Molecular characteristics of Echinococcus multilocularis FABP1 and its regulatory functions on murine macrophages.

Fatty acid binding proteins (FABPs) have emerged as attractive vaccination candidates for several platyhelminth species. To explore the physiological functions of Echinococcus multilocularis (E. multilocularis) FABP, the molecular characteristics of EmFABP1 were analyzed by online software, and the regulatory roles of rEmFABP1 protein in murine macrophages were further investigated. The emfabp1 gene encodes 133 amino acids with the characteristic ß-barrel shape of the cytoplasmic FABP family. Natural EmFABP1 protein is predominantly expressed in protoscoleces tegument and germinal layer cells and is also detected in cyst fluid and exosomes of E. multilocularis. rEmFABP1 protein demonstrated a notable suppression of phagocytic activity and nitric oxide production in murine macrophages. Additionally, the protein was observed to promote apoptosis and regulate cytokine expression in macrophages. These findings suggested that E. multilocularis FABP1 is critical in modifying macrophage physiological processes and that this protein may have immunomodulatory roles during infection.

Bioinformatic prediction and identification of immunogenic epitopes of the antigenic 14-3-3 protein of Echinococcus multilocularis.

INTRODUCTION: Alveolar echinococcosis is a high-risk parasitic disease caused by the larval stage of Echinococcus multilocularis. The study aimed to predict and identify the dominant Th1/Th2 and B cell epitopes of the antigen protein 14-3-3 beta:alpha from Echinococcus multilocularis. METHODS: A comparison of the four amino acid sequences of 14-3-3 beta:alpha was respectively derived from Echinococcus multilocularis, Rattus norvegicus, Canis lupus familiaris, and Homo sapiens was carried out by CLUSTALW to provide a basis for excluding similar epitopes. The amino acid sequence information was analyzed by SOPMA and the homology model was established by Swiss-Model. IEDB and SYFPEITHI were used to predict T cell epitopes. According to the Bcepred and ABCpred, the B cell epitopes were comprehensively predicted and analyzed. The dominant epitopes were validated by Lymphocyte Proliferation, ELISA, ELISpot, and Flow cytometry. RESULTS: Eight potential epitopes of 14-3-3 from Echinococcus multilocularis were screened according to the results of prediction and analysis: 14-3-31-15, 14-3-36-21, 14-3-371-86, 14-3-3144-157, 14-3-3145-166, 14-3-3146-160, 14-3-3153-161, and 14-3-3164-177. The 3D structure model of the protein was constructed and the location distribution of potential epitope was ascertained. Respectively, the epitopes of the dominant antigen of B cells were validated as 14-3-3145-166 and 14-3-3164-177; the Th1 dominant antigen epitopes were 14-3-36-21, 14-3-3145-166; and the Th2 dominant epitopes was 14-3-3145-166. CONCLUSION: In this study, two dominant antigen epitopes of B cells, two Th1 dominant antigen epitopes, and one Th2 dominant antigen epitope were validated. Our work provides a basis for the subsequent development of efficient and safe vaccines targeting epitopes of Echinococcus multilocularis.

Evaluation of fecal immunoassays for canine Echinococcus infection in China.

Human echinococcosis is present worldwide but it is in China that disease prevalence is the highest. In western China, especially in the Tibetan Plateau, the burden of echinococcosis is the most important. Dogs are a major definitive host of Echinococcus and monitoring the presence of Echinococcus worms in dogs is therefore essential to efficiently control the disease. Detection kits based on three different technologies including sandwich ELISA, (indirect) ELISA, and gold immunodiffusion, are currently marketed and used in China. The objective of this work was to assess the efficacy of these kits, in particular with respect to sensitivity and specificity. Four fecal antigen detection kits for canine infection reflecting the three technologies were obtained from companies and tested in parallel on 220 fecal samples. The results indicate that the performance is lower than expected, in particular in terms of sensitivity. The best results were obtained with the sandwich ELISA technology. The gold immunofiltration yielded the poorest results. In all cases, further development is needed to improve the performance of these kits which are key components for the control of echinococcosis.

Agranulocytosis leads to intestinal Echinococcus multilocularis oncosphere invasion and hepatic metacestode development in naturally resistant Wistar rats.

