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Background: Schistosomiasis is a zoonotic parasitic disorder induced by the infestation of schistosomes, a genus of trematodes. MicroRNAs (miRNAs) in egg-derived exosomes are crucial for modulating the host's immune responses and orchestrating the pathophysiological mechanisms. Although the exosomes secreted by S. japonicum contain abundant miRNAs, the specific roles of these miRNAs in the pathogenesis of schistosomiasis-induced hepatic fibrosis are yet to be comprehensively elucidated. The egg exosomes of S. japonicum secrete miRNA-30, a novel miRNA., Methods: In vitro , the effect of miRNA-30 was evaluated by transfecting HSCs with miRNA mimics. The target gene biosignature for miRNA-30 was predicted using the miRDB software. The effect of miRNA-30 in hepatic fibrosis was evaluated by either elevating its expression in healthy mice or by inhibiting its activity in infected mice by administration of recombinant adeno-associated virus serotype eight vectors expressing miRNA-30 or miRNA sponges., Results: This novel miRNA can activate hepatic stellate cells (HSCs), the prinary effector cells of hepatic fibrosis, in vitro , i.e., it significantly increases the fibrogenic factors Col1 ( α1 ), Col3 ( α1 ), and α-SMA at both mRNA and protein levels. In addition, miRNA-30 may activate HSCs by targeting the host RORA gene. In addition, in vivo experiments were conducted by administering a recombinant adeno-associated viral vector to modulate the expression levels of miRNA-30. The overexpression of miRNA-30 in healthy mice significantly elevated the expression of Col1 ( α1 ), Col3 ( α1 ), and α -SMA at both the transcriptomic and proteomic scales. This overexpression was coupled with a pronounced augmentation in the hepatic hydroxyproline content. Conversely, the in vivo silencing of miRNA-30 in infected mice induced a considerable reduction in the size of hepatic granulomas and areas of collagen deposition. Hence, in vivo , modulation of miRNA-30 expression may play a pivotal role in ameliorating the severity of hepatic fibrosis in mice afflicted with S. japonica ., Conclusions: The study results suggest that miRNA-30 may augment schistosomiasis-induced hepatic fibrosis through a probable interaction with the host RORA. Our study may improve the current theoretical framework regarding cross-species regulation by miRNAs of hepatic fibrosis in schistosomiasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Hu, Zhou, Jiang, Wang, Zhang, Shen, Yu and Cao.)

Schistosomiasis is a parasitic disease characterized by liver fibrosis, a process driven by the activation of hepatic stellate cells (HSCs) and subsequent collagen production. Previous studies from our laboratory have demonstrated the ability of Schistosoma japonicum protein P40 (SjP40) to inhibit HSCs activation and exert an antifibrotic effect. In this study, we aimed to elucidate the molecular mechanism underlying the inhibitory effect of recombinant SjP40 (rSjP40) on HSCs activation. Using a cell model in which rSjP40 inhibited LX-2 cell activation, we performed RNA-seq analyses and identified ATF3 as the most significantly altered gene. Further investigation revealed that rSjP40 inhibited HSCs activation partly by suppressing ATF3 activation. Knockdown of ATF3 in mouse liver significantly alleviated S. japonicum-induced liver fibrosis. Moreover, our results indicate that ATF3 is a direct target of microRNA-494-3p, a microRNA associated with anti-liver fibrosis effects. rSjP40 was found to downregulate ATF3 expression by upregulating microRNA-494-3p in LX-2 cells. This downregulation led to the inhibition of the expression of liver fibrosis proteins α-SMA and COL1A1, ultimately alleviating liver fibrosis caused by S. japonicum., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Schistosoma infection is a significant public health issue, affecting over 200 million individuals and threatening 700 million people worldwide. The species prevalent in China is Schistosoma japonicum. Recent studies showed that both gut microbiota and metabolome are closely related to schistosomiasis caused by S. japonicum, but clinical study is limited and the underlying mechanism is largely unclear. This study aimed to explore alterations as well as function of gut microbiota and metabolite profile in the patients with S. japonicum infection., Methods: This study included 20 patients diagnosed with chronic schistosomiasis caused by S. japonicum, eight patients with advanced schistosomiasis caused by S. japonicum and 13 healthy volunteers. The fresh feces of these participators, clinical examination results and basic information were collected. 16S ribosomal RNA gene sequencing was used to investigate gut microbiota, while ultraperformance liquid chromatography-mass spectrometry (UHPLC-MS) was applied to explore the metabolome of patients in different stages of schistosomiasis., Results: The study found that gut microbiota and metabolites were altered in patients with different stages of S. japonicum infection. Compared with healthy control group, the gut microbial diversity in patients with chronic S. japonicum infection was decreased significantly. However, the diversity of gut microbiota in patients with chronic schistosomiasis was similar to that in patients with advanced schistosomiasis. Compared with uninfected people, patients with schistosomiasis showed decreased Firmicutes and increased Proteobacteria. As disease progressed, Firmicutes was further reduced in patients with advanced S. japonicum infection, while Proteobacteria was further increased. In addition, the most altered metabolites in patients with S. japonicum infection were lipids and lipid-like molecules as well as organo-heterocyclic compounds, correlated with the clinical manifestations and disease progress of schistosomiasis caused by S. japonicum., Conclusions: This study suggested that the gut microbiota and metabolome altered in patients in different stages of schistosomiasis, which was correlated with progression of schistosomiasis caused by S. japonicum. This inter-omics analysis may shed light on a better understanding of the mechanisms of the progression of S. japonicum infection and contribute to identifying new potential targets for the diagnosis and prognosis of S. japonicum infection. However, a large sample size of validation in clinic is needed, and further study is required to investigate the underlying mechanism., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Schistosomes undergo complicated migration in final hosts during infection, associated with differential immune responses. It has been shown that CD4 + T cells play critical roles in response to Schistosoma infections and accumulated documents have indicated that miRNAs tightly regulate T cell activity. However, miRNA profiles in host T cells associated with Schistosoma infection remain poorly characterized. Therefore, we undertook the study and systematically characterized T cell miRNA profiles from the livers and blood of S. japonicum infected C57BL/6J mice at 14- and 21-days post-infection. We observed 508 and 504 miRNAs, in which 264 miRNAs were co-detected in T cells isolated from blood and livers, respectively. The comparative analysis of T cell miRNAs from uninfected and infected C57BL/6J mice blood showed that miR-486b-5p/3p expression was significantly downregulated and linked to various T cell immune responses and miR-375-5p was highly upregulated, associated with Wnt signaling and pluripotency, Delta notch signaling pathways, etc. Whereas hepatic T cells showed miR-466b-3p , miR-486b-3p , miR-1969 , and miR-375 were differentially expressed compared to the uninfected control. The different expressions of some miRNAs were further corroborated in isolated T cells from mice and in vitro cultured EL-4 cells treated with S. japonicum worm antigens by RT-qPCR and similar results were found. In addition, bioinformatics analysis combined with RT-qPCR validation of selected targets associated with the immune system and parasite-caused infectious disease showed a significant increase in the expression of Ctla4 , Atg5 , Hgf , Vcl and Arpc4 and a decreased expression of Fermt3 , Pik3r1 , Myd88 , Nfkbie , Ppp1r12a , Ppp3r1 , Nfyb , Atg12 , Ube2n , Tyrobp , Cxcr4 and Tollip . Overall, these results unveil the comprehensive repertoire of T cell miRNAs during S. japonicum infection, suggesting that the circulatory (blood) and liver systems have distinct miRNAs landscapes that may be important for regulating T cell immune response. Altogether, our findings indicated a dynamic expression pattern of T cell miRNAs during the hepatic stages of S. japonicum infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Giri, Li, Fang, Qiu, Yan, Pakharukova and Cheng.)

