An improved medium for in vitro studies of female reproduction and oviposition in Schistosoma japonicum.

BACKGROUND: Schistosomiasis is a disease primarily caused by eggs laid by pathogens called schistosomes. Among the schistosome species infecting humans, Schistosoma japonicum possesses the largest fecundity; each adult female produces an average of 3500 eggs per day. The lack of proper culture conditions supporting continuous oviposition in vitro has precluded detailed investigation of mechanisms regulating sexual maturation and egg production in Schistosoma japonicum. METHODS: We optimized in vitro culture conditions by replacing reagents that are part of the classical ABC169 medium. Fast Blue BB staining and 4',6-diamidino-2-phenylindole (DAPI) labeling were applied to observe the sexual development status of the females. In vitro RNA interference (RNAi) technology was used to validate the capability of the modified medium. The detection of male ß-alanyl-tryptamine (BATT) was conducted using liquid chromatography-mass spectrometry (LC-MS). RESULTS: Both m-AB169 (1640) and AB169 (1640) media are capable of facilitating the sexual development of paired virgin female S. japonicum, as well as sustaining the mature reproductive organs and egg production of adult S. japonicum for at least 22 days in vitro. M-AB169 (1640) provided a more stable condition for supporting the sexual maturity of female S. japonicum, as evidenced by the consistent initiation of egg production compared with AB169 (1640). Through a comparative analysis of S. japonicum and S. mansoni in diverse media, we demonstrated that these closely related species display distinct demands for their sexual development and egg production, suggesting a potential influence of nutritional factors on the observed variations in host ranges among different schistosome species. Importantly, we successfully identified the presence of the pheromone ß-alanyl-tryptamine (BATT) in S. japonicum, previously identified in S. mansoni, highlighting its conserved role in schistosome reproductive development. Through the employment of double-stranded RNA (dsRNA) treatment to silence two genes that are involved in either the male (gli1, glioma-associated oncogene homolog 1) or female (vf1, vitellogenic factor 1) side in male-induced female reproductive development of S. mansoni, we confirmed that the combination of m-AB169 (1640) and RNAi technology has the capacity to facilitate in vitro studies of S. japonicum's reproductive and oviposition processes. CONCLUSIONS: We developed a novel medium, m-AB169 (1640), that not only maintains the mature reproductive organs and continuous oviposition of adult female Schistosoma japonicum for up to 22 days but also supports the reproductive development and subsequent egg-laying of virgin females after pairing with male worms. This study provides a valuable in vitro platform for functional studies of the mechanisms underlying the fascinating biology of the female sexual development and egg production of S. japonicum, which may accelerate the development of new strategies targeting schistosome egg production.

Metagenomic analysis of the intestinal microbiome reveals the potential mechanism involved in Bacillus amyloliquefaciens in treating schistosomiasis japonica in mice.

Schistosomiasis japonica is one of the neglected tropical diseases characterized by chronic hepatic, intestinal granulomatous inflammation and fibrosis, as well as dysbiosis of intestinal microbiome. Previously, the probiotic Bacillus amyloliquefaciens has been shown to alleviate the pathological injuries in mice infected with Schistosoma japonicum by improving the disturbance of the intestinal microbiota. However, the underlying mechanisms involved in this process remain unclear. In this study, metagenomics sequencing and functional analysis were employed to investigate the differential changes in taxonomic composition and functional genes of the intestinal microbiome in S. japonicum-infected mice treated with B. amyloliquefaciens. The results revealed that intervention with B. amyloliquefaciens altered the taxonomic composition of the intestinal microbiota at the species level in infected mice and significantly increased the abundance of beneficial bacteria. Moreover, the abundance of predicted genes in the intestinal microbiome was also significantly changed, and the abundance of xfp/xpk and genes translated to urease was significantly restored. Further analysis showed that Limosilactobacillus reuteri was positively correlated with several KEGG Orthology (KO) genes and metabolic reactions, which might play important roles in alleviating the pathological symptoms caused by S. japonicum infection, indicating that it has the potential to function as another effective therapeutic agent for schistosomiasis. These data suggested that treatment of murine schistosomiasis japonica by B. amyloliquefaciens might be induced by alterations in the taxonomic composition and functional gene of the intestinal microbiome in mice. We hope this study will provide adjuvant strategies and methods for the early prevention and treatment of schistosomiasis japonica. IMPORTANCE: Targeted interventions of probiotics on gut microbiome were used to explore the mechanism of alleviating schistosomiasis japonica. Through metagenomic analysis, there were significant changes in the composition of gut microbiota in mice infected with Schistosoma japonicum and significant increase in the abundance of beneficial bacteria after the intervention of Bacillus amyloliquefaciens. At the same time, the abundance of functional genes was found to change significantly. The abundance of genes related to urease metabolism and xfp/xpk related to D-erythrose 4-phosphate production was significantly restored, highlighting the importance of Limosilactobacillus reuteri in the recovery and abundance of predicted genes of the gut microbiome. These results indicated potential regulatory mechanism between the gene function of gut microbiome and host immune response. Our research lays the foundation for elucidating the regulatory mechanism of probiotic intervention in alleviating schistosomiasis japonica, and provides potential adjuvant treatment strategies for early prevention and treatment of schistosomiasis japonica.

