NMDAR antagonists suppress tumor progression by regulating tumor-associated macrophages.

Neurotransmitter receptors are increasingly recognized to play important roles in anti-tumor immunity. The expression of the ion channel N-methyl-D-aspartate receptor (NMDAR) on macrophages was reported, but the role of NMDAR on macrophages in the tumor microenvironment (TME) remains unknown. Here, we show that the activation of NMDAR triggered calcium influx and reactive oxygen species production, which fueled immunosuppressive activities in tumor-associated macrophages (TAMs) in the hepatocellular sarcoma and fibrosarcoma tumor settings. NMDAR antagonists, MK-801, memantine, and magnesium, effectively suppressed these processes in TAMs. Single-cell RNA sequencing analysis revealed that blocking NMDAR functionally and metabolically altered TAM phenotypes, such that they could better promote T cell- and Natural killer (NK) cell-mediated anti-tumor immunity. Treatment with NMDAR antagonists in combination with anti-PD-1 antibody led to the elimination of the majority of established preclinical liver tumors. Thus, our study uncovered an unknown role for NMDAR in regulating macrophages in the TME of hepatocellular sarcoma and provided a rationale for targeting NMDAR for tumor immunotherapy.

Estradiol mitigates stress-induced cardiac injury and inflammation by downregulating ADAM17 via the GPER-1/PI3K signaling pathway.

Stress-induced cardiovascular diseases characterized by inflammation are among the leading causes of morbidity and mortality in postmenopausal women worldwide. Estradiol (E2) is known to be cardioprotective via the modulation of inflammatory mediators during stress. But the mechanism is unclear. TNFα, a key player in inflammation, is primarily converted to its active form by 'A Disintegrin and Metalloprotease 17' (ADAM17). We investigated if E2 can regulate ADAM17 during stress. Experiments were performed using female FVB wild-type (WT), C57BL/6 WT, and G protein-coupled estrogen receptor 1 knockout (GPER-1 KO) mice and H9c2 cells. The study revealed a significant increase in cardiac injury and inflammation during isoproterenol (ISO)-induced stress in ovariectomized (OVX) mice. Additionally, ADAM17's membrane content (mADAM17) was remarkably increased in OVX and GPER-1 KO mice during stress. However, in vivo supplementation of E2 significantly reduced cardiac injury, mADAM17, and inflammation. Also, administering G1 (GPER-1 agonist) in mice under stress reduced mADAM17. Further experiments demonstrated that E2, via GPER-1/PI3K pathway, localized ADAM17 at the perinuclear region by normalizing ß1AR-Gαs, mediating the switch from ß2AR-Gαi to Gαs, and reducing phosphorylated kinases, including p38 MAPKs and ERKs. Thus, using G15 and LY294002 to inhibit GPER-1 and its down signaling molecule, PI3K, respectively, in the presence of E2 during stress resulted in the disappearance of E2's modulatory effect on mADAM17. In vitro knockdown of ADAM17 during stress significantly reduced cardiac injury and inflammation, confirming its significant inflammatory role. These interesting findings provide novel evidence that E2 and G1 are potential therapeutic agents for ADAM17-induced inflammatory diseases associated with postmenopausal females.

CsHscB Derived from a Liver Fluke Clonorchis Sinensis Ameliorates Cholestatic Hepatic Fibrosis in a Mouse Model of Sclerosing Cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by inflammatory fibrosis usually involving the whole biliary tree. However, there are very limited treatment options to treat this disease. Our previous study found a lipid-protein rCsHscB from a liver fluke - Clonorchis sinensis, which had full capacities of immune regulation. Therefore, we investigated the role of rCsHscB in a mouse model of sclerosing cholangitis induced by xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to explore whether this protein had potential therapeutic value for PSC. METHODS: Mice were fed 0.1% DDC for 4 weeks and treated with CsHscB (30 µg/mouse, intraperitoneal injection, once every 3 days); the control group was given an equal amount of PBS or CsHscB under normal diet conditions. All the mice were sacrificed at 4 weeks for the evaluation of biliary proliferation, fibrosis, and inflammation. RESULTS: rCsHscB treatment attenuated DDC-induced liver congestion and enlargement and significantly decreased the upregulation of serum AST and ALT levels. The administration of rCsHscB to DDC-fed mice significantly decreased cholangiocyte proliferation and pro-inflammatory cytokine production compared to mice fed with DDC alone. Also, rCsHscB treatment showed a decreased expression of α-SMA in the liver and other markers of liver fibrosis (Masson staining, Hydroxyproline content, and collagen deposit). More interestingly, DDC-fed mice treated with rCsHscB showed a significant up-regulation of PPAR-γ expression, which was similar to control mice, indicating the involvement of PPAR-γ signaling in the protective action of rCsHscB. CONCLUSION: Overall, our data show that rCsHscB attenuates the progression of cholestatic fibrosis induced by DDC and supports the potential for manipulating the parasite-derived molecule to treat certain immune-mediated disorders.