Objective: To screen differentially expressed genes (DEGs) associated with chronic schistosomiasis japonica-induced hepatic fibrosis and analyze their functions., Methods: The dataset of gene expression profiles of patients with chronic schistosomiasis japonica-induced hepatic fibrosis was downloaded from the Gene Expression Omnibus (GEO) database, and DEGs were screened using R package. The biological functions of DEGs were characterized using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In addition, the protein-protein interaction (PPI) network of DEGs was created to screen the hub genes., Results: A total of 62 DEGs were identified, including 12 down-regulated genes and 50 up-regulated genes. GO enrichment analysis showed that DEGs were mainly enriched in 116 biological processes, including fatty acid, sulfur compound, acyl-coenzyme A and thioester metabolism; 19 cellular components, including mitochondrial matrix, outer mitochondrial membrane and organelle outer membrane; and 7 molecular functions, including insulin-like growth factor binding and oxidoreductase activity. KEGG pathway enrichment analysis that the DEGs were significantly enriched in phosphatidylinositol-3-kinase/serine/threonine protein kinase (PI3K/Akt), mitogen-activated protein kinase (MAPK), calcium metabolism and cyclic adenosine monophosphate (cAMP) signaling. PPI network analysis identified six hub genes involved in the development of chronic schistosomiasis japonica-induced hepatic fibrosis, including ACACA , ACSL1 , GPAM , THRSP , PLIN1 and DGAT2 , and ACSL1 , ACACA and PLIN1 were the top 3 hub genes., Conclusions: ACSL1 , ACACA and PLIN1 may be the hub genes associated with the development of chronic schistosomiasis japonica-induced hepatic fibrosis, and abnormal lipid metabolism mediated by these DEGs may play an important role in the development of chronic schistosomiasis japonica-induced hepatic fibrosis.

B cells played an important role in Schistosoma infection-induced diseases. TLR7 is an intracellular member of the innate immune receptor. The role of TLR7 on B cells mediated immune response is still unclear. Here, C57BL/6 mice were percutaneously infected by S. japonicum for 5-6 weeks. The percentages and numbers of B cells increased in the infected mice (p < 0.05), and many activation and function associated molecules were also changed on B cells. More splenic cells of the infected mice expressed TLR7, and B cells were served as the main cell population. Moreover, a lower level of soluble egg antigen (SEA) specific antibody and less activation associated molecules were found on the surface of splenic B cells from S. japonicum infected TLR7 gene knockout (TLR7 KO) mice compared to infected wild type (WT) mice (p < 0.05). Additionally, SEA showed a little higher ability in inducing the activation of B cells from naive WT mice than TLR7 KO mice (p < 0.05). Finally, the effects of TLR7 on B cells are dependent on the activation of NF-κB p65. Altogether, TLR7 was found modulating the splenic B cell responses in S. japonicum infected C57BL/6 mice., Competing Interests: The authors have declared that no competing interests exist.

Background: CD4 + T helper (Th) cells play critical roles in both host humoral and cellular immunity against parasitic infection and in the immunopathology of schistosomiasis. T follicular helper (Tfh) cells are a specialized subset of Th cells involved in immunity against infectious diseases. However, the role of Tfh cells in schistosome infection is not fully understood. In this study, the dynamics and roles of Tfh cell regulation were examined. We demonstrated that granulocytic myeloid-derived suppressor cells (G-MDSC) can suppress the proliferation of Tfh cells., Methods: The levels of Tfh cells and two other Th cells (Th1, Th2) were quantitated at different Schistosoma japonicum infection times (0,3, 5, 8, 13 weeks) using flow cytometry. The proliferation of Tfh cells stimulated by soluble egg antigen (SEA) and soluble worm antigen (SWA) in vivo and in vitro were analyzed. Tfh cells were co-cultured with MDSC to detect the proliferation of Tfh cells labelled by 5(6)-carboxyfluorescein diacetate N-succinimidyl ester. We dynamically monitored the expression of programmed cell death protein 1 (PD-1) on the surface of Tfh cells and programmed cell death ligand 1 (PD-L1) on the surface of MDSC at different infection times (0, 3, 5, 8 weeks). Naïve CD4 + T cells (in Tfh cell differentiation) were co-cultured with G-MDSC or monocytic MDSC in the presence, or in the absence, of PD-L1 blocking antibody., Results: The proportion of Tfh cells among CD4 + T cells increased gradually with time of S. japonicum infection, reaching a peak at 8 weeks, after which it decreased gradually. Both SEA and SWA caused an increase in Tfh cells in vitro and in vivo. It was found that MDSC can suppress the proliferation of Tfh cells. The expression of PD-1 on Tfh cells and PD-L1 from MDSC cells increased with prolongation of the infection cycle. G-MDSC might regulate Tfh cells through the PD-1/PD-L1 pathway., Conclusions: The reported study not only reveals the dynamics of Tfh cell regulation during S. japonicum infection, but also provides evidence that G-MDSC may regulate Tfh cells by PD-1/PD-L1. This study provides strong evidence for the important role of Tfh cells in the immune response to S. japonicum infection., (© 2021. The Author(s).)

Background: Th cells (helper T cells) have multiple functions in Schistosoma japonicum ( S. japonicum ) infection. Inducible co-stimulator (ICOS) is induced and expressed in activated T lymphocytes, which enhances the development of B cells and antibody production through the ICOS/ICOSL pathway. It remains unclear about the role and possible regulating mechanism of ICOS + Th cells in the spleen of S. japonicum -infected C57BL/6 mice., Methods: C57BL/6 mice were infected with cercariae of S. japonicum through the abdomen. The expression of ICOS, activation markers, and the cytokine production on CD4 + ICOS + Th cells were detected by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR). Moreover, the differentially expressed gene data of ICOS + and ICOS - Th cells from the spleen of infected mice were obtained by mRNA sequencing. Besides, Western blot and chromatin immunoprecipitation (ChIP) were used to explore the role of Ikzf2 on ICOS expression., Results: After S. japonicum infection, the expression of ICOS molecules gradually increased in splenic lymphocytes, especially in Th cells ( P < 0.01). Compared with ICOS - Th cells, more ICOS + Th cells expressed CD69, CD25, CXCR5, and CD40L ( P  < 0.05), while less of them expressed CD62L ( P < 0.05). Also, ICOS + Th cells expressed more cytokines, such as IFN-γ, IL-4, IL-10, IL-2, and IL-21 ( P < 0.05). RNA sequencing results showed that many transcription factors were increased significantly in ICOS + Th cells, especially Ikzf2 ( P < 0.05). And then, the expression of Ikzf2 was verified to be significantly increased and mainly located in the nuclear of ICOS + Th cells. Finally, ChIP experiments and dual-luciferase reporter assay confirmed that Ikzf2 could directly bind to the ICOS promoter in Th cells., Conclusion: In this study, ICOS + Th cells were found to play an important role in S. japonicum infection to induce immune response in the spleen of C57BL/6 mice. Additionally, Ikzf2 was found to be one important transcription factor that could regulate the expression of ICOS in the spleen of S. japonicum -infected C57BL/6 mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xie, Wei, Peng, Xie, Li, Fang, Shi, Yang, Huang, Xie, Pan, Tian and Huang.)

Background: Hepatic stellate cells (HSCs) are one of the main cell types involved in liver fibrosis induced by many factors, including schistosomes. Previous studies in our lab have shown that recombinant P40 protein from Schistosoma japonicum (rSjP40) can inhibit HSC activation in vitro. Let-7b is a member of the let-7 microRNA family and plays an inhibitory role in a variety of diseases and inflammatory conditions. In this study, we investigated the role of let-7b in the inhibition of HSC activation by rSjP40., Methods: Expression of let-7b was detected by quantitative real-time PCR. A dual luciferase assay was used to confirm direct interaction between let-7b and collagen I. We also used western blot to assess protein levels of TGFβRI and collagen type I α1 (COL1A1)., Results: We found that rSjP40 up-regulates expression of let-7b in HSCs. Let-7b inhibits collagen I expression by directly targeting the 3'UTR region of the collagen I gene. Furthermore, we discovered that let-7b inhibitor partially restores the loss of collagen I expression caused by rSjP40., Conclusion: Our research clarifies the role of let-7b in the inhibition of HSC activation by rSjP40 and will provide new insights and ideas for the inhibition of HSC activation and treatment of liver fibrosis., Competing Interests: The authors have declared that no competing interests exist.

Schistosomiasis is a parasitic helminth disease that can cause organ lesions leading to health damage. During a schistosome infection, schistosome eggs can flow into the liver along the portal vein. Numerous inflammatory cells gather around the eggs, causing granulomas and fibrosis in the liver. In this process, many molecules are involved in the initiation and regulation of the fibrous scar formation. However, the precise molecular mechanisms responsible for the progression of granuloma formation and fibrosis initiation caused by schistosome infection have not been extensively studied. In this study, C57BL/6 wild-type mice and Stat3 flox/flox Alb-Cre mice were infected with cercariae of Schistosoma japonicum Liver injury, effector molecule levels, and RNA transcriptome resequencing of liver tissue were detected at 4, 5, and 6 weeks postinfection. We investigated the role of STAT3 (signal transducer and activator of transcription 3) in Schistosoma -induced liver injury in mice. After 6 weeks postinfection, there was obvious liver fibrosis. A sustained pathological process (inflammation, oxidative stress, proliferation, and apoptosis) occurred in S. japonicum -induced liver fibrosis initiation. Meanwhile, we observed activation of the STAT3 pathway in hepatic injury during S. japonicum infection by RNA transcriptome resequencing. Liver deficiency of phospho-STAT3 alleviated infection-induced liver dysfunction, hepatic granuloma formation, and fibrosis initiation. It also promoted STAT3-dependent apoptosis and reduced liver inflammation, oxidative stress, and proliferation. Our results suggest that STAT3 signal pathway and its mediating inflammation, oxidative stress, proliferation, and apoptosis are involved in S. japonicum -induced liver injury and may be a new potential guideline for the treatment of schistosomiasis., (Copyright © 2021 American Society for Microbiology.)