Schistosome egg-derived extracellular vesicles deliver Sja-miR-71a inhibits host macrophage and neutrophil extracellular traps via targeting Sema4D.

BACKGROUND: Macrophages and neutrophils are rapidly recruited around Schistosome eggs to form granulomas. Extracellular traps (ETs) of macrophages and neutrophils are part of the pathogen clearance armamentarium of leukocytes. Schistosome eggs possess the ability to resist attack by the host's immune cells and survive by employing various immune evasion mechanisms, including the release of extracellular vesicles (EVs). However, the specific mechanisms by which Schistosome egg-derived EVs (E-EVs) evade the immune response and resist attack from macrophage and neutrophil ETs remain poorly understood. In this study, we aimed to investigate the association between E-EVs and macrophage/neutrophil ETs. METHODS: EVs were isolated from the culture supernatant of S. japonicum eggs and treated macrophages and neutrophils with E-EVs and Sja-miR-71a. The formation of ETs was then observed. Additionally, we infected mice with S. japonicum, administered HBAAV2/9-Sja-miR-71a, and the formation of macrophage ETs (METs) and neutrophil ETs (NETs) in the livers was measured. Sema4D-knockout mice, RNA sequencing, and trans-well assay were used to clarify Sja-miR-71a in E-EVs inhibits METs and NETs formation via the Sema4D/ PPAR-γ/ IL-10 axis. RESULTS: Our findings revealed that E-EVs were internalized by macrophages and neutrophils, leading to the inhibition of METs and NETs formation. The highly expressed Sja-miR-71a in E-EVs targeted Sema4D, resulting in the up-regulation of IL-10 and subsequent inhibition of METs and NETs formation. Sema4D knockout up-regulated IL-10 expression and inhibited the formation of METs and NETs. Furthermore, we further demonstrated that Sja-miR-71a inhibits METs and NETs formation via the Sema4D/ PPAR-γ/ IL-10 axis. CONCLUSIONS: In summary, our findings provide new insights into the immune evasion abilities of Schistosome eggs by demonstrating their ability to inhibit the formation of METs and NETs through the secretion of EVs. This study enhances our understanding of the host-pathogen interaction and may have implications for the development of novel therapeutic approaches. Video Abstract.

Inhibition of signal peptidase complex expression affects the development and survival of Schistosoma japonicum.

Background: Schistosomiasis, the second most neglected tropical disease defined by the WHO, is a significant zoonotic parasitic disease infecting approximately 250 million people globally. This debilitating disease has seriously threatened public health, while only one drug, praziquantel, is used to control it. Because of this, it highlights the significance of identifying more satisfactory target genes for drug development. Protein translocation into the endoplasmic reticulum (ER) is vital to the subsequent localization of secretory and transmembrane proteins. The signal peptidase complex (SPC) is an essential component of the translocation machinery and functions to cleave the signal peptide sequence (SP) of secretory and membrane proteins entering the ER. Inhibiting the expression of SPC can lead to the abolishment or weaker cleavage of the signal peptide, and the accumulation of uncleaved protein in the ER would affect the survival of organisms. Despite the evident importance of SPC, in vivo studies exploring its function have yet to be reported in S. japonicum. Methods: The S. japonicum SPC consists of four proteins: SPC12, SPC18, SPC22 and SPC25. RNA interference was used to investigate the impact of SPC components on schistosome growth and development in vivo. qPCR and in situ hybridization were applied to localize the SPC25 expression. Mayer's carmalum and Fast Blue B staining were used to observe morphological changes in the reproductive organs of dsRNA-treated worms. The effect of inhibitor treatment on the worm's viability and pairing was also examined in vitro. Results: Our results showed that RNAi-SPC delayed the worm's normal development and was even lethal for schistosomula in vivo. Among them, the expression of SPC25 was significantly higher in the developmental stages of the reproductive organs in schistosomes. Moreover, SPC25 possessed high expression in the worm tegument, testes of male worms and the ovaries and vitellarium of female worms. The SPC25 knockdown led to the degeneration of reproductive organs, such as the ovaries and vitellarium of female worms. The SPC25 exhaustion also reduced egg production while reducing the pathological damage of the eggs to the host. Additionally, the SPC-related inhibitor AEBSF or suppressing the expression of SPC25 also impacted cultured worms' pairing and viability in vitro. Conclusions: These data demonstrate that SPC is necessary to maintain the development and reproduction of S. japonicum. This research provides a promising anti-schistosomiasis drug target and discovers a new perspective on preventing worm fecundity and maturation.

