Expression profile analysis of the transient receptor potential (TRPM) channel, a possible target of praziquantel in Schistosoma japonicum.

The WHO considers schistosomiasis, which is controlled by the mass administration of the drug praziquantel (PZQ), to be a neglected tropical disease. Despite its clinical use for over four decades, PZQ remains the only choice of chemotherapy against this disease. Regarding the previous studies that demonstrated that PZQ activates the transient receptor potential (TRP) channel in Schistosoma mansoni (Sm.TRPMPZQ), the expression profile of the ortholog of this channel gene (Smp_246790.5) in S. japonicum (EWB00_008853) (Sj.TRPMPZQ) was analyzed. The relative expression of this gene in various stages of the parasite lifecycle was analyzed by quantitative real-time reverse transcription-PCR (qRT-PCR), and the expression of Sj.TRPMPZQ was observed by immunohistochemical staining using anti-serum against the recombinant Sj.TRPMPZQ protein. qRT-PCR revealed the significantly lower mRNA expression in the snail stage in comparison to other stages (p < 0.01). The relative quantity of the Sj.TRPMPZQ expression for paired females, unpaired males, and eggs was 60%, 56%, and 68%, respectively, in comparison to paired males that showed the highest expression (p < 0.05). Interestingly, immunostaining demonstrated that Sj.TRPMPZQ is expressed in the parenchyma which contains muscle cells, neuronal cells and tegument cells in adult worms. This may support the two major effects of PZQ-worm paralysis and tegument disruption-induced by channel activation. Moreover, the channel was expressed in both the eggshell and the miracidia inside, but could not be observed in sporocyst. These results suggest that the expression of Sj.TRPMPQZ corresponds to the known sensitivity of S. japonicum to PZQ.

Schistosome egg antigen stimulates the secretion of miR-33-carrying extracellular vesicles from macrophages to promote hepatic stellate cell activation and liver fibrosis in schistosomiasis.

Schistosomiasis is a serious and neglected disease with a high prevalence in tropical and subtropical countries. The primary pathology of hepatic schistosomiasis caused by Schistosoma japonicum (S. japonicum) or Schistosoma mansoni (S. mansoni) infection is egg-induced granuloma and subsequent fibrosis in the liver. Activation of hepatic stellate cells (HSCs) is the central driver of liver fibrosis. Macrophages (Mφ), making up 30% of cells in hepatic granulomas, directly or indirectly regulate HSC activation by paracrine mechanisms, via secreting cytokines or chemokines. Currently, Mφ-derived extracellular vesicles (EVs) are broadly involved in cell communication with adjacent cell populations. However, whether Mφ-derived EVs could target neighboring HSCs to regulate their activation during schistosome infection remains largely unknown. Schistosome egg antigen (SEA) is considered to be the main pathogenic complex mixture involved in liver pathology. Here, we demonstrated that SEA induced Mφ to produce abundant extracellular vesicles, which directly activated HSCs by activating their autocrine TGF-ß1 signaling. Mechanistically, EVs derived from SEA-stimulated Mφ contained increased miR-33, which were transferred into HSCs and subsequently upregulated autocrine TGF-ß1 in HSCs through targeting and downregulating SOCS3 expression, thereby promoting HSC activation. Finally, we validated that EVs derived from SEA-stimulated Mφ utilized enclosed miR-33 to promote HSC activation and liver fibrosis in S. japonicum-infected mice. Overall, our study indicates that Mφ-derived EVs play important roles in the paracrine regulation of HSCs during the progression of hepatic schistosomiasis, representing a potential target for the prevention of liver fibrosis in hepatic schistosomiasis.

Praziquantel promotes protection against Schistosoma japonicum infection in mice.

