digestion. After repeated washing and centrifugation, the BECs were resuspended using RPMI-1640 medium (with 5% serum) and filtered through a 75μm aperture metal sieve. Cell number and viability were assessed using a 0.4% trypan blue solution (Procell, Wuhan, China) and BECs with 3 ×105 were plated incubated at 37°C with 5% CO 2 in 6 well tissue culture plates. To investigate CsEVs functions, The BECs were plated and treated with the CsEVs (50 μg/mL), rCsEVs (50 μg/mL) or PBS cultures for 2 h or 18 h at 37°C with 5% CO 2 , respectively. C. sinensis-caused liver fibrosis mouse model TLR3-/- and WT mice were inoculated with 200 C. sinensis metacercariae by gavage in 200 μL of PBS (pH 7.4), with mice administered 200 μL of PBS as the control. Poly (I:C)-intervened WT mice were injected intraperitoneally twice with poly (I:C) (10 mg/kg) (InvivoGen, California, US) at 0, 10, and 20 dpi. Weight and mortality rates were monitored daily until sacrifice. Mice were euthanized at 7, 15, and 35 dpi to examine the liver lesions and intrahepatic parasite burdens. Mouse faeces were collected daily after infection. Rice grain sized faeces were placed on a slide, added 50 μL of saline then covered with a coverslip to examine the eggs under the microscope. The liver tissues were isolated and used for cytokine detection, quantitative real-time PCR (RT-qPCR), western blotting, histological observation, Masson staining, and immunohistochemical staining. CsEVs injection The mice of CsEVs group were intravenously injected with CsEVs (50 μg/mice, 50 μL) or PBS (50 μL) at the indicated time (1d, 3d,5d,7d) [15]. At the 8d, the mice were euthanized, and the livers were collected to detect TLR3 mRNA expression, cytokine secretion, liver lesions and collagen deposition. RT-qPCR analysis Total RNAs from WT mouse livers and BECs was extracted using the TRIzol reagent and the cDNA was synthesized from the total RNA using reverse transcriptase prior to the PCR step. Then the mRNA levels were determined by RT-qPCR followed the conditions and procedures of Green qPCR Mix (Monad, Suzhou, China). The primer sequences used were TLR3 F: 5-AAGACAGAGACTGGGTCTGGG-3, R:5- AAGGACGCCTGCTTCAAAGT-3; and GAPDH F: 5-CCATGTTTGTGATGGGTGTG-3, R: 5-CCTTCTTG ATGTCATCATAC-3 [16]. The data were normalized to GAPDH and the mRNA level of all experimental groups was presented in terms of numerical vales calculated using (delta)(delta)C/t equation. Cytokine detection Liver tissues of TLR3-/- and WT mice were lysed into cell suspensions, and the supernatant was stored for cytokine detection. WT and TLR3-/- mouse BECs were incubated with CsEVs (50 μg/mL) for 18 h, and poly (I:C) (30 μg/mL) was used as the positive control [17]. To investigate the role of p38 and ERK signaling pathways in regulating cytokine production, the WT BECs were pretreated with p38 or ERK inhibitors (Sigma-Aldrich, Missouri, US) for 60 min at 37°C, with untreated cells as the control. Then the cells were co-stimulated with CsEVs for 18 h. The secretion levels of cytokines in the liver tissues and BECs supernatant were detected using ELISA kits (IL-4/IL-6/IFN-γ/TGF-β1/TNF, Thermo Scientific, Massachusetts, US) [17]. Histology observation Liver tissues from the same positions were removed and embedded in paraffin. The tissue sections were sliced to a thickness of 3 μm, deparaffinized with xylene, and stained with hematoxylin and eosin. Hepatic injury and inflammation were thoroughly documented under a microscope and evaluated using the hepatic histological activity index (HAI) [18,19]. Masson staining The paraffin-embedded liver tissues were subjected to the same procedure until routine deparaffinization, and the tissue sections were stained with Masson’s Trichrome Stain Kit (Solarbio, Beijing, China). The positive area of collagen fibers was scanned and quantified using the Image-Pro Plus software (Media Cybernetics, Massachusetts, USA). Immunohistochemistry Liver tissue was sliced into 5 μm sections and analyzed using routine immunohistochemistry. After peroxidase removal and antigen repair, the liver tissue sections were treated with FBS at room temperature for 30 min and then incubated overnight with CK-19 and α-SMA antibodies at 4°C, followed by incubation with HRP-conjugated antibodies. After counterstaining with hematoxylin, the tissue sections were mounted with neutral gum. The positive area of immunohistochemistry