Susceptibility to Echinococcus multilocularis infection considerably varies among intermediate (mostly rodents) and dead-end host species (e.g. humans and pig), in particular regarding intestinal oncosphere invasion and subsequent hepatic metacestode development. Wistar rats are highly resistant to infection and subsequent diseases upon oral inoculation with E. multilocularis eggs, however, after immunosuppressive treatment with dexamethasone, rats become susceptible. To address the role of the cellular innate immunity, Wistar rats were individually or combined depleted of natural killer (NK) cells, macrophages (MΦ) and granulocytes (polymorphonuclear cells, PMN) prior to E. multilocularis egg inoculation. Although NK cell and MΦ depletion did not alter the resistance status of rats, the majority of PMN-depleted animals developed liver metacestodes within 10 weeks, indicating that PMN are key players in preventing oncosphere migration and/or development in Wistar rats. In vitro studies indicated that resistance is not caused by neutrophil reactive oxygen species or NETosis. Also, light microscopical examinations of the small intestine showed that oral inoculation of E. multilocularis eggs does not elicit a mucosal neutrophil response, suggesting that the interaction of oncospheres and neutrophils may occur after the former have entered the peripheral blood. We suggest to consider granulocytes as mediators of resistance in more resistant species, such as humans.

Digest the Sugar, Kill the Parasite: A New Experimental Concept in Treating Alveolar Echinococcosis.

INTRODUCTION: The E. multilocularis laminated layer (LL) is a heavily glycosylated parasitic structure that plays an important role in protecting the larval stage (metacestode) of this parasite from physiological and immunological host reactions. We elaborated an experimental design with the idea to modify the (glycan) surface of the LL by a targeted digestion. This should allow the host defense to more easily recognize and attack (or kill) the parasite by immune-mediated effects. METHODS: Experimentally, E. multilocularis (clone H95) metacestodes were cultured in vitro with or without addition of α1-3,4,6-galactosidase or ß1-3-galactosidase in the medium. Morphological changes were subsequently measured by microscopy at different time points. Parasites were then recovered at day 5 and reinjected into mice for assessing their viability and infectious status. For finally recovered parasites, the respective load was assessed ex vivo by wet weight measurement, and host-related PD1 and IL-10 levels were determined as the key immunoregulators by using flow cytometry. RESULTS: Our experiments demonstrated that the parasite vesicular structure can be directly destroyed by adding galactosidases into the in vitro culture system, resulting in the fact that the parasite metacestode vesicles could not anymore infect and develop in mice after this glycan digestion. Moreover, when compared to the mice inoculated with E. multilocularis metacestode without galactosidases, PD1 expression was upregulated in CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with ß1-3-galactosidase, with a lower IL-10 secretion from CD4+ Teffs; there was no difference of PD1 and IL-10 expression levels regarding CD4+ Teff from mice inoculated with E. multilocularis metacestode pretreated with α1-3,4,6-galac-tosidase. DISCUSSION: We raised our hypothesis that this "aborting" effect may be linked to an altered PD1 and IL-10 response fine-tuning between immunopathology and immune protection. These findings justify a continuation of these experiments upon therapeutical in vivo administration of the enzymes.

Immunohistological detection of small particles of Echinococcus multilocularis and Echinococcus granulosus in lymph nodes is associated with enlarged lymph nodes in alveolar and cystic echinococcosis.

BACKGROUND: Alveolar (AE) and cystic echinococcosis (CE) in humans are caused by the metacestode of the tapeworms Echinococcus multilocularis and Echinococcus granulosus sensu lato (s.l.). Immunohistochemistry with the monoclonal antibodies (mAb) Em2G11, specific for AE, and the mAb EmG3, specific for AE and CE, is an important pillar of the histological diagnosis of these two infections. Our aim was to further evaluate mAb EmG3 in a diagnostic setting and to analyze in detail the localization, distribution, and impact of small particles of Echinococcus multilocularis (spems) and small particles of Echinococcus granulosus s.l. (spegs) on lymph nodes. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the mAb EmG3 in a cohort of formalin-fixed, paraffin embedded (FFPE) specimens of AE (n = 360) and CE (n = 178). These samples originated from 156 AE-patients and 77 CE-patients. mAb EmG3 showed a specific staining of the metacestode stadium of E. multilocularis and E. granulosus s.l. and had a higher sensitivity for spems than mAb Em2G11. Furthermore, we detected spegs in the surrounding host tissue and in almost all tested lymph nodes (39/41) of infected patients. 38/47 lymph nodes of AE showed a positive reaction for spems with mAb EmG3, whereas 29/47 tested positive when stained with mAb Em2G11. Spegs were detected in the germinal centers, co-located with CD23-positive follicular dendritic cells, and were present in the sinuses. Likewise, lymph nodes with spems and spegs in AE and CE were significantly enlarged in size in comparison to the control group. CONCLUSIONS/SIGNIFICANCE: mAb EmG3 is specific for AE and CE and is a valuable tool in the histological diagnosis of echinococcosis. Based on the observed staining patterns, we hypothesize that the interaction between parasite and host is not restricted to the main lesion since spegs are detected in lymph nodes. Moreover, in AE the number of spems-affected lymph nodes is higher than previously assumed. The enlargement of lymph nodes with spems and spegs points to an immunological interaction with the small immunogenic particles (spems and spegs) of Echinococcus spp.

