Clonorchis sinensis infection induces pathological changes in feline bile duct epithelium and alters biliary microbiota composition.

BACKGROUND: Clonorchis sinensis is a zoonotic liver fluke that inhabits the bile ducts of the human liver for prolonged periods, leading to cholangiocarcinoma. Recent research indicates associations between altered biliary microbiota and bile duct disorders. However, the impacts of C. sinensis infection on bile duct epithelium and subsequent effects on biliary microbiota remain unknown. METHODS: Feline bile duct samples were collected from both uninfected and C. sinensis-infected cats. Histopathological examination was performed to assess epithelial changes, fibrosis, mucin and cell proliferation using hematoxylin-eosin staining and immunohistochemistry. Additionally, biliary microbiota composition was analyzed through 16S rRNA gene sequencing. Statistical analyses were conducted to compare the microbial diversity and relative abundance between infected and uninfected samples. RESULTS: Histopathological analysis of infected feline bile ducts revealed prominent epithelial hyperplasia characterized by increased cell proliferation. Moreover, periductal fibrosis and collagen fibrosis were observed in infected samples compared to uninfected controls. Biliary microbial richness decreased with disease progression compared to uninfected controls. Streptococcus abundance positively correlated with disease severity, dominating communities in cancer samples. Predictive functional analysis suggested that C. sinensis may promote bile duct lesions by increasing microbial genes for carbohydrate metabolism, replication, and repair. CONCLUSIONS: This study provides comprehensive insights into the pathological effects of C. sinensis infection on feline bile duct epithelium and its influence on biliary microbiota composition. These novel findings provide insight into C. sinensis pathogenesis and could inform therapeutic development against human clonorchiasis. Further research is warranted to elucidate the underlying mechanisms driving these changes and their implications for host-parasite interactions.

Title: L'infection par Clonorchis sinensis induit des changements pathologiques dans l'épithélium des voies biliaires félines et modifie la composition du microbiote biliaire. Abstract: Contexte : Clonorchis sinensis est une douve zoonotique du foie qui habite les voies biliaires du foie humain pendant des périodes prolongées, conduisant au cholangiocarcinome. Des recherches récentes indiquent des associations entre une altération du microbiote biliaire et des pathologies des voies biliaires. Cependant, les impacts de l'infection par C. sinensis sur l'épithélium des voies biliaires et les effets ultérieurs sur le microbiote biliaire restent inconnus. Méthodes : Des échantillons de voies biliaires félines ont été prélevés sur des chats non infectés et infectés par C. sinensis. Un examen histopathologique a été réalisé pour évaluer les modifications épithéliales, la fibrose, la mucine et la prolifération cellulaire à l'aide de la coloration à l'hématoxyline-éosine et de l'immunohistochimie. De plus, la composition du microbiote biliaire a été analysée par séquençage du gène de l'ARNr 16S. Des analyses statistiques ont été menées pour comparer la diversité microbienne et l'abondance relative entre les échantillons infectés et non infectés. Résultats : L'analyse histopathologique des voies biliaires félines infectées a révélé une hyperplasie épithéliale importante caractérisée par une prolifération cellulaire accrue. De plus, une fibrose péricanalaire et une fibrose du collagène ont été observées dans les échantillons infectés par rapport aux témoins non infectés. La richesse microbienne biliaire diminue avec la progression de la maladie par rapport aux témoins non infectés. L'abondance des streptocoques est positivement corrélée à la gravité de la maladie, dominant les communautés dans les échantillons avec cancer. L'analyse fonctionnelle prédictive suggère que C. sinensis pourrait favoriser les lésions des voies biliaires en augmentant les gènes microbiens pour le métabolisme des glucides, la réplication et la réparation. Conclusions : Cette étude fournit des informations complètes sur les effets pathologiques de l'infection à C. sinensis sur l'épithélium des voies biliaires félines et son influence sur la composition du microbiote biliaire. Ces nouvelles découvertes donnent un aperçu sur la pathogenèse de C. sinensis et pourraient éclairer le développement thérapeutique contre la clonorchiase humaine. Des recherches supplémentaires sont nécessaires pour élucider les mécanismes sous-jacents à l'origine de ces changements et leurs implications sur les interactions hôte-parasite.

Liver Flukes: Clonorchis and Opisthorchis.