The role of glutamate receptors in the regulation of the tumor microenvironment.

Glutamate, as one of the most important carbon sources in the TCA cycle, is central in metabolic processes that will subsequently influence tumor progression. Several factors can affect the expression of glutamate receptors, playing either a tumor-promoting or tumor-suppressor role in cancer. Thus, the activation of glutamate receptors by the ligand could play a role in tumor development as ample studies have demonstrated the expression of glutamate receptors in a broad range of tumor cells. Glutamate and its receptors are involved in the regulation of different immune cells' development and function, as suggested by the receptor expression in immune cells. The activation of glutamate receptors can enhance the effectiveness of the effector's T cells, or decrease the cytokine production in immunosuppressive myeloid-derived suppressor cells, increasing the antitumor immune response. These receptors are essential for the interaction between tumor and immune cells within the tumor microenvironment (TME) and the regulation of antitumor immune responses. Although the role of glutamate in the TCA cycle has been well studied, few studies have deeply investigated the role of glutamate receptors in the regulation of cancer and immune cells within the TME. Here, by a systematic review of the available data, we will critically assess the physiopathological relevance of glutamate receptors in the regulation of cancer and immune cells in the TME and provide some unifying hypotheses for futures research on the role of glutamate receptors in the immune modulation of the tumor.

Estrogen downregulates CD73/adenosine axis hyperactivity via adaptive modulation PI3K/Akt signaling to prevent myocarditis and arrhythmias during chronic catecholamines stress.

BACKGROUND: During myocardial damage, the sex hormone estrogen and CD73, the main enzyme that converts AMP into adenosine, are cardioprotective molecules. However, it is unclear how these two molecules work together to provide cardioprotection. The current study aimed to elucidate the interaction between estrogen and CD73 under chronic stress. METHODS: Ovariectomy and SHAM operations were done on FVB wild-type (WT) female mice. Two weeks after the operation, the mice were treated with daily isoproterenol (10 mg/kg/day) injections for 14 days. The effect of E2 on relevant cardiac injury biomarkers (BNP, ANP), myocardial morphology (cardiomyocyte surface area), electrocardiography, CD73 protein expression and activity, and macrophage (CD86 + and CD206 +) infiltrations were assessed. In vitro, H9C2 cells were treated with 1 nM of estrogen and 10 mM APCP (CD73 inhibitor α, ß-methylene adenosine-5'-diphosphate), 10 µM isoproterenol and 20 µm LY294002 (PI3K inhibitor) for 24 h and western blot was done to elucidate the mechanism behind the effect of estrogen on the CD73/adenosine axis. RESULTS: Estrogen deficiency during chronic catecholamine stress caused myocardial injury, thereby triggering the hyperactivity of the CD73/adenosine axis, which aggravated myocarditis, adverse remodeling, and arrhythmias. However, estrogen normalizes CD73/Adenosine axis via the upregulation of PI3K/Akt pathways to prevent adverse outcomes during stress. In vivo results showed that the inhibition of PI3K significantly decreased PI3K/Akt pathways while upregulating the CD73/adenosine axis and apoptosis. CONCLUSION: Estrogen's pleiotropy cardioprotection mechanism during stress includes its normalization of the CD73/Adenosine axis via the PI3K/Akt pathway. Video Abstract.

The association between physical activity and mental health in medical postgraduates in China during COVID-19 pandemic.