Microtus fortis is the only mammalian host that exhibits intrinsic resistance against Schistosoma japonicum infection. However, the underlying molecular mechanisms of this resistance are not yet known. Here, we perform the first de novo genome assembly of M. fortis, comprehensive gene annotation analysis, and evolution analysis. Furthermore, we compare the recovery rate of schistosomes, pathological changes, and liver transcriptomes between M. fortis and mice at different time points after infection. We observe that the time and type of immune response in M. fortis are different from those in mice. M. fortis activates immune and inflammatory responses on the 10th day post infection, such as leukocyte extravasation, antibody activation, Fc-gamma receptor-mediated phagocytosis, and the interferon signaling cascade, which play important roles in preventing the development of schistosomes. In contrast, an intense immune response occurrs in mice at the late stages of infection and could not eliminate schistosomes. Infected mice suffer severe pathological injury and continuous decreases in cell cycle, lipid metabolism, and other functions. Our findings offer new insights into the intrinsic resistance mechanism of M. fortis against schistosome infection. The genome sequence also provides the basis for future studies of other important traits in M. fortis., (Copyright © 2021 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Immunopathological mechanisms of schistosomiasis, a debilitating parasitic disease, are still unclear. In this study, we investigated the involvement of CX3C chemokine ligand 1 (CX3CL1) and its sole receptor CX3CR1 in the development of liver fibrosis in schistosomiasis. The animal model of schistosomiasis was established by infection of C57BL/6 mice with Schistosoma japonicum cercariae; mice injected with carbon tetrachloride (CCl 4 ) were used as positive control of liver injury. After 4 and 8 weeks, the degree of liver lesions was assessed by hematoxylin and eosin staining, serum levels of hyaluronic acid (HA) were analyzed by a chemiluminescence immunoassay, liver fibrosis was evaluated by immunohistochemistry analysis of α-smooth muscle actin (α-SMA) expression, and CX3CL1 and CX3CR1 expression in the liver was measured by immunohistochemistry and real-time PCR. The results showed that at 8 weeks after Schistosoma infection, serum HA levels were increased and α-SMA-expressing cells appeared in the liver, indicating fibrogenesis. CX3CL1- and CX3CR1-positive cells were observed in the outer layer of granulomas formed around Schistosoma eggs in liver tissues, which was consistent with the significant upregulation of hepatic CX3CL1 and CX3CR1 mRNA expression at 4 and 8 weeks post-infection. Furthermore, correlation analysis revealed positive association between CX3CL1 and CX3CR1 expression and serum HA levels at 8 weeks post-infection, indicating a link between fibrogenesis and the CX3CL1/CX3CR1 axis in schistosomiasis. In conclusion, our data suggest the involvement of CX3CL1 and CX3CR1 in the progression of liver fibrosis caused by Schistosoma infection.

Objective: The activation of hepatic stellate cells (HSCs) is a key event in schistosome-induced liver fibrosis. Previous studies have shown that soluble egg antigens and the recombinant P40 protein from Schistosoma japonicum eggs inhibit HSC activation. In the present study, we observed the direct effect of the S. japonicum recombinant (r)SjE16 protein on HSCs., Methods: The sequence of SjE16 was analyzed by bioinformatics. Then western blotting, quantitative PCR, and MTT assays were performed to observe the effects of rSjE16 on HSCs., Results: The SjE16 protein has no signal peptide or transmembrane region. rSjE16 significantly inhibited expression levels of α-smooth muscle actin and collagen I protein in LX-2 cells. rSjE16 also significantly increased the expression levels of interleukin (IL)-6 and IL-8, and enhanced the expression of matrix metalloproteinase (MMP)-2, MMP-9, and peroxisome proliferator-activated receptor-γ in LX-2 cells. LX-2 cell viability was not inhibited by rSjE16., Conclusion: rSjE16 may be involved in the progression of HSC activation via a complex molecular mechanism, which requires further study to fully understand.

Schistosomiasis occurs in 240 million people worldwide and is a major public health concern. Thus, early diagnosis and monitoring of schistosomiasis progression are needed to treat patients. Cell-free DNA (cfDNA) is present as fragments of parasite-derived DNA in host body fluids. Detection of this cfDNA in host blood may be a promising diagnostic marker of schistosomiasis. Therefore, in this study, we investigated the potential of internal transcribed spacer 2 (ITS2), a molecular taxonomy and barcoding marker, in diagnosing schistosomiasis using infected rabbit and mice sera. A 192 bp fragment of ITS2 was detected in the serum-isolated DNA from the infected host on different days after infection. We also determined the sensitivity of detecting ITS2 in mice with varying numbers of cercaria: cfDNA was present even in mice with low abundance of the parasite. Overall, our results show that cfDNA may be a potential tool for the early diagnosis and therapeutic evaluation of S. japonicum infection., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

Background: Recent investigations indicate that schistosome infection is closely associated with aberrant glycolipid metabolism. However, the actual glycolipid metabolism gene expression, as well as the possible pathways that regulate glycolipid metabolism in the schistosome-infected liver, has not been extensively explored., Methods: In this study, we evaluated the dynamic expression of glycolipid metabolism-associated genes and proteins in the livers from mice infected with Schistosoma japonicum at the indicated time points using real-time PCR and immunofluorescence. Then, cultures of macrophages were treated with schistosome soluble egg antigen (SEA) to detect the expression levels of genes associated with glucose and lipid metabolism in order to identify macrophages metabolic characteristics in response to these antigens. Furthermore, SEA-stimulated macrophages were co-cultures with hepatocytes and detected the effects of macrophages on the gene expression of hepatocytes metabolism., Results: The expression of glycolysis-related genes (Ldha, Glut4, Pkm2, Glut1, Pfkfb3, Aldoc, HK2, Pfk) in the liver were upregulated but the gluconeogenesis gene (G6pc) was downregulated during S. japonicum infection. In addition, the mRNA levels of fatty acid (FA) oxidation-related genes (Ucp2, Atp5b, Pparg) in the liver were significantly upregulated; however, the FA synthesis genes (Fas, Acc, Scd1, Srebp1c) and lipid uptake gene (Cd36) were downregulated post-S. japonicum-infection. In consistence with these data, stimulation with SEA in vitro significantly enhanced the gene expression that involved in glycolysis and FA oxidation, but decreased genes related to gluconeogenesis, FA synthesis and lipid uptake in macrophages. The levels of phosphorylated AMPK, AKT and mTORC1 were increased in macrophages after SEA stimulation. Inhibition of phosphorylated AMPK, AKT and mTORC1 promoted SEA-treated macrophages to produce glucose. In addition, suppression of phosphorylated-AMPK, but not phosphorylated-AKT and phosphorylated-mTOR, induced the lipid accumulation in SEA-stimulated macrophages. Furthermore, SEA-treated macrophages significantly reduced the expression of Acc mRNA in hepatocytes in vitro., Conclusions: These findings reveal S. japonicum infection induces dynamic changes in the expression levels of genes involved in catabolism (glucose uptake, glycolysis and fatty acid oxidation) and suppressing anabolism (glycogen synthesis) in the liver, which could occur via macrophages' metabolic states, particularly those involved in the AMPK, AKT and mTORC1 pathways.