Clonorchis sinensis, Opisthorchis viverrini and O. felineus are liver flukes of human and animal pathogens occurring across much of Europe and Asia. Nevertheless, they are often underestimated compared to other, better known neglected diseases in spite of the fact that many millions of people are infected and hundreds of millions are at risk. This is possibly because of the chronic nature of the infection and disease and that it takes several decades prior to a life-threatening pathology to develop. Several studies in the past decade have provided more information on the molecular biology of the liver flukes which clearly lead to better understanding of parasite biology, systematics and population genetics. Clonorchiasis and opisthorchiasis are characterized by a chronic infection that induces hepatobiliary inflammation, especially periductal fibrosis, which can be detected by ultrasonography. These chronic inflammations eventually lead to cholangiocarcinoma (CCA), a usually fatal bile duct cancer that develops in some infected individuals. In Thailand alone, opisthorchiasis-associated CCA kills up to 20,000 people every year and is therefore of substantial public health importance. Its socioeconomic impacts on impoverished families and communities are considerable. To reduce hepatobiliary morbidity and CCA, the primary intervention measures focus on control and elimination of the liver fluke. Accurate diagnosis of liver fluke infections in both human and other mammalian, snail and fish intermediate hosts is important for achieving these goals. While the short-term goal of liver fluke control can be achieved by praziquantel chemotherapy, a comprehensive health education package targeting school children is believed to be more beneficial for a long-term goal/solution. It is recommended that transdisciplinary research or multisectoral control approach including one health and/or eco health intervention strategy should be applied to combat the liver flukes and hence contribute to reduction of CCA in endemic areas.

Clonorchis sinensis aggravated liver fibrosis by activating PARP-1 signaling to induce parthanatos via DNA damage.

Clonorchis sinensis is an important food-borne zoonotic parasite that is highly associated with liver fibrosis and cholangiocarcinoma. Further understanding of the pathogenesis of C. sinensis, especially liver fibrosis, could help us develop novel strategies for controlling clonorchiasis. Poly (ADP-ribose) polymerase-1 (PARP-1) can induce cellular parthanatos which is reported to be involved in liver fibrosis. Currently, whether C. sinensis could activate PARP-1 signaling to induce parthanatos or whether parthanatos play a role in C. sinensis-induced liver fibrosis is not clear. In the present study, the expression of PARP-1 and parthanatos indicators were detected in C. sinensis-infected mouse liver and in human intrahepatic biliary epithelial cells (HiBEpiCs) incubated with excretory/secretory products (ESPs) of C. sinensis. To explore the role of PARP-1 in C. sinensis infection, PARP-1 inhibitor NMS-P118 was used to block PARP-1 expression in vivo and vitro. The mortality rate, body weight, worm load, liver and bile duct lesions as well as PARP-1 and parthanatos indicators in C57BL/6 mice infected with C. sinensis, or in HiBEpiCs incubated with C. sinensis ESPs and NMS-P118 were analyzed and compared to the group without NMS-P118. The results showed that C. sinensis infection induced the activation of PARP-1 signaling as well as the translocation of AIF and MIF into the nucleus in mouse liver. ESPs of C. sinensis could induce PARP-1 up-regulation, ATP depletion and DNA damage in HiBEpiCs, indicating that C. sinensis could induce parthanatos. Inhibiting PARP-1 with NMS-P118 significantly reduced liver fibrosis and the number of larvae, increased the survival rate and body weight gain of the mice infected with C. sinensis. In addition, NMS-P118 decreased the expression of PARP-1 and alleviated ATP depletion as well as DNA damage in HiBEpiCs incubated with ESPs of C. sinensis. Our data indicated that C. sinensis and its ESPs could activate PARP-1 signaling to induce cellular parthanatos. NMS-P118 treatment alleviated liver fibrosis and promoted survival of the mice by inhibiting PARP-1, which suggested that PARP-1 could be used as a potential therapeutic target against clonorchiasis.