Background: Chinese medical postgraduates (CMPs) are a special subpopulation that has a great risk of mental health due to high workload, and heavy academic and clinical pressure during the COVID-19 pandemic. Physical activity has been demonstrated to be positive for the mental health of human being. However, little is known about the risks to mental health among CMPs as well as the potential effects of physical activity on mental health in CMPs during the COVID-19 pandemic. Aims: In the present study, the aims are to (1) investigate the levels of PA and mental health that CMPs had; (2) to identify the potential factors that contribute to mental health among CMPs; (3) to explore the potential effects of PA on the mental health in CMPs. Methods: The survey was conducted online across 25 provinces of mainland China in 28 medical colleges or universities with structured questionnaires. Physical Activity Rating Scale-3 (PARS-3), Depression, Anxiety, Stress Scales (DASS-21), and General Self-Efficacy Scale (GSES) were employed to assess the levels of PA, Negative emotional states, and self-efficacy of the participants, respectively. Resilience was evaluated using Connor-Davidson Resilience Scale (CD-RISC). The chi-square and Wilcoxon rank-sum tests were used to compare inter-group differences in demographic data and mental health conditions. Spearman's rank correlation test and partial correlation analysis were used to assess associations between exercise scores and mental health. Results: We found some socio-demographic variables such as the location, education levels of parents and the levels of degrees they are training had potential effects on outcomes of mental health among 2,217 CMPs (P < 0.05); furthermore, we also found that PA was negatively correlated with the negative emotion (r = -0.045, P < 0.05) such as depression (r = -0.052, P < 0.05), anxiety and stress, (r = -0.051, P < 0.05) but positively correlated with the self-efficacy (r = 0.143, P < 0.001) and resilience (r = 0.192, P < 0.001) among Chinese postgraduate medical students. Conclusion: We concluded that for Chinese postgraduate medical students, taking part in physical activity would depress negative emotions such as depression, anxiety, and stress, but improve their self-efficacy and resilience, which will benefit them in completing their studies and training.

Clonorchis sinensis infection induces hepatobiliary injury via disturbing sphingolipid metabolism and activating sphingosine 1-phosphate receptor 2.

Clonorchis sinensis (C. sinensis) infection induces severe hepatobiliary injuries, which can cause inflammation, periductal fibrosis, and even cholangiocarcinoma. Sphingolipid metabolic pathways responsible for the generation of sphingosine-1-phosphate (S1P) and its receptor S1P receptors (S1PRs) have been implicated in many liver-related diseases. However, the role of S1PRs in C. sinensis-mediated biliary epithelial cells (BECs) proliferation and hepatobiliary injury has not been elucidated. In the present study, we found that C. sinensis infection resulted in alteration of bioactive lipids and sphingolipid metabolic pathways in mice liver. Furthermore, S1PR2 was predominantly activated among these S1PRs in BECs both in vivo and in vitro. Using JTE-013, a specific antagonist of S1PR2, we found that the hepatobiliary pathological injuries, inflammation, bile duct hyperplasia, and periductal fibrosis can be significantly inhibited in C. sinensis-infected mice. In addition, both C. sinensis excretory-secretory products (CsESPs)- and S1P-induced activation of AKT and ERK1/2 were inhibited by JTE-013 in BECs. Therefore, the sphingolipid metabolism pathway and S1PR2 play an important role, and may serve as potential therapeutic targets in hepatobiliary injury caused by C. sinensis-infection.

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.

IL-10 regulates Th17 response to inhibit hepatobiliary injury caused by Clonorchis sinensis infection in C57BL/6J mice.

Clonorchiasis caused by Clonorchis sinensis is a mainly foodborne parasitic disease. It can lead to hepatobiliary duct inflammation, fibrosis, obstructive jaundice, liver cirrhosis, and even cholangiocarcinoma. Interleukin (IL)-10 is an immune-regulatory cytokine which plays an immunosuppressive role during infection. Our previous study found that IL-10 was increased in mice with C. sinensis infection. However, the role and mechanism of IL-10 playing in hepatobiliary injury induced by C. sinensis infection remain unknown. Herein, Il10+/+ mice and Il10+/- C57BL/6J mice were infected with C. sinensis. It was found that IL-10 deficiency aggravated biliary hyperplasia and exacerbated periductal fibrosis induced by C. sinensis infection. Moreover, IL-10 deficiency increased CD4+T cells and CD8+T cells but not macrophages in the liver of mice with infection. There were no apparent differences in Th1 and Treg cells between Il10+/+ and Il10+/- mice infected with C. sinensis. However, the proportion of Th17 cells in CD4+T cells in Il10+/- infected mice was significantly higher than that in Il10+/+ infected mice. IL-10 deficiency also enhanced the increase of Th17 cells induced by ESPs stimulation in vitro. Taken together, our results suggest that IL-10 plays a protective role in hepatobiliary injury in C57BL/6J mice induced by C. sinensis infection via inhibiting Th17 cells, which could deepen our understanding of the immunopathology of clonorchiasis.

