Knocking Down Gm16685 Decreases Liver Granuloma in Murine Schistosomiasis Japonica.

Long noncoding RNAs (lncRNAs) can regulate key genes and pathways in liver disease development. Moreover, macrophages are speculated to play an important role in regulating granulomatous inflammation during schistosomiasis. However, the role of lncRNAs in the formation of liver granulomas by influencing the polarization of macrophages in Schistosoma japonicum infection is unclear. Our study aimed to determine whether lncRNAs can play a role in S. japonicum-induced hepatic egg granulomas and elucidate their effect on macrophages. We established S. japonicum infection models and screened the target lncRNA Gm16685 highly expressed in schistosomiasis mice using high-throughput sequencing. Hematoxylin and eosin staining revealed that the knockdown of Gm16685 reduced the area of egg granulomas. Moreover, M1 macrophage factor genes were significantly downregulated in Gm16685 knockdown livers. Meanwhile, M2 macrophage factor genes were significantly upregulated, which was consistent with the protein detection results. Hepatocytes, hepatic stellate cells, and macrophages were isolated from mouse models infected with S. japonicum, with Gm16685 being significantly upregulated in macrophages. Moreover, the knockdown of Gm16685 in RAW264.7 cells revealed similar results to in liver tissue. RNA fluorescence in situ hybridization (FISH) and nucleocytoplasmic separation experiments revealed that Gm16685 was predominantly localized in the cytoplasm of cells. We found that miR-205-5p was upregulated after Gm16685 was knocked down. After overexpression of miR-205-5p, the expression of Gm16685 and inflammatory factors was significantly downregulated. These results indicate that Gm16685 can participate in the pathogenesis of hepatic disease in schistosomiasis and promote M1 macrophage polarization by regulating miR-205-5p. Thus, our study may provide a new target for schistosomiasis japonica treatment.

Pyp25α is required for male gametocyte exflagellation.

Malaria, a mosquito-borne infectious disease, is caused by the unicellular apicomplexan protozoa of the genus Plasmodium. For malaria parasite transmission, the essential sexual stage includes production of gametocytes through gametocytogenesis in vertebrate hosts and formation of gametes from gametocytes through gametogenesis in mosquito vectors. Whereas each female gametocyte forms a single immotile macrogamete, a male gametocyte produces eight flagella-like microgametes in a process called exflagellation. We identified a conserved protein named as Py05543 (Pyp25α), required for male gametocyte exflagellation in Plasmodium yoelii, which is the ortholog of PFL1770c (PF3D7_1236600). Interestingly, PF3D7_1236600 was previously phenotypically screened to be gametocyte-essential genes during gametocytogenesis of Plasmodium falciparum, using piggyBac transposon-mediated insertional mutagenesis. In this study, using CRISPR/Cas9-mediated genome editing, the Pyp25α¯ (KO) parasite line was successfully established. We found that the KO parasites proliferated asexually in mouse blood normally. In addition, compared with that of the parental parasites, the KO parasites displayed similar levels of gametocytes formation. Unexpectedly, the KO parasites showed considerable deficiency in exflagellation of male gametes, by observing exflagellation centre formation. Taken together, our data suggested that Pyp25α gene, the ortholog of PF3D7_1236600, was nonessential for the growth of asexual parasites, required for male gametocyte exflagellation in P. yoelii.

IL-37 alleviates liver granuloma caused by Schistosoma japonicum infection by inducing alternative macrophage activation.

