Opisthorchis viverrini excretory-secretory products suppress GLUT8 of cholangiocytes.

Opisthorchis viverrini infection and the subsequent bile duct cancer it induces remains a significant public health problem in Southeast Asia. Opisthorchiasis has been reported to cause reduced plasma glucose levels among infected patients. The underlying mechanism for this phenomenon is unclear. In the present study, evidence is presented to support the hypothesis that O. viverrini exploits host cholangiocyte glucose transporters (GLUTs) in a similar manner to that of rodent intestinal nematodes, to feed on unabsorbed glucose in the bile for survival. GLUT levels in a cholangiocyte H69 cell line co-cultured with excretory-secretory products of O. viverrini were examined using qPCR and immunoblotting. GLUT 8 mRNA and expressed proteins were found to be downregulated in H69 cells in the presence of O. viverrini. This suggests that O. viverrini alters glucose metabolism in cells within its vicinity by limiting transporter expression resulting in increased bile glucose that it can utilize and potentially explains the previously reported anti-insulin effect of opisthorchiasis.

Trichinella spiralis-Secreted Products Promote Collagen Capsule Formation through TGF-ß1/Smad3 Pathway.

Trichinella spiralis (T. spiralis) muscle larvae colonize in the host's skeletal muscle cells, which are surrounded by collagen capsules. The mechanism underlying muscle stage larva-induced collagen capsule formation remains unknown. To clarify the mechanism, a T. spiralis muscular-infected mouse model was established by a single lateral tail vein injection with 20,000 T. spiralis newborn larvae (NBL). The infected mice were treated with or without SB525334 (TGF-ß1 receptor type I inhibitor). Diaphragms were obtained post-infection, and the expression levels of the TGF-ß1/Smad3 pathway-related genes and collagen genes (type IV and VI) were observed during the process of collagen capsule formation. The changes in myoblasts under stimulation of the excretory-secretory (ES) products of NBL with or without SB525334 were further investigated. Results showed that the expression levels of type IV collagen gene, type VI collagen gene, Tgfb1, and Smad3 were significantly increased in infected mice muscle cells. The expression levels of all the above genes were enhanced by the products of NBL in myoblast cells. These changes were reversed by co-treatment with SB525334 in vivo and in vitro. In conclusion, the TGF-ß1/Smad3 pathway can be activated by T. spiralis infection in muscle cells. The activated TGF-ß1/Smad3 pathway can stimulate the secretion of collagens by myocytes and plays a promoting role in the process of collagen capsule formation. The research has the limitation that the protein identification of the products of NBL has yet to be performed. Therefore, the specific components in the T. spiralis ES products that induce collagen synthesis should be further investigated.

The Protective Effect of the Soluble Egg Antigen of Schistosoma japonicum in A Mouse Skin Transplantation Model.

Background: Organ transplantation is currently an effective method for treating organ failure. Long-term use of immunosuppressive drugs has huge side effects, which severely restricts the long-term survival of patients. Schistosoma can affect the host's immune system by synthesizing, secreting, or excreting a variety of immunomodulatory molecules, but its role in transplantation was not well defined. In order to explore whether Schistosoma-related products can suppress rejection and induce long-term survival of the transplant, we used soluble egg antigen (SEA) of Schistosoma japonicum in mouse skin transplantation models. Materials and methods: Each mouse was intraperitoneally injected with 100 µg of SEA three times a week for four consecutive weeks before allogenic skin transplant. Skin transplants were performed on day 0 to observe graft survival. Pathological examination of skin grafts was conducted 7 days post transplantation. The skin grafts were subjected to mRNA sequencing. Bioinformatics analysis was conducted and the expression of hub genes was verified by qPCR. Flow cytometry analysis was performed to evaluate the immune status and validate the results from bioinformatic analysis. Results: The mean survival time (MST) of mouse skin grafts in the SEA-treated group was 11.67 ± 0.69 days, while that of the control group was 8.00 ± 0.36 days. Pathological analysis showed that Sj SEA treatment led to reduced inflammatory infiltration within skin grafts 7 days after allogenic skin transplantation. Bioinformatics analysis identified 86 DEGs between the Sj SEA treatment group and the control group, including 39 upregulated genes and 47 downregulated genes. Further analysis revealed that Sj SEA mediated regulation on cellular response to interferon-γ, activation of IL-17 signaling and chemokine signaling pathways, as well as cytokine-cytokine receptor interaction. Flow cytometry analysis showed that SEA treatment led to higher percentages of CD4+IL-4+ T cells and CD4+Foxp3+ T cells and decreased CD4+IFN-γ+ T cells in skin transplantation. Conclusion: Sj SEA treatment suppressed rejection and prolonged skin graft survival by regulating immune responses. Sj SEA treatment might be a potential new therapeutic strategy to facilitate anti-rejection therapy and even to induce tolerance.