FXR deficiency in hepatocytes disrupts the bile acid homeostasis and inhibits autophagy to promote liver injury in Schistosoma japonicum-infected mice.

BACKGROUND: Schistosomiasis, with 250 million people affected, is characterized by its serious hepatic inflammatory response and fibrosis formation, which could lead to dangerous complications, such as portal hypertension, splenomegaly and even ascites. But until now, the pathogenesis of schistosomiasis remains largely unknown. Farnesoid X Receptor (FXR), a bile acid-activated nuclear transcription factor mainly expresses in hepatocytes in the liver, can regulate liver diseases by controlling bile acid metabolism. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that the expression of FXR was decreased in the liver of infected mice as shown by western blot and RT-qPCR assays. Furthermore, hepatocyte-specific FXR-deficient mice (FXRflox/floxAlbCre, FXR-HKO) were generated and infected with ~16 cercariae of S. japonicum for five weeks. We found that FXR deficiency in hepatocytes promoted the progression of liver injury, aggravated weight loss and death caused by infection, and promoted inflammatory cytokines production, such as IL-6, IL-1ß, TNF-α, IL-4, IL-10, and IL-13. Surprisingly, hepatic granulomas and fibrosis were not affected. In addition, using UPLC-MS/MS spectrometry, it was found that S. japonicum infection resulted in elevated bile acids in the liver of mice, which was more obvious in FXR-deficient mice. Meanwhile, autophagy was induced in littermate control mice due to the infection, but it was significantly decreased in FXR-HKO mice. CONCLUSIONS/SIGNIFICANCE: All these findings suggest that FXR deficiency in hepatocytes disrupts bile acid homeostasis and inhibits autophagy, which may aggravate the damages of hepatocytes caused by S. japonicum infection. It highlights that FXR in hepatocytes plays a regulatory role in the progression of schistosomiasis.

ß2-Adrenergic Receptor Enhances the Alternatively Activated Macrophages and Promotes Biliary Injuries Caused by Helminth Infection.

The autonomic nervous system has been studied for its involvement in the control of macrophages; however, the mechanisms underlying the interaction between the adrenergic receptors and alternatively activated macrophages (M2) remain obscure. Using FVB wild-type and beta 2 adrenergic receptors knockout, we found that ß2-AR deficiency alleviates hepatobiliary damage in mice infected with C. sinensis. Moreover, ß2-AR-deficient mice decrease the activation and infiltration of M2 macrophages and decrease the production of type 2 cytokines, which are associated with a significant decrease in liver fibrosis in infected mice. Our in vitro results on bone marrow-derived macrophages revealed that macrophages from Adrb2-/- mice significantly decrease M2 markers and the phosphorylation of ERK/mTORC1 induced by IL-4 compared to that observed in M2 macrophages from Adrb2+/+ . This study provides a better understanding of the mechanisms by which the ß2-AR enhances type 2 immune response through the ERK/mTORC1 signaling pathway in macrophages and their role in liver fibrosis.

Csi-let-7a-5p delivered by extracellular vesicles from a liver fluke activates M1-like macrophages and exacerbates biliary injuries.