CRISPR/Cas9-mediated gene knockout of Sj16 in Schistosoma japonicum eggs upregulates the host-to-egg immune response.

Schistosomiasis is an important, neglected tropical disease. Schistosoma japonicum can evade host attacks by regulating the host's immunity, causing continuous infection. However, interactions between the host's immune system and S. japonicum are unclear. Our previous research found that the Sj16 protein isolated from S. japonicum has an anti-inflammatory effect in the host. However, the role of Sj16 in the regulation of host immunity in S. japonicum infection is not clear. Here, we applied the CRISPR/Cas9 technique to knockout Sj16 in S. japonicum eggs and investigated the effect of Sj16 in regulating host immunity. We found egg viability decreased after Sj16 knockout. In addition, we found granulomatous inflammation increased, the T-cell immune response enhanced and the immune microenvironment changed in mice model injected with Sj16-knockout eggs by tail vein. These findings suggested that S. japonicum could regulate host immunity through Sj16 to evade the host immune attack and cause continuous infection. In addition, we confirmed the application of CRISPR/Cas9-mediated gene reprogramming for functional genomics in S. japonicum.

miR-182-5p attenuates Schistosoma japonicum-induced hepatic fibrosis by targeting tristetraprolin.

Egg granuloma formation in the liver is the main pathological lesion caused by Schistosoma japonicum infection, which generally results in liver fibrosis and may lead to death in advanced patients. MicroRNAs (miRNAs) regulate the process of liver fibrosis, but the putative function of miRNAs in liver fibrosis induced by S. japonicum infection is largely unclear. Here, we detect a new miRNA, miR-182-5p, which shows significantly decreased expression in mouse livers after stimulation by soluble egg antigen (SEA) of S. japonicum or S. japonicum infection. Knockdown or overexpression of miR-182-5p in vitro causes the increased or decreased expression of tristetraprolin (TTP), an important immunosuppressive protein in the process of liver fibrosis. Furthermore, knockdown of miR-182-5p in vivo upregulates TTP expression and significantly alleviates S. japonicum-induced hepatic fibrosis. Our data demonstrate that downregulation of miR-182-5p increases the expression of TTP in mouse livers following schistosome infection, which leads to destabilization of inflammatory factor mRNAs and attenuates liver fibrosis. Our results uncover fine-tuning of liver inflammatory reactions related to liver fibrosis caused by S. japonicum infection and provide new insights into the regulation of schistosomiasis-induced hepatic fibrosis.

The Potential Role of MicroRNA-124-3p in Growth, Development, and Reproduction of Schistosoma japonicum.

The microRNA-124-3p plays an important role in regulating development and neurogenesis. Previous microRNA sequencing analyses of Schistosoma japonicum revealed sja-miR-124-3p differential expression patterns in schistosomes from different hosts and at different developmental stages. This study explores the regulatory role of sja-miR-124-3p in S. japonicum development and reproduction. Quantitative reverse-transcription PCR (qRT-PCR) showed that the expression level of sja-miR-124-3p in S. japonicum from resistant hosts, such as Microtus fortis, and unsuitable hosts, such as rats and water buffalo, was significantly higher than that in mice and yellow cattle at the same developmental stage. Overexpressing sja-miR-124-3p in infected mice led to a hepatic egg reduction rate of 36.97%, smaller egg granulomas in the livers, increased liver weight, subsided hepatocyte necrosis, and diminished inflammatory cell infiltration. The width of female worms increased but decreased in males. The vitelline cells were irregular, swollen, or fused. The teguments and ventral sucker of males and females were swollen and broken, but the morphological changes were particularly notable in males. qRT-PCR and dual-luciferase reporter assay system were used to confirm the in-silico-predicted target genes, S. japonicum DEAD-box ATP-dependent RNA helicase 1 (sjDDX1) and DNA polymerase II subunit 2 (sjPOLE2). Our results showed that RNA interference (RNAi)-mediated sjDDX1 silencing in mice provided a 24.55% worm reduction rate and an 18.36% egg reduction rate, but the difference was not significant (p > 0.05). Thus, our findings suggest that sja-miR-124-3p has an important role in growth, development, and reproduction in S. japonicum. All these results will greatly contribute toward providing important clues for searching vaccine candidates and new drug targets against schistosomiasis.