BACKGROUND: Hepatic macrophages regulate liver granuloma formation and fibrosis caused by infection with Schistosoma japonicum, with the manner of regulation dependent on macrophage activation state. Interleukin (IL)-37 may have immunomodulatory effects on macrophages. However, whether IL-37 can affect liver granuloma formation and fibrosis by affecting the polarization of macrophages in S. japonicum infection remains unclear. The aim of this study was to investigate IL-37-affected macrophage polarization in liver granuloma formation and fibrosis in S. japonicum infection. METHODS: An enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of IL-37 in the serum of patients with acute S. japonicum infection and in the serum of healthy people. Recombinant IL-37 (rIL-37), CPP-IgG2Fc-IL-37 and no CPP-IgG2Fc-IL-37 proteins were injected into S. japonicum-infected mice every 3 days for a total of 6 times from day 24 post infection onwards. Subsequently, ELISA, quantitative reverse transcription-PCR, fluorescence-activated cell sorting and western blot were used to analyze whether IL-37 inhibits the formation of liver granulomas and the development of liver fibrosis by regulating the phenotypic transition of macrophages. Finally, the three IL-37 proteins and SIS3, a Smad3 inhibitor, were co-cultured in mouse peritoneal macrophages to explore the mechanism underlying the promotion of the polarization of M0 macrophages to the M2 phenotype by IL-37. RESULTS: Serum IL-37 levels were upregulated in schistosomiasis patients, and this increased level of IL-37 protein apparently alleviated the liver granuloma of mice in infection models. It also could induce liver and peritoneal macrophages to polarize to the M2 phenotype in S. japonicum-infected mice. The S. japonicum-infected mice injected with CPP-IgG2Fc-IL-37 group exhibited the most obvious improvement in inflammatory reaction against the liver granuloma. The number and ratio of M2 macrophages in the liver and peritoneal cavity were significantly higher in the three IL-37 protein groups, especially in the CPP-IgG2Fc-IL-37 group, compared to the controls. Similar results were also found regarding liver function damage. IL-37 induced macrophage M2 polarization by promoting AMP-activated protein kinase (AMPK) phosphorylation in vitro. Among all groups, the activation of AMPK was most significant in the CPP-IgG2Fc-IL-37 group, and it was found that SMAD3 could enhance the anti-inflammatory function of IL-37. CONCLUSIONS: The results show that IL-37 was able to promote the polarization of macrophages to the M2 phenotype, thereby inhibiting the development of schistosomiasis. In comparison to the rIL-37 protein, the CPP-IgG2Fc-IL-37 protein has the advantages of being effective in small doses and having fewer side effects and a better efficacy.

JQ-1 ameliorates schistosomiasis liver granuloma in mice by suppressing male and female reproductive systems and egg development of Schistosoma japonicum.

Schistosomiasis is a serious and widespread parasitic disease caused by infection with Schistosoma. Because the parasite's eggs are primarily responsible for schistosomiasis dissemination and pathogenesis, inhibiting egg production is a potential approach to control the spread and severity of the disease. The bromodomain and extra-terminal (BET) proteins represent promising targets for the development of epigenetic drugs against Schistosoma. JQ-1 is a selective inhibitor of the BET protein family. In the present study, JQ-1 was applied to S. japonicum in vitro. By using laser confocal scanning microscopy and EdU incorporation assays, we showed that application of JQ-1 to worms in vitro affected egg laying and the development of both the male and female reproductive systems. JQ-1 also inhibited the expression of the reproductive-related genes SjPlk1 and SjNanos1 in S. japonicum. Mice infected with S. japonicum were treated with JQ-1 during egg granuloma formation. JQ-1 treatment significantly reduced the size of the liver granulomas and levels of serum alanine aminotransferase and aspartate aminotransferase in mice and suppressed both egg laying and the development of male and female S. japonicum reproductive systems in vivo. Moreover, the mRNA expression levels of some proinflammatory cytokines were decreased in the parasites. Our findings suggest that JQ-1 treatment attenuates S. japonicum egg-induced hepatic granuloma due at least in part to suppressing the development of the reproductive system and egg production of S. japonicum. These findings further suggest that JQ-1 or other BET inhibitors warrant additional study as a new approach for the treatment or prevention of schistosomiasis.

Calcium/calmodulin-dependent protein kinase IV promotes imiquimod-induced psoriatic inflammation via macrophages and keratinocytes in mice.