Praziquantel (PZQ) is the first line drug for the treatment of schistosomiasis. Several studies have confirmed that PZQ regulates host immunity, and we have recently found that pretreatment with PZQ enhances resistance against Schistosoma japonicum infection in buffaloes. We speculate that PZQ induces physiological changes in mice that prevent S. japonicum infection. To test this hypothesis and provide a practical measure to prevent S. japonicum infection, we determined the effective dose (the minimum dose), protection period and onset time of protection by comparing the worm burden, female worm burden and egg burden in PZQ-pretreated mice and blank control mice. Morphological differences between parasites were observed by measuring the total worm length, oral sucker, ventral sucker and ovary. The levels of cytokines, nitrogen monoxide (NO), 5-hydroxytryptamine (5-HT) and specific antibodies were measured using kits or soluble worm antigens. Hematological indicators on day 0 were analyzed in mice that received PZQ on days -15, -18, -19, -20, -21 and -22. The PZQ concentrations in plasma and blood cells were monitored using high performance liquid chromatography (HPLC). The effective dose was found to be two oral administrations (interval of 24 h) at 300 mg/kg body weight (BW) or one injection at 200 mg/kg BW, and the protection period of PZQ injection was 18 days. The optimal preventive effect was observed at two days post-administration, with a >92% worm reduction rate and significant worm reduction until 21 days after administration. Adult worms from PZQ-pretreated mice were runtish showing a shorter length, smaller organs and fewer eggs in the uteri of females. Detection of cytokines, NO, 5-HT and hematological indicators showed that PZQ induced immune-physiological changes, including higher levels of NO, IFN-γ and IL-2, and a lower level of TGF-ß. No significant difference in the anti-S. japonicum specific antibody levels was observed. The PZQ concentrations in plasma and blood cells 8 and 15 days post-administration were lower than the detection limit. Our results confirmed that pretreatment with PZQ promotes the protection of mice against S. japonicum infection within 18 days. Although we observed some immune-physiological changes in the PZQ-pretreated mice, the exact mechanisms involved in the preventive effect require further study.

TLR7 controls myeloid-derived suppressor cells expansion and function in the lung of C57BL6 mice infected with Schistosoma japonicum.

Toll-like receptors (TLRs) play an important role in the induction of innate and adaptive immune responses against Schistosoma japonicum (S. japonicum) infection. However, the role of Toll-like receptor 7 (TLR7) in the mouse lung during S. japonicum infection and the myeloid-derived suppressor cells (MDSCs) affected by the absence of TLR7 are not clearly understood. In this study, the results indicated that the MDSCs were accumulated and the proportion and activation of CD4+ and CD8+ T cells were decreased in the lung of mice at 6-7 weeks after S. japonicum infection. Then, the expression of TLR7 was detected in isolated pulmonary MDSCs and the results showed that the expression of TLR7 in MDSCs was increased after infection. Furthermore, TLR7 agonist R848 could down-regulate the induction effect of the soluble egg antigen (SEA) on pulmonary MDSCs in vitro. Meanwhile, TLR7 deficiency could promote the pulmonary MDSCs expansion and function by up-regulating the expression of PD-L1/2 and secreting of IL-10 in the mice infected with S. japonicum. Mechanistic studies revealed that S. japonicum infection and the antigen effects are mediated by NF-κB signaling. Moreover, TLR7 deficiency aggravates S. japonicum infection-induced damage in the lung, with more inflammatory cells infiltration, interstitial dilatation and granuloma in the tissue. In summary, this study indicated that TLR7 signaling inhibits the accumulation and function of MDSCs in S. japonicum infected mouse lung by down-regulating the expression of PD-L1/2 and secreting of IL-10, via NF-κB signaling.

[Advances in studies on schistosome-host interactions mediated by extracellular vesicles].

Extracellular vesicles (EVs) are minute particles secreted by the cells of living organisms, which can encapsulate various bioactive molecules for long-distance transport to present biological functions. With the recent studies on parasite-host interactions, EVs, as a carrier for long-distance transport of worm-derived molecules, have been paid much attention during the across-species regulation of hosts. During schistosome infections, adult worms and eggs have been found to mediate hosts via secretion of EVs. This review presents the advances in the studies on schistosome-host interactions mediated by EVs.

A new ferritin SjFer0 affecting the growth and development of Schistosoma japonicum.