Emerging roles for extracellular vesicles in Schistosoma infection.

The co-evolution of Schistosoma and its host necessitates the use of extracellular vesicles (EVs) generated by different lifecycle stages to manipulate the host immune system to achieve a delicate balance between the survival of the parasite and the limited pathology of the host. EVs are phospholipid bilayer membrane-enclosed vesicles capable of transferring a complex mixture of proteins, lipids, and genetic materials to the host. They are nano-scale-sized vesicles involved in cellular communication. In this review, the author summarized the proteins involved in the biogenesis of schistosome-derived EVs and their cargo load. miRNAs are one cargo molecule that can underpin EVs functions and significantly affect parasite/host interactions and immune modulation. They skew macrophage polarization towards the M1 phenotype and downregulate Th2 immunity. Schistosoma can evade the host immune system's harmful effects by utilizing this strategy. In order to compromise the protective effect of Th2, EVs upregulate T regulatory cells and activate eosinophils, which contribute to granuloma formation. Schistosomal EVs also affect fibrosis by acting on non-immune cells such as hepatic stellate cells. These vesicles drew attention to translational applications in diagnosis, immunotherapy, and potential vaccines. A deep understanding of the interaction of schistosome-derived EVs with host cells will significantly increase our knowledge about the dynamics between the host and the worm that may aid in controlling this debilitating disease.

Schistosoma japonicum translationally controlled tumour protein, which is associated with the development of female worms, as a target for control of schistosomiasis.

Schistosomiasis is a globally important helminthic disease of both humans and animals, and is the second most common parasitic disease after malaria. Although praziquantel is extensively used for treatment of parasitic diseases, drug resistance has been reported. Therefore, new drugs and effective vaccines are needed for continuous control of schistosomiasis. Eggs produced by schistosomes are responsible for the occurrence and spread of schistosomiasis. Revealing the reproductive mechanism of schistosomes will help to control this disease. In this study, the proteomic profiles of single-sex infected female worms and bisexual infected mature female worms of Schistosoma japonicum at 18, 21, 23 and 25 days p.i. were identified with isobaric tags for relative quantitation-coupled liquid chromatography-tandem mass spectrometry. Differentially expressed proteins were subsequently used for bioinformatic analysis. Six highly expressed differentially expressed proteins in mature female worms were selected and long-term interference with small interfering RNA (siRNA) was conducted to determine biological functions. SiRNA against S. japonicum translationally controlled tumour protein (SjTCTP) resulted in the most significant effect on the growth and development of MF worms. Sjtctp mRNA expression gradually increased over time with a high level of expression maintained at 25-42 days p.i., while levels were significantly higher in mature female worms than male and SF worms. The subsequent animal immune protection experiments showed that recombinant SjTCTP (rSjTCTP) reduced the number of adults by 44.7% (P < 0.01), average egg burden per gram of liver by 57.94% (P < 0.01), egg hatching rate by 47.57% (P < 0.01), and oviposition of individual females by 43.16%. rSjTCTP induced higher levels of serum IgG, IL-2, and IL-10 in mice. Collectively, these results show that SjTCTP is vital to reproduction of female worms and, thus, is a candidate antigen for immune protection.

Identification of a Schistosoma japonicum MicroRNA That Suppresses Hepatoma Cell Growth and Migration by Targeting Host FZD4 Gene.