Parasite worm antigens instruct macrophages to release immunoregulatory extracellular vesicles.

Emerging evidence suggests that immune cells not only communicate with each other through cytokines, chemokines, and cell surface receptors, but also by releasing small membranous structures known as extracellular vesicles (EVs). EVs carry a variety of different molecules that can be taken up by recipient cells. Parasitic worms are well known for their immunomodulatory properties, but whether they can affect immune responses by altering EV-driven communication between host immune cells remains unclear. Here we provide evidence that stimulation of bone marrow-derived macrophages (BMDMs) with soluble products of Trichuris suis (TSPs), leads to the release of EVs with anti-inflammatory properties. Specifically, we found that EVs from TSP-pulsed BMDMs, but not those from unstimulated BMDMs can suppress TNFα and IL-6 release in LPS-stimulated BMDMs and BMDCs. However, no polarization toward M1 or M2 was observed in macrophages exposed to EVs. Moreover, EVs enhanced reactive oxygen species (ROS) production in the exposed BMDMs, which was associated with a deregulated redox homeostasis as revealed by pathway analysis of transcriptomic data. Proteomic analysis identified cytochrome p450 (CYP450) as a potential source of ROS in EVs from TSP-pulsed BMDMs. Finally, pharmacological inhibition of CYP450 activity could suppress ROS production in those BMDMs. In summary, we find that TSPs can modulate immune responses not only via direct interactions but also indirectly by eliciting the release of EVs from BMDMs that exert anti-inflammatory effects on recipient cells.

Analysis of daily variation in the release of faecal eggs and coproantigen of Fasciola hepatica in naturally infected dairy cattle and the impact on diagnostic test sensitivity.

The liver fluke, Fasciola hepatica (F. hepatica) is a widespread parasite infection in dairy cattle in Victoria, South-eastern Australia. Robust diagnosis of fluke infection is needed in dairy cattle to identify sub-clinical infections which often go unnoticed, causing significant production losses. We tested the coproantigen ELISA (cELISA) and the FlukeFinder faecal egg count kit® on naturally infected cows in a fluke endemic region of Victoria. The aim of the study was to investigate the variation in the release of coproantigens and eggs into faeces over a 5-day period, at the morning (AM) and afternoon (PM) milkings, and to assess the impact of the timing of faecal sample collection on diagnostic test sensitivity. Ten cows were enrolled into the study based on positive F. hepatica faecal egg counts (LFEC) and faecal samples from the ten cows were collected twice daily, at the 7-9 AM and 4-6 PM milking, for five consecutive days. At the conclusion of the sampling period, the cows were euthanized and F. hepatica burden determined at necropsy. A moderate negative correlation between cow age and cELISA optical density (OD) was observed using data from all samples (R -0.63; 95 % CI -0.68 to -0.57). Over the 5-day sampling period, we observed within-animal variation between days for both the cELISA OD (2.6-8.9 fold) and LFEC (5-16 fold), with more variation in values observed in the PM samples for both tests. The correlation with total fluke burden was higher in the AM sampling using both the cELISA and LFEC (R 0.64 and 0.78, respectively). The sensitivity was 100 % for the cELISA using various cut offs from the literature (0.014 OD, 0.030 OD, and 1.3 % or 1.6 % of the positive control). The sensitivity of the FlukeFinder kit® (based on 588 faecal samples and not accounting for lack of independence in the data) was 88 % (95 % CI 85 %-90 %). Seventy one false negatives were recorded from the 588 LFEC tests all of which were observed in the cows with fluke burdens <14 flukes; 42 of the 71 false negative LFECs occurred in one individual cow which had the lowest burden of nine flukes. In dairy cows, the cut-off for production losses due to fasciolosis is estimated at> 10 fluke. Both the cELISA and the LFEC identified all cows that had burdens equal to or greater than this cut-off. Five of the ten cows also exhibited relatively high paramphistome egg counts.