CaMK4 has an important function in autoimmune diseases, and the contribution of CaMK4 in psoriasis remains obscure. Here, we show that CaMK4 expression is significantly increased in psoriatic lesional skin from psoriasis patients compared to healthy human skin as well as inflamed skin from an imiquimod (IMQ)-induced mouse model of psoriasis compared to healthy mouse skin. Camk4-deficient (Camk4-/-) mice treated with IMQ exhibit reduced severity of psoriasis compared to wild-type (WT) mice. There are more macrophages and fewer IL-17A+γδ TCR+ cells in the skin of IMQ-treated Camk4-/- mice compared to IMQ-treated WT mice. CaMK4 inhibits IL-10 production by macrophages, thus allowing excessive psoriatic inflammation. Deletion of Camk4 in macrophages alleviates IMQ-induced psoriatic inflammation in mice. In keratinocytes, CaMK4 inhibits apoptosis as well as promotes cell proliferation and the expression of pro-inflammatory genes such as S100A8 and CAMP. Taken together, these data indicate that CaMK4 regulates IMQ-induced psoriasis by sustaining inflammation and provides a potential target for psoriasis treatment.

JQ-1 ameliorates schistosomiasis liver fibrosis by suppressing JAK2 and STAT3 activation.

Schistosomiasis is a serious parasitic infection caused by Schistosoma. The parasite deposits eggs in the host liver, causing inflammation that activates hepatic stellate cells (HSCs), which leads to liver fibrosis. Currently, there is no effective therapy for liver fibrosis; thus, treatments are urgently needed. Therefore, in the present study, mice infected with Schistosoma japonicum were treated with JQ-1, a small-molecule bromodomain inhibitor with reliable anti-tumor and anti-inflammatory activities. The fibrotic area of the liver measured by computer-assisted morphometric analysis and the expression levels of the cytoskeletal protein alpha smooth muscle actin (α-SMA) and of collagen assessed by quantitative PCR, Western blot and immunohistochemistry were significantly decreased in the liver following JQ-1 treatment compared with vehicle-treated controls. Total RNA was extracted from the liver of JQ-1-treated Schistosoma-infected mice for RNA-sequencing analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that JQ-1 affected biological processes and the expression of cellular components known to play key roles in the transdifferentiation of HSCs to myofibroblasts. In vitro treatment with JQ-1 of JS-1 cells, a mouse HSC line, indicated that JQ-1 significantly inhibited JS-1 proliferation but had no effect on JS-1 activity, senescence, or apoptosis. Western blot results showed that JQ-1 inhibited the expression levels of phosphorylated JAK2 and phosphorylated STAT3 without altering expression levels of these non-phosphorylated proteins. Taken together, these findings suggested that JQ-1 treatment ameliorated S. japonicum egg-induced liver fibrosis, at least in part, by suppressing HSC activation and proliferation through the inhibition of JAK2/STAT3 signaling. These results lay a foundation for the development of novel approaches to treat and control liver fibrosis caused by S. japonicum.

Imbalance of uterine innate lymphoid cells is involved in the abnormal pregnancy induced by Toxoplasma gondii infection.

Toxoplasma gondii (T. gondii) is a ubiquitous intracellular protozoan parasite that causes adverse pregnancy outcomes. Innate lymphoid cells (ILCs) are critical mediators of mucosal immunity, and have been reported to play an important role in uterine vascular adaptation for successful pregnancy. However, the specific role of ILCs in T. gondii-infection-induced adverse pregnancy outcomes remains elusive. In the present study, we found that T. gondii infection caused the imbalance of uterine ILC cells (uILCs). It was characterized by substantially lower expression of the transcription factor GATA-3 and RORγt and higher expression of T-bet in uILCs. Consistent with the transcription factor changes, uILCs from T. gondii-infected mice produced much less IL-5 and IL-17 and substantially more IFN-γ and TNF-α than did uILCs from uninfected mice. Notably, IL-12, IL-18, and their receptors were increased in the uterus of T. gondii-infected mice. In vitro experiments showed that IL-12 and IL-18 treatment reduced the percentages of uILC2 and uILC3 and increased the percentages of uILC1. Conclusion, our data suggest that alterations in uILC composition may disrupt the balance of immune microenvironment after T. gondii infection and contribute to the adverse pregnancy outcomes caused by T. gondii infection.