FASN-mediated fatty acid biosynthesis remodels immune environment in Clonorchis sinensis infection-related intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer and is highly lethal. Clonorchis sinensis (C. sinensis) infection is an important risk factor for iCCA. Here we investigated the clinical impact and underlying molecular characteristics of C. sinensis infection-related iCCA. METHODS: We performed single-cell RNA sequencing, whole-exome sequencing, RNA sequencing, metabolomics and spatial transcriptomics in 251 patients with iCCA from three medical centers. Alterations in metabolism and the immune microenvironment of C. sinensis-related iCCAs were validated through an in vitro co-culture system and in a mouse model of iCCA. RESULTS: We revealed that C. sinensis infection was significantly associated with iCCA patients' overall survival and response to immunotherapy. Fatty acid biosynthesis and the expression of fatty acid synthase (FASN), a key enzyme catalyzing long-chain fatty acid synthesis, were significantly enriched in C. sinensis-related iCCAs. iCCA cell lines treated with excretory/secretory products of C. sinensis displayed elevated FASN and free fatty acids. The metabolic alteration of tumor cells was closely correlated with the enrichment of tumor-associated macrophage (TAM)-like macrophages and the impaired function of T cells, which led to formation of an immunosuppressive microenvironment and tumor progression. Spatial transcriptomics analysis revealed that malignant cells were in closer juxtaposition with TAM-like macrophages in C. sinensis-related iCCAs than non-C. sinensis-related iCCAs. Importantly, treatment with a FASN inhibitor significantly reversed the immunosuppressive microenvironment and enhanced anti-PD-1 efficacy in iCCA mouse models treated with excretory/secretory products from C. sinensis. CONCLUSIONS: We provide novel insights into metabolic alterations and the immune microenvironment in C. sinensis infection-related iCCAs. We also demonstrate that the combination of a FASN inhibitor with immunotherapy could be a promising strategy for the treatment of C. sinensis-related iCCAs. IMPACT AND IMPLICATIONS: Clonorchis sinensis (C. sinensis)-infected patients with intrahepatic cholangiocarcinoma (iCCA) have a worse prognosis and response to immunotherapy than non-C. sinensis-infected patients with iCCA. The underlying molecular characteristics of C. sinensis infection-related iCCAs remain unclear. Herein, we demonstrate that upregulation of FASN (fatty acid synthase) and free fatty acids in C. sinensis-related iCCAs leads to formation of an immunosuppressive microenvironment and tumor progression. Thus, administration of FASN inhibitors could significantly reverse the immunosuppressive microenvironment and further enhance the efficacy of anti-PD-1 against C. sinensis-related iCCAs.

Clonorchis sinensis calcium-binding protein Cs16 causes acute hepatic injury possibly by reprogramming the metabolic pathway of bone marrow-derived monocytes.

Introduction: Clonorchis sinensis infection results in various complications in the liver and biliary systems and is a neglected tropical disease in Eastern Asia. In this study, we report that C. sinensis calcium-binding protein Cs16 activates host immune cells and induces immunopathology in liver. Methods: Immunohistochemistry was used to detect the localization of Cs16 in C. sinensis adult worms. ELISA was used to detect the serum levels of anti-Cs16 IgG antibody in infected humans and mice. Bile duct injection model was used to figure out the role of Cs16 in vivo. RT-qPCR and ELISA were used to detect the cytokine production from Cs16-treated BMMs in vitro. Seahorse assay was used to detect the metabolic pathway of Cs16-treated BMMs in vitro. Result: Cs16 localizes in the tegument and gut of C. sinensis. Humans and mice with C. sinensis infection exhibited increased levels of anti-Cs16-specific antibody. Using the bile duct injection technique, we found that Cs16 induced obvious inflammation and hepatic necrosis in vivo. Cs16 treatment caused the upregulation of inflammatory cytokines in innate immune cells. Moreover, Cs16-treated monocytes relied more on the glycolytic metabolic pathway. Discussion: Our findings suggest that Cs16 is a potential pathogenic factor derived from C. sinensis adult worm. By reprogramming the metabolic pathway of innate immune cells, Cs16 triggers pro-inflammatory responses in the liver, and therefore, Cs16 is a potential target for the prevention and treatment of clonorchiasis.

Interleukin-33 deficiency prevents biliary injuries and repairments caused by Clonorchis sinensis via restraining type 2 cytokines.

BACKGROUND: Clonorchiasis caused by Clonorchis sinensis is a zoonotic parasitic disease characterized by cholangitis, biliary proliferation, biliary fibrosis, and even cholangiocarcinoma. Our previous study showed that the expression of interleukin (IL)-33 is increased in both humans and mice infected by C. sinensis, suggesting that IL-33 is potentially involved in the pathogenesis of clonorchiasis. However, the roles and potential mechanism of IL-33 underlying remain unknown. METHODS: Wild-type (WT) and IL-33 knockout (KO) mice (BALB/c female mice) were orally infected with 45 metacercariae of C. sinensis for 8 weeks. Biliary injuries and fibrosis were extensively evaluated. Hepatic type II cytokines (IL-4, IL-13, and IL-10) were detected by ELISA. RESULTS: For wild-type mice, we found that the mice infected with C. sinensis showed severe biliary injuries and fibrosis compared with the normal mice that were free from worm infection. In addition, the levels of type II cytokines such as IL-4, IL-13, and IL-10 in infected wild-type mice were significantly higher than in the control mice without infection (P < 0.05). However, IL-33 deficiency (IL-33 KO) prevents the augmentation of biliary injuries and fibrosis caused by C. sinensis infection. Furthermore, the increased levels of these type II cytokines induced by worm infection were also reversed in IL-33 KO mice. CONCLUSION: Our present study demonstrates that IL-33 contributes to the pathogenesis of C. sinensis-induced biliary injuries and repair, which can potentially orchestrate type 2 responses. These findings highlight the pathophysiological role of IL-33 in the progression of clonorchiasis.