Wnts are secreted signaling molecules that are implicated in a variety of growth-related processes. Frizzled proteins have been identified as receptors for Wnt ligands in vertebrates and invertebrates, but a functional role for dioecious flatworm Frizzleds has not been determined. To evaluate the endogenous role of Frizzled proteins during development, we have identified and characterized a Schistosoma japonicum frizzled gene (Sjfz7). We found that Sjfz7 encodes a 698 amino acid protein with typical characteristics of Frizzled proteins. The immunohistochemical localization pattern showed that Sjfz7 protein was extensively distributed in almost all tissues of S. japonicum, including subtegumental muscle cells, parenchymal cells, intestinal epithelial cells and male and female germ cells. This indicated that Sjfz7-mediated Wnt signaling might be associated with the development of musculature, intestinal tract and reproductive organs in schistosome. Comparing mRNA levels between frizzled family members showed that Sjfz7 mRNA was consistently higher in the developmental stages analyzed, suggesting that Sjfz7 may be responsible for more functional tasks than other frizzled family members. Comparing frizzled mRNA levels between not fully developed and normal worms suggested that Wnt signaling might be abnormal in not fully developed worms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

According to conservative estimates, >230 million people are infected with schistosomiasis,which becomes one of the most common parasitic diseases. This study focuses on investigating in vivo and in vitro effects of mmu-miR-92a-2-5p in Schistosoma japonicum-induced liver fibrosis by targeting TLR2. Through bioinformatic analysis, the overexpression of TLR2 and the down-regulation of mmu-miR-92a-2-5p were revealed in the progression of S. japonicum-induced liver fibrosis. BALB/C mice were taken advantage to construct normal control and schistosomiasis liver fibrosis (SLF) model. The mice in model groups were transfected recombinant lentivirus (Lenti-mmu-miR-92a-2-5p or Lenti-NC) to alter the expression of mmu-miR-92a-2-5p in vivo. HE and Masson staining were employed to observe the pathological changes and collagenous fibrosis. QRT-PCR showed that mmu-miR-92a-2-5p was decreased while TLR2 was elevated in the infected groups. However, lenti-mmu-miR-92a-2-5p group could inhibit liver fibrosis. Then the effect of mmu-miR-92a-2-5p on S. japonicum-induced liver fibrosis including cell apoptosis rates, proliferation and proteins related to liver fibrosis was examined in NIH-3T3 mouse embryonic fibroblasts. Moreover, the association between mmu-miR-92a-2-5p and TLR2 was detected by dual-luciferase reporter gene assay and the expression of cytokines IL-4, IFN-γ and TNF-α in SLF model was detected by ELISA. Further, the knockout of TLR2 in C57BL/6J mice was used to confirm the association between mmu-miR-92a-2-5p and TLR2. Thus, these findings demonstrated that mmu-miR-92a-2-5p inhibited S. japonicum-induced liver fibrosis by targeting TLR2 in vitro and in vivo., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the effect of Schistosoma japonicum glutathione S-transferase (SjGST) on the developmental stages of the parasite. We found that the mRNA levels of GST were higher in schistosomula obtained from the host and the eggs than that in other developmental stages. SjGST was mainly distributed in the egg shells, teguments of the worms, and part of the parenchyma of the worms. GST knockdown with RNA interference in S. japonicum worms resulted in a silencing rate higher than 80%. The egg reduction rate (18%) and abnormal egg ratio (28%) were significantly higher (P < 0.05) in the GST-silenced group than in the negative control group. These results indicate that SjGST plays an important role in the fecundity of S. japonicum, specifically in egg formation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

BACKGROUND The prevalence and intensity of schistosomiasis infection in China has decreased markedly in recent years. Therefore, more accurate methods are critically needed to ensure further control of low-intensity schistosomiasis infection. For chronic schistosomiasis patients, the detection of schistosome eggs in colorectal mucosa tissues is commonly used. This work aimed to explore differences in sensitivity of the Schistosoma japonicum (S. japonicum) retrotransposon (SjR2) gene in colon tissue from S. japonicum infected hosts and to develop an ideal method for genetic diagnosis of low-intensity schistosomiasis. MATERIAL AND METHODS Serum and colon samples were collected from mice at different time points, either post-infection (PI) or post-treatment (PT). Colorectal biopsy specimens from outpatients with schistosomiasis were collected. All samples from mice and patients, including serum as well as colon tissue containing eggs and tissue containing no eggs, were examined using the polymerase chain reaction technique. RESULTS The results showed that the SjR2 gene could be detected in all colon tissue containing at least one egg, except for when the egg was completely degraded. The positive rate of gene detection in serum was low. The results from egg-free colon tissue from around the eggs were more consistent with the actual parasitism in vivo. CONCLUSIONS The results indicate that detection of the gene in colon tissue located within a 0.5 cm distance from the eggs would be a practical and ideal method for genetic diagnosis of schistosomiasis. After the colorectal biopsy, this method can be a sensitive assisted examination to the clinical diagnosis of low-intensity schistosomiasis infection.

Schistosomiasis is one of the world's major public health problems in terms of morbidity and mortality, causing granulomatous inflammation and cumulative fibrosis. This study explored in vivo and vitro effects of miR-29b-3p in granulomatous liver fibrosis by targeting COL1A1 and COL3A1 in Schistosoma japonicum infection. Thirty male Balb/c mice were assigned to normal control and model (percutaneous infection of cercariae of S. japonicum) groups. NIH-3T3 mouse embryonic fibroblasts were designated into blank, NC, miR-29b-3p mimic, TGF-β1, TGF-β1 + NC, and TGF-β1 + miR-29b-3p mimic groups. HE and Masson staining were employed to observe the pathological changes and collagenous fibrosis. The expression of α-SMA, COL1A1, COL3A1, TIMP-1 was determined by immunohistochemistry. The RT-qPCR, Western blotting and immunofluorescence staining were conducted to determine expression of miR-29b-3p, COL1A1, and COL3A1. CCK-8 assay and flow cytometry were performed to evaluate viability and apoptosis. The relative expression of miR-29b-3p decreased in the model group. The model group showed marked fibrosis in liver tissues. The expression of α-SMA, COL1A1, COL3A1, TIMP-1 was higher in the model group than that in the normal control group. Dual luciferase reporter gene assay revealed that miR-29b-3p directly targeted COL1A1 and COL3A1. Compared with the blank, NC, TGF-β1 and TGF-β1 + NC groups, the miR-29b-3p mimic group exhibited up-regulated expression of miR-29b-3p and MMP-9 but down-regulated expression of TIMP-1, HSP47, α-SMA, COL1A1, and COL3A1; while lower cell viability but higher apoptosis rate showed. It indicated that miR-29b-3p prevents S. japonicum-induced liver fibrosis by inhibiting COL1A1 and COL3A1., (© 2017 Wiley Periodicals, Inc.)

MicroRNAs (miRNAs) are short, non-coding RNAs that repress the translation of target gene transcripts. They have been implicated in various activities such as cell proliferation, survival, differentiation, migration and metabolism. We report here the first known miRNome and transcriptome analysis of human livers displaying advanced fibrosis due to Schistosoma japonicum infection. We present evidence that hsa-miR-150-5p, hsa-miR-10a-5p, hsa-miR-199a-3p, hsa-miR-4521, hsa-miR-222/221, hsa-miR-663b and hsa-miR-143-3p (associated without correction) play an important role in hepatic fibrosis by acting on metabolism, organization of the extracellular matrix proteins, lipid mobilization and limitation of oxidative damage stress., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Transforming growth factor-β (TGF-β) signaling pathway is documented to participate in liver fibrosis via multifactorial mechanisms. microRNA Let-7b (Let-7b) has been proved to alleviate cell fibrosis through regulating TGF-β receptor I (TβRI), but whether it is involved in Schistosomiasis liver fibrosis (SLF) has not been determined. In the present, SLF mice model was used to investigate Let-7b's function and mechanism in SLF. We found that hepatic let-7b expression was continuously declined in SLF, accompanied by the induction of TGF-β pathway molecules (TGF-β1, TβRI), profibrogenic mediators (α-SMA, colla I), and Th1/Th2 cells response factors (IFN-γ, IL-4). When recombinant Lentivirus of let-7b (Lenti-let-7b) was transfected into S. japonicum-infected mice, the mice hepatic fibrosis was distinctly ameliorated, and TGF-β1, TβRI, α-SMA, and colla I expressions were remarkly decreased, mice serum IL-4 and IFN-γ levels were reduced. Similarly, over-expression of let-7b down-regulated the expression of TβRI in THP-1 cells transfected with let-7b mimics, while TβRI was up-regulated after treated with let-7b inhibitor. These findings suggested that let-7b is a negative regulator to SLF through downregulating TβRI, and inhibits Th1 and Th2 type cell immune response. This provides a novel potential therapeutic strategy for SFL prevention., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hepatic stellate cells (HSCs) are considered as the main effector cells in vitamin A metabolism and liver fibrosis, as well as in hepatic immune regulation. Recently, researches have revealed that HSCs have plasticity and heterogeneity, which depend on their lobular location and whether liver is normal or injured. This research aimed to explore the biological characteristics and heterogeneity of HSCs in mice with Schistosoma japonicum (S. japonicum) infection, and determine the subpopulation of HSCs in pathogenesis of hepatic fibrosis caused by S. japonicum infection. Results revealed that HSCs significantly increased the expressions of MHC II and fibrogenic genes after S. japonicum infection, and could be classified into MHC II + HSCs and MHC II - HSCs subsets. Both two HSCs populations suppressed the proliferation of activated CD4 + T cells, whereas only MHC II - HSCs displayed a myofibroblast-like phenotype. In response to IFN-γ, HSCs up-regulated the expressions of MHC II and CIITA, while down-regulated the expression of fibrogenic gene Col1. In addition, praziquantel treatment decreased the expressions of fibrogenic genes in MHC II - HSCs. These results confirmed that HSCs from S. japonicum-infected mice have heterogeneity. The MHC II - α-SMA + HSCs were major subsets of HSCs contributing to liver fibrosis and could be considered as a potential target of praziquantel anti-fibrosis treatment., (Copyright © 2017. Published by Elsevier B.V.)