Chronic infection with liver flukes (such as Clonorchis sinensis) can induce severe biliary injuries, which can cause cholangitis, biliary fibrosis, and even cholangiocarcinoma. The release of extracellular vesicles by C. sinensis (CsEVs) is of importance in the long-distance communication between the hosts and worms. However, the biological effects of EVs from liver fluke on biliary injuries and the underlying molecular mechanisms remain poorly characterized. In the present study, we found that CsEVs induced M1-like activation. In addition, the mice that were administrated with CsEVs showed severe biliary injuries associated with remarkable activation of M1-like macrophages. We further characterized the signatures of miRNAs packaged in CsEVs and identified a miRNA Csi-let-7a-5p, which was highly enriched. Further study showed that Csi-let-7a-5p facilitated the activation of M1-like macrophages by targeting Socs1 and Clec7a; however, CsEVs with silencing Csi-let-7a-5p showed a decrease in proinflammatory responses and biliary injuries, which involved in the Socs1- and Clec7a-regulated NF-κB signaling pathway. Our study demonstrates that Csi-let-7a-5p delivered by CsEVs plays a critical role in the activation of M1-like macrophages and contributes to the biliary injuries by targeting the Socs1- and Clec7a-mediated NF-κB signaling pathway, which indicates a mechanism contributing to biliary injuries caused by fluke infection. However, molecules other than Csi-let-7a-5p from CsEVs that may also promote M1-like polarization and exacerbate biliary injuries are not excluded.

Amlexanox and Forskolin Prevents Isoproterenol-Induced Cardiomyopathy by Subduing Cardiomyocyte Hypertrophy and Maladaptive Inflammatory Responses.

Chronic catecholamine stress (CCS) induces the occurrence of cardiomyopathy-pathological cardiac hypertrophy (PCH), which is characterized by left ventricular systolic dysfunction (LVSD). Recently, mounting evidence has implicated myocardial inflammation in the exacerbation of pathological cardiac remodeling. However, there are currently no well-defined treatment interventions or regimes targeted at both the attenuation of maladaptive myocardial hypertrophy and inflammation during CCS to prevent PCH. G protein-coupled receptor kinase 5 (GRK5) and adenylyl cyclases (ACs)-cAMP mediates both cardiac and inflammatory responses. Also, GRK5 and ACs are implicated in stress-induced LVSD. Herein, we aimed at preventing PCH during CCS via modulating adaptive cardiac and inflammatory responses by inhibiting GRK5 and/or stimulating ACs. Isoproterenol-induced cardiomyopathy (ICM) was modeled using 0.5 mg/100 g/day isoproterenol injections for 40 days. Alterations in cardiac and inflammatory responses were assessed from the myocardia. Similarities in the immunogenicity of cardiac troponin I (cTnI) and lipopolysaccharide under CCS were assessed, and Amlexanox (35 µM/ml) and/or Forskolin (10 µM/ml) were then employed in vitro to modulate adaptive inflammatory responses by inhibiting GRK5 or activating ACs-cAMP, respectively. Subsequently, Amlexanox (2.5 mg/100 g/day) and/or Forskolin (0.5 mg/100 g/day) were then translated into in vivo during CCS to modulate adaptive cardiac and inflammatory responses. The effects of Amlexanox and Forskolin on regulating myocardial systolic functions and inflammatory responses during CCS were ascertained afterward. PCH mice had excessive myocardial hypertrophy, fibrosis, and aggravated LVSD, which were accompanied by massive CD68+ inflammatory cell infiltrations. In vitro, Forskolin-AC/cAMP was effective than Amlexanox-GRK5 at downregulating proinflammatory responses during stress; nonetheless, Amlexanox and Forskolin combination demonstrated the most efficacy in modulating adaptive inflammatory responses. Individually, the translated Amlexanox and Forskolin treatment interventions were ineffective at subduing the pathological remodeling and sustaining cardiac function during CCS. However, their combination was potent at preventing LVSD during CCS by attenuating maladaptive myocardial hypertrophy, fibrosis, and inflammatory responses. The treatment intervention attained its potency mainly via Forskolin-ACs/cAMP-mediated modulation of cardiac and inflammatory responses, coupled with Amlexanox inhibition of GRK5 mediated maladaptive cascades. Taken together, our findings highlight the Amlexanox and Forskolin combination as a potential therapeutic intervention for preventing the occurrence of pathological cardiac hypertrophy during chronic stress.

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.

Molecular Mechanisms of Clonorchis sinensis-Host Interactions and Implications for Vaccine Development.