MicroRNA-497 induced by Clonorchis sinensis enhances the TGF-ß/Smad signaling pathway to promote hepatic fibrosis by targeting Smad7.

BACKGROUND: Various stimuli, including Clonorchis sinensis infection, can cause liver fibrosis. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) with massive production of extracellular matrix (ECM). Our previous study showed that the TGF-ß1-induced Smad signaling pathway played a critical role in the activation of HSCs during liver fibrosis induced by worm infection; however, the mechanisms that modulate the TGF-ß/Smad signaling pathway are still poorly understood. Accumulating evidence demonstrates that miRNAs act as an important regulator of activation of HSCs during liver fibrosis. METHODS: The target of miR-497 was determined by bioinformatics analysis combined with a dual-luciferase activity assay. LX-2 cells were transfected with miR-497 inhibitor and then stimulated with TGF-ß1 or excretory/secretory products of C. sinensis (CsESPs), and activation of LX-2 was assessed using qPCR or western blot. In vivo, the mice treated with CCl4 were intravenously injected with a single dose of adeno-associated virus serotype 8 (AAV8) that overexpressed anti-miR-497 sequences or their scramble control for 6 weeks. Liver fibrosis and damage were assessed by hematoxylin and eosin (H&E) staining, Masson staining, and qPCR; the activation of the TGF-ß/Smad signaling pathway was detected by qPCR or western blot. RESULTS: In the present study, the expression of miR-497 was increased in HSCs activated by TGF-ß1 or ESPs of C. sinensis. We identified that Smad7 was the target of miR-497 using combined bioinformatics analysis with luciferase activity assays. Transfection of anti-miR-497 into HSCs upregulated the expression of Smad7, leading to a decrease in the level of p-Smad2/3 and subsequent suppression of the activation of HSCs induced by TGF-ß1 or CsESPs. Furthermore, miR-497 inhibitor delivered by highly-hepatotropic (rAAV8) inhibited TGF-ß/smads signaling pathway by targeting at Smad7 to ameliorate CCL4-induced liver fibrosis. CONCLUSIONS: The present study demonstrates that miR-497 promotes liver fibrogenesis by targeting Smad7 to promote TGF-ß/Smad signaling pathway transduction both in vivo and in vitro, which provides a promising therapeutic strategy using anti-miR-497 against liver fibrosis.

Clonorchis sinensis-Derived Protein Attenuates Inflammation and New Bone Formation in Ankylosing Spondylitis.

Helminth infections and their components have been shown to have the potential to modulate and attenuate immune responses. The objective of this study was to evaluate the potential protective effects of Clonorchis sinensis-derived protein (CSp) on ankylosing spondylitis (AS). Cytotoxicity of CSp at different doses was assessed by MTS and flow cytometry before performing experiments. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were obtained from AS patients. Inflammatory cytokine-producing cells were analyzed using flow cytometry. The levels of INF- γ , IL-17A, TNF-α, and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). SKG mice were treated with CSp or vehicles. Inflammation and new bone formation were evaluated using immunohistochemistry, positron emission tomography (PET), and micro-computed tomography (CT). Treatment with CSp resulted in no reduced cell viability of PBMCs or SFMCs until 24 h. In experiments culturing PBMCs and SFMCs, the frequencies of IFN- γ and IL-17A producing cells were significantly reduced after CSp treatment. In the SKG mouse model, CSp treatment significantly suppressed arthritis, enthesitis, and enteritis. Micro-CT analysis of hind paw revealed reduced new bone formation in CSp-treated mice than in vehicle-treated mice. We provide the first evidence demonstrating that CSp can ameliorate clinical signs and cytokine derangements in AS. In addition, such CSp treatment could reduce the new bone formation of AS.

Sodium-bile acid co-transporter is crucial for survival of a carcinogenic liver fluke Clonorchis sinensis in the bile.