BACKGROUND: Schistosomiasis, an acute and chronic parasitic disease, causes substantial morbidity and mortality in tropical and subtropical regions of the world. Iron is an essential constituent of numerous macromolecules involving in important cellular reactions in virtually all organisms. Trematodes of the genus Schistosoma live in iron-rich blood, feed on red blood cells and store abundant iron in vitelline cells. Ferritins are multi-meric proteins that store iron inside cells. Three ferritin isoforms in Schistosoma japonicum are known, namely SjFer0, SjFer1 and SjFer2; however, their impact on the growth and development of the parasites  is still unknown. In this study we report on and characterize the ferritins in S. japonicum. METHODS: A phylogenetic tree of the SjFer0, SjFer1 and SjFer2 genes was constructed to show the evolutionary relationship among species of genus Schistosoma. RNA interference in vivo was used to investigate the impact of SjFer0 on schistosome growth and development.  Immunofluorescence assay was applied  to localize the expression of the ferritins. RNA-sequencing was performed  to characterize the iron transport profile after RNA interference. RESULTS: SjFer0 was found to have low similarity with SjFer1 and SjFer2 and contain an additional signal peptide sequence. Phylogenetic analysis revealed that SjFer0 can only cluster with some ferritins of other trematodes and tapeworms, suggesting that this ferritin branch might be unique to these parasites. RNA interference in vivo showed that SjFer0 significantly affected the growth and development of schistosomula but did not affect egg production of adult female worms. SjFer1 and SjFer2 had no significant impact on growth and development. The immunofluorescence study showed that SjFer0 was widely expressed in the somatic cells and vitelline glands but not in the testicle or ovary. RNA-sequencing indicated  that, in female, the ion transport process and calcium ion binding function were downregulated after SjFer0 RNA interference. Among the differentially downregulated genes, Sj-cpi-2, annexin and insulin-like growth factor-binding protein may be accounted for the suppression of schistosome growth and development. CONCLUSIONS: The results indicate that SjFer0 affects the growth and development of schistosomula but does not affect egg production of adult female worms. SjFer0 can rescue the growth of the fet3fet4 double mutant Saccharomyces cerevisiae (strain DEY1453), suggesting being able to promote iron absorption. The RNA interference of SjFer0 inferred that  the suppression of worm growth and development may via  down-regulating Sj-cpi-2, annexin, and IGFBP.

Neutrophils and schistosomiasis: a missing piece in pathology.

Schistosomiasis is a chronic human parasitic disease that causes serious health problems worldwide. The disease-associated liver pathology is one of the hallmarks of infections by Schistosoma mansoni and Schistosoma japonicum, and is accountable for the debilitating condition found in infected patients. In the past few years, investigative studies have highlighted the key role played by neutrophils and the influence of inflammasome signalling pathway in different pathological conditions. However, it is noteworthy that the study of inflammasome activation in neutrophils has been overlooked by reports concerning macrophages and monocytes. This interplay between neutrophils and inflammasomes is much more poorly investigated during schistosomiasis. Herein, we reviewed the role of neutrophils during schistosomiasis and addressed the potential connection between these cells and inflammasome activation in this context.

Calpain inhibitor suppresses both extracellular vesicle-mediated secretion of miRNAs and egg production from paired adults of Schistosoma japonicum.

Extracellular vesicles (EVs) have been reported to be secreted from Schistosoma japonicum at all developmental stages. However, the reproduction and communication mechanisms between the paired adults through the EVs in dioecious Trematoda have not been reported. In this study, EVs containing many exosome-like vesicles and microvesicles were observed in the supernatants of paired adults cultured in vitro, and abundant selected miRNAs were contained in them. In particular, the female-specific miR-bantam was present only in vesicles and was hardly secreted outside the vesicles. In this study, we found that male-female pairing induced secretion of miR-3479 and miR-bantam in EVs, but not of male-specific miR-61. Furthermore, ingestion of mouse erythrocytes also increased the production of miRNAs in paired adult and single female worms. Vesicles were found in the tegument of females treated with erythrocytes under electron microscopy. After the paired worms were treated with several inhibitors against the secretion of EVs, only calpain inhibitor (calpeptin) significantly reduced the amount of miRNA in EVs. Furthermore, the worms treated with only calpeptin inhibited egg production in vitro. Together, these results indicate that qualitative miRNA production through EVs regulated by calpain plays a role in egg production in S. japonicum.