Recent advances on innate immune pathways related to host-parasite cross-talk in cystic and alveolar echinococcosis.

Cystic echinococcosis (CE) and alveolar echinococcosis (AE) are life-threatening parasitic infections worldwide caused by Echinococcus granulosus (sensu lato) and E. multilocularis, respectively. Very little is known about the factors affecting innate susceptibility and resistance to infection with Echinococcus spp. Although benzimidazolic drugs against CE and AE have definitively improved the treatment of these cestodes; however, the lack of successful control campaigns, including the EG95 vaccine, at a continental level indicates the importance of generating novel therapies. This review represents an update on the latest developments in the regulatory functions of innate immune pathways such as apoptosis, toll-like receptors (TLRs), and inflammasomes against CE and AE. We suggest that apoptosis can reciprocally play a bi-functional role among the host-Echinococcus metabolite relationships in suppressive and survival mechanisms of CE. Based on the available information, further studies are needed to determine whether the orchestrated in silico strategy for designing inhibitors and interfering RNA against anti-apoptotic proteins and TLRs would be effective to improve new treatments as well as therapeutic vaccines against the E. granulosus and E. multilocularis.

Expansion of Host Regulatory T Cells by Secreted Products of the Tapeworm Echinococcus multilocularis.

Background: Alveolar echinococcosis (AE), caused by the metacestode larval stage of the fox-tapeworm Echinococcus multilocularis, is a chronic zoonosis associated with significant modulation of the host immune response. A role of regulatory T-cells (Treg) in generating an immunosuppressive environment around the metacestode during chronic disease has been reported, but the molecular mechanisms of Treg induction by E. multilocularis, particularly parasite immunoregulatory factors involved, remain elusive so far. Methodology/Principal Findings: We herein demonstrate that excretory/secretory (E/S) products of the E. multilocularis metacestode promote the formation of Foxp3+ Treg from CD4+ T-cells in vitro in a TGF-ß-dependent manner, given that this effect was abrogated by treatment with antibody to mammalian TGF-ß. We also show that host T-cells secrete elevated levels of the immunosuppressive cytokine IL-10 in response to metacestode E/S products. Within the E/S fraction of the metacestode we identified an E. multilocularis activin A homolog (EmACT) that displays significant similarities to mammalian Transforming Growth Factor-ß (TGF-ß/activin subfamily members. EmACT obtained from heterologous expression failed to directly induce Treg expansion from naïve T cells but required addition of recombinant host TGF-ß to promote CD4+ Foxp3+ Treg conversion in vitro. Furthermore, like in the case of metacestode E/S products, EmACT-treated CD4+ T-cells secreted higher levels of IL-10. These observations suggest a contribution of EmACT to in vitro expansion of Foxp3+ Treg by the E. multilocularis metacestode. Using infection experiments we show that intraperitoneally injected metacestode tissue expands host Foxp3+ Treg, confirming the expansion of this cell type in vivo during parasite establishment. Conclusion/Significance: In conclusion, we herein demonstrate that E. multilocularis larvae secrete factors that induce the secretion of IL-10 by T-cells and contribute to the expansion of TGF-b-driven Foxp3+ Treg, a cell type that has been reported crucial for generating a tolerogenic environment to support parasite establishment and proliferation. Among the E/S factors of the parasite we identified a factor with structural and functional homologies to mammalian activin A which might play an important role in these activities.

Echinococcus multilocularis vesicular fluid induces the expression of immune checkpoint proteins in vitro.