Opisthorchis viverrini antigens up-regulates the expression of CD80 and MHC class II in JAWSII mouse dendritic cells and promotes IL-10 and TGF-ß secretions.

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4+ T cells was determined. Significantly increased levels of immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-ß) were found. The up-regulation of these cytokines may indicate the response of regulatory T cells (Treg) to CS antigen-stimulated JAWSII DC. These findings may lead to a better understanding of the role that DCs play in O. viverrini infection.

The role of let-7b in the inhibition of hepatic stellate cell activation by rSjP40.

BACKGROUND: Hepatic stellate cells (HSCs) are one of the main cell types involved in liver fibrosis induced by many factors, including schistosomes. Previous studies in our lab have shown that recombinant P40 protein from Schistosoma japonicum (rSjP40) can inhibit HSC activation in vitro. Let-7b is a member of the let-7 microRNA family and plays an inhibitory role in a variety of diseases and inflammatory conditions. In this study, we investigated the role of let-7b in the inhibition of HSC activation by rSjP40. METHODS: Expression of let-7b was detected by quantitative real-time PCR. A dual luciferase assay was used to confirm direct interaction between let-7b and collagen I. We also used western blot to assess protein levels of TGFßRI and collagen type I α1 (COL1A1). RESULTS: We found that rSjP40 up-regulates expression of let-7b in HSCs. Let-7b inhibits collagen I expression by directly targeting the 3'UTR region of the collagen I gene. Furthermore, we discovered that let-7b inhibitor partially restores the loss of collagen I expression caused by rSjP40. CONCLUSION: Our research clarifies the role of let-7b in the inhibition of HSC activation by rSjP40 and will provide new insights and ideas for the inhibition of HSC activation and treatment of liver fibrosis.

Identification of an enolase gene and its physiological role in Spirometra mansoni.

Enolase is a crucial enzyme involved in the glycolytic pathway and gluconeogenesis in parasites. It also has been reported to function as a plasminogen receptor and may be involved in tissue invasion. In this study, the biochemical properties of the enolase of Spirometra mansoni (Smenolase) were investigated. The Smenolase gene was found to cluster closely with the enolase genes of Clonorchis sinensis and Echinococcus granulosus, and some functional motifs were identified as conserved. Smenolase was confirmed to be a component of the secretory/excretory products (ESPs) and a circulating antigen of spargana. Recombinant Smenolase expressed in vitro was able to bind to human plasminogen. Smenolase was detected in the eggs, testicles, and vitellaria of adult worms and the tegument of spargana. The transcription level of Smenolase was highest at the gravid proglottid stage. When spargana were cultured with glucose of different concentration in vitro, it was observed that the expression levels of Smenolase in the low-glucose groups were consistent with that of Smenolase in vivo. These results indicate that Smenolase is a critical enzyme involved in supplying energy to support the development and reproduction of the parasite, and it may also play a role in sparganum invasion.

Mapping Rora expression in resting and activated CD4+ T cells.

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.

A Bead-Based Assay for the Detection of Antibodies against Trichinella spp. Infection in Humans.

