Renyong Lin

The genome of the hydatid tapeworm Echinococcus granulosus

Cystic echinococcosis (hydatid disease), caused by the tapeworm E. granulosus, is responsible for considerable human morbidity and mortality. This cosmopolitan disease is difficult to diagnose, treat and control. We present a draft genomic sequence for the worm comprising 151.6 Mb encoding 11,325 genes. Comparisons with the genome sequences from other taxa show that E. granulosus has acquired a spectrum of genes, including the EgAgB family, whose products are secreted by the parasite to interact and redirect host immune responses. We also find that genes in bile salt pathways may control the bidirectional development of E. granulosus, and sequence differences in the calcium channel subunit EgCavβ1 may be associated with praziquantel sensitivity. Our study offers insights into host interaction, nutrient acquisition, strobilization, reproduction, immune evasion and maturation in the parasite and provides a platform to facilitate the development of new, effective treatments and interventions for echinococcosis control.

Immunology and immunodiagnosis of cystic echinococcosis: an update.

Cystic echinococcosis (CE) is a cosmopolitan zoonosis caused by the larval cystic stage of the dog tapeworm Echinococcus granulosus. This complex multicellular pathogen produces various antigens which modulate the host immune response and promote parasite survival and development. The recent application of modern molecular and immunological approaches has revealed novel insights on the nature of the immune responses generated during the course of a hydatid infection, although many aspects of the Echinococcus-host interplay remain unexplored. This paper summarizes recent developments in our understanding of the immunology and diagnosis of echinococcosis, indicates areas where information is lacking, and suggests possible new strategies to improve serodiagnosis for practical application.

Th17/Treg imbalance in patients with liver cystic echinococcosis.

Echinococcosis is a chronic parasitic infectious disease regulated by T‐cell subsets. CD4+ CD25+ FoxP3 +  regulatory T (Treg) cells and Th17 cells have been described as two distinct subsets and have the opposite effect on inflammation. Th17/Treg balance controls inflammation and may play an important role in the pathogenesis of immune evasion. To assess whether this balance was broken, we detected Th17/Treg functions in different levels including cell frequencies, related cytokines secretion and key transcription factors in patients with cystic echincoccosis and healthy controls. The results demonstrated that patients with cystic echinococcosis revealed significant increase in peripheral Treg number, related cytokines (IL‐10 and TGF‐β1) and transcription factor (Foxp3) levels and moderate decrease in Th17 number, related cytokines (IL‐17 and IL‐23) and transcription factor (RORγt) levels as compared with controls. Results indicated that Th17/Treg functional imbalance exists in patients with chronic cystic echinococcosis, suggesting a potential role for Th17/Treg imbalance in the pathogenesis of immune evasion in echinococcosis.

TGF-β and TGF-β/Smad Signaling in the Interactions between Echinococcus multilocularis and Its Hosts

Alveolar echinococcosis (AE) is characterized by the development of irreversible fibrosis and of immune tolerance towards Echinococcus multilocularis (E. multilocularis). Very little is known on the presence of transforming growth factor-β (TGF-β) and other components of TGF-β/Smad pathway in the liver, and on their possible influence on fibrosis, over the various stages of infection. Using Western Blot, qRT-PCR and immunohistochemistry, we measured the levels of TGF-β1, TGF-β receptors, and down-stream Smads activation, as well as fibrosis marker expression in both a murine AE model from day 2 to 360 post-infection (p.i.) and in AE patients. TGF-β1, its receptors, and down-stream Smads were markedly expressed in the periparasitic infiltrate and also in the hepatocytes, close to and distant from AE lesions. Fibrosis was significant at 180 days p.i. in the periparasitic infiltrate and was also present in the liver parenchyma, even distant from the lesions. Over the time course after infection TGF-β1 expression was correlated with CD4/CD8 T-cell ratio long described as a hallmark of AE severity. The time course of the various actors of the TGF-β/Smad system in the in vivo mouse model as well as down-regulation of Smad7 in liver areas close to the lesions in human cases highly suggest that TGF-β plays an important role in AE both in immune tolerance against the parasite and in liver fibrosis.

Transcriptional Profiles of Cytokine/Chemokine Factors of Immune Cell-Homing to the Parasitic Lesions: A Comprehensive One-Year Course Study in the Liver of E. multilocularis-Infected Mice

Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-β. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.

Echinococcus granulosus infection reduces airway inflammation of mice likely through enhancing IL-10 and down-regulation of IL-5 and IL-17A

BackgroundCystic echinococcosis (CE) is a near cosmopolitan zoonosis caused by the larval stage of the dog tapeworm Echinococcus granulosus. E. granulosus infection induces a polarized T-helper type 2 (Th2) systematic immune response in its intermediate hosts. However, it is not known whether the infection modulates lung inflammation by regulating local immune response. In this study, we examined the effects of E. granulosus infection on mouse ovalbumin (OVA)-induced asthma model.MethodsBALB/c mice were intraperitoneally transplanted with 50 small E. granulosus cysts cultured in vitro. At 3 months post-inoculation, the mice were sensitized and challenged with ovalbumin (OVA). For histopathological studies, hematoxylin eosin and periodic acid schiff staining was used to examine the inflammatory cells infiltration and goblet cells hyperplasia, respectively. Cytokine levels were measured by mouse cytometric bead array (CBA) Kit and quantitative RT-PCR and other molecular biological approaches. Airway hyperresponsiveness was assessed in response to increasing doses of methacholine. Serum immunoglobulins were determined by ELISA.ResultsE. granulosus infection significantly increased Th2 and Treg cytokine levels in serum and lung tissues, but down-regulated the expression of IL-5 in the lungs and IL-17A in serum and lung tissues of asthmatic mice sensitized and challenged with OVA. Histological staining of lung tissues showed that E. granulosus infection significantly reduced the severity of OVA-induced airway inflammation including reduction of eosinophil cell infiltration and mucus production. The E. granulosus infection also reduced eosinophil accumulation induced by OVA in bronchoalveolar lavage fluid (BALF) and also ameliorated airway hyperresponsiveness, a hallmark symptom of asthma.ConclusionsE. granulosus infection remarkably reduces the severity