TLR7 modulating B-cell immune responses in the spleen of C57BL/6 mice infected with Schistosoma japonicum.

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.

Reduction of autofluorescence in whole adult worms of Schistosoma japonicum for immunofluorescence assay.

Immunofluorescence assay is one of methods to understand the spatial biology by visualizing localization of biomolecules in cells and tissues. Autofluorescence, as a common phenomenon in organisms, is a background signal interfering the immunolocalization assay of schistosome biomolecules, and may lead to misinterpretation of the biomolecular function. However, applicable method for reducing the autofluorescence in Schistosoma remains unclear. In order to find a suitable method for reducing autofluorescence of schistosomes, different chemical reagents, such as Sudan black B (SBB), trypan blue (TB), copper sulfate (CuSO4), Tris-glycine (Gly), and ammonia/ethanol (AE), at different concentrations and treatment time were tested, and SBB and CuSO4 were verified for the effect of blocking autofluorescence in immunofluorescence to localize the target with anti-SjCRT antibody. By comparing the autofluorescence characteristics of different conditions, it was found that SBB, TB and CuSO4 had a certain degree of reducing autofluorescence effect, and the best effect in females was using 50 mM CuSO4 for 6 h and in males was 0.5% SBB for 6 h. Furthermore, we have applied the optimized conditions to the immunofluorescence of SjCRT protein, and the results revealed that the immunofluorescence signal of SjCRT was clearly visible without autofluorescence interference. We present an effective method to reduce autofluorescence in male and female worm of Schistosoma japonicum for immunofluorescence assay, which could be helpful to better understand biomolecular functions. Our method provides an idea for immunofluorescence assay in other flukes with autofluoresence.

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.

GENE EXPRESSION LEVEL, IMMUNOLOCALIZATION, AND FUNCTION OF FATTY ACID-BINDING PROTEIN FROM SCHISTOSOMA JAPONICUM.

The Schistosoma japonicum fatty acid-binding protein (FABP) is used in the cell membrane to absorb and transport fatty acids, which cannot be resynthesized by the organism and combined with hydrophobic ligands. Among the 5 stages of the worm life cycle examined, FABP messenger ribonucleic acid (mRNA) expression was highest in male adult worms, followed by the liver-stage schistosome, and was the lowest in the lung-stage schistosome. The fabp gene-coding region was cloned and expressed to obtain recombinant S. japonicum FABP (rSjFABP) with a molecular weight of approximately 18 kDa. Mice were then immunized against rSjFABP to prepare anti-FABP serum. Using immunohistochemical techniques, FABP protein was found to localize to the eggshell, parenchyma, and digestive tract. Double-stranded RNA-mediated knockdown of FABP mRNA by RNA interference decreased the number of transcripts by >70%. Moreover, the egg production rate decreased, whereas the abnormal egg ratio was significantly increased in the FABP-silenced group compared with the negative control group (P < 0.05). These results demonstrate that FABP localizes in adults and in various stages. FABP contributes to the egg-laying capacity of adults, which may be related to the reproductive function of S. japonicum.

Morphology and activities of cell populations of haemocytes in Oncomelania hupensis following Schistosoma japonicum infection.

Oncomelania hupensis is the only obligatory intermediate host of Schistosoma japonicum, the pathogen of zoonosis schistosomiasis. Haemocytes play a critical role in the cellular immune defence of O. hupensis against S. japonicum challenge. Here, the morphology and classification of haemocytes of O. hupensis were investigated by Giemsa staining and light microscopy, combining with the scanning and transmission electron microscopy and flow cytometry. Granulocytes and hyalinocytes were confirmed as two main types of haemocytes, account for ~ 10% and ~ 90% of all haemocytes, with size varying in 4.3-10.9 µm and 0.4-30.8 µm, respectively. Subpopulations can be identified further by granule feature, shape, size, and surface and inner structure of cells. The heterogeneity in morphology implied varied developmental process and function of haemocyte subpopulations. After the S. japonicum challenge, haemocytes of O. hupensis respond to S. japonicum invasion immediately. The dynamic change of haemocyte subpopulations indicates that the small hyalinocyte could differentiate into a larger one or granulocyte after S. japonicum challenge, and the granulocytes and larger hyalinocytes play leading roles in early defence reaction, but in different ways. Phagocytosis and apoptosis of haemocytes in O. hupensis were proved to be related to immune defence against S. japonicum, with the combined effect of granulocytes and larger hyalinocytes. However, the main pathway of each subpopulation to take effect in different periods need further investigation.

