Inositol serves as a natural inhibitor of mitochondrial fission by directly targeting AMPK.

Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.

Deregulated transcription factors in cancer cell metabolisms and reprogramming.

Metabolic reprogramming is an important cancer hallmark that plays a key role in cancer malignancies and therapy resistance. Cancer cells reprogram the metabolic pathways to generate not only energy and building blocks but also produce numerous key signaling metabolites to impact signaling and epigenetic/transcriptional regulation for cancer cell proliferation and survival. A deeper understanding of the mechanisms by which metabolic reprogramming is regulated in cancer may provide potential new strategies for cancer targeting. Recent studies suggest that deregulated transcription factors have been observed in various human cancers and significantly impact metabolism and signaling in cancer. In this review, we highlight the key transcription factors that are involved in metabolic control, dissect the crosstalk between signaling and transcription factors in metabolic reprogramming, and offer therapeutic strategies targeting deregulated transcription factors for cancer treatment.

Immune response induced by oral delivery of Bacillus subtilis spores expressing enolase of Clonorchis sinensis in grass carps (Ctenopharyngodon idellus).

Clonorchiasis, caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensisis (C.sinensis), remains a common public health problem. New effective prevention strategies are still urgent to control this food-borne infectious disease. The previous studies suggested Bacillus subtilis (B. subtilis) spores was an ideal vaccines delivery system, and the C.sinensis enolase (CsENO) was a potential vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgM levels by ELISA in sera, intestinal mucus and skin mucus in grass carps (Ctenopharyngodon idella) through oral administration with B. subtilis spores surface expressing CsENO. In addition, immune-related genes expression was also measured by qRT-PCR. Grass carps orally treated with B. subtilis spores or normal forages were used as controls. The results of ELISA manifested that specific IgM levels of grass carps in CsENO group in sera, intestine mucus and skin mucus almost significantly increased from week 4 post the first oral administration when compared to the two control groups. The levels of specific IgM reached its peak in intestine mucus firstly, then in sera, and last in skin mucus. qRT-PCR results showed that 5 immune-related genes expression had different degree of rising trend in CsENO group when compared to the two control groups. Our study demonstrated that orally administrated with B. subtilis spores expressing CsENO induced innate and adaptive immunity, systemic and local mucosal immunity, and humoral and cellular immunity. Our work may pave the way to clarify the exact mechanisms of protective efficacy elicited by B. subtilis spores expressing CsENO and provide new ideas for vaccine development against C. sinensis infection.

Oral delivery of Bacillus subtilis spores expressing cysteine protease of Clonorchis sinensis to grass carp (Ctenopharyngodon idellus): Induces immune responses and has no damage on liver and intestine function.

Clonorchis sinensis (C. sinensis) is a fish-borne trematode. Human can be infected by ingestion of C. sinensis metacercariae parasitized in grass carp (Ctenopharyngodon idella). For induction of effective oral immune responses, spores of Bacillus subtilis (B. subtilis) WB600 were utilized as vehicle to delivery CsCP (cysteine protease of C. sinensis) cooperated with CotC (B.s-CotC-CP), one of coat proteins, to the gastrointestinal tract. After routine culture of 8-12 h in LB medium, B. subtilis containing CotC-CsCP was transferred into the sporulation culture medium. SDS-PAGE, western blotting and the growth curve indicated that the best sporulation time of recombinant WB600 was 24-30 h at 37 °C with continuous shaking (250 rpm). Grass carp were fed with three levels of B.s-CotC-CP (1 × 106, 1 × 107, and 1 × 108 CFU g-1) incorporated in the basal pellets diet. The commercial pellets or supplemented with spores just expressing CotC (1 × 107 CFU g-1) were served as control diet. Our results showed that grass carp orally immunized with the feed-based B.s-CotC-CP developed a strong specific immune response with significantly (P < 0.05) higher levels of IgM in samples of serum, bile, mucus of surface and intestinal compared to the control groups. Abundant colonization spores expressing CsCP were found in hindgut that is conducive to absorption and presentation of antigen. Moreover, B. subtilis spores appeared to show no sign of toxicity or damage in grass carp. Our cercariae challenge experiments suggested that oral administration of spores expressing CsCP could develop an effective protection against C. sinensis in fish body. Therefore, this study demonstrated that the feed-based recombinant spores could trigger high levels of mucosal and humoral immunity, and would be a promising candidate vaccine against C. sinensis metacercariae formation in freshwater fish.