Human trichinellosis can be diagnosed by a combination of medical history, clinical presentation, and laboratory findings, and through detection of anti-Trichinella IgG in the patient's sera. ELISA using excretory-secretory (E/S) antigens of Trichinella spiralis larvae is currently the most used assay to detect Trichinella spp. antibodies. Bead-based assay can detect antibodies to multiple antigens concurrently; the ability to detect antibody to T. spiralis using a bead assay could be useful for diagnosis and surveillance. We developed and evaluated a bead assay to detect and quantify total IgG or IgG4 Trichinella spp. antibodies in human serum using T. spiralis E/S antigens. The sensitivity and specificity of the assay were determined using serum from 110 subjects with a confirmed diagnosis of trichinellosis, 140 subjects with confirmed infections with other tissue-dwelling parasites, 98 human serum samples from residents of the United States with no known history of parasitic infection, and nine human serum samples from residents of Egypt with negative microscopy for intestinal parasites. Sensitivity and specificity were 93.6% and 94.3% for total IgG and 89.2% and 99.2% for IgG4, respectively. Twelve percent of sera from patients with confirmed schistosomiasis reacted with the IgG Trichinella bead assay, as did 11% of sera from patients with neurocysticercosis. The Trichinella spp. bead assay to detect IgG total antibody responses has a similar performance as the Trichinella E/S ELISA. The Trichinella spp. bead assay shows promise as a method to detect trichinellosis with a possibility to be used in multiplex applications.

Transcriptome and Secretome Analysis of Intra-Mammalian Life-Stages of Calicophoron daubneyi Reveals Adaptation to a Unique Host Environment.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke.

Characterization and localization of antigens for serodiagnosis of human paragonimiasis.

Paragonimiasis is a foodborne trematode infection that affects 23 million people, mainly in Asia. Lung fluke infections lead frequently to chronic cough with fever and hemoptysis, and are often confused with lung cancer or tuberculosis. Paragonimiasis can be efficiently treated with praziquantel, but diagnosis is often delayed, and patients are frequently treated for other conditions. To improve diagnosis, we selected five Paragonimus kellicotti proteins based on transcriptional abundance, recognition by patient sera, and conservation among trematodes and expressed them as His-fusion proteins in Escherichia coli. Sequences for these proteins have 76-99% identity with amino acid sequences for orthologs in the genomes of Paragonimus westermani, Paragonimus heterotremus, and Paragonimus miyazakii. Immunohistology studies showed that antibodies raised to four recombinant proteins bound to the tegument of adult P. kellicotti worms, at the parasite host interface. Only a known egg antigen was absent from the tegument but present in developing and mature eggs. We evaluated the diagnostic potential of these antigens by Western blot with sera from patients with paragonimiasis (from MO and the Philippines), fascioliasis, and schistosomiasis, and with sera from healthy North American controls. Two recombinant proteins (a cysteine protease and a myoglobin) showed the highest sensitivity and specificity as diagnostic antigens, and they detected antibodies in sera from paragonimiasis patients with early or mature infections. In contrast, antibodies to egg yolk ferritin appeared to be specific marker for patients with adult fluke infections that produce eggs. Our study has identified and localized antigens that are promising for serodiagnosis of human paragonimiasis.

Anisakis simplex (s.l.) resistance to the action of gastric enzymes depends upon previous treatments applied to infected fish mince and affects antigen release.

BACKGROUND: Freezing is considered the most suitable technological treatment to avoid Anisakis infection from eating raw or undercooked fish but modifications of their cuticles upon freezing may reduce their resistance to gastric fluids, provoking a greater release of allergens. This work aimed to study the relationship between freezing-induced modifications of Anisakis simplex s.l., antigen recognition, and resistance to oral and gastric digestion in spiked fish mince. RESULTS: (i) Differences between non-treated larvae and larvae that survived freezing / thawing were studied in terms of respiratory capacity, survival in simulated gastric fluid (SGF), recognition of antigens and allergens. (ii) Untreated (i.e. chilled) mince containing live larvae, mince frozen at two freezing rates, with a negative (uninfected) mince and a positive mince (infected with broken larvae) as controls, were subjected to the oral and gastric phases of a simulated digestion process. Anisakis able to survive freezing showed lower resistance to gastric fluid (i.e. faster mortality as compared to controls). Untreated larvae released significantly more antigens than freeze-surviving larvae but only after 96 h in SGF. In treatments rendering complete larvae mortality, the highest loss of larvae integrity was found upon fast freezing. There was a positive correlation between antigen release and the number of ruptures of larvae after the oral digestion phase, whereas a more complex trend was observed after oral plus gastric digestion phases. CONCLUSION: These results suggest a new factor to consider for sensitized patients and suggest that the numbers of L3 should be reduced before industrial freezing to minimize risk. © 2020 Society of Chemical Industry.