Previous studies have demonstrated miRNAs derived from plants and parasites can modulate mammalian gene expression and cell phenotype in a cross-kingdom manner, leading to occurrence of diseases or strengthening resistance of host to diseases such as cancer. In this study, we identified a schistosome miRNA (named Sja-miR-71a) through screening of 57 Schistosoma japonicum miRNAs that exerts antitumor activity in vitro and in vivo models. We demonstrated presence of this parasite miRNA in liver cells during infection. We showed that Sja-miR-71a arrested cell cycle at G0/G1 phase of hepatoma cell lines and inhibited cell proliferation in vitro. The HepG2 transfected with Sja-miR-71a mimics displayed significant reduction of migration and colony formation. Further, growth of the tumor cells transfected with the Sja-miR-71a mimics was obviously suppressed in a xenograft mouse model. Mechanically, we found the antitumor activity of Sja-miR-71a was through targeting a host gene encoding Frizzled Class Receptor 4 (FZD4), as FZD4 small interfering RNAs (siRNAs) generated a similar inhibitory effect on the tumor. These data indicated that Sja-miR-71a is a tumor suppressor miRNA and suggested this parasite-derived miRNA as a potential therapeutic target for cancer.

miR-181a regulates the host immune response against Schistosoma japonicum infection through the TLR4 receptor pathway.

BACKGROUND: Schistosomiasis japonica is a serious zoonotic parasitic disease. Preliminary studies have shown that the expression of microRNA-181a (miR-181a) in the liver, lung and spleen tissues of susceptible host BALB/c mice and resistant host reed vole (Microtus fortis) 10 days post-infection (dpi) with Schistosoma japonicum was significantly different from pre-infection levels. This difference suggests the possibility that miR-181a expression may be related to the regulation of the hosts' early immune response against S. japonicum infection and thereby affect the development and survival of parasites in their final hosts. METHODS: BALB/c mice, M. fortis, Toll-like receptor 4 (TLR4)-deficient mice and wild-type mice (C57BL/6) were infected with S. japonicum, and differences in miR-181a expression between BALB/c mice and M. fortis over different time points post-infection (0, 3, 7, 10 and 14 dpi) were compared. MiR-181a mimic, miR-181a inhibitor and irrelevant miRNA, as well as lipopolysaccharide (LPS), a TLR4 receptor ligand, were used to transfect mouse RAW264.7 macrophages. The expression levels of the TLR4 pathway-related cytokines interleukin (IL)-1ß, tumor necrosis factor α (TNF-α) and IL-6 were detected by quantitative PCR analysis. RESULTS: The expression of miR-181a was significantly upregulated in the serum and liver of mice infected with S. japonicum and downregulated in the serum and liver of M. fortis. T-helper cell (Th1)-type cytokines, such as TNF-α, IL-6 and IL-1ß, and Th2-type cytokines, such as IL-10 and IL-4, were differentially expressed in M. fortis and BALB/c mice in the early stage of infection. The expression level of miR-181a in the serum was threefold higher in TLR4-deficient mice than in wild-type mice 10 dpi with S. japonicum. The expression of IL-1ß, TNF-α and IL-6 decreased in RAW264.7 cells transfected with miR-181a mimic and increased in cells transfected with miR-181a inhibitor. miR-181a expression was downregulated and the expressions of TLR4 and three TLR4 pathway-related cytokines (IL-1ß, IL-6, and TNF-α) were upregulated in RAW264.7 macrophages stimulated with the TLR4 receptor ligand LPS. CONCLUSION: These results suggest the possibility of mutual regulation between miR-181a and the TLR4 signaling pathway during S. japonicum infection. miR-181a may regulate the expression of pro-inflammatory factors through the TLR4 receptor pathway and participate in the immunomodulatory effect of anti-S. japonicum infection.

Schistosoma japonicum peptide SJMHE1 inhibits acute and chronic colitis induced by dextran sulfate sodium in mice.

BACKGROUND: Harnessing helminth-based immunoregulation is a novel therapeutic strategy for many immune dysfunction disorders, including inflammatory bowel diseases (IBDs). We previously identified a small molecule peptide from Schistosoma japonicum and named it SJMHE1. SJMHE1 can suppress delayed-type hypersensitivity, collagen-induced arthritis and asthma in mice. In this study, we assessed the effects of SJMHE1 on dextran sulfate sodium (DSS)-induced acute and chronic colitis. METHODS: Acute and chronic colitis were induced in C57BL/6 mice by DSS, following which the mice were injected with an emulsifier SJMHE1 or phosphate-buffered saline. The mice were then examined for body weight loss, disease activity index, colon length, histopathological changes, cytokine expression and helper T (Th) cell subset distribution. RESULTS: SJMHE1 treatment significantly suppressed DSS-induced acute and chronic colitis, improved disease activity and pathological damage to the colon and modulated the expression of pro-inflammatory and anti-inflammatory cytokines in splenocytes and the colon. In addition, SJMHE1 treatment reduced the percentage of Th1 and Th17 cells and increased the percentage of Th2 and regulatory T (Treg) cells in the splenocytes and mesenteric lymph nodes of mice with acute colitis. Similarly, SJMHE1 treatment upregulated the expression of interleukin-10 (IL-10) mRNA, downregulated the expression of IL-17 mRNA and modulated the Th cell balance in mice with chronic colitis. CONCLUSIONS: Our data show that SJMHE1 provided protection against acute and chronic colitis by restoring the immune balance. As a small molecule, SJMHE1 might be a novel agent for the treatment of IBDs without immunogenicity concerns.