Video Microscopic Analysis of Invasion of Toxoplasma gondii into Peritoneal Macrophages.

Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect all nucleated cells through active invasion. Some non-canonical pathways for T. gondii infection of macrophages have recently been reported. We report a new mode of T. gondii invasion using a time-lapse imaging system, in which T. gondii tachyzoites are engulfed by a tube-like structure on peritoneal macrophage phagosomes and then escape from the phagosomes. Escaped parasites re-invade macrophages through intercellular junctions between their apical end and host cell membranes. We call this invasion pathway of T. gondii "pseudopod-assisted invasion" (PAI). The completion of this invasion process depends on parasitic motility and secretion of adhesins from parasitic micronemes. Our results provide new information about T. gondii infection and establish another platform for studying interactions between T. gondii and macrophages.

Seroprevalence of Toxoplasma gondii Infection in Pet Dogs in Anhui Province, China.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite, which can infect all nucleated cells in a variety of vertebrate animals, including human, causing toxoplasmosis. Although a number of studies have reported on the seroprevalence of T. gondii infection in dogs in China, however, information about T. gondii infection in pet dogs in Anhui, China is not available. METHODS: The modified agglutination test (MAT) was used to detect antibodies in sera samples from 468 pet dogs at Anhui Province in China from November 2013 to April 2017. RESULTS: 18.6% animals were T. gondii seropositive, indicating a slightly higher prevalence of T. gondii infection in pet dogs in Anhui, China in comparison with other provinces in China. CONCLUSION: Our present study provided epidemiological data on T. gondii seroprevalence in pet dogs in Anhui, China for the effective prevention and control of the parasite prevalence in this area.

Prp5-Spt8/Spt3 interaction mediates a reciprocal coupling between splicing and transcription.

Transcription and pre-mRNA splicing are coupled to promote gene expression and regulation. However, mechanisms by which transcription and splicing influence each other are still under investigation. The ATPase Prp5p is required for pre-spliceosome assembly and splicing proofreading at the branch-point region. From an open UV mutagenesis screen for genetic suppressors of prp5 defects and subsequent targeted testing, we identify components of the TBP-binding module of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, Spt8p and Spt3p. Spt8Δ and spt3Δ rescue the cold-sensitivity of prp5-GAR allele, and prp5 mutants restore growth of spt8Δ and spt3Δ strains on 6-azauracil. By chromatin immunoprecipitation (ChIP), we find that prp5 alleles decrease recruitment of RNA polymerase II (Pol II) to an intron-containing gene, which is rescued by spt8Δ. Further ChIP-seq reveals that global effects on Pol II-binding are mutually rescued by prp5-GAR and spt8Δ. Inhibited splicing caused by prp5-GAR is also restored by spt8Δ. In vitro assays indicate that Prp5p directly interacts with Spt8p, but not Spt3p. We demonstrate that Prp5p's splicing proofreading is modulated by Spt8p and Spt3p. Therefore, this study reveals that interactions between the TBP-binding module of SAGA and the spliceosomal ATPase Prp5p mediate a balance between transcription initiation/elongation and pre-spliceosome assembly.

JQ1, a bromodomain inhibitor, suppresses Th17 effectors by blocking p300-mediated acetylation of RORγt.