SLAM family receptors control pro-survival effectors in germinal center B cells to promote humoral immunity.

Expression of the signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is critical for the germinal center (GC) reaction and T cell-dependent antibody production. However, when SAP is expressed normally, the role of the associated SLAM family receptors (SFRs) in these processes is nebulous. Herein, we established that in the presence of SAP, SFRs suppressed the expansion of the GC reaction but facilitated the generation of antigen-specific B cells and antibodies. SFRs favored the generation of antigen-reactive B cells and antibodies by boosting expression of pro-survival effectors, such as the B cell antigen receptor (BCR) and Bcl-2, in activated GC B cells. The effects of SFRs on the GC reaction and T cell-dependent antibody production necessitated expression of multiple SFRs, both in T cells and in B cells. Hence, while in the presence of SAP, SFRs inhibit the GC reaction, they are critical for the induction of T cell-mediated humoral immunity by enhancing expression of pro-survival effectors in GC B cells.

Schistosoma japonicum SjE16.7 Protein Promotes Tumor Development via the Receptor for Advanced Glycation End Products (RAGE).

Schistosome infection contributes to cancer development, but the mechanisms are still not well-understood. SjE16.7 is an EF-hand calcium-binding protein secreted from Schistosoma japonicum eggs. It is a neutrophil attractant and macrophage activator and, as such, plays an important role in the inflammatory granuloma response in schistosomiasis. Here, we show that SjE16.7 binds to host cells by interacting with receptors for advanced glycation end products (RAGE). This ligation leads to activation of the NF-κB signaling pathway, an increase in the generation of reactive oxygen species, and production of the pro-inflammatory cytokines IL-6 and TNF-α. Using a mouse model of colorectal cancer, we demonstrate that intraperitoneal injection of SjE16.7 promotes colorectal cancer progression along with systemic myeloid cell accumulation. Thus, our results identify a new helminth antigen contributing to tumor development in the mammalian host.

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.

Schistosoma antigens as activators of inflammasome pathway: from an unexpected stimulus to an intriguing role.

Parasites of the genus Schistosoma are organisms capable of living for decades within the definitive host. They interfere with the immune response by interacting with host's receptors. In this review, we discuss from the first reports to the most recent discoveries regarding the ability of Schistosoma antigens in triggering intracellular receptors and inducing inflammasome activation.

Schistosoma mansoni immunomodulatory molecule Sm16/SPO-1/SmSLP is a member of the trematode-specific helminth defence molecules (HDMs).

BACKGROUND: Sm16, also known as SPO-1 and SmSLP, is a low molecular weight protein (~16kDa) secreted by the digenean trematode parasite Schistosoma mansoni, one of the main causative agents of human schistosomiasis. The molecule is secreted from the acetabular gland of the cercariae during skin invasion and is believed to perform an immune-suppressive function to protect the invading parasite from innate immune cell attack. METHODOLOGY/PRINCIPAL FINDINGS: We show that Sm16 homologues of the Schistosomatoidea family are phylogenetically related to the helminth defence molecule (HDM) family of immunomodulatory peptides first described in Fasciola hepatica. Interrogation of 69 helminths genomes demonstrates that HDMs are exclusive to trematode species. Structural analyses of Sm16 shows that it consists predominantly of an amphipathic alpha-helix, much like other HDMs. In S. mansoni, Sm16 is highly expressed in the cercariae and eggs but not in adult worms, suggesting that the molecule is of importance not only during skin invasion but also in the pro-inflammatory response to eggs in the liver tissues. Recombinant Sm16 and a synthetic form, Sm16 (34-117), bind to macrophages and are internalised into the endosomal/lysosomal system. Sm16 (34-117) elicited a weak pro-inflammatory response in macrophages in vitro but also suppressed the production of bacterial lipopolysaccharide (LPS)-induced inflammatory cytokines. Evaluation of the transcriptome of human macrophages treated with a synthetic Sm16 (34-117) demonstrates that the peptide exerts significant immunomodulatory effects alone, as well as in the presence of LPS. Pathways most significantly influenced by Sm16 (34-117) were those involving transcription factors peroxisome proliferator-activated receptor (PPAR) and liver X receptors/retinoid X receptor (LXR/RXR) which are intricately involved in regulating the cellular metabolism of macrophages (fatty acid, cholesterol and glucose homeostasis) and are central to inflammatory responses. CONCLUSIONS/SIGNIFICANCE: These results offer new insights into the structure and function of a well-known immunomodulatory molecule, Sm16, and places it within a wider family of trematode-specific small molecule HDM immune-modulators with immuno-biotherapeutic possibilities.