Infections caused by Clonorchis sinensis remain a significant public health challenge for both humans and animals, causing pyogenic cholangitis, cholelithiasis, cholecystitis, biliary fibrosis, and even cholangiocarcinoma. However, the strategies used by the parasite and the immunological mechanisms used by the host have not yet been fully understood. With the advances in technologies and the accumulated knowledge of host-parasite interactions, many vaccine candidates against liver flukes have been investigated using different strategies. In this review, we explore and analyze in-depth the immunological mechanisms involved in the pathogenicity of C. sinensis. We highlight the different mechanisms by which the parasite interacts with its host to induce immune responses. All together, these data will allow us to have a better understanding of molecular mechansism of host-parasite interactions, which may shed lights on the development of an effective vaccine against C. sinensis.

Recombinant CsHscB of carcinogenic liver fluke Clonorchis sinensis induces IL-10 production by binding with TLR2.

BACKGROUND: Clonorchis sinensis, a fluke dwelling in the intrahepatic bile ducts causes clonorchiasis, which affect about 15 million people wide-distributed in eastern Asia. During C. sinensis infection, worm-host interaction results in activation of patterns recognition receptors (PRRs) such as Toll-like receptors (TLRs) and further triggers immune responses, which determines the outcome of the infection. However, the mechanisms by which pathogen-associated molecules patterns from C. sinensis interact with TLRs were poorly understood. In the present study, we assumed that the molecules from C. sinensis may regulate host immune responses via TLR2 signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we have identified a ~34 kDa CsHscB from C. sinensis which physically bound with TLR2 as demonstrated by molecular docking and pull-down assay. We also found that recombinant CsHscB (rCsHscB) potently activates macrophage to express various proteins including TLR2, CD80, MHCII, and cytokines like IL-6, TNF-α, and IL-10, but rCsHscB failed to induce IL-10 in macrophages from Tlr2-/- mice. Moreover, ERK1/2 activation was required for rCsHscB-induced IL-10 production in macrophages. In vivo study revealed that rCsHscB triggered a high production of IL-10 in the wild-type (WT) but not in Tlr2-/- mice. Consistently, the phosphorylation of ERK1/2 was also attenuated in Tlr2-/- mice compared to the WT mice, after the treatment with rCsHscB. CONCLUSIONS/SIGNIFICANCE: Our data thus demonstrate that rCsHscB from C. sinensis interacts with TLR2 to be endowed with immune regulatory activities, and may have some therapeutic implications in future beyond parasitology.

TLR4 Deficiency Exacerbates Biliary Injuries and Peribiliary Fibrosis Caused by Clonorchis sinensis in a Resistant Mouse Strain.

Mice with different genetic backgrounds have various susceptibilities to infection with Clonorchis sinensis, although the mechanisms underlying are largely unknown. Toll-like receptor 4 (TLR4) as one of the most important pattern recognition receptors (PPRs) is essential for the invasion, survival, pathogenesis, and elimination of worms. The roles played by TLR4 in C. sinensis infection may vary due to the different genetic backgrounds of mice. In the present study, a relatively resistant mouse strain-C57BL/10 to C. sinensis was used for investigation on the possible roles of TLR4 in the biliary injuries and peribiliary fibrosis. TLR4 wild type (TLR4 wild ) and TLR4 defective (TLR4 def ) mice were orally infected with 45 metacercariae of C. sinensis, and all C. sinensis-infected mice and non-infected groups were anesthetized on day 28 post-infection. The liver and serum from each mouse were collected for assessment of the biliary injuries and biliary fibrosis. Meanwhile, hepatic leukocytes were isolated and detected for the activation of M1 or M2 macrophage using flow cytometry. The hepatic type 1 immune response and type 2 immune responses -relative molecules were also evaluated using ELISA and quantitative PCR. The data showed that TLR4 def aggravated liver inflammatory cell infiltrations, bile duct proliferation, biliary and hepatocellular injuries, and ECM deposition in C. sinensis-infected mice, compared with TLR4 wild mice when they were intragastrically administered with the same amounts of C. sinensis metacercaria. Furthermore, the M2-like macrophages and type 2 immune responses were significantly predominant induced in TLR4 def mice, compared with that of TLR4 wild mice following C. sinensis infection. But the type 1 immune response were significantly decreased in TLR4 def mice, compared with TLR4 wild mice after C. sinensis infection. These data demonstrate that TLR4 deficiency exacerbates biliary injuries and peribiliary fibrosis caused by C. sinensis in C57BL/10 strain mice, which is contributed by augments of type 2 immune responses and decrease pro-inflammatory responses.