BACKGROUND AND PURPOSE: Th17 cells play critical roles in chronic inflammation, including fibrosis. Histone acetyltransferase p300, a bromodomain-containing protein, acetylates RORγt and promotes Th17 cell development. The bromodomain inhibitor JQ1 was shown to alleviate Th17-mediated pathologies, but the underlying mechanism remains unclear. We hypothesized that JQ1 suppresses the response of Th17 cells by impairing p300-mediated acetylation of RORγt. EXPERIMENTAL APPROACH: The effect of JQ1 on p300-mediated acetylation of RORγt was investigated in HEK293T (overexpressing Flag-p300 and Myc-RORγt) and human Th17 cells through immunoprecipitation and western blotting. To determine the regions of p300 responsible for JQ1-mediated suppression of HAT activity, we performed HAT assays on recombinant p300 fragments with/without the bromodomain, after exposure to JQ1. Additionally, the effect of JQ1 on p300-mediated acetylation of RORγt and Th17 cell function was verified in vivo, using murine Schistosoma-induced fibrosis models. Liver injury was assessed by histopathological examination and measurement of serum enzyme levels. Expression of Th17 effectors was detected by qRT-PCR, whereas IL-17- and RORγt-positive granuloma cells were detected by FACS. KEY RESULTS: JQ1 impaired p300-mediated RORγt acetylation in human Th17 and HEK293T cells. JQ1 failed to suppress the acetyltransferase activity of p300 fragments lacking the bromodomain. JQ1 treatment attenuated Schistosoma-induced fibrosis in mice, by inhibiting RORγt acetylation and IL-17 expression. CONCLUSIONS AND IMPLICATIONS: JQ1 impairs p300-mediated RORγt acetylation, thus reducing the expression of RORγt target genes, including Th17-specific cytokines. JQ1-mediated inhibition of p300 acetylase activity requires the p300 bromodomain. Strategies targeting p300 may provide new therapeutic approaches for controlling Th17-related diseases.

Role of the Pumilio gene in the reproductive system of Schistosoma japonicum.

The elimination of schistosomiasis, a parasitic disease caused by Schistosoma and a major source of morbidity and mortality in developing countries, faces serious challenges. Although the pumilio protein regulates the reproductive organ development in many species, its role in Schistosoma japonicum is unknown. Thus, this study investigated the function of pumilio in S. japonicum reproduction. The complete coding sequences of S. japonicum Pumilio1 (SjPum1) and SjPum2 genes were cloned and characterized. The full-length open-reading frame SjPum1 (2613 nucleotides) and SjPum2 (4479 nucleotides) genes were obtained. Bioinformatics analysis showed that those genes belonged to the PUF (pumilio and FBF) family. Quantitative polymerase chain reaction analyses revealed that SjPum1 and SjPum2 were differentially expressed throughout the S. japonicum life cycle and were highly expressed in reproductive organs. In situ hybridization results showed that mRNA expression of SjPum2 was higher than that of SjPum1 in the ovary and testis. Knocking down SjPum2 using RNA interference techniques to explore potential reproductive functions showed that compared with the control (untransfected or scrambled mRNA-transfected) worms, the morphology of both male and female reproductive organs was altered, the number of eggs produced by paired females was significantly decreased, and the transcription levels of caspase 3 and caspase 7 genes related to apoptosis were significantly increased. The transcription level of Nanos1 gene which related to reproduction was also significantly increased. Therefore, SjPum2 may play a role in the reproductive development of S. japonicum.

Prohibitin 1 interacts with signal transducer and activator of transcription 3 in T-helper 17 cells.