AIMS: Alveolar echinococcosis is a severe chronic helminthic infection that mimics a tumour-like disease. This study aimed at investigating in vitro interactions between Echinococcus multilocularis vesicular fluid (VF) and different immune checkpoints (PD-1/PD-L1, CTLA-4, LAG-3 and TIM-3). METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMC) from healthy blood donors were isolated by Ficoll. Natural killer (NK) cells were selected. Each type of cell was stimulated individually with E. multilocularis-VF. Expression of the different immune checkpoints was measured by flow cytometry on day 3 and day 6; all supernatants were used for immunoassays. Cells and supernatants from 22 healthy donors were analysed. A significant increase of PD-1, PD-L1, LAG-3 and TIM-3 was observed upon E. multilocularis-VF exposure for NK cells on day 3 (P < .05, Wilcoxon signed-rank test). A significant increase of PD-L1 and CTLA-4 was observed upon E. multilocularis-VF exposure for T cells on day 6 (P < .05, Wilcoxon signed-rank test), which was associated with increased levels of Th1 and Th2 cytokines P < .05, Wilcoxon signed-rank test). CONCLUSION: These preliminary data suggest that immune checkpoints could be a way for E. multilocularis to modulate the host immune response during alveolar echinococcosis.

Dual Role of Hepatic Macrophages in the Establishment of the Echinococcus multilocularis Metacestode in Mice.

Echinococcus multilocularis larvae, predominantly located in the liver, cause a tumor-like parasitic disease, alveolar echinococcosis (AE), that is characterized by increased infiltration of various immune cells, including macrophages, around the lesion that produces an "immunosuppressive" microenvironment, favoring its persistent infection. However, the role of hepatic macrophages in the host defense against E. multilocularis infection remains poorly defined. Using human liver tissues from patients with AE and a hepatic experimental mouse model of E. multilocularis, we investigated the phenotype and function of hepatic macrophages during the parasite infection. In the present study, we found that a large number of CD68+ macrophages accumulated around the metacestode lesion in the liver of human AE samples and that both S100A9+ proinflammatory (M1 phenotype) and CD163+ anti-inflammatory (M2 phenotype) macrophages were significantly higher in close liver tissue (CLT) than in distant liver tissue (DLT), whereas M2 macrophages represent the dominant macrophage population. Furthermore, E. multilocularis-infected mice exhibited a massive increase in macrophage (F4/80+) infiltration in the liver as early as day 5, and the infiltrated macrophages were mainly monocyte-derived macrophages (CD11bhi F4/80int MoMFs) that preferentially differentiated into the M1 phenotype (iNOS+) at the early stage of E. multilocularis infection and then polarized to anti-inflammatory macrophages of the M2 phenotype (CD206+) at the chronic stage of infection. We further showed that elimination of macrophages by treatment of mice with clodronate-liposomes before E. multilocularis infection impaired worm expulsion and was accompanied by a reduction in liver fibrosis, yielding a high parasite burden. These results suggest that hepatic macrophages may play a dual role in the establishment and development of E. multilocularis metacestodes in which early larvae clearance is promoted by M1 macrophages while persistent metacestode infection is favored by M2 macrophages.

Species specificity preliminary evaluation of an IL-4-based test for the differential diagnosis of human echinococcosis.

The diagnosis of cystic echinococcosis (CE) is based on imaging, while serology is a complementary test of particular use when imaging is inconclusive. Serology has several limitations. Among them, false-positive results are often obtained in subjects with alveolar echinococcosis (AE), rendering difficult the differential diagnosis. We set up an immune assay based on IL-4-specific production after stimulating whole blood with an antigen B (AgB)-enriched fraction from E granulosus that associates with CE and CE cysts in active stage. We aimed to evaluate potential cross-reactivity of this test using samples from patients with AE. Twelve patients with AE were recruited; IL-4 levels ranged from 0 to 0.07 pg/mL. Based on the previously identified cut-off of 0.39 pg/mL using samples from patients with CE, none of samples from AE patients scored positive. In contrast, almost 80% of samples from AE patients scored positive in serology tests based on different E granulosus-derived antigenic preparations. Our preliminary data show that this experimental whole-blood assay has no cross-reactivity in our cohort of patients with AE, in turn indicating a high specificity of the assay for CE diagnosis. This result supports further work towards the development of improved diagnostic tests for CE.