Comparative analysis of immune effects in mice model: Clonorchis sinensis cysteine protease generated from recombinant Escherichia coli and Bacillus subtilis spores.

Clonorchiasis remains a nonnegligible public health problem in endemic areas. Cysteine protease of Clonorchis sinensis (CsCP) plays indispensable roles in the parasitic physiology and pathology, and has been exploited as a promising drug and vaccine candidate. In recent years, development of spore-based vaccines against multiple pathogens has attracted many investigators' interest. In previous studies, the recombinant Escherichia coli (BL21) and Bacillus subtilis spores expressing CsCP have been successfully constructed, respectively. In this study, the immune effects of CsCP protein purified from recombinant BL21 (rCsCP) and B. subtilis spores presenting CsCP (B.s-CsCP) in Balb/c mice model were conducted with comparative analysis. Levels of specific IgG, IgG1 and IgG2a were significantly increased in sera from both rCsCP and B.s-CsCP intraperitoneally immunized mice. Additionally, recombinant spores expressing abundant fusion CsCP (0.03125 pg/spore) could strongly enhance the immunogenicity of CsCP with significantly higher levels of IgG and isotypes. Compared with rCsCP alone, intraperitoneal administration of mice with spores expressing CsCP achieved a better effect of fighting against C. sinensis infection by slowing down the process of fibrosis. Our results demonstrated that a combination of Th1/Th2 immune responses could be elicited by rCsCP, while spores displaying CsCP prominently induced Th1-biased specific immune responses, and the complex cytokine network maybe mediates protective immune responses against C. sinensis. This work further confirmed that the usage of B. subtilis spores displaying CsCP is an effective way to against C. sinensis.

Clonorchis sinensis lysophospholipase inhibits TGF-ß1-induced expression of pro-fibrogenic genes through attenuating the activations of Smad3, JNK2, and ERK1/2 in hepatic stellate cell line LX-2.

Liver fibrosis is a wound healing response associated with chronic liver injury. Hepatic stellate cells (HSCs) activation is a key event in the development of liver fibrosis. Since helminths have the ability to live for decades in the host by establishing an adaptive relationship in the interplay with its hosts, we hypothesize that whether Clonochis sinensis LysophospholipaseA (CsLysoPLA), a component of excretory/secretory proteins, can attenuate the fibrogenic response by inhibiting activation of LX-2 cells, thereby balancing the pro-fibrotic and anti-fibrotic response during the Clonochis sinensis (C. sinensis) infection. In the present study, LX-2 cells were stimulated with CsLysoPLA in the presence of TGF-ß1, and the expressions of collagen type I (COL1A1), α-smooth muscle actin (α-SMA), and matrix metalloproteinase 2 (MMP2) were decreased. In addition, CsLysoPLA significantly inhibited the proliferation and migration of LX-2 cells stimulated by TGF-ß1. Pretreatment of LX-2 cells with CsLysoPLA attenuated the phosphorylation of Smad3 as well as JNK2 and ERK1/2 in response to the stimulation of TGF-ß1. For the first time, our results showed an anti-fibrogenic effect of CsLysoPLA by attenuating the response of LX-2 cells to TGF-ß1 through inhibiting the activations of Smad3, ERK1/2, and JNK2.

Oral delivery of Bacillus subtilis spore expressing enolase of Clonorchis sinensis in rat model: induce systemic and local mucosal immune responses and has no side effect on liver function.

Caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensis, human clonorchiasis remains a major public health problem in China. In previous study, we had expressed enolase from C. sinensis (CsENO) on the surface of Bacillus subtilis spore and the recombinant spore induced a pronounced protection in terms of reduced worm burden and eggs per gram feces, suggesting B. subtilis spore as an ideal vehicle for antigen delivery by oral treatment and CsENO as a promising vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgG and IgA levels both in serum and in intestinal mucus from rats orally administrated with B. subtilis spore surface expressing CsENO by ELISA. Lysozyme levels in serum and in intestinal mucus were analyzed too. In addition, IgA-secreting cells in intestine epithelium of the rats were detected by immunohistochemistry assay. The intestinal villi lengths of duodenum, jejunum, and ileum were also measured. Rats orally treated with B. subtilis spore or normal saline were used as controls. Our results showed that, compared with the control groups, oral administration of B. subtilis spore expressing CsENO induced both systemic and local mucosal immune response. The recombinant spores also enhanced non-specific immune response in rats. The spores had no side effect on liver function. Moreover, it might facilitate food utilization and digestion of the rats. Our work will pave the way to clarify the involved mechanisms of protective efficacy elicited by B. subtilis spore expressing CsENO and encourage us to carry out more assessment trails of the oral treated spore to develop vaccine against clonorchiasis.

Clonorchis sinensis ferritin heavy chain triggers free radicals and mediates inflammation signaling in human hepatic stellate cells.

Clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis, is associated with hepatobiliary damage, inflammation, periductal fibrosis, and the development of cholangiocarcinoma. Hepatic stellate cells respond to liver injury through production of proinflammatory mediators which drive fibrogenesis; however, their endogenous sources and pathophysiological roles in host cells were not determined. C. sinensis ferritin heavy chain (CsFHC) was previously confirmed as a component of excretory/secretory products and exhibited a number of extrahepatic immunomodulatory properties in various diseases. In this study, we investigated the expression pattern and biological role of CsFHC in C. sinensis. CsFHC was expressed throughout life stages of C. sinensis. More importantly, we found that treatment of human hepatic stellate cell line LX-2 with CsFHC triggered the production of free radicals via time-dependent activation of NADPH oxidase, xanthine oxidase, and inducible nitric oxide synthase. The increase in free radicals substantially promoted the degradation of cytosolic IκBα and nuclear translocation of NF-κB subunits (p65 and p50). CsFHC-induced NF-κB activation was markedly attenuated by preincubation with specific inhibitors of corresponding free radical-producing enzyme or the antioxidant. In addition, CsFHC induced an increased expression level of proinflammatory cytokines, IL-1ß and IL-6, in NF-κB-dependent manner. Our results indicate that CsFHC-triggered free radical-mediated NF-κB signaling is an important factor in the chronic inflammation caused by C. sinensis infection.

The serological diagnosis of human clonorchiasis by time-resolved fluoroimmunoassay based on GST2-specific IgG4 detection.

Due to its delayed fluorescence of a lanthanide chelate, high accuracy and low background the broad linear range, long fluorescent life-time and large Stoke's shift of europium chelates, the time-resolved fluorescence has been developed for higher sensitive immunoassay. In this article, a simple, sensitive and specific method-time-resolved fluoroimmunoassay (TRFIA) was adopted for immunoassay of clonorchiasis, and recombinant glutathione transferases 2 of Clonorchis sinensis (rCsGST2) was used as a diagnostic antigen. To evaluate this novel assay for clinical applications, 409 serum samples were investigated. The diagnostic accuracy of the antigen was evaluated by receiver-operating characteristic (ROC) analysis. The area under the ROC curve (AUC) was 0.965, 95% confidence interval (CI, 0.946, 0.985). To eliminate the random influence of ambient temperature, test parameters, photometric instruments and so on, the cut-off value was expressed as ratios between the fluorescence of sample and that of a well-defined negative control serum, and the deduced cut-off value was 9.3605. At the optimum cut-off criteria, the technique has a sensitivity of 95.80%, specificity of 93.60%. And the cross reactivity revealed that its cross reactivity with Schistosoma japonicum, round worm, hook worm, whip worm, and Toxoplasma gondii was 9.3, 8.3, 7.6, 9.8, and 5.0%, respectively. Kappa score of agreement between TRFIA and microscopic examination of stools was 0.892, P < 0.05. These combined results showed that our method is feasible and could be used for the clinical determination of clonorchiasis.