The role of hydrogen sulfide (H 2 S) in portal hypertension (PH)-induced esophagus-gastric junction vascular lesions in rabbits was observed. The rabbit PH models were established. The animals were randomly divided into the following groups: normal, PH, PH+sodium hydrosulfide (PH+S), PH+propargylglycine (PH+PPG). The plasma H 2 S levels, apoptosis of esophageal-gastric junction vascular smooth muscle cells, and the expression of nuclear transcription factor-κB (NF-κB), p-AKT, IκBa and Bcl-2 were detected. The cystathionine γ lyase (cystathionine-gamma-splitting enzyme, CSE) in the junction vascular tissue was measured. The results showed that the plasma H 2 S levels and the CSE expression levels had statistically significant difference among different groups (P<0.05). As compared with PH group, plasma H 2 S levels were declined obviously (11.9±4.2 vs. 20.6±4.5, P<0.05), and CSE expression levels in the junction vascular tissue were notably reduced (1.7±0.6 vs. 2.8±0.8, P<0.05), apoptosis rate of vascular smooth muscle cells per unit area was significantly decreased (0.10±0.15 vs. 0.24±0.07, P<0.05), and the expression levels of p-AKT and NF-κB were significantly decreased (2.31±0.33 vs. 3.04±0.38, P<0.05; 0.33±0.17 vs. 0.51±0.23, P<0.05), however, IκBa and Bcl-2 expression increased obviously (5.57±0.17 vs. 3.67±0.13, P<0.05; 0.79±0.29 vs. 0.44±0.36, P<0.05) in PH+PPG group. As compared with PH group, H 2 S levels were notably increased (32.7±7.3 vs. 20.6±4.5, P<0.05), the CSE levels in the junction vascular tissue were significantly increased (6.3±0.7 vs. 2.8±0.8, P<0.05), apoptosis rate of vascular smooth muscle cells per unit area was significantly increased (0.35±0.14 vs. 0.24±0.07, P<0.05), and the expression levels of p-AKT and NF-κB were significantly increased (4.29±0.49 vs. 3.04±0.38, P<0.05; 0.77±0.27 vs. 0.51±0.23, P<0.05), yet IκBa and Bcl-2 expression decreased significantly (3.23±0.24 vs. 3.67±0.13, P<0.05; 0.31±0.23 vs. 0.48±0.34, P<0.05) in PH+S group. It is concluded that esophagus-gastric junction vascular lesions happen under PH, and apoptosis of smooth muscle cells is declined. H 2 S can activate NF-κB by the p-AKT pathway, leading to the down-regulation of Bcl-2, eventually stimulating apoptosis of vascular smooth muscle cells, easing PH. H 2 S/CSE system may play an important role in remission of PH via the AKT-NF-κB pathway.

Liver granulomatous inflammation and fibrosis were the primary pathological changes observed during Schistosoma japonicum (S. japonicum) infection. In the present study, the characteristics of IL-9 were investigated in the liver of S. japonicum infection C57BL/6 mice. Immunofluorescence, qRT-PCR, and ELISA results demonstrated that the expression of IL-9 significantly increased after infection (P < 0.01). FACS results indicated that the peak of IL-9 + Th9 cells in the liver mononuclear cells appeared at the early phase of infection (week 5), except that Th9 cells, CD8 + Tc cells, NKT and γδT cells could secrete IL-9 in this model. Although IL-9 neutralization has a limited effect on liver granulomatous inflammation, it could decrease the level of fibrosis-associated factor, PC-III, in the serum of infected mice (P < 0.05). Taken together, our results indicated that IL-9 was an important type of cytokine involved in the progression of S. japonicum infection-induced hepatic damage.

Dendritic cells are the initiation and key point of immune response and play a role in immune regulation. So we explored the mechanisms involved in immune regulation of dendritic cells (DCs) against schistosomiasis using mice infected with Schistosoma japonicum. Splenic DCs from normal mice and mice with acute and chronic S. japonicum infection were sorted by flow cytometry. The numbers and functions of differentially expressed genes (DEGs) in DCs were determined by high-throughput analysis. All DEGs with transcription-level fold changes of ≥2 were selected and matched to corresponding genes in databases. Annotations and cluster analysis of DEGs were performed to compare differences between groups. Six important DEGs about immune regulation-CD86, TLR2, DC-SIGN, Capase3, PD-L2, and IL-7r were selected, and their transcription levels at different stages of schistosomisis were validated by qPCR. The Venn diagram of DEGs implied some genes are functional at all stages during S. japonicum infection, while others are only involved at certain stages. GO and KEGG pathway annotations indicated that these DEGs mainly belong to biological regulation, regulation of biological process, regulation of cellular process, antigen processing and presentation, cell adhesion molecules, cytokine-cytokine receptor interaction and Toll-like receptor signaling. Cluster analysis revealed immune regulation existed in splenic DCs. The results above indicated that the mechanisms underlying immune regulation to S. japonicum infection in mice are very complex. The present high-throughput dynamic analysis of DEGs in splenic DCs provides valuable insights into the molecular mechanisms underlying immune regulation in S. japonicum infection., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Studies have shown that cysteine protease inhibitors from some parasites have immunosuppressive effects on the host. We previously have cloned a novel cysteine protease inhibitor from Schistosoma japonicum and purified its recombinant version (protein named rSj-C). Its possible inhibitory effect on the host immune response has not been described.This study shows that rSj-C inhibits lysosomal cysteine protease of murine dendritic cells (DCs). After DCs were incubated with rSj-C and then with soluble adult worm antigen (AWA) of S. japonicum, the mean fluorescence intensity of MHC class II antigens on the surface of DCs decreased significantly by flow cytometry. These results indirectly proved that rSj-C can suppress exogenous-antigen presentation by DCs. The flow cytometric assay revealed that in comparison with control groups, the proportion of CD4 + CD25 + Foxp3 + T cells among CD4 + CD25 + T cells of Schistosom-infected mice increased significantly 8 weeks after the infected mice were injected with rSj-C (p ˂ 0.05). Additionally, the expression levels of cytokines IL-4 and TGF-β produced by T cells increased significantly as compared with these levels in the normal group (p ˂ 0.05). These results clearly show that the cysteine protease inhibitor from S. japonicum is a new parasite-derived immunosuppressive factor.

Natural killer cells (NK cells) and natural killer T cells (NKT cells) play a role in anti-infection, anti-tumor, transplantation immunity, and autoimmune regulation. However, the role of NK and NKT cells during Schistosoma japonicum (S. japonicum) infection has not been widely reported, especially regarding lung infections. The aim of this study was to research the NK and NKT cell response to S. japonicum infection in the lungs of mice. Using immunofluorescent histological analysis, NK and NKT cells were found near pulmonary granulomas. Moreover, flow cytometry revealed that the percentage and number of pulmonic NK cells in S. japonicum-infected mice were significantly increased (P < 0.05). However, the percentage and cell number of NKT cells were decreased compared to those of normal mice (P < 0.05). The expression of CD69 on pulmonic NK and NKT cells was increased after infection (P < 0.05), and CD25 expression increased only on NKT cells (P < 0.05). Intracellular cytokine staining showed a higher percentage of IFN-γ + and lower percentage of IL-5 + pulmonic NK cells (P < 0.05) compared to controls. However, the percentage of IL-17 + , IL-10 + , and IL-5 + pulmonic NKT cells significantly increased (P < 0.05). Additionally, there was a significant decrease in NKG2A/C/E (CD94) expression and an increase of NKG2D (CD314) expression on pulmonic NKT cells (P < 0.05), which might serve as a mechanism for NKT cell activation during S. japonicum infection.