Immunotherapy of alveolar echinococcosis via PD-1/PD-L1 immune checkpoint blockade in mice.

The growth potential of the tumour-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly dependent upon the nature/function of the periparasitic adaptive host immune-mediated processes. PD-1/PD-L1 pathway (programmed cell death 1), which inhibits lymphocytic proliferation in tumour development, is over-expressed at the chronic stage of AE. We tested the impact of a PD-1/PD-L1 pathway blockade on the outcome of both chronic AE (intraperitoneal metacestode inoculation, secondary AE and SAE) and acute AE (peroral egg infection, primary AE and PAE). To assess the parasite proliferation potential, we measured parasite mass weight for SAE and liver lesion number for PAE. In both models, the parasite load was significantly decreased in response to anti-PD-L1 antibody treatment. In SAE, anti-PDL1 administration was associated with increased Th1 response parameters and decreased Treg responses, while in PAE anti-PDL1 administration was associated with fewer lesions in the liver and decreased Treg/Th2 responses. Our findings highly suggested that a PD-1/PD-L1 pathway blockade triggered the host immune responses in favour of an immune-mediated control of E. multilocularis proliferation. Based on this, future studies that combine PD-1/PD-L1 blockade with a parasitostatic albendazole medication may yield in a putatively curative therapeutic approach to control alveolar echinococcosis.

Foxp3+ T Regulatory Cells as a Potential Target for Immunotherapy against Primary Infection with Echinococcus multilocularis Eggs.

Alveolar echinococcosis (AE) is a lethal disease caused by infection with the metacestode stage of the helminth Echinococcus multilocularis, which develops into a tumorlike mass in susceptible intermediate hosts. The growth potential of this parasite stage is directly linked to the nature of the surrounding periparasitic immune-mediated processes. In a first step (experiment 1), mice were orally infected with E. multilocularis eggs, to be used for assessing the hepatic expression profiles of 15 selected cytokine and chemokine genes related to acquired immunity from 21 to 120 days postinfection. The early stage of infection in immunocompetent animals was marked by a mixed Th1/Th2 immune response, as characterized by the concomitant presence of gamma interferon (IFN-γ) and interleukin-4 (IL-4) and their related chemokines. At the late stage of AE, the profile extended to a combined tolerogenic mode including Foxp3, IL-10, and transforming growth factor beta (TGF-ß) as key components. In a second step (experiment 2), the effect of T regulatory cell (Treg) deficiency on metacestode growth was assessed in E. multilocularis-infected DEREG (depletion of regulatory T cells) mice upon induction of Treg deficiency with diphtheria toxin (DT). The parasite lesions were significantly smaller in the livers of treated mice than in corresponding control groups. Foxp3+ Tregs appear to be one of the key players in immune-regulatory processes favoring metacestode survival by affecting antigen presentation and suppressing Th1-type immune responses. For these reasons, we suggest that affecting Foxp3+ Tregs could offer an attractive target in the development of an immunotherapy against AE.

Identification of emu-TegP11, an EF-hand domain-containing tegumental protein of Echinococcus multilocularis.

Tegumental proteins (TegPs) are a group of proteins that coat on the surface of worms, mainly being involved in ion uptake and immune evasion. Echinococcus species have many TegPs, but none of them have been characterized and their role remains unclear. The genome-wide analysis revealed that there were at least 14 tegp genes (tegp1 - 14) in Echinococcus species, the majority of which were found to contain an EF-hand domain or a dynein light chain-like domain or both. Despite low identity, all TegP11 proteins from 25 flatworms were conserved in structure. Echinococcus multilocularis TegP11 (emu-TegP11) was verified to be secreted by extracellular vesicles and to be localized in different spatiotemporal patterns in protoscoleces. Moreover, emu-TegP11 was also shown to have weak or no Ca2+-binding capacity. In treated macrophages, emu-TegP11 interfered with the small RNA-induced silencing pathway via inducing ectopic expression of some key component genes. Additionally, emu-TegP11 remarkably promoted NO secretion possibly by upregulation of inos gene expression (p < 0.05). It was further shown that emu-TegP11 acted as a suppressor of inflammation, with il-12B and il-1ß being significantly down-regulated (p < 0.01), and il-10 and il-4 being significantly upregulated (p < 0.05). The study demonstrates a regulatory role of emu-TegP11, likely acting as a immunomodulator to be involved in regulation of host immune system during Echinococcus infection.