T-helper 17 (Th17) cells are involved in the occurrence and development of several inflammation-associated diseases. Interleukin (IL)-17, the main cytokine secreted by differentiated Th17 cells, mediates immunoreactions and plays important roles in immunological diseases, including psoriasis, rheumatic arthritis, and inflammatory bowel disease. The maturation and stabilization of the differentiated Th17 cell phenotype are associated with the expression of IL-17A, which is induced by the activation of signal transducer and activator of transcription 3 (STAT3). Prohibitin 1 (PHB1) also plays an essential role in T-cell activation. However, the molecular mechanisms underlying Th17 cell differentiation and the role of PHB1 have not yet been completely elucidated, and this information would greatly facilitate the development of therapeutic strategies to prevent or treat immune-associated diseases. Here, we found that STAT3 expression was correlated with PHB1 mRNA expression during Th17 cell differentiation. Double-labeling immunofluorescence assay results showed that exogenous PHB1 and STAT3 proteins were not only located primarily in the nucleus but also in the cytoplasm of human Th17 cells. Co-immunoprecipitation assays of Th17 cells revealed that PHB1 interacted with STAT3 and with activated STAT3 phosphorylated at Ser727 but not at Tyr705. Knocking down PHB1 with specific short hairpin RNAs attenuated both STAT3 and IL-17 expression levels as well as IL-17 secretion in Th17 cells. These results indicate that PHB1 and STAT3 interact to affect IL-17 secretion in Th17 cells and provide important insights for modulating Th17-mediated pathogenic immune responses.

microRNA-196a Overexpression Inhibits Apoptosis in Hemin-Induced K562 Cells.

microRNAs (miRNAs) have a crucial role in erythropoiesis. However, the understanding of the apoptosis of erythroid lineage remains poorly understood. Hence, an additional examination is required. K562 cell lines can be differentiated into early erythrocytes by hemin and the model of early erythrocytes can be established, consequently. miR-196a has been proven to take part in antiapoptosis in many cell lines. However, the role of miR-196a associated with the apoptosis in hemin-induced K562 cells remains unclear. To study the potential function of miR-196a involved in the common progenitor of erythroblasts, miR-196a mimics and microRNA-small hairpin negative control (miRNA-ShNC) were transfected into hemin-induced K562 cells with lentiviruses. After that, the viability of the transfected hemin-induced K562 cells was tested by CCK-8 assay, and the alteration of cell cycle and apoptosis rate were detected by flow cytometry. Furthermore, bioinformatics and dual-luciferase report system verified that p27kip1 is a target gene of miR-196a. Additionally, the expression of some proteins associated with cell cycle and apoptosis was tested by Western blotting assays. It was found that after overexpressing miR-196a, the proliferation of hemin-induced K562 cells was promoted while the apoptosis inhibited. Furthermore, miR-196a combines with the 3'UTR of p27kip1 directly. Additionally, the relationship between miR-196a and the protein level of p27kip1 is negative. After restoring the expression of p27kip1, the growth rate of hemin-induced K562 cells was not as high as before and the inhibition of apoptosis was alleviated. The present study validates that miR-196a overexpression inhibits apoptosis in hemin-induced K562 cells through downregulating p27kip1.

An Efficient Schistosoma japonicum Bivalent Membrane Protein Antigen DNA Vaccine Against Schistosomiasis in Mice.

BACKGROUND Schistosomiasis is one of the most important infectious parasitic diseases in the world. The most important was to control schistosomiasis is through a combination of medical therapy and immunization. The membrane antigens Tsp2 and 29 from Schistosoma are promising anti-schistosomiasis vaccine candidates. MATERIAL AND METHODS In this study, the pcDNA3.1(+)-SjTsp2, pcDNA3.1(+)-Sj29, and pcDNA3.1 (+)-SjTsp2-29 eukaryotic expression vectors were successfully constructed as DNA vaccines, and the protective abilities of these vaccines were evaluated in mice. RESULTS The results showed that vaccination with SjTsp2, Sj29, and SjTsp2-29 reduced parasite burden and hepatic pathology compared to the control group, and the protective effect of the bivalent SjTsp2-29 DNA vaccine was better than that of the univalent SjTsp2 or Sj29 DNA vaccines. We also found high levels of IgG, IgG1, and IgG2a against SjTsp2, Sj29, and SjTsp2-29 DNA vaccines, with high expression of IFN-γ and no IL-4 in the mice. CONCLUSIONS The double-membrane antigen DNA vaccine SjTsp2-29 elicited protection against Schistosoma infection and might serve as a vaccine candidate.