Schistosome eggs stimulate reactive oxygen species production to enhance M2 macrophage differentiation and promote hepatic pathology in schistosomiasis.

Schistosomiasis is a neglected tropical disease of public health concern. The most devastating pathology in schistosomiasis japonica and mansoni is mainly attributed to the egg-induced granulomatous response and secondary fibrosis in host liver, which may lead to portal hypertension or even death of the host. Schistosome eggs induce M2 macrophages-rich granulomas and these M2 macrophages play critical roles in the maintenance of granuloma and subsequent fibrosis. Reactive oxygen species (ROS), which are highly produced by stimulated macrophages during infection and necessary for the differentiation of M2 macrophages, are massively distributed around deposited eggs in the liver. However, whether ROS are induced by schistosome eggs to subsequently promote M2 macrophage differentiation, and the possible underlying mechanisms as well, remain to be clarified during S. japonicum infection. Herein, we observed that extensive expression of ROS in the liver of S. japonicum-infected mice. Injection of ROS inhibitor in infected mice resulted in reduced hepatic granulomatous responses and fibrosis. Further investigations revealed that inhibition of ROS production in S. japonicum-infected mice reduces the differentiation of M2, accompanied by increased M1 macrophage differentiation. Finally, we proved that S. japonicum egg antigens (SEA) induce a high level of ROS production via both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and mitochondria in macrophages. Our study may help to better understand the mechanism of schistosomiasis japonica-induced hepatic pathology and contribute to the development of potential therapeutic strategies by interfering with ROS production.

Whole-Proteome Differential Screening Identifies Novel Vaccine Candidates for Schistosomiasis japonica.

Schistosomiasis remains a leading cause of chronic morbidity in endemic regions despite decades of widespread mass chemotherapy with praziquantel. Using our whole proteome differential screening approach, and plasma and epidemiologic data from a longitudinal cohort of individuals living in a Schistosoma japonicum-endemic region of the Philippines, we interrogated the parasite proteome to identify novel vaccine candidates for Schistosoma japonicum. We identified 16 parasite genes which encoded proteins that were recognized by immunoglobulin G or immunoglobulin E antibodies in the plasma of individuals who had developed resistance to reinfection, but were not recognized by antibodies in the plasma of individuals who remained susceptible to reinfection. Antibody levels to Sj6-8 and Sj4-1 measured in the entire cohort (N = 505) 1 month after praziquantel treatment were associated with significantly decreased risk of reinfection and lower intensity of reinfection over 18 months of follow-up.

Schistosoma japonicum cystatin suppresses osteoclastogenesis via manipulating the NF­κB signaling pathway.

Abnormal osteoclastic activation and secretion of cysteine proteinases result in excessive bone resorption, which is one of the primary factors in the development of bone metabolic disorders, such as rheumatoid arthritis and osteoporosis. Mammalian cystatins have been demonstrated to restrain osteoclastic bone resorption and to alleviate severe osteolytic destruction via blocking the activity of cysteine proteinases. However, the specific effects of parasite cystatins on the formation and function of osteoclasts remain unclear. The purpose of the current study was to explore the effects of cystatins from Schistosoma japonicum (Sj­Cys) on macrophage colony­stimulating factor (M­CSF) and receptor activator of NF­κB ligand (RANKL)­induced osteoclast differentiation, as well as the underlying molecular mechanisms. Recombinant Sj­Cys (rSj­Cys) dose­dependently restrained osteoclast formation, with a half­maximal inhibitory concentration (IC50) value of 0.3 µM, and suppressed osteoclastic bone resorptive capability in vitro. The findings were based on tartrate resistant acid phosphatase (TRAP) staining and bone resorption assays, respectively. However, the cell viability assay showed that the repression of rSj­Cys on osteoclast formation did not depend on effects on cell viability or apoptosis. Based on the results of reverse transcription­quantitative PCR and western blot analysis, it was found that rSj­Cys downregulated the expression levels of osteoclastogenesis­related genes and proteins, by interfering with M­CSF and RANKL­induced NF­κB signaling and downstream transcription factors during early­phase osteoclastogenesis. Overall, the results of the present study revealed that rSj­Cys exerted an inhibitory role in osteoclast differentiation and could be a prospective biotherapeutic candidate for the treatment and prevention of bone metabolic disorders.