Environmental risk factors and changing spatial patterns of human seropositivity for Echinococcus spp. in Xiji County, Ningxia Hui Autonomous Region, China.

BACKGROUND: Human echinococcoses are parasitic helminth infections that constitute a serious public health concern in several regions across the world. Cystic (CE) and alveolar echinococcosis (AE) in China represent a high proportion of the total global burden of these infections. This study was conducted to predict the spatial distribution of human seropositivity for Echinococcus species in Xiji County, Ningxia Hui Autonomous Region (NHAR), with the aim of identifying communities where targeted prevention and control efforts are required. METHODS: Bayesian geostatistical models with environmental and demographic covariates were developed to predict spatial variation in the risk of human seropositivity for Echinococcus granulosus (the cause of CE) and E. multilocularis (the cause of AE). Data were collected from three cross-sectional surveys of school children conducted in Xiji County in 2002-2003, 2006-2007 and 2012-2013. Environmental data were derived from high-resolution satellite images and meteorological data. RESULTS: The overall seroprevalence of E. granulosus and E. multilocularis was 33.4 and 12.2%, respectively, across the three surveys. Seropositivity for E. granulosus was significantly associated with summer and winter precipitation, landscape fragmentation variables and the extent of areas covered by forest, shrubland, water and bareland/artificial surfaces. Seropositivity for E. multilocularis was significantly associated with summer and winter precipitations, landscape fragmentation variables and the extent of shrubland and water bodies. Spatial correlation occurred over greater distances for E. granulosus than for E. multilocularis. The predictive maps showed that the risk of seropositivity for E. granulosus expanded across Xiji during the three surveys, while the risk of seropositivity for E. multilocularis became more confined in communities located in the south. CONCLUSIONS: The identification of high-risk areas for seropositivity for these parasites, and a better understanding of the role of the environment in determining the transmission dynamics of Echinococcus spp. may help to guide and monitor improvements in human echinococcosis control strategies by allowing targeted allocation of resources.

Immunological features and efficacy of the recombinant subunit vaccine LTB-EMY162 against Echinococcus multilocularis metacestode.

Alveolar echinococcosis is a zoonotic disease caused by the infection of the larval stage Echinococcus multilocularis with worldwide distribution especially in the northwest China. It is important to develop a well-tolerated immunoprophylaxis against E. multilocularis for alveolar echinococcosis control. In this study, a prokaryotic expression system for recombinant immunogen LTB-EMY162 was established, and the immunological features, sensitized lymphocyte, IL-4/IFN-γ secreted, prophylactic effect, and therapeutic effect were also evaluated. Arctic Express (DE3) system, Ni2+-charged and molecular sieve chromatography were used to obtain a high-purity 29 kDa protein. The ELISA and lymphocyte proliferation assay showed that LTB-EMY162 induced high-titer specific IgG against EMY162 and E. multilocularis protoscoleces protein in BALB/c mice and promoted sensitized T lymphocyte cell proliferation, and LTB-EMY162 stimulated Th cell to secrete IL-4 and IFN-γ and induced a Th1/Th2 mixed type immunological response. We also found that LTB-EMY162 significantly inhibited the cysts formation by challenging with 1000 E. multilocularis protoscoleces. The growth of protoscoleces and cysts were also significantly decreased by treating with LTB-EMY162 in 1000 protoscoleces intraperitoneal injection therapeutic mice model. In conclusion, we have constructed a subunit vaccine LTB-EMY162 which has prevention and therapeutic effect against E. multilocularis infection.

MicroRNA expression profile in RAW264·7 macrophage cells exposed to Echinococcus multilocularis metacestodes.

MicroRNAs (miRNAs) are short noncoding RNAs, involved in the regulation of parasite diseases. However, a role of miRNAs in Echinococcus multilocularis infection remains largely unknown. In this study, we first found the expression levels of key genes involved in miRNA biogenesis and function, including Ago2, Xpo5, Tarbp2 and DgcR8, were obviously altered in the macrophage RAW264·7 cells exposed to E. multilocularis metacestodes. Compared with the control, 18 and 32 known miRNAs were found to be differentially expressed (P 2) in the macrophages exposed to E. multilocularis metacestodes for 6 and 12 h, respectively. Among these, several are known to be involved in regulating cytokine activities and immune responses. Quantitative real-time polymerase chain reaction results showed that the expression of nine selected miRNAs was consistent with the sequencing data at each treatment time points. Moreover, there were statistically significant correlations between the expression levels of miRNAs and their corresponding targeted genes. Our data give us some clues to pinpoint a role of miRNAs in the course of infection and immunity of E. multilocularis.