Protocol to analyze the role of various metabolites in impacting global RNA 5-methylcytosine levels in cultured cells by dot blot.

RNA 5-methylcytosine (m5C) modification critically impacts many biological processes. Here, we provide a protocol to analyze the role of various metabolites in impacting global RNA m5C levels in cultured cells by dot blot. We describe steps for treating cultured cells with various metabolites; extracting, quantifying, and denaturing RNA samples; and performing dot blot to detect global RNA m5C levels in cultured cells. We then detail procedures to verify the input loading by methylene blue staining and quantify using ImageJ. For complete details on the use and execution of this protocol, please refer to Chen et al.1.

IMPA1-derived inositol maintains stemness in castration-resistant prostate cancer via IMPDH2 activation.

Acquisition of prostate cancer stem cells (PCSCs) manifested during androgen ablation therapy (ABT) contributes to castration-resistant prostate cancer (CRPC). However, little is known about the specific metabolites critically orchestrating this process. Here, we show that IMPA1-derived inositol enriched in PCSCs is a key metabolite crucially maintaining PCSCs for CRPC progression and ABT resistance. Notably, conditional Impa1 knockout in the prostate abrogates the pool and properties of PCSCs to orchestrate CRPC progression and prolong the survival of TRAMP mice. IMPA1-derived inositol serves as a cofactor that directly binds to and activates IMPDH2, which synthesizes guanylate nucleotides for maintaining PCSCs with ARlow/- features leading to CRPC progression and ABT resistance. IMPA1/inositol/IMPDH2 axis is upregulated in human prostate cancer, and its overexpression predicts poor survival outcomes. Genetically and pharmacologically targeting the IMPA1/inositol/IMPDH2 axis abrogates CRPC and overcomes ABT resistance in various CRPC xenografts, patient-derived xenograft (PDX) tumor models, and TRAMP mouse models. Our study identifies IMPDH2 as an inositol sensor whose activation by inositol represents a key mechanism for maintaining PCSCs for CRPC and ABT resistance.

Molecular characterization and expression of Rab7 from Clonorchis sinensis and its potential role in autophagy.

Accumulating evidences suggest that Rab7 GTPase is important for the normal progression of autophagy. However, the role of Rab7 GTPase in regulation of autophagy in Clonorchis sinensis is not known. In this study, a gene encoding Rab7 was isolated from C. sinensis adult cDNA. Recombinant CsRab7 was expressed and purified from Escherichia coli. CsRab7 transcripts were detected in the cDNA of adult worm, metacercaria, cercaria, and egg of C. sinensis, and were highly expressed in the metacercaria. Immunohistochemical localization results revealed that CsRab7 was specifically deposited on the vitellarium and eggs of adult worm. Furthermore, EGFP signal of CsRab7WT and the active mutant CsRab7Q67L were associated with autophagic vesicles in transiently transfected 293T cells. It is concluded from the present study that CsRab7 GTPase possibly contributes to the development of C. sinensis and that the autophagy pathway could be an important site of action with respect to the developmental role of CsRab7 in C. sinensis.

Current status and progress in the omics of Clonorchis sinensis.

Clonorchis sinensis (C. sinensis) is a fish-borne trematode that inhabits the bile duct of mammals including humans, cats, dogs, rats, and so on. In the complex life cycle of C. sinensis, the worm develops successively in two intermediate hosts in fresh water and one definitive host. What's more, it undergoes eight developmental stages with a distinct morphology. Clonorchiasis, caused by C. sinensis infection, is an important food-borne parasitic disease and one of the most common zoonoses. C. sinensis infection could result in hyperplasia of the bile duct epithelium, obstructive jaundice, gall-stones, cholecystitis and cholangitis, even liver cirrhosis and cholangiocarcinoma. Thus, clonorchiasis is a serious public health problem in endemic areas. Integrated strategies should be adopted in the prevention and control of clonorchiasis due to the epidemiological characteristics. The recent advances in high-throughput technologies have made available the profiling of multiple layers of a biological system, genomics, transcriptomics, proteomics, and metabolomics. These data can help us to get more information about the development, physiology, metabolism, and reproduction of the parasite as well as pathogenesis and parasite-host interactions in clonorchiasis. In the present study, we summarized recent progresses in omics studies on C. sinensis providing insights into the studies and future directions on treating and preventing C. sinensis associated diseases.