Background: More than 220 million people worldwide are chronically infected with schistosomes, causing severe disease or even death. The major pathological damage occurring in schistosomiasis is attributable to the granulomatous inflammatory response and liver fibrosis induced by schistosome eggs. The inflammatory response is tightly controlled and parallels immunosuppressive regulation, constantly maintaining immune homeostasis and limiting excessive immunopathologic damage in important host organs. It is well known that the activation of programmed death 1 (PD-1) signaling causes a significant suppression of T cell function. However, the roles of PD-1 signaling in modulating CD4+ T cell responses and immunopathology during schistosome infection, have yet to be defined., Methodology/principal Findings: Here, we show that PD-1 is upregulated in CD4+ T cells in Schistosoma japonicum (S. japonicum)-infected patients. We also show the upregulation of PD-1 expression in CD4+ T cells in the spleens, mesenteric lymph nodes, and livers of mice with S. japonicum infection. Finally, we found that the blockade of PD-1 signaling enhanced CD4+ T helper 2 (Th2) cell responses and led to more severe liver immunopathology in mice with S. japonicum infection, without a reduction of egg production or deposition in the host liver., Conclusions/significance: Overall, our study suggests that PD-1 signaling is specifically induced to control Th2-associated inflammatory responses during schistosome infection and is beneficial to the development of PD-1-based control of liver immunopathology., Competing Interests: The authors have declared that no competing interests exist.

Schistosoma infection is a major cause of morbidity and mortality worldwide. Schistosomiasis japonica is endemic in mainland China along the Yangtze River, typically distributed in two geographical categories of lake and mountainous regions. Study on schistosome genetic diversity is of interest in respect of understanding parasite biology and transmission, and formulating control strategy. Certain genetic variations may be associated with adaptations to different ecological habitats. The aim of this study is to gain insight into Schistosoma japonicum genetic variation, evolutionary origin and associated causes of different geographic lineages through examining homozygous Single Nucleotide Polymorphisms (SNPs) based on resequenced genome data. We collected S. japonicum samples from four sites, three in the lake regions (LR) of mid-east (Guichi and Tonglin in Anhui province, Laogang in Hunan province) and one in mountainous region (MR) (Xichang in Sichuan province) of south-west of China, resequenced their genomes using Next Generation Sequencing (NGS) technology, and made use of the available database of S. japonicum draft genomic sequence as a reference in genome mapping. A total of 14,575 SNPs from 2059 genes were identified in the four lineages. Phylogenetic analysis confirmed significant genetic variation exhibited between the different geographical lineages, and further revealed that the MR Xichang lineage is phylogenetically closer to LR Guich lineage than to other two LR lineages, and the MR lineage might be evolved from LR lineages. More than two thirds of detected SNPs were nonsynonymous; functional annotation of the SNP-containing genes showed that they are involved mainly in biological processes such as signaling and response to stimuli. Notably, unique nonsynonymous SNP variations were detected in 66 genes of MR lineage, inferring possible genetic adaption to mountainous ecological condition., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Interferon-inducible GTPase LRG-47 (also named immune-related GTPase M, Irgm1) is a member of the p47 GTPase family that has been shown to regulate host resistance to intracellular pathogens. Little knowledge has been known about the role of LRG-47 in host's responses to extracellular pathogens. To investigate possible roles of LRG-47 in the course of Schistosoma japonicum infection, LRG-47-deficient (LRG-47(-/-)) and wild-type (WT) mice were challenged with cercariae of S. japonicum, and the cellular and humoral responses in mice were analyzed. At the acute stage of S. japonicum infection, in contrast to WT mice, LRG-47(-/-) mice showed the significantly decreased egg burden, low schistosome-specific antibody response, and the decreased Th1 and increased Tc1 responses. Additionally, Schistosoma japonicum-specific egg antigen immunization also produced the similar humoral and cellular immune responses as S. japonicum infection. Taken together, these data suggested that the deficiency of LRG-47 might affect host's CD4(+) T cell immune response via the weakening of IFN-γ downstream signaling; however, the specific function of LRG-47 on dealing with extracellular worm needs to be further studied.

Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42-44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica.

Schistosomiasis is a neglected tropical disease that affects more than 200 million people worldwide. The main disease-causing agents, Schistosoma japonicum, S. mansoni and S. haematobium, are blood flukes that have complex life cycles involving a snail intermediate host. In Asia, S. japonicum causes hepatointestinal disease (schistosomiasis japonica) and is challenging to control due to a broad distribution of its snail hosts and range of animal reservoir hosts. In China, extensive efforts have been underway to control this parasite, but genetic variability in S. japonicum populations could represent an obstacle to eliminating schistosomiasis japonica. Although a draft genome sequence is available for S. japonicum, there has been no previous study of molecular variation in this parasite on a genome-wide scale. In this study, we conducted the first deep genomic exploration of seven S. japonicum populations from mainland China, constructed phylogenies using mitochondrial and nuclear genomic data sets, and established considerable variation between some of the populations in genes inferred to be linked to key cellular processes and/or pathogen-host interactions. Based on the findings from this study, we propose that verifying intraspecific conservation in vaccine or drug target candidates is an important first step toward developing effective vaccines and chemotherapies against schistosomiasis.

Natural killer (NK) cells are classic innate immune cells that play roles in many types of infectious disease. Recently, some new characteristics of NK cells were discovered. In this study, C57BL/6 mice were infected with Schistosoma japonicum for 5-6 weeks and lymphocytes were isolated from the spleen to detect some of the NK cell characteristics by multiparametric flow cytometry. The results revealed that the S. japonicum infection induced a large amount of NK cells, although the percentage of NK cells was not increased significantly. At the same time, the results showed that infected mouse splenic NK cells expressed increased levels of CD25 and CD69 and produced more IL-2, IL-4, and IL-17 and less IFN-γ after stimulation with PMA and ionomycin. This meant that NK cells played a role in S. japonicum infection. Moreover, decreased NKG2A/C/E (CD94) expression levels were detected on the surface of NK cells from infected mouse spleens, which might serve as a NK cell activation mechanism. Additionally, high levels of IL-10, but not PD-1, were expressed on the infected mouse NK cells, which implied that functional exhaustion might exist in the splenic NK cells from S. japonicum-infected mice. Collectively, our results suggest that NK cells play important roles in the course of S. japonicum infection.

Although dendritic cells (DCs) have been widely demonstrated to play essential roles in initiation of Th2 responses in helminth infections and allergic reactions, the mechanisms remain uncertain largely because DCs do not produce IL-4. In present investigation, we have uncovered a novel subset of DCs from mice infected with Th2-provoking pathogens Schistosoma japonica, which independently promoted Th2 cells via IL-4-dependent pathway. These DCs contained similar levels of IL-4 mRNA and higher levels of IL-12p40 mRNA comparing to basophils, correlating to their Th2-promoting and Th1-promoting dual polarization capacities. Characterized by expression of FcεRI(+), these DCs were induced independent of T cells. Further investigations revealed that Th2-promoting FcεRI(+) DCs were monocyte-derived inflammatory DCs, which were sufficient to induce Th2 cells in vivo. Egg Ags together with GM-CSF or IL-3 alone were able to stimulate the generation of Th2-promoting FcεRI(+) DCs from bone marrow cells in vitro. To our knowledge, our data for the first time demonstrate that IL-4-producing DCs are induced under some Th2-provoking situations, and they should play important roles in initiation of Th2 response., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The aim of this study was to determine whether two polymorphisms in the gene encoding IL13 previously associated with Schistosoma hematobium (S. hematobium) and S. mansoni infection are associated with S. japonicum infection. Single nucleotide polymorphisms (SNPs) rs1800925 (IL13/-1112C>T) and rs20541 (IL13R130Q) were genotyped in 947 unrelated individuals (307 chronically infected, 339 late-stage with liver fibrosis, 301 uninfected controls) from a schistosomiasis-endemic area of Hubei province in China. Regression models were used to evaluate allelic and haplotypic associations with chronic and late-stage schistosomiasis adjusted for non-genetic covariates. Expression of IL-13 was measured in S. japonicun-infected liver fibrosis tissue and normal liver tissue from uninfected controls by immunohistochemistry (IHC). The role of rs1800925 in IL-13 transcription was further determined by Luciferase report assay using the recombinant PGL4.17-rs180092 plasmid. We found SNP rs1800925T was associated with late-stage schistosomiasis caused by S. japonicum but not chronic schistosomiasis (OR = 1.39, 95%CI = 1.02-1.91, p = 0.03) and uninfected controls (OR = 1.49, 95%CI = 1.03-2.13, p = 0.03). Moreover, the haplotype rs1800925T-rs20541C increased the risk of disease progression to late-stage schistosomiasis (OR = 1.46, p = 0.035), whereas haplotype rs1800925C-rs20541A showed a protective role against development of late-stage schistosomiasis (F = 0.188, OR = 0.61, p = 0.002). Furthermore, S. japonicum-induced fibrotic liver tissue had higher IL13 expression than normal liver tissue. Plasmid PGL4.17-rs1800925T showed a stronger relative luciferase activity than Plasmid PGL4.17-rs1800925C in 293FT, QSG-7701 and HL-7702 cell lines. In conclusion, the functional IL13 polymorphism, rs1800925T, previously associated with risk of schistosomiasis, also contributes to risk of late-stage schistosomiasis caused by S. japonicum.

T cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) has been regarded as an important regulatory factor in both adaptive and innate immunity. Recently, Tim-3 was reported to be involved in Th2-biased immune responses in mice infected with Schistosoma japonicum, but the exact mechanism behind the involvement of Tim-3 remains unknown. The present study aims to understand the role of Tim-3 in the immune response against S. japonicum infection. Tim-3 expression was determined by flow cytometry, and increased Tim-3 expression was observed on CD4(+) and CD8(+) T cells, NK1.1(+) cells, and CD11b(+) cells from the livers of S. japonicum-infected mice. However, the increased level of Tim-3 was lower in the spleen than in the liver, and no increase in Tim-3 expression was observed on splenic CD8(+) T cells or CD11b(+) cells. The schistosome-induced upregulation of Tim-3 on natural killer (NK) cells was accompanied by reduced NK cell numbers in vitro and in vivo. Tim-3 antibody blockade led to upregulation of inducible nitric oxide synthase and interleukin-12 (IL-12) mRNA in CD11b(+) cells cocultured with soluble egg antigen and downregulation of Arg1 and IL-10, which are markers of M2 macrophages. In summary, we observed schistosome-induced expression of Tim-3 on critical immune cell populations, which may be involved in the Th2-biased immune response and alternative activation of macrophages during infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Water buffalo are less susceptible to Schistosoma japonicum infection than yellow cattle. The factors that affect such differences in susceptibility remain unknown. A Bos taurus genome-wide gene chip was used to analyze gene expression profiles in the peripheral blood of water buffalo and yellow cattle pre- and post-infection with S. japonicum. This study showed that most of the identified differentially expressed genes (DEGs) between water buffalo and yellow cattle pre- and post-infection were involved in immune-related processes, and the expression level of immune genes was lower in water buffalo. The unique DEGs (390) in yellow cattle were mainly associated with inflammation pathways, while the unique DEGs (2,114) in water buffalo were mainly associated with immune-related factors. The 83 common DEGs may be the essential response genes during S. japonicum infection, the highest two gene ontology (GO) functions were associated with the regulation of fibrinolysis. The pathway enrichment analysis showed that the DEGs constituted similar immune-related pathways pre- and post-infection between the two hosts. This first analysis of the transcriptional profiles of natural hosts has enabled us to gain new insights into the mechanisms that govern their susceptibility or resistance to S. japonicum infections.

Background: Recent advances in studies of the Schistosoma japonicum genome have opened new avenues for the elucidation of parasite biology and the identification of novel targets for vaccines, drug development and early diagnostic tools., Results: In this study, we surveyed the S. japonicum genome database for genes encoding nucleases. A total of 130 nucleases of 3 classes were found. Transcriptional analysis of these genes using a genomic DNA microarray revealed that the majority of the nucleases were differentially expressed in parasites of different developmental stages or different genders, whereas no obvious transcriptional variation was detected in parasites from different hosts. Further analysis of the putative DNases of S. japonicum revealed a novel DNase II homologue (Sjda) that contained a highly conserved catalytic domain. A recombinant Sjda-GST protein efficiently hydrolysed genomic DNA in the absence of divalent iron. Western-blot and immunofluorescence assays showed that Sjda was mainly expressed on the teguments of female adult parasites and induced early humoral immune responses in infected mice., Conclusions: A novel DNase II homologue, Sjda, was identified in S. japonicum. Sjda was mainly distributed on the teguments of adult female parasites and possessed a typical divalent iron-independent DNA catalytic activity. This protein may play an important role in the host-parasite interaction.

The current status of schistosomiasis in highly endemic areas is difficult to determine by ovum detection because of the superficially low parasite load after mass drug administration, whereas the parasite transmission rates are still high. Cell-free parasite DNA is fragments of parasite-derived DNA existing in the host's body fluids. We conducted population-based studies to test the presence of cell-free schistosome DNA in endemic areas of Sorsogon Province, the Philippines. Schistosome DNA in the serum and urine of Kato-Katz (KK)-positive subjects was detected by PCR (100% sensitivity). Schistosome DNA was also detected from KK-negative subjects (9/22 serum and 10/41 urine samples). Schistosome DNA was found to be network echogenic pattern (NW)-positive (serum 53.3%, urine 42.9%) or NW-negative (serum 25.5%, urine 20.8%) and enzyme-linked immunosorbent assay (ELISA)-positive (serum 47.1%, urine 40%) or ELISA-negative (serum 33.3%, urine 13.3%). These results indicate that cell-free schistosome DNA is a promising diagnostic marker for active schistosome infection in the case of light infection., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Chronic infection with Schistosoma japonicum is an important cause of hepatic fibrosis (HF). Human 9q33.3 is one of the most important loci for stress-related diseases. We examined the potential associations of 43 single-nucleotide polymorphisms (SNPs) with S. japonicum infection and HF in epidemic region in China. We identified a SNP (rs10118570 GG in mitogen-activated protein kinase associated protein 1, MAPKAP1) contributes to anti-infection (adjusted OR = 0.35) and anti-fibrogenesis (adjusted RR = 0.44) in the discovery study. Replicative and combined studies showed consistent protective quality for this genotype (replicative: adjusted OR = 0.37 for anti-infection, and adjusted RR = 0.40 for anti-fibrogenesis; Combined: adjusted OR = 0.45 for anti-infection, and adjusted RR = 0.42 for anti-fibrogenesis). Univariate and multivariate analysis in the discovery, replicative and combined studies, suggested that durations (years), splenomegaly, serum ALB and rs10118570 were independent predictors influencing the fibrogenesis. The analysis of gene-gene interaction showed rs10118570 functions independently. We conclude that MAPKAP1 may represent a novel anti-infection and anti-fibrogenesis genomic locus in chronic schistosomiasis japonica. And rs10118570 may be a potential biomarker and target for the treatment of this life-threatening ancient disease.

Boswellic acid (BA)-containing extracts such as BSE have anti-inflammatory and immunomodulatory activity. In chronic schistosomiasis, the hepatic granuloma and fibrosis induced by egg deposition in the liver is the most serious pathological manifestations. However, little is known regarding the role of BAs in Schistosoma japonicum (S. japonicum) egg-induced liver granuloma and fibrosis. In order to investigate the effect of a water-soluble complex preparation of BSE, BSE-CD, on S. japonicum egg-induced liver pathology, liver granuloma and fibrosis were induced by infecting C57BL/6 mice with 18-22 cercariae of S. japonicum. S. japonicum cercariae infected mice were injected with BSE-CD at the onset of egg granuloma formation (early phase BSE-CD treatment after 4 weeks infection) or after the formation of liver fibrosis (late phase BSE-CD treatment after 7 weeks infection). Our data show that treatment of infected mice with BSE-CD significantly reduced both the extent of hepatic granuloma and fibrosis. Consistent with an inhibition of NF-κB signaling as evidenced by reduced IκB kinase (IKK) activation, the mRNA expression of VEGF (vascular endothelial growth factor, VEGF), TNF-α (tumor necrosis factor-alpha TNF-α) and MCP-1 (monocyte chemotactic protein 1, MCP-1) was decreased. Moreover, immunohistochemical analysis (IHC) revealed that the content of α-SMA in liver tissue of BSE-CD treated mice was dramatically decreased. Our findings suggest that BSE-CD treatment attenuates S. japonicum egg-induced hepatic granulomas and fibrosis, at least partly due to reduced NF-κB signaling and the subsequently decreased expression of VEGF, TNF-α, and MCP-1. Suppression of the activation of hepatic stellate cells (HSC) may also be involved in the therapeutic efficacy of BSE-CD.