Potential impact of flooding on schistosomiasis in Poyang Lake regions based on multi-source remote sensing images.

BACKGROUND: Flooding is considered to be one of the most important factors contributing to the rebound of Oncomelania hupensis, a small tropical freshwater snail and the only intermediate host of Schistosoma japonicum, in endemic foci. The aim of this study was to assess the risk of intestinal schistosomiasis transmission impacted by flooding in the region around Poyang Lake using multi-source remote sensing images. METHODS: Normalized Difference Vegetation Index (NDVI) data collected by the Landsat 8 satellite were used as an ecological and geographical suitability indicator of O. hupensis habitats in the Poyang Lake region. The expansion of the water body due to flooding was estimated using dual-polarized threshold calculations based on dual-polarized synthetic aperture radar (SAR). The image data were captured from the Sentinel-1B satellite in May 2020 before the flood and in July 2020 during the flood. A spatial database of the distribution of snail habitats was created using the 2016 snail survey in Jiangxi Province. The potential spread of O. hupensis snails after the flood was predicted by an overlay analysis of the NDVI maps in the flood-affected areas around Poyang Lake. The risk of schistosomiasis transmission was classified based on O. hupensis snail density data and the related NDVI. RESULTS: The surface area of Poyang Lake was approximately 2207 km2 in May 2020 before the flood and 4403 km2 in July 2020 during the period of peak flooding; this was estimated to be a 99.5% expansion of the water body due to flooding. After the flood, potential snail habitats were predicted to be concentrated in areas neighboring existing habitats in the marshlands of Poyang Lake. The areas with high risk of schistosomiasis transmission were predicted to be mainly distributed in Yongxiu, Xinjian, Yugan and Poyang (District) along the shores of Poyang Lake. By comparing the predictive results and actual snail distribution, we estimated the predictive accuracy of the model to be 87%, which meant the 87% of actual snail distribution was correctly identified as snail habitats in the model predictions. CONCLUSIONS: Data on water body expansion due to flooding and environmental factors pertaining to snail breeding may be rapidly extracted from Landsat 8 and Sentinel-1B remote sensing images. Applying multi-source remote sensing data for the timely and effective assessment of potential schistosomiasis transmission risk caused by snail spread during flooding is feasible and will be of great significance for more precision control of schistosomiasis.

Afforestation suppresses Oncomelania hupensis snail density through influencing algae in beaches of the Dongting Lake.