NSUN2 is a glucose sensor suppressing cGAS/STING to maintain tumorigenesis and immunotherapy resistance.

Glucose metabolism is known to orchestrate oncogenesis. Whether glucose serves as a signaling molecule directly regulating oncoprotein activity for tumorigenesis remains elusive. Here, we report that glucose is a cofactor binding to methyltransferase NSUN2 at amino acid 1-28 to promote NSUN2 oligomerization and activation. NSUN2 activation maintains global m5C RNA methylation, including TREX2, and stabilizes TREX2 to restrict cytosolic dsDNA accumulation and cGAS/STING activation for promoting tumorigenesis and anti-PD-L1 immunotherapy resistance. An NSUN2 mutant defective in glucose binding or disrupting glucose/NSUN2 interaction abolishes NSUN2 activity and TREX2 induction leading to cGAS/STING activation for oncogenic suppression. Strikingly, genetic deletion of the glucose/NSUN2/TREX2 axis suppresses tumorigenesis and overcomes anti-PD-L1 immunotherapy resistance in those cold tumors through cGAS/STING activation to facilitate apoptosis and CD8+ T cell infiltration. Our study identifies NSUN2 as a direct glucose sensor whose activation by glucose drives tumorigenesis and immunotherapy resistance by maintaining TREX2 expression for cGAS/STING inactivation.

Identification of primordial germ cell-like cells as liver metastasis initiating cells in mouse tumour models.

Liver metastasis, characterized by the spread of tumors to the liver from other areas, represents a deadly disease with poor prognosis. Currently, there is no effective therapeutic strategies and/or agents to combat liver metastasis primarily due to the insufficient understanding of liver metastasis. To develop a promising strategy for targeting liver metastasis, understanding of a cell origin responsible for liver metastasis and how this cell can be pharmacologically eliminated are therefore crucial. Using diverse tumor models including p53 -/- genetic mouse model and syngeneic tumor models, we identified primordial germ cell (PGC)-like tumor cells, which are enriched in earliest liver micro-metastasis (up to 99%), as a cell origin of liver metastasis. PGC-like tumor cells formed earliest micro-metastasis in liver and gradually differentiated into non-PGC-like tumor cells to constitute late macro-metastasis in the course of tumor metastasis. The liver metastasis-initiating cells (PGC-like tumor cells) display cell renewal and differentiation capabilities, resemble primordial germ cells (PGCs) in morphology and PGC marker gene expression, and express higher level of the genes linked to metastasis and immune escape compared with non-PGC-like tumor cells. Of note, Stellarhigh PGC-like tumor cells, but not Stellarlow non-PGC-like cells, sorted from primary tumors of p53 -/- mice readily form liver metastasis. Depletion of PGC-like tumor cells through genetic depletion of any of key germ cell genes impairs liver metastasis, while increased PGC-like tumor cells by SMAD2 knockout is correlated with markedly enhanced liver metastasis. Finally, we present the proof of principle evidence that pharmacologically targeting BMP pathways serves as a promising strategy to eliminate PGC-like tumor cells leading to abrogating liver metastasis. Collectively, our study identifies PGC-like tumor cells as a cell origin of liver metastasis, whose depletion by genetically targeting core PGC developmental genes or pharmacologically inhibiting BMP pathways serves a promising strategy for targeting liver metastasis.

Amino acids serve as an important energy source for adult flukes of Clonorchis sinensis.