A Biological and Immunological Characterization of Schistosoma Japonicum Heat Shock Proteins 40 and 90α.

We characterized Schistosoma japonicum HSP40 (Sjp40) and HSP90α (Sjp90α) in this study. Western blot analysis revealed both are present in soluble egg antigens and egg secretory proteins, implicating them in triggering the host immune response after secretion from eggs into host tissues. These observations were confirmed by immunolocalization showing both HSPs are located in the Reynolds' layer within mature eggs, suggesting they are secreted by miracidia and accumulate between the envelope and the eggshell. Both HSPs are present in the musculature and parenchyma of adult males and in the vitelline cells of females; only Sjp90α is present on the tegument of adults. Sjp40 was able to enhance the expression of macrophages, dendritic cells, and eosinophilic cells in mouse liver non-parenchymal cells, whereas rSjp90α only stimulated the expression of dendritic cells. T helper 1 (Th1), Th2, and Th17 responses were increased upon rSjp40 stimulation in vitro, but rSjp90 only stimulated an increased Th17 response. Sjp40 has an important role in reducing the expression of fibrogenic gene markers in hepatic stellate cells in vitro. Overall, these findings provide new information on HSPs in S. japonicum, improving our understanding of the pathological roles they play in their interaction with host immune cells.

NLRP6 Plays an Important Role in Early Hepatic Immunopathology Caused by Schistosoma mansoni Infection.

Schistosomiasis is a debilitating parasitic disease that affects more than 200 million people worldwide and causes approximately 280,000 deaths per year. Inside the definitive host, eggs released by Schistosoma mansoni lodge in the intestine and especially in the liver where they induce a granulomatous inflammatory process, which can lead to fibrosis. The molecular mechanisms initiating or promoting hepatic granuloma formation remain poorly understood. Inflammasome activation has been described as an important pathway to induce pathology mediated by NLRP3 receptor. Recently, other components of the inflammasome pathway, such as NLRP6, have been related to liver diseases and fibrotic processes. Nevertheless, the contribution of these components in schistosomiasis-associated pathology is still unknown. In the present study, using dendritic cells, we demonstrated that NLRP6 sensor is important for IL-1ß production and caspase-1 activation in response to soluble egg antigens (SEA). Furthermore, the lack of NLRP6 has been shown to significantly reduce periovular inflammation, collagen deposition in hepatic granulomas and mRNA levels of α-SMA and IL-13. Livers of Nlrp6-/- mice showed reduced levels of CXCL1/KC, CCL2, CCL3, IL-5, and IL-10 as well as Myeloperoxidase (MPO) and Eosinophilic Peroxidase (EPO) enzymatic activity. Consistently, the frequency of macrophage and neutrophil populations were lower in the liver of NLRP6 knockout mice, after 6 weeks of infection. Finally, it was further demonstrated that the onset of hepatic granuloma and collagen deposition were also compromised in Caspase-1-/- , IL-1R-/- and Gsdmd-/- mice. Our findings suggest that the NLRP6 inflammasome is an important component for schistosomiasis-associated pathology.