The liver fluke Clonorchis sinensis inhabits the bile ducts, where bile concentration disparities across the fluke cell membrane can cause bile intoxication. Sodium-bile acid co-transporter (SBAT) plays a crucial role in bile acid recycling. The process by which SBAT imports bile acids is electrically coupled to sodium ion co-transportation. Here, we report that the SBAT of C. sinensis (CsSBAT) is involved in bile acid transportation. CsSBAT cDNA encoded a putative polypeptide of 546 amino acid residues. Furthermore, CsSBAT consisted of ten putative transmembrane domains, and its 3D structure was predicted to form panel and core domains. The CsSBAT had one bile acid- and three Na+-binding sites, enabling coordination of a symport process. CsSBAT was mainly localized in the mesenchymal tissue throughout the fluke body and sparsely localized in the basement of the tegument, intestinal epithelium, and excretory bladder wall. Bile acid permeated into the adult flukes in a short time and remained at a low concentration level. Bile acid accumulated inside the mesenchymal tissue when CsSBAT was inhibited using polyacrylic acid-tetradeoxycholic acid conjugate. The accumulated bile acid deteriorated the C. sinensis adults leading to death. CsSBAT silencing shortened the lifespan of the fluke when it was placed into bile. Taken together, we propose that CsSBAT transports bile acids in the mesenchymal tissue and coordinate with outward transporters to maintain bile acid homeostasis of C. sinensis adults, contributing to C. sinensis survival in the bile environment.

Dopaminergic antagonists inhibit bile chemotaxis of adult Clonorchis sinensis and its egg production.

Human clonorchiasis, caused by Clonorchis sinensis, is endemic in East Asian countries. C. sinensis metacercariae excyst in the duodenum of mammalian hosts, migrate to the intrahepatic bile duct, and mature into adults in the milieu of bile. We have previously shown that newly excysted juvenile C. sinensis move chemotactically toward bile and bile acids. Here, the chemotactic behavior of adult C. sinensis (CsAd) toward bile and bile acids was investigated. CsAds moved toward 0.05-5% bile and were most attracted to 0.5% bile but moved away from 10% bile. Upon exposure to 1-10% bile, CsAds eventually stopped moving and then died quickly. Among bile acids, CsAds showed strong chemotaxis toward cholic acid (CA) and deoxycholic acid. On the contrary, CsAds repelled from lithocholic acid (LCA). Moreover, at higher than 10 mM LCA, CsAds became sluggish and eventually died. Dopamine D1 receptor antagonists (LE-300 and SKF-83566), D2/3 receptor antagonists (raclopride and its derivative CS-49612), and a dopamine re-uptake inhibitor inhibited CA-induced chemotaxis of CsAds almost completely. Clinically used antipsychotic drugs, namely chlorpromazine, haloperidol, and clozapine, are dopaminergic antagonists and are secreted into bile. They completely inhibited chemotaxis of CsAds toward CA. At the maximum doses used to treat patients, the three tested medicines only expelled 2-12% of CsAds from the experimentally infected rabbits, but reduced egg production by 64-79%. Thus, antipsychotic medicines with dopaminergic antagonism could be considered as new anthelmintic candidates for human C. sinensis infections.

The impact of Clonorchis sinensis infection on immune response in mice with type II collagen-induced arthritis.

BACKGROUND: Clonorchis sinensis infection could trigger strong immune responses in mice and humans. However, whether the C.sinensis infection has an impact on arthritis is unknown. Here we investigated the effect of C.sinensis infection on type II collagen-induced arthritis in BALB/c mice. RESULTS: The mice were firstly infected with 45 C.sinensis metacercariae by oral gavage. Four weeks later, arthritis in mice was induced by type II collagen. Joint inflammation with severe redness and swelling in hind paws was observed in type II collagen-induced arthritis (CIA) mice. Besides, the physical activity was significantly reduced, but the respiratory exchange ratio was increased in CIA mice. Compared with CIA mice, C.sinensis infection could increase the severity of arthritis in CIA mice, based on the results of disease score and pathological changes. Compared to CIA mice, increased neutrophils and Ly6Chi monocytes, decreased B cells and CD4+T cells, were found in C.sinensis infected CIA mice. Besides these, C.sinensis infected mice also displayed significantly higher levels of serum IL-4 and IL-17 than those in CIA mice. CONCLUSIONS: Taken together, our data suggest that C.sinensis infection have a bad effect on arthritis, and could induce the abnormality of the immune response in mice with CIA.

Expanded complement of Niemann-Pick type C2-like protein genes in Clonorchis sinensis suggests functions beyond sterol binding and transport.