Clonorchiasis, caused by chronic infection with Clonorchis sinensis (C. sinensis), is an important food-borne parasitic disease that seriously afflicts more than 35 million people globally, resulting in a socioeconomic burden in endemic regions. C. sinensis adults long-term inhabit the microaerobic and limited-glucose environment of the bile ducts. Energy metabolism plays a key role in facilitating the adaptation of adult flukes to crowded habitat and hostile environment. To understand energy source for adult flukes, we compared the component and content of free amino acids between C. sinensis-infected and uninfected bile. The results showed that the concentrations of free amino acids, including aspartic acid, serine, glycine, alanine, histidine, asparagine, threonine, lysine, hydroxylysine, and urea, were significantly higher in C. sinensis-infected bile than those in uninfected bile. Furthermore, exogenous amino acids could be utilized by adult flukes via the gluconeogenesis pathway regardless of the absence or presence of exogenous glucose, and the rate-limiting enzymes, such as C. sinensis glucose-6-phosphatase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate carboxylase, exhibited high expression levels by quantitative real-time PCR analysis. Interestingly, no matter whether exogenous glucose was present, inhibition of gluconeogenesis reduced the glucose and glycogen levels as well as the viability and survival time of adult flukes. These results suggest that gluconeogenesis might play a vital role in energy metabolism of C. sinensis and exogenous amino acids probably serve as an important energy source that benefits the continued survival of adult flukes in the host. Our study will be a cornerstone for illuminating the biological characteristics of C. sinensis and the host-parasite interactions.

Bacillus subtilis spore with surface display of paramyosin from Clonorchis sinensis potentializes a promising oral vaccine candidate.

BACKGROUND: Clonorchiasis caused by Clonorchis sinensis has become increasingly prevalent in recent years. Effective prevention strategies are urgently needed to control this food-borne infectious disease. Previous studies indicated that paramyosin of C. sinensis (CsPmy) is a potential vaccine candidate. METHODS: We constructed a recombinant plasmid of PEB03-CotC-CsPmy, transformed it into Bacillus subtilis WB600 strain (B.s-CotC-CsPmy), and confirmed CsPmy expression on the spore surface by SDS-PAGE, Western blotting and immunofluorescence assay. The immune response and protective efficacy of the recombinant spore were investigated in BALB/c mice after intragastrical or intraperitoneal immunization. Additionally, biochemical enzyme activities in sera, the intestinal histopathology and gut microflora of spore-treated mice were investigated. RESULTS: CsPmy was successfully expressed on the spore surface and the fusion protein on the spore surface with thermostability. Specific IgG in sera and intestinal mucus were increased after intraperitoneal and intragastrical immunization. The sIgA level in intestinal mucus, feces and bile of B.s-CotC-CsPmy orally treated mice were also significantly raised. Furthermore, numerous IgA-secreting cells were detected in intestinal mucosa of intragastrically immunized mice. No inflammatory injury was observed in the intestinal tissues and there was no significant difference in levels of enzyme-indicated liver function among the groups. Additionally, the diversity and abundance of gut microbiota were not changed after oral immunization. Intragastric and intraperitoneal immunization of B.s-CotC-CsPmy spores in mice resulted in egg reduction rates of 48.3 and 51.2% after challenge infection, respectively. Liver fibrosis degree in B.s-CotC-CsPmy spores treated groups was also significantly reduced. CONCLUSIONS: CsPmy expressed on the spore surface maintained its immunogenicity. Both intragastrical and intraperitoneal immunization with B.s-CotC-CsPmy spores induced systemic and local mucosal immune response in mice. Although both intragastric and intraperitoneal immunization elicited a similar protective effect, intragastric immunization induced stronger mucosal immune response without side effects to the liver, intestine and gut microbiota, compared with intraperitoneal immunization. Oral immunization with B. subtilis spore expressing CsPmy on the surface was a promising, safe and needle-free vaccination strategy against clonorchiasis.

Clonorchis sinensis adult-derived proteins elicit Th2 immune responses by regulating dendritic cells via mannose receptor.