STAT3 Promotes Schistosome-Induced Liver Injury by Inflammation, Oxidative Stress, Proliferation, and Apoptosis Signal Pathway.

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 Stat3flox/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.

RNA sequencing analysis of altered expression of long noncoding RNAs associated with Schistosoma japonicum infection in the murine liver and spleen.

BACKGROUND: Schistosomiasis is a chronic, debilitating infectious disease caused by members of the genus Schistosoma. Previous findings have suggested a relationship between infection with Schistosoma spp. and alterations in the liver and spleen of infected animals. Recent reports have shown the regulatory role of noncoding RNAs, such as long noncoding RNAs (lncRNAs), in different biological processes. However, little is known about the role of lncRNAs in the mouse liver and spleen during Schistosoma japonicum infection. METHODS: In this study, we identified and investigated lncRNAs using standard RNA sequencing (RNA-Seq). The biological functions of the altered expression of lncRNAs and their target genes were predicted using bioinformatics. Ten dysregulated lncRNAs were selected randomly and validated in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) experiments. RESULTS: Our study identified 29,845 and 33,788 lncRNAs from the liver and spleen, respectively, of which 212 were novel lncRNAs. We observed that 759 and 789 of the lncRNAs were differentially expressed in the respective organs. The RT-qPCR results correlated well with the sequencing data. In the liver, 657 differentially expressed lncRNAs were predicted to target 2548 protein-coding genes, whereas in the spleen 660 differentially expressed lncRNAs were predicted to target 2673 protein-coding genes. Moreover, functional annotation showed that the target genes of the differentially expressed lncRNAs were associated with cellular processes, metabolic processes, and binding, and were significantly enriched in metabolic pathways, the cell cycle, ubiquitin-mediated proteolysis, and pathways in cancer. CONCLUSIONS: Our study showed that numerous lncRNAs were differentially expressed in S. japonicum-infected liver and spleen compared to control liver and spleen; this suggested that lncRNAs may be involved in pathogenesis in the liver and spleen during S. japonicum infection.

Schistosoma japonicum cathepsin B2 (SjCB2) facilitates parasite invasion through the skin.

Cercariae invasion of the human skin is the first step in schistosome infection. Proteases play key roles in this process. However, little is known about the related hydrolytic enzymes in Schistosoma japonicum. Here, we investigated the biochemical features, tissue distribution and biological roles of a cathepsin B cysteine protease, SjCB2, in the invasion process of S. japonicum cercariae. Enzyme activity analysis revealed that recombinant SjCB2 is a typical cysteine protease with optimum temperature and pH for activity at 37°C and 4.0, respectively, and can be totally inhibited by the cysteine protease inhibitor E-64. Immunoblotting showed that both the zymogen (50 kDa) and mature enzyme (30.5 kDa) forms of SjCB2 are expressed in the cercariae. It was observed that SjCB2 localized predominantly in the acetabular glands and their ducts of cercariae, suggesting that the protease could be released during the invasion process. The protease degraded collagen, elastin, keratin, fibronectin, immunoglobulin (A, G and M) and complement C3, protein components of the dermis and immune system. In addition, proteomic analysis demonstrated that SjCB2 can degrade the human epidermis. Furthermore, it was showed that anti-rSjCB2 IgG significantly reduced (22.94%) the ability of the cercariae to invade the skin. The cysteine protease, SjCB2, located in the acetabular glands and their ducts of S. japonicum cercariae. We propose that SjCB2 facilitates skin invasion by degrading the major proteins of the epidermis and dermis. However, this cysteine protease may play additional roles in host-parasite interaction by degrading immunoglobins and complement protein.

Meta-analyses of Schistosoma japonicum infections in wild rodents across China over time indicates a potential challenge to the 2030 elimination targets.