Larval Echinococcus multilocularis infection reduces dextran sulphate sodium-induced colitis in mice by attenuating T helper type 1/type 17-mediated immune reactions.

The tumour-like growth of larval Echinococcus multilocularis tissue (causing alveolar echinococcosis, AE) is directly linked to the nature/orientation of the periparasitic host immune-mediated processes. Parasite-mediated immune suppression is a hallmark triggering infection outcome in both chronic human and murine AE. So far, little is known about secondary systemic immune effects of this pathogen on other concomitant diseases, e.g. endogenous gut inflammation. We examined the influence of E. multilocularis infection on murine dextran sodium sulphate (DSS) -induced colitis. At 3 months after E. multilocularis infection (chronic stage), the mice were challenged with 3% DSS in the drinking water for 5 days plus subsequently with tap water (alone) for another 4 days. After necropsy, fixed tissues/organs were sectioned and stained with haematoxylin & eosin for assessing inflammatory reactions. Cytokine levels were measured by flow cytometry and quantitative RT-PCR. Colitis severity was assessed (by board-certified veterinary pathologists) regarding (i) colon length, (ii) weight loss and (iii) a semi-quantitative score of morphological changes. The histopathological analysis of the colon showed a significant reduction of DSS-induced gut inflammation by concomitant E. multilocularis infection, which correlated with down-regulation of T helper type 1 (Th1)/Th17 T-cell responses in the colon tissue. Echinococcus multilocularis infection markedly reduced the severity of DSS-induced gut inflammation upon down-regulation of Th1/Th17 cytokine expression and attenuation of CD11b+ cell activation. In conclusion, E. multilocularis infection remarkably reduces DSS-induced colitis in mice by attenuating Th1/Th17-mediated immune reactions.

Suppression of nemo-like kinase by miR-71 in Echinococcus multilocularis.

Echinococcus multilocularis metacestodes are a causative pathogen for alveolar echinococcosis in human beings, and have been found to express miRNAs including emu-miR-71. miR-71 is evolutionarily conserved and highly expressed across platyhelminths, but little is known about its role. Here it was shown that emu-miR-71 was differentially expressed in protoscoleces and was unlikely to be expressed in neoblasts. The results of the luciferase assay indicated that emu-miR-71 was able to bind in vitro to the 3'-UTR of emu-nlk, encoding a key regulator of cell division, causing significant downregulation of luciferase activity (p < 0.01) compared to the negative control and the construct with mutations in the binding site. Consistent with the decreased luciferase activity, transfection of emu-miR-71 mimics into protoscoleces notably repressed emu-NLK (p < 0.05). These results demonstrate the suppression of emu-nlk by emu-miR-71, potentially involved in the protoscolex development.

T-cell tolerance and exhaustion in the clearance of Echinococcus multilocularis: role of inoculum size in a quantitative hepatic experimental model.

The local immune mechanisms responsible for either self-healing or sustained chronic infection are not clear, in the development of E. multilocularis larvae. Here, we developed a suitable experimental model that mimics naturally infected livers, according to the parasite load. We demonstrated that local cellular immunity and fibrogenesis are actually protective and fully able to limit metacestode growth in the liver of low or medium dose-infected mice (LDG or MDG), or even to clear it, while impairment of cellular immunity is followed by a more rapid and severe course of the disease in high dose-infected mice (HDG). And recruitment and/ or proliferation of memory T cells (including CD4 Tem, CD8 Tcm and CD8 Tem) and imbalance of T1/T2/T17/Treg-type T cells in liver were not only associated with clearance of the parasite infection in LDG, but also with increased hepatic injury in HDG; in particular the dual role of CD8 T cells depending on the parasite load and the various stages of metacestode growth. Besides, we first demonstrate the association between LAG3- or 2B4-expressing T cells exhaustion and HD inocula in late stages. Our quantitative experimental model appears fully appropriate to study immunomodulation as a therapeutic strategy for patients with Alveolar Echinococcosis.