was digitized and analyzed using the Image-Pro Plus software. Antibody information is presented in S1 Table. Immunofluorescence Adult C. sinensis was isolated, transferred to a cell culture dish, and incubated in 1640 medium for 12 h. After washing with sterile PBS, the worms were fixed and permeabilized, and dsRNAs was labeled with J2 antibody and FITC-labeled fluorescent secondary antibody. The nuclei were stained with Hoechst (Sigma-Aldrich, Missouri, US), and the subcellular localization of dsRNA was observed using the Live Cell Imaging System (Olympus, Tokyo, Japan). Antibody information is presented in S1 Table. Western blot Liver tissues and BECs were collected and resuspended in RIPA lysis buffer containing PMSF (1:100) (Boster Bio, California, USA). The SDS-PAGE and membrane transfer tests were executed as previously reported [17]. The membranes were incubated overnight at 4°C with primary antibodies (Phosphorylated protein p65, ERK, Smad2/3, p38 and Total protein p65, ERK, Smad2/3, p38). Membranes were incubated with secondary antibody for 1 h at room temperature after three washes with PBST. An ECL-Chemiluminescence meter was used to visualize the protein (Clinx Science Instruments Co., Ltd., Shanghai, China). The protein expression level was quantified using ImageJ (National Institutes of Health, Bethesda, Maryland, USA) and the relative gray values of phospho-p65/GAPDH, phospho-Smad2/3/GAPDH, phospho-ERK/GAPDH and phospho-p38/GAPDH were calculated using Excel (Microsoft Corp., Redmond, WA, USA) software, respectively [9]. Antibody information is presented in S1 Table. Dot Immunobinding Assay (DIBA) The dot immunoassay was performed according to the previous protocol [20]. Briefly, the sheared nitrocellulose membranes (NCM) were placed into reaction wells and 50 μL of CsEVs (1 mg or 0.1mg/mL), Poly (1:C) (30 μg/mL), and PBS were dropped onto NCM, and acted at room temperature for 30 min, and the remaining liquid was discarded. Then NCM was sealed with 5% milk, and then incubated overnight with J2 antibody at 4°C. The membrane was incubated at room temperature with HRP-link antibodies for 30 min after three washes with PBS, and the results were viewed using an imaging system. Antibody information is presented in S1 Table. Statistical analysis GraphPad Prism Software (version 6.01) was used to conduct Tukey tests (T-tests) and two-way ANOVA on the data set and generated pictures (GraphPad Software Inc, California, US). The experiment data was obtained from three independent experiments, with the results expressed as the mean ± SEM. Significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001.

Discussion TLR activation provides the first line of defense in the anti-pathogen immune response. The role of TLR3 in liver diseases promptes us to investigate whether it is involved in the liver fibrosis process caused by C. sinensis [21–23]. To elucidate the potential mechanism of TLR3 in C. sinensis-induced liver fibrosis, liver fibrosis modeling assays were performed using TLR3-deficient or normal C57/BL6 mice in our research. The experimental data from both types of mice helped us to explore the role of TLR3 in liver fibrosis caused C. sinensis and the mechanism of host-parasite interaction. It is noteworthy that TLR3 deficiency caused severe clonorchiasis with lower survival quality, higher liver damage, and more severe liver fibrosis. This study suggests that TLR3-based treatments have a great potential for applications in C. sinensis and other parasitic liver fibrosis. EVs produced by C. sinensis, carrying parasite information, activating the innate immune response of mouse macrophages and BECs, produce proinflammatory cytokines [9,15]. The bile ducts are the normal parasitic site of C. sinensis, and the immune response of BECs TLRs play the important role during C. sinensis infection [9,24]. We used CsEVs and BECs to simulate the interaction process between C. sinensis and host in vitro, and found that C. sinensis releases large amounts of dsRNA via CsEVs, which were recognized by host TLR3 and activated the innate immune response. The deregulation of response of TLRs to PAMPs in BECs can lead to various liver diseases [25]. The IL-6 and TNF secreted by BECs and macrophages are the vital cytokines in C. sinensis induced liver injure and fibrosis [15,24]. The present data indicated that BECs TLR3 deficiency