The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers α-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers α-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide (O2(-)) production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, α-SMA, collagen I, and TIMP-1 by inhibition of O2(-) production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on O2(-) production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cholesteryl ester transfer protein (CETP)-deficiency manifests a unique plasma lipoprotein profile without other apparent symptoms. It is highly common in East Asia while rather rare anywhere else. A potential environmental screening factor(s) may therefore contribute to this eccentric distribution, such as its selective advantage against a regional illness, most likely an infectious disease, in relation to plasma lipoproteins. Blood flukes use the host plasma lipoproteins as nutrient sources through the lipoprotein receptor-like systems. Its Asian-specific species, Schistosoma (S) japonicum, which has been endemic in East Asia, takes up cholesteryl ester (CE) from high-density lipoprotein (HDL) for the embryonation of their eggs to miracidia, a critical step of the hepatic pathogenesis of this parasite, but poorly from HDL of CETP-deficiency. CD36-related protein (CD36RP) was cloned from the adults and the eggs of S. japonicum, with 1880-bp encoding 506 amino-acid residues exhibiting the CD36 domains and two transmembrane regions. Its extracellular domain selectively bound human HDL but neither LDL nor CETP-deficiency HDL, and the antibody against the extracellular domain suppressed the selective HDL-CE uptake and embryonation of the eggs. When infected with S. japonicum, wild-type mice developed less hepatic granulomatosis than CETP-transgenic mice by the ectopic egg embryonation. CD36RP is thus a candidate receptor of S. japonicum to facilitate uptake of HDL-CE necessary for egg embryonation. Abnormal HDL caused by CETP-deficiency retards this process and thereby protects the patients from development of hepatic lesions. S. japonicum infection is a potential screening factor for high prevalence of CETP deficiency in East Asia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In order to explore the high performance bivalent DNA-based vaccine against schistosomes, SjFABP and Sj26GST were selected and used to construct a vaccine. Two strategies were used to construct the bivalent DNA vaccine. In the first strategy, a plasmid encoding antigen in the secreted form was used, while in the other, a plasmid encoding a truncated form of SjFABP and Sj26GST targeted to the cell surface was used. Various parameters, including antibody and cytokine response, proliferation, histopathological examination, and characterization of T cell subsets were used to evaluate the type of immune response and the level of protection against challenge infection. Injection with secreted pIRES-sjFABP-sj26GST significantly increased the levels of antibody, splenocyte proliferation, and production of IFN-γ, compared with membrane-anchored groups. Analysis of splenic T cell subsets showed that the secreted vaccine significantly increased the percentage of CD3(+)CD4(+) and CD3(+)CD8(+) T cells. Liver immunopathology (size of liver granulomas) was significantly reduced in the secreted group compared with the membrane-anchored groups. Moreover, challenge experiments showed that the worm and egg burdens were significantly reduced in animals immunized with recombinant vaccines. Most importantly, secreted Sj26GST-SjFABP markedly enhanced protection, by reducing worm and egg burdens by 31.8% and 24.78%, respectively, while the membrane-anchored group decreased worm and egg burdens by 24.80% and 18.80%, respectively. Taken together, these findings suggest that the secretory vaccine is more promising than the membrane-anchored vaccine, and provides support for the development and application of this vaccine.

Toll-like receptor 2 (TLR2) was shown to be an important immune receptor involved in the recognition of schistosome antigens, especially soluble egg antigen (SEA). In mice models with Schistosoma japonicum acute infection, we observed enhanced T cell-mediated immune responses in TLR2 knock out (TLR2(-/-)) mice compared with B6 mice. In Schistosoma japonicum chronic infection models, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) expression as well as TLR2 expression gradually increased in B6 mice, while only PD-L2 expression significantly decreased in TLR2(-/-) mice. Meanwhile, Programmed Death 1(PD-1) expression on CD4(+)T cells was down-regulated in TLR2(-/-) mice after a large number of egg appeared. We also found that stimulation with schistosome antigens, especially SEA, could up-regulate PD-L2 expression on BMDCs in a TLR2-dependent manner in vitro. Schistosome antigens primed-BMDCs with impaired expression of TLR2 or PD-L2 could induce CD4(+)T cells to produce low level of IL-10 or high level of IFN-γ. Our results indicated that TLR2 signaling can direct PD-L2 expression on DCs, which binds to PD-1 mainly on CD4(+)T cells, to help inhibit T cells response in Schistosoma japonicum infection.

The severity of schistosome egg-induced hepatic granulomatous pathology depends markedly on the nature of the host immune responses. In this study, we used LMM and microarray analysis to compare gene expression profiles of histologically distinct zones within, and directly proximal to, hepatic granulomas that developed in C57BL/6 mice infected with Schistosoma japonicum. There was significant up-regulation of type-1, type-2, and type-17 immune-associated genes within the granuloma core (adjacent to eggs), followed by increased expression of type-2 and fibrotic genes at the outer zones of granulomas. Neutrophil-associated genes were also found to be expressed differentially in the core and at the peripheral zone of granulomas, present at 7 weeks p.i., demonstrating a significant role of neutrophils in S. japonicum granulomatous pathology. The release of NETs was observed microscopically in granulomas obtained from the livers of infected mice and when human neutrophils were incubated in vitro in the presence of S. japonicum eggs. These finding are the first to suggest a novel, dual role for neutrophils in the mediation of tissue damage and repair in S. japonicum egg-induced hepatic granulomatous lesions. Together, these results provide an overview of the local events occurring within the granuloma microenvironment.

Background: Schistosomiasis japonica remains a significant public health problem in China and Southeast Asian countries. The most typical and serious outcome of the chronic oriental schistosomiasis is the progressive granuloma and fibrosis in the host liver, which has been a major medical challenge. However, the molecular mechanism underling the hepatic pathogenesis is still not clear., Methodology and Principal Findings: Using microarrays, we quantified the temporal gene expression profiles in the liver of Schistosoma japonicum-infected BALB/c mice at 15, 30, and 45 day post infection (dpi) with that from uninfected mice as controls. Gene expression alternation associated with liver damage was observed in the initial phase of infection (dpi 15), which became more magnificent with the onset of egg-laying. Up-regulated genes were dominantly associated with inflammatory infiltration, whereas down-regulated genes primarily led to the hepatic functional disorders. Simultaneously, microRNA profiles from the same samples were decoded by Solexa sequencing. More than 130 miRNAs were differentially expressed in murine liver during S. japonicum infection. MiRNAs significantly dysregulated in the mid-phase of infection (dpi 30), such as mmu-miR-146b and mmu-miR-155, may relate to the regulation of hepatic inflammatory responses, whereas miRNAs exhibiting a peak expression in the late phase of infection (dpi 45), such as mmu-miR-223, mmu-miR-146a/b, mmu-miR-155, mmu-miR-34c, mmu-miR-199, and mmu-miR-134, may represent a molecular signature of the development of schistosomal hepatopathy. Further, a dynamic miRNA-gene co-expression network in the progression of infection was constructed., Conclusions and Significance: This study presents a global view of dynamic expression of both mRNA and miRNA transcripts in murine liver during S. japonicum infection, and highlights that miRNAs may play a variety of regulatory roles in balancing the immune responses during the development of hepatic pathology. The data provide robust information for further researches on the pathogenesis and molecular events of hepatopathy induced by schistosome eggs.

Objective: To investigate the differences of mRNA quantitation and protein expression of vascular growth factors including platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) in intestinal tissues in colorectal carcinoma patients with and without schistosomiasis., Methods: Thirty colorectal carcinoma patients with schistosomiasis and 30 colorectal carcinoma patients without schistosomiasis were included in this study. The mRNA quantitation and protein expression of PD-ECGF and VEGF in the normal tissue, peri-carcinoma tissue as well as carcinoma tissue obtained from surgical specimens were detected by qRT-PCR and Western blot., Results: The mRNA relative quantitations of PD-ECGF in normal tissue, peri-carcinoma tissue and carcinoma tissue in the colorectal carcinoma patients with schistosomiasis were 1.726, 1.766 and 2.729 times to those in the colorectal carcinoma patients without schistosomiasis, respectively. The corresponding ones of VEGF were 2.138, 1.831 and 3.376 times, respectively. The protein expression levels of PD-ECGF and VEGF in normal tissue, peri-carcinoma tissue and carcinoma tissue were higher in the colorectal carcinoma patients with schistosomiasis than in the colorectal carcinoma patients without schistosomiasis., Conclusions: The expressions of vascular growth factors including PD-ECGF and VEGF are higher in the colorectal carcinoma patients with schistosomiasis than in the colorectal carcinoma patients without schistosomiasis. Therefore, schistosomiasis may be one of the risk factors of colorectal cancer.