BACKGROUND: The parasitic flatworm Clonorchis sinensis inhabits the biliary tree of humans and other piscivorous mammals. This parasite can survive and thrive in the bile duct, despite exposure to bile constituents and host immune attack. Although the precise biological mechanisms underlying this adaptation are unknown, previous work indicated that Niemann-pick type C2 (NPC2)-like sterol-binding proteins might be integral in the host-parasite interplay. Expansions of this family in some invertebrates, such as arthropods, have shown functional diversification, including novel forms of chemoreception. Thus, here we curated the NPC2-like protein gene complement in C. sinensis, and predicted their conserved and/or divergent functional roles. METHODS: We used an established comparative genomic-bioinformatic approach to curate NPC2-like proteins encoded in published genomes of Korean and Chinese isolates of C. sinensis. Protein sequence and structural homology, presence of conserved domains and phylogeny were used to group and functionally classify NPC2-like proteins. Furthermore, transcription levels of NPC2-like protein-encoding genes were explored in different developmental stages and tissues. RESULTS: Totals of 35 and 32 C. sinensis NPC2-like proteins were predicted to be encoded in the genomes of the Korean and Chinese isolates, respectively. Overall, these proteins had low sequence homology and high variability of sequence alignment coverage when compared with curated NPC2s. Most C. sinensis proteins were predicted to retain a conserved ML domain and a conserved fold conformation, with a large cavity within the protein. Only one protein sequence retained the conserved amino acid residues required in bovine NPC2 to bind cholesterol. Non-canonical C. sinensis NPC2-like protein-coding domains clustered into four distinct phylogenetic groups with members of a group frequently encoded on the same genome scaffolds. Interestingly, NPC2-like protein-encoding genes were predicted to be variably transcribed in different developmental stages and adult tissues, with most being transcribed in the metacercarial stage. CONCLUSIONS: The results of the present investigation confirms an expansion of NPC2-like proteins in C. sinensis, suggesting a diverse array of functions beyond sterol binding and transport. Functional explorations of this protein family should elucidate the mechanisms enabling the establishment and survival of C. sinensis and related flukes in the biliary systems of mammalian hosts.

Chemotactic migration of newly excysted juvenile Clonorchis sinensis is suppressed by neuro-antagonists.

The metacercariae of the Clonorchis sinensis liver fluke excyst in the duodenum of mammalian hosts, and the newly excysted juveniles (CsNEJs) migrate along the bile duct via bile chemotaxis. Cholic acid is a major component of bile that induces this migration. We investigated the neuronal control of chemotactic behavior of CsNEJs toward cholic acid. The migration of CsNEJs was strongly inhibited at sub-micromolar concentration by dopamine D1 (LE-300 and SKF-83566), D2 (spiramide, nemonapride, and sulpiride), and D3 (GR-103691 and NGB-2904) receptor antagonists, as well as a dopamine reuptake inhibitor (BTCP). Neuropeptides, FMRFamide, peptide YY, and neuropeptide Y were also potent inhibitors of chemotaxis. Meanwhile, serotonergic, glutamatergic, and cholinergic inhibitors did not affect chemotaxis, with the exception of fluoxetine and CNQX. Confocal immunofluorescence analysis indicated that dopaminergic and cholinergic neurons were colocalized in the somatic muscle tissues of adult C. sinensis. Our findings suggest that dopaminergic neurons and neuropeptides play a major role in the chemotactic migration of CsNEJs to bile, and their inhibitors or modulators could be utilized to prevent their migration from the bile duct.

Membrane-bound IgE on B cells is increased during Clonorchis sinensis infection.

A high level of serum IgE is a hallmark of helminthic disease. Secretory IgE can bind FcεRI or FcεRII/CD23. The combination of IgE and FcεRI, a high-affinity interaction, has long received attention and is believed to facilitate helminth control, while the properties of CD23-bound IgE have long been unexplored. Here, we established a Clonorchis sinensis (C. sinensis) infection model with different mouse strains and investigated membrane-bound IgE on B cells during infection. We show that after infection, the increase in CD23 expression on B cells was obvious, even in relatively resistant C57BL/6 mice, as well as in susceptible BALB/c and FVB mice. Although the serum IgE amount was lower in C57BL/6 mice than in BALB/c and FVB mice, the level of IgE binding to peripheral B cells was also elevated. Additionally, the IgE on B cells was soon undetectable in vitro due to dissociable binding. The results of the present study demonstrate the dramatic increase in CD23-bound IgE on B cells after C. sinensis infection. The significance of CD23-bound IgE in Ag transport and presentation has gained consideration in allergy development for its potential ability to promote the Th2 response. Therefore, even though the association of IgE and CD23 is not as substantial as that of IgE and FcεRI, membrane-bound IgE on B cells may be worth further study regarding clonorchiasis and other parasitic infections.