BACKGROUND: Oncomelania snails serve as the sole intermediate host for Schistosoma japonicum, one of the most important neglected tropical diseases in the world. Afforestation suppression of the Oncomelania hupensis snail has been a long-term effective national strategy to decrease snail density in China. Many previous studies have made clear that vegetation (biotic factors) and soil (abiotic factors) were the basic requirements for snail survival on beaches. Moreover, a lot of research on snail control has been focused on the specific influencing environmental factors for snail survival, such as the vegetation community structure, species composition, diversity index, and the physical and chemical properties of the soil. Most of the existing research has studied the influence of a single factor on snail population density. Conversely, there have been only a few studies focused on the food sources and food composition of the snails. The current research situation on snail control has indicated that the mechanisms underlying ecological snail control have not been systematically characterized. The question of whether biotic or abiotic factors were more important in influencing snail survival remains unclear. Afforestation on beaches has significantly suppressed snail density in China so far. In this study, we proposed that the reduction of snail density was not affected by a single factor but by the interactions of multiple related factors introduced by afforestation. Moreover, different biotic and abiotic factors have significantly different effects on snail control. Therefore the goal of this study was to evaluate the relative importance and interactions of related biotic and abiotic factors on snail density. Methods: Four major vegetation communities: Sedge, Reed, Artificial poplar (3 years of age) and Artificial poplar (5 years of age), on the beaches of the Yangtze River in China were selected for vegetation and snail surveys, as well as for soil sampling. Structural Equation Model (SEM) analysis was used to assess the interactions of biotic and abiotic factors in the context of snail ecology. The soil properties were considered as abiotic factors, while algae of Chlorophyta, Cyanophyta and Bacillariophyta phyla were considered to be biotic factors. In the path analysis, the total effect between the variables was the sum of the direct and indirect effects. RESULTS: The snail density had significant correlations with soil properties, such as water content, bulk density, capillary porosity and pH value, as well as with all three types of soil algae, Chlorophyta, Cyanophyta, and Bacillariophyta. Snail density had a direct negative relationship with capillary porosity and soil bulk density, an indirect negative relationship with soil pH value and an indirect positive relationship with soil water content via soil algae. Meanwhile, as an important food source for the snail, the Chlorophyta, Cyanophyta and Bacillariophyta algae had a significant positive correlation with snail density. High soil pH had a negative impact on Chlorophyta, Bacillariophyta, while soil water content had a positive impact on Chlorophyta, and soil bulk density had a negative impact on Cyanophyta. In addition, the soil pH value and soil bulk density both had negative correlations with soil water content. CONCLUSION: Afforestation of the beach environment can significantly reduce the snail population density by altering ecological factors. Soil algae (biological factors) might be the key element that drives ecological snail control. As important habitat determinants, the impact of the properties of the soil (non-biological factors) on the snail population was largely mediated through soil algae.

Granuloma formation and tissue pathology in Schistosoma japonicum versus Schistosoma mansoni infections.

Schistosomiasis is the most important helminth disease in the world from a public health perspective. S mansoni and S japonicum account for the majority of global intestinal schistosomiasis cases, and the pathogenesis is widely assumed to be fundamentally similar. However, the majority of research on schistosomiasis has been carried out on S mansoni and comparisons between the two species are rarely made. Here, we will discuss aspects of both older and recent literature where such comparisons have been made, with a particular focus on the pathological agent, the host granulomatous response to the egg. Major differences between the two species are apparent in features such as egg production patterns and cellular infiltration; however, it is also clear that even subtle differences in the cascade of various cytokines and chemokines contribute to the different levels of pathology observed between these two main species of intestinal schistosomiasis. A better understanding of such differences at species level will be vital when it comes to the development of new treatment strategies and vaccines.

Loss of natural resistance to schistosome in T cell deficient rat.

Schistosomiasis is among the major neglected tropical diseases and effective prevention by boosting the immune system is still not available. T cells are key cellular components governing adaptive immune response to various infections. While common laboratory mice, such as C57BL/6, are highly susceptible to schistosomiasis, the SD rats are extremely resistant. However, whether adaptive immunity is necessary for such natural resistance to schistosomiasis in rats remains to be determined. Therefore, it is necessary to establish genetic model deficient in T cells and adaptive immunity on the resistant SD background, and to characterize liver pathology during schistosomiasis. In this study we compared experimental schistosomiasis in highly susceptible C57BL/6 (B6) mice and in resistant SD rats, using cercariae of Schistosoma japonicum. We observed a marked T cell expansion in the spleen of infected B6 mice, but not resistant SD rats. Interestingly, CD3e-/- B6 mice in which T cells are completely absent, the infectious burden of adult worms was significantly higher than that in WT mice, suggesting an anti-parasitic role for T cells in B6 mice during schistosome infection. In further experiments, we established Lck deficient SD rats by using CRISPR/Cas9 in which T cell development was completely abolished. Strikingly, we found that such Lck deficiency in SD rats severely impaired their natural resistance to schistosome infection, and fostered parasite growth. Together with an additional genetic model deficient in T cells, the CD3e-/- SD rats, we confirmed the absence of T cell resulted in loss of natural resistance to schistosome infection, but also mitigated liver immunopathology. Our further experiments showed that regulatory T cell differentiation in infected SD rats was significantly decreased during schistosomiasis, in contrast to significant increase of regulatory T cells in infected B6 mice. These data suggest that T cell mediated immune tolerance facilitates persistent infection in mice but not in SD rats. The demonstration of an important role for T cells in natural resistance of SD rats to schistosomiasis provides experimental evidences supporting the rationale to boost T cell responses in humans to prevent and treat 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.