resulted in significantly increased IL-6 and TNF secretion induced by CsEVs, which may be an important reason for more severe liver damage and liver fibrosis in TLR3-/- mice infected with C. sinensis. And this inference was confirmed in the CsEVs injection mice. TLR3-/- mice injection with CsEVs resulted in higher IL-6 and TNF expression, leading to more severe biliary injures. On the other hand, the infection intensity of helminths is closely related to the severity of liver lesions caused by them [26]. We found that an important result of TLR3 deficiency was a significantly higher number of infected worms, which may be another important reason for more severe liver damage and liver fibrosis in TLR3-/- mice infected with C. sinensis. C. sinensis infection significantly activates the TGF-β/Smad pathway, leading to liver fibrosis [7,27]. The recombinant worm proteins rCsMF6p/HDM promote immune response and cell differentiation through the MAPK pathway [28]. However, the interaction between TGF-β/Smad and p38 in liver fibrosis induced by C. sinensis has not been clarified. Isorhamnetin protects against liver fibrosis via inhibition of TGF-β1-mediate Smad3 and p38 MAPK signaling pathways [29]. Drug-containing serum of rhubarb-astragalus reduce the protein expression of TGF-β1 and p38 MAPK and mRNA expression of SMA-α, Smad2 and Smad3 in HK-2 cells caused by the increase of TGF-β1, and the same results are found in the treatment of p38 inhibitors[30]. These studies indicate that TGF-β/Smad pathway and p38 pathway have the mutual regulatory role in the regulation of the epithelial-mesenchymal transformation and liver fibrosis. Our study clarified the mechanism by which TLR3-p38/ERK regulated cytokine expression promoted inflammation and damage, and also found that TLR3 deletion leaded to increased activation of TGF-β/Smad pathway in C. sinensis-induced liver fibrosis. We infer that TLR3-p38/ERK regulated cytokine expression may be one of the factors inducing TGF-β/Smad pathway activation which needs to be further explored in future research. Based on this concept, we achieved promising results with TLR3 agonists for the treatment of C. sinensis. Poly (I:C), a viral dsRNA mimetic, is the most commonly used TLR3 [31]. In parasite control, poly (I:C) is used as a vaccine adjuvant to induce a multifunctional CD4+ T cell response and enhance antibody production against Plasmodium falciparum [32,33]. Total Leishmania antigens-poly (I:C) immunization resultes in good protection in mice, which is associated with decreased footpad swelling, histopathological alterations in the footpads, and parasite burdens [31]. There is no evidence that poly (I:C) can be used to control or treat liver flukes. In this study, we used poly (I:C) to treat liver fibrosis caused by C. sinensis. Poly (I:C) intervention significantly blocked the acute phase of death in mice, reduced the number of intrahepatic parasites, and alleviated C. sinensis-induced liver fibrosis, which demonstrated that poly (I:C) has great potential for application in the treatment of clonorchiasis caused by C. sinensis. Opisthorchis viverrini infection induces liver fibrosis and even cholangiocarcinoma, causing the serious disease burden in Southeast Asian countries [34]. Whether the positive contribution of poly (I:C) is also applicable to the pathogenic process of O. viverrini deserves further investigation in future studies. In summary, a new role for TLR3 in controlling C. sinensis-induced liver fibrosis was identified (Fig 10). TLR3 deficiency resulted in severe clonorchiasis in C. sinensis-infected mice compared with WT mice. Poly (I:C) is a promising drug for clonorchiasis treatment caused by C. sinensis. Our results will help in understanding the molecular mechanisms governing the host’s immune responses to C. sinensis infections and provide new information about clonorchiasis treatment. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 10. TLR3 inhibit IL-6 and TNF production via p38/ERK signaling pathways alleviate C. sinensis-induced liver fibrosis. https://doi.org/10.1371/journal.pntd.0011325.g010

Acknowledgments We thank Fucheng Ma, Heng Yang, Ran Wei and Zhu Ming for their excellent help in the laboratory work and the animal experiments.

[END]
---
[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0011325

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/