Bile acids drive chemotaxis of Clonorchis sinensis juveniles to the bile duct.

Clonorchiasis is a neglected tropical disease caused by Chinese liver fluke, Clonorchis sinensis infection. C. sinensis is a biological carcinogen causing cholangiocarcinoma in humans. In the mammalian host, C. sinensis newly excysted juveniles (CsNEJs) migrate from the duodenum into the bile duct. Bile drives the chemotactic behavior of CsNEJs. Little is known about which components of bile induce the chemotaxis. We designed a chemotaxis assay panel and measured the chemotactic behavior of CsNEJs in response to bile or bile acids. The CsNEJs migrated toward 0.1-1% bile but away from 5-10% bile. The CsNEJs showed strong chemoattraction to cholic acid ≥25 mM, but chemorepulsion to lithocholic acid ≥0.25 mM. To the CsNEJs, mixture of cholic acid and lithocholic acid was chemoattractive at a ratio greater than 25:1 but chemorepulsive at one smaller than that. Regarding migration in the mammalian hosts, high concentration of lithocholic acid in the gallbladder bile may repel CsNEJs from entering it. However, bile in the hepatic bile duct has a chemoattractive strength of cholic acid but a trace amount of lithocholic acid. Collectively, our results explain why the CsNEJs migrate principally to the hepatic bile ducts, bypassing the gallbladder.

Immune responses induced by co-infection with Capillaria hepatica in Clonorchis sinensis-infected rats.

Clonorchis sinensis and Capillaria hepatica are zoonotic parasites that mainly infect the liver and cause serious liver disorders. However, immunological parameters induced by co-infection with these parasites remain unknown. In this study, for the first time, we investigated immunological profiles induced by co-infection with C. hepatica (CH) in C. sinensis (CS)-infected rats (Sprague-Dawley). Rats were infected primarily with 50 metacercariae of C. sinensis; 4 weeks later, they were subsequently infected with 1000 infective C. hepatica eggs. Significantly higher levels of C. sinensis- or C. hepatica-specific IgG antibodies were found in the sera of rats. Interestingly, no cross-reacting antibody was observed between C. sinensis and C. hepatica infections. Significantly raised eosinophil levels were found in the blood of C. sinensis/C. hepatica co-infected rats (CS + CH) compared to the blood of rats infected singly with C. sinensis. Co-infected rats showed significantly higher levels of lymphocyte proliferation and cytokine production compared to a single C. sinensis infection. The worm burden of C. sinensis was significantly reduced in co-infected rats compared to the single C. sinensis infection. These results indicate that the eosinophils, lymphocyte proliferation and cytokine production induced by subsequent infection with C. hepatica in C. sinensis-infected rats might contribute to the observed C. sinensis worm reduction.

TLR2 signal influences the iNOS/NO responses and worm development in C57BL/6J mice infected with Clonorchis sinensis.

BACKGROUND: Although the responses of inducible nitric oxide synthase (iNOS) and associated cytokine after Clonorchis sinensis infection have been studied recently, their mechanisms remain incompletely understood. In this study, we investigated the effects of toll-like receptor 2 (TLR2) signals on iNOS/nitric oxide (NO) responses after C. sinensis infection. We also evaluated the correlations between iNOS responses and worm development, which are possibly regulated by TLR2 signal. METHODS: TLR2 wild-type and mutant C57BL/6 J mice were infected with 60 C. sinensis metacercariae, and the samples were collected at 30, 60, 90 and 120 days post-infection (dpi). The total serum NO levels were detected using Griess reagent after nitrate was reduced to nitrite. Hepatic tissue samples from the infected mice were sliced and stained with hematoxylin and eosin (HE) to observe worm development in the intrahepatic bile ducts. The iNOS mRNA transcripts in the splenocytes were examined by real time reverse transcriptase polymerase chain reaction (qRT-PCR), and iNOS expression was detected by immunohistochemistry. RESULTS: Developing C. sinensis juvenile worms were more abundant in the intrahepatic bile ducts of TLR2 mutant mice than those of TLR2 wild-type mice. However, no eggs were found in the faeces of both mice samples. The serum levels of total NO significantly increased in TLR2 mutant mice infected with C. sinensis at 30 (t (5) = 2.595, P = 0.049), 60 (t (5) = 7.838, P = 0.001) and 90 dpi (t (5) = 3.032, P = 0.029). Meanwhile, no changes occurred in TLR2 wild-type mice compared with uninfected controls during the experiment. The iNOS expression in splenocytes showed unexpected higher background levels in TLR2 mutant mice than those in TLR2 wild-type mice. Furthermore, the iNOS mRNA transcripts in splenocytes were significantly increased in the TLR2 wild-type mice infected with C. sinensis at 30 (t (5) = 5.139, P = 0.004), 60 (t (5) = 6.138, P = 0.002) and 90 dpi (t (5) = 6.332, P = 0.001). However, the rising of iNOS transcripts dropped under the uninfected control level in the TLR2 mutant mice at 120 dpi (t (5) = -9.082, P < 0.0001). Both total NO and iNOS transcripts were significantly higher in the TLR2 mutant mice than those in the TLR2 wild-type mice at 30 (t (5) = 3.091/2.933, P = 0.027/0.033) and 60 dpi (t (5) = 2.667/6.331, P = 0.044/0.001), respectively. In addition, the remarkable increase of iNOS expressions was immunohistochemically detected in the splenic serial sections of TLR2 wild-type mice at 30 and 60 dpi. However, the expressions of iNOS were remarkably decreased in the splenocytes of both TLR2 wild-type and mutant mice at 120 dpi. CONCLUSIONS: These results demonstrate that TLR2 signal plays an important role in the regulation of iNOS expression after C. sinensis infection. TLR2 signal is also beneficial to limiting worm growth and development and contributing to the susceptibility to C. sinensis in which the iNOS/NO reactions possibly participate.