The Differential and Dynamic Progression of Hepatic Inflammation and Immune Responses During Liver Fibrosis Induced by Schistosoma japonicum or Carbon Tetrachloride in Mice.

Liver fibrosis can result from various causes and could progress to cirrhosis and cancer; however, there are no effective treatments due to that its molecular mechanism is unclear. liver fibrosis model made by Schistosoma japonicum (S. japonicum) infection or Carbon tetrachloride (CCl4) intraperitoneal injection is a conventional model used in liver fibrosis-related studies for mechanism or pharmaceutical research purposes. But the differences in the pathological progression, immune responses and the underlying mechanism between the two liver fibrosis model have not been carefully compared and characterized, which hinders us from correctly understanding and making better use of the two models. In the present study, the pathological changes to the liver, and the cytokines, inflammatory factors, macrophages, and lymphocytes subsets involved were analyzed in the liver fibrosis model of S. japonicum infection or CCl4 intraperitoneal injection. Additionally, the pathological progression, immune responses and the underlying injury mechanism in these two models were compared and characterized. The results showed that the changing trend of interleukin-13 (IL-13), transforming growth factor beta (TGF-ß), inflammatory factors, and M1, M2 macrophages, were consistent with the development trend of fibrosis regardless of whether liver fibrosis was caused by S. japonicum or CCl4. For lymphocyte subsets, the proportions of CD3+ T cells and CD4+ T cells decreased gradually, while proportion of CD8+ T cells peaked at 6 weeks in mice infected with S. japonicum and at 12 weeks in mice injected with CCl4. With prolonged S. japonicum infection time, Th1 (CD4+IFN-γ+) immunity converted to Th2 (CD4+IL-4+)/Th17 (CD4+IL-17+) with weaker regulatory T cell (Treg) (CD4+CD25+FOXP3+) immunity. However, in liver fibrosis caused by CCl4, Th1 cells occupied the dominant position, while proportions of Th2, Th17, and Treg cells decreased gradually. In conclusion, liver fibrosis was a complex pathological process that was regulated by a series of cytokines and immune cells. The pathological progressions and immune responses to S. japonicum or CCl4 induced liver fibrosis were different, possibly because of their different injury mechanisms. The appropriate animal model should be selected according to the needs of different experiments and the pathogenic factors of liver fibrosis in the study.

Geographical survey of the intermediate host of Schistosoma japonicum: Toward precise management of Oncomelania hupensis.

Schistosomiasis caused by Schistosoma japonicum is a public health concern in China, and Hubei is one of the most affected provinces. Although the routine surveillance since the mid 1950s has generated substantial data pertaining to the habitats of the intermediate snail host, Oncomelania hupensis, its spatiotemporal distribution is not known. A review of local chronicles on the annual records of schistosomiasis control program was conducted to retrospectively collect information about O. hupensis habitats. The habitats were mapped by a field survey in 2016. We categorized the habitats into five evolutionary types, namely, Type I, current habitat; Types II-IV, historical habitat; and Type V, suspected habitat according to habitat development. The shape of habitats was determined using geographical information systems. A visual database was established and managed on the ArcGIS platform. A total of 43 472 O. hupensis habitats, covering an area of approximately 430 000 hectares, were identified through the study. Over 60% of these habitats have been eliminated. The highest number of O. hupensis habitats was recorded in 1975; however, most of them were preserved until 1995. Our study, for the first time, sheds light on the spatiotemporal distribution of O. hupensis in the most affected province in China. The data will be valuable for policy making and for formulating strategies to eliminate schistosomiasis in Hubei Province.