Potential role of IL-37 signaling pathway in feedback regulation of autoimmune Hashimoto thyroiditis.

IL-37, the anti-inflammatory cytokine of the IL-1 family, plays several key roles in the regulation of autoimmune diseases. Yet, its role in Hashimoto's thyroiditis (HT) is not clear. In the present study, we found that, in tissues from HT patients, most of the follicular epithelial cells were positive for both IL-37 and single Ig IL-1-related receptor (SIGIRR) by immunohistochemical staining, while the infiltrating lymphocytes and other inflammatory cells hardly expressed any. Meanwhile, mRNA expression levels of IL-37 in peripheral blood mononuclear cells (PBMC) of HT patients were significantly higher than those in normal controls measured by quantitative real-time PCR. Finally, we studied the possible role of IL-37 in IFN-γ-stimulated rat FRTL-5 cells. The results showed that IL-1ß, TNF-α, and MCP-1 mRNA levels were significantly decreased, while the expression of IL-4 mRNA was dramatically up-regulated in IFN-γ-stimulated rat thyroid cell line FRTL-5 pre-treated with IL-37. The current study, for the first time, demonstrated that the IL-37 network is involved in Hashimoto's thyroiditis, and IL-37 signaling pathway may ameliorate the excessive autoimmune responses in this chronic lymphocytic thyroiditis.

Differential gene expression, including Sjfs800, in Schistosoma japonicum females at pre-pairing, initial pairing and oviposition.

BACKGROUND: Schistosomiasis is a prevalent but neglected tropical disease caused by parasitic trematodes of the genus Schistosoma, with the primary disease-causing species being S. haematobium, S. mansoni and S. japonicum. Male-female pairing of schistosomes is necessary for sexual maturity and the production of a large number of eggs, which are primarily responsible for schistosomiasis dissemination and pathology. METHODS: Here, we used microarray hybridization, bioinformatics, quantitative PCR, in situ hybridization and gene silencing assays to identify genes that play critical roles in S. japonicum reproduction biology, particularly in vitellarium development, a process that affects male-female pairing, sexual maturation and subsequent egg production. RESULTS: Microarray hybridization analyses generated a comprehensive set of genes differentially transcribed before and after male-female pairing. Although the transcript profiles of females were similar 16 and 18 days after host infection, marked gene expression changes were observed at 24 days. The 30 most abundantly transcribed genes on day 24 included those associated with vitellarium development. Among these, the gene for female-specific 800 (fs800) was substantially upregulated. Our in situ hybridization results in female S. japonicum indicated that Sjfs800 mRNA was observed only in the vitellarium, localized in mature vitelline cells. Knocking down the Sjfs800 gene in female S. japonicum by approximately 60% reduced the number of mature vitelline cells, decreased rates of pairing and oviposition, and decreased the number of eggs produced in each male-female pairing by about 50%. CONCLUSIONS: These results indicate that Sjfs800 may play a role in vitellarium development and egg production in S. japonicum and suggest that Sjfs800 regulation may provide a novel approach for the prevention or treatment of schistosomiasis.

B1 cells protect against Schistosoma japonicum-induced liver inflammation and fibrosis by controlling monocyte infiltration.