Local immune response to primary infection and re-infection by Clonorchis sinensis in FVB mice.

Although Clonorchis sinensis lives in the bile duct, few studies have investigated the local immune response in the liver and bile duct. To investigate the local immune response to C. sinensis, we investigated the activation and recruitment of various immune cells and cytokine levels in the liver and bile duct lymph nodes (BLN) in FVB mice after primary infection and re-infection. Male 4-week-old FVB mice were divided into 6 experimental groups: uninfected controls, primary infection lasting 1week (PI 1w), primary infection lasting 4weeks (PI 4w), praziquantel treatment after PI 4w (Tx), re-infection lasting 1week after Tx (RI 1w), and re-infection lasting 4weeks after Tx (RI 4w). Recovery rates were 80.0% and 73.0% in PI 1w and PI 4w mice, respectively, but significantly decreased during re-infection to 26.6% in RI 1w and 13.3% in RI 4w. This result suggested that the mice were resistant to re-infection. In the liver, Kupffer cells were augmented 70-fold in PI 1w mice (P<0.001). Kupffer cells expressed Th2-related cytokines (IL-10 and IL-13) during primary infection. In addition, serum levels of C. sinensis-specific IgG1 and IgG2a strongly increased in RI 1w mice. Secretion of C. sinensis-specific IgE reached a plateau at 4weeks after primary infection, and remained elevated in all infected groups. In conclusion, during infection with C. sinensis, Kupffer cells likely act as antigen-presenting cells, stimulating the Th2 cytokine production system.

Peroxiredoxin 6 expression is inversely correlated with nuclear factor-κB activation during Clonorchis sinensis infestation.

Clonorchis sinensis is a carcinogenic human liver fluke. Its infection promotes persistent oxidative stress and chronic inflammation environments in the bile duct and surrounding liver tissues owing to direct contact with worms and their excretory-secretory products (ESPs), provoking epithelial hyperplasia, periductal fibrosis, and cholangiocarcinogenesis. We examined the reciprocal regulation of two ESP-induced redox-active proteins, NF-κB and peroxiredoxin 6 (Prdx6), during C. sinensis infection. Prdx6 overexpression suppressed intracellular free-radical generation by inhibiting NADPH oxidase2 and inducible nitric oxide synthase activation in the ESP-treated cholangiocarcinoma cells, substantially attenuating NF-κB-mediated inflammation. NF-κB overexpression decreased Prdx6 transcription levels by binding to two κB sites within the promoter. This transcriptional repression was compensated for by other ESP-induced redox-active transcription factors, including erythroid 2-related factor 2 (Nrf2), hypoxia inducible factor 1α (HIF1α), and CCAAT/enhancer-binding protein ß (C/EBPß). Distribution of immunoreactive Prdx6 and NF-κB was distinct in the early stages of infection in mouse livers but shared concomitant localization in the later stages. The intensity and extent of their immunoreactive staining in infected mouse livers are proportional to lesion severity and infection duration. The constitutive elevations of Prdx6 and NF-κB during C. sinensis infection may be associated with more severe persistent hepatobiliary abnormalities mediated by clonorchiasis.