BACKGROUND: Clonorchis sinensis (C. sinensis) is the most widespread human liver fluke in East Asia including China and Korea. Clonorchiasis as a neglected tropical zoonosis, leads to serious economic and public health burden in China. There are considerable evidences for an etiological relation between chronic clonorchiasis and liver fibrosis in human beings. Liver fibrosis is a highly conserved and over-protected response to hepatic tissue injury. Immune cells including CD4+ T cell as well as dendritic cell (DC), and pro-fibrogenic cytokines like interleukin 4 (IL-4), IL-13 have been identified as vital manipulators in liver fibrogenesis. Our previous studies had a mere glimpse of T helper type 2 (Th2) dominant immune responses as key players in liver fibrosis induced by C. sinensis infection, but little is known about the involved mechanisms in this pathological process. METHODOLOGY/PRINCIPAL FINDINGS: By flow cytometry (FACS), adult-derived total proteins of C. sinensis (CsTPs) down-regulated the expression of surface markers CD80, CD86 and major histocompatibility complex class II (MHC-II) on lipopolysaccharide (LPS) induced DC. ELISA results demonstrated that CsTPs inhibited IL-12p70 release from LPS-treated bone marrow-derived dendritic cells (BMDC). IL-10 level increased in a time-dependent manner in LPS-treated BMDCs after incubation with CsTPs. CD4+ T cells incubated with LPS-treated BMDCs plus CsTPs could significantly elevate IL-4 level by ELISA. Meanwhile, elevated expression of pro-fibrogenic mediators including IL-13 and IL-4 were detected in a co-culture system of LPS-activated BMDCs and naive T cells containing CsTPs. In vivo, CsTPs-immunized mice enhanced expression of type 2 cytokines IL-13, IL-10 and IL-4 in both splenocytes and hepatic tissue. Exposure of BMDCs to CsTPs activated expression of mannose receptor (MR) but not toll like receptor 2 (TLR2), TLR4, C-type lectin receptor DC-SIGN and Dectin-2 on the cell surface by RT-PCR and FACS. Blockade of MR almost completely reversed the capacity of CsTPs to suppress LPS-induced BMDCs surface markers CD80, CD86 and MHC-II expression, and further made these BMDCs fail to induce a Th2-skewed response as well as Th2 cell-associated cytokines IL-13 and IL-4 release in vitro. CONCLUSIONS/SIGNIFICANCE: Collectively, we validated that CsTPs could suppress the maturation of BMDCs in the presence of LPS via binding MR, and showed that the CsTPs-pulsed BMDCs actively polarized naive T helper cells to Th2 cells though the production of IL-10 instead of IL-12. CsTPs endowed host with the capacity to facilitate Th2 cytokines production including IL-13 and IL-4 in vitro and vivo. The study might provide useful information for developing potential therapeutic targets against the disease.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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 α-smooth muscle actin (α-SMA) and collagen type I (Collagen-I), and promoted cell migration. CONCLUSIONS: 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.

Sequence analysis and characterization of pyruvate kinase from Clonorchis sinensis, a 53.1-kDa homopentamer, implicated immune protective efficacy against clonorchiasis.

BACKGROUND: Clonorchis sinensis, the causative agent of clonorchiasis, is classified as one of the most neglected tropical diseases and affects more than 15 million people globally. This hepatobiliary disease is highly associated with cholangiocarcinoma. As key molecules in the infectivity and subsistence of trematodes, glycolytic enzymes have been targets for drug and vaccine development. Clonorchis sinensis pyruvate kinase (CsPK), a crucial glycolytic enzyme, was characterized in this research. RESULTS: Differences were observed in the sequences and spatial structures of CsPK and PKs from humans, rats, mice and rabbits. CsPK possessed a characteristic active site signature (IKLIAKIENHEGV) and some unique sites but lacked the N-terminal domain. The predicted subunit molecular mass (Mr) of CsPK was 53.1 kDa. Recombinant CsPK (rCsPK) was a homopentamer with a Mr. of approximately 290 kDa by both native PAGE and gel filtration chromatography. Significant differences in the protein and mRNA levels of CsPK were observed among four life stages of C. sinensis (egg, adult worm, excysted metacercaria and metacercaria), suggesting that these developmental stages may be associated with diverse energy demands. CsPK was widely distributed in adult worms. Moreover, an intense Th1-biased immune response was persistently elicited in rats immunized with rCsPK. Also, rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CONCLUSIONS: The sequences and spatial structures, molecular mass, and expression profile of CsPK have been characterized. rCsPK was indicated to be a homopentamer. Rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CsPK is worthy of further study as a promising target for drug and vaccine development.