During Schistosoma infection, lack of B cells results in more severe granulomas, inflammation, and fibrosis in the liver, but the mechanisms underlying this pathology remain unclear. This study was to clarify the mechanisms underpinning the immunomodulation of B cells in mice infected with Schistosoma japonicum (S. japonicum). We found that B cell deficiency led to aggravated liver pathology, as demonstrated by increases in the size of the egg-associated granulomas, alanine transaminase levels, and collagen deposition. Compared with infected wild-type (WT) mice, infected B cell-deficient (µMT) mice showed increased infiltration of Ly6Chi monocytes and higher levels of proinflammatory cytokines and chemokines. Furthermore, B1 cells were increased significantly in the liver of WT mice following S. japonicum infection. Adoptively transferring B1 cells, but not B2 cells, to µMT mice significantly reduced liver pathology and liver infiltration of Ly6Chi monocytes. Additionally, secretion of IL-10 from hepatic B cells increased significantly in infected WT mice and this IL-10 was mainly derived from B1 cells. Adoptively transferring B1 cells purified from WT mice, but not from IL-10-deficient mice, to µMT mice significantly reduced liver pathology and liver infiltration of Ly6Chi monocytes. These reductions were accompanied by decreases in the expression levels of chemokines and inflammatory cytokines. Taken together, these data indicated that after S. japonicum infection, an increased number of hepatic B1 cells secrete IL-10, which inhibits the expression of chemokines and cytokines and suppresses the infiltration of Ly6Chi monocytes into the liver thereby alleviating liver early inflammation and late fibrosis.

Functions of the Vasa gene in Schistosoma japonicum as assessed by RNA interference.

Vasa, an enzyme belonging to the helicase family, contributes to the regulation of reproductive system development in many species. Thus, we hypothesized that the Vasa3 gene may function in the reproductive system of the parasite Schistosoma japonicum (S. japonicum), which is a major causative agent of schistosomiasis. It is a severe disease globally affecting humans and animals. To test this hypothesis, we firstly conducted whole mount in situ hybridization analyses and found that the S. japonicum Vasa3 (SjVasa3) gene was expressed mainly in the reproductive organs. We then explored the reproductive functions of Vasa3 in S. japonicum using RNA interference (RNAi) techniques. Coupled schistosomes collected from mice 28days post infection (dpi) were transfected three times with SjVasa3-specific small interfering RNA (siRNA) and cultured in vitro for up to 10days. As measured by quantitative PCR (qPCR) and Western blot analysis, levels of SjVasa3 mRNA and protein in Vasa siRNA treated worms were significantly reduced compared with untreated and scrambled siRNA treated worms. Confocal laser scanning microscopy (CLSM) images showed markedly siRNA induced changes in the morphology of the reproductive organs, especially in the female ovary, vitellarium and the male testes. SjVasa3 gene silencing also significantly reduced egg production. These data demonstrate that SjVasa3 is essential in reproductive organ development and egg production in S. japonicum, and could be a potential target for developing novel compounds to treat schistosomiasis.

Exogenous tumor necrosis factor-alpha could induce egress of Toxoplasma gondii from human foreskin fibroblast cells.

Toxoplasma gondii is an intra-cellular protozoan parasite that can infect almost all nucleated cells, eliciting host immune responses against infection. Host tissue damage is mainly caused by cellular lysis when T. gondii egresses from infected cells. However, the effects of cytokines released by host immune cells on egression of T. gondii remain elusive. This study aimed to investigate the role of tumor necrosis factor-alpha (TNF-α) on the egress of T. gondii from infected human foreskin fibroblast (HFF) cells and to elucidate the underlying mechanisms that regulate TNF-α-induced egress. Using flow cytometry to count tachyzoites of T. gondii released into cell culture medium, we found that egress of T. gondii from infected HFF cells could be induced by 10 ng/mL TNF-α in a time-dependent manner. Pre-treatment of infected HFF cells with BAPTA-AM to chelate intra-parasitic calcium could greatly inhibit TNF-α-induced egress. Similar results were obtained when using cytochalasin D to block parasite motility before the TNF-α-induced egress assay. In addition, blocking host apoptosis by Z-VAD-FMK could decrease TNF-α induced egress, while blocking necroptosis by necrostatin-1 has little impact on TNF-α-induced egress. The egressed tachyzoites displayed a normal growth rate and lost no virulence. Our results suggest that host cytokines could influence the cellular lytic processes of T. gondii, providing new insights into the relationship between host TNF-α and T. gondii pathogenesis.