China once suffered greatly from schistosomiasis japonica, a major zoonotic disease. Nearly 70 years of multidisciplinary efforts have achieved great progress in disease control, with infections in both humans and bovines significantly reduced to very low levels. However, reaching for the target of complete interruption of transmission at the country level by 2030 still faces great challenges, with areas of ongoing endemicity and/or re-emergence within previously 'eliminated' regions. The objectives of this study were, by using meta-analytical methods, to estimate the overall prevalence of Schistosoma japonicum infections in abundant commensal rodent species in mainland China after the introduction of praziquantel for schistosomiasis treatment in humans and bovines in 1980s. In doing so we thereby aimed to further assess the role of wild rodents as potential reservoirs in ongoing schistosome transmission. Published studies on infection prevalence of S. japonicum in wild rodents in mainland China since 1980 were searched across five electronic bibliographic databases and lists of article references. Eligible studies were selected based on inclusion and exclusion criteria. Risks of within and across study biases, and the variations in prevalence estimates attributable to heterogeneities were assessed. The pooled infection prevalence and its 95% confidence intervals (CIs) were calculated with the Freeman-Tukey double arcsine transformation. We identified a total of 37 relevant articles involving 61 field studies which contained eligible data on 8,795 wild rodents across mainland China. The overall pooled infection prevalence was 3.86% (95% CI: 2.16-5.93%). No significant change in the overall pooled prevalence was observed between 1980-2003 (n = 23 studies) and 2004-current (n = 38 studies). However, whilst the estimated prevalence decreased over time in the marshland and lake regions, there was an apparent increase in prevalence within hilly and mountainous regions. Among seven provinces, a significant prevalence reduction was only seen in Jiangsu where most endemic settings are classified as the marshland and lakes. These estimates changed over season, ranging from 0.58% in spring to 22.39% in winter, in association with increases in rodent density. This study systematically analyzed S. japonicum infections in wild rodents from the published literature over the last forty years after the introduction of praziquantel for schistosomiasis treatment in humans and bovines in 1980s. Although numbers of schistosomiasis cases in humans and bovines have been greatly reduced, no such comparable overall change of infection prevalence in rodents was detected. Furthermore, there appeared to be an increase in S. japonicum prevalence in rodents over time within hilly and mountainous regions. Rodents have been projected to become the dominant wildlife in human-driven environments and the main reservoir of zoonotic diseases in general within tropical zones. Our findings thus suggest that it is now necessary to include monitoring and evaluation of potential schistosome infection within rodents, particularly in hilly and mountainous regions, if we are ever to reach the new 2030 elimination goals and to maximize the impact of future public, and indeed One Health, interventions across, regional, national and international scales.

Analyses of the expression, immunohistochemical properties and serodiagnostic potential of Schistosoma japonicum peroxiredoxin-4.

BACKGROUND: Schistosoma japonicum, which inhabits the mesenteric vein of the mammalian hosts for about 20 to 30 years, is subjected to the oxidative stresses from the host defense mechanism during their intra-mammalian stages. To counteract this host immune attack, the parasite utilizes their antioxidant system for survival inside the host. Peroxiredoxins (Prxs), thiol-specific antioxidant proteins, play an essential role for protecting the parasite against oxidative stress by reducing hydrogen peroxide to water. Only three types of 2-Cys Prxs have been previously characterized in S. japonicum whereas a fourth Prx has been identified for Schistosoma mansoni as Prx-4. A sequence coding homologous to this gene in the S. japonicum database was identified, characterized and expressed as recombinant SjPrx-4 protein (rSjPrx-4). Furthermore, rSjPrx-4 was evaluated in this study for its diagnostic potentials in detecting S. japonicum infection in humans. RESULTS: The gene found in the parasite genome contained 2 active-site cysteines with conserved sequences in the predicted amino acid (AA) sequence and showed 75% identity with that of the previously characterized Prx (TPx-1) of S. japonicum. The gene was expressed in different stages of schistosome life-cycle with highest transcription level in the adult male. The gene was cloned into a plasmid vector and then transfected into Escherichia coli for expression of rSjPrx-4. Anti-rSjPrx-4 mouse sera recognized native SjPrx-4 in egg and adult worm lysate by western blotting. The result of a mixed function oxidation assay in which rSjPrx-4 prevented the nicking of DNA from hydroxyl radicals confirmed its antioxidant activity. Subsequently, immunolocalization analysis showed the localization of SjPrx-4 inside the egg, on the tegument and in the parenchyma of the adult worm. Enzyme-linked immunosorbent assay results showed that rSjPrx-4 has 83.3% sensitivity and 87.8% specificity. Its diagnostic potential was further evaluated in combination with recombinant SjTPx-1 protein, yielding an improved sensitivity and specificity of 90% and 92.7%, respectively. CONCLUSIONS: These results suggest that SjPrx-4 plays a role as an antioxidant dealing with oxidative stresses of S. japonicum, and its diagnostic potential improved by coupling it with SjTPx-1 is a proof for developing a serological test with better diagnostic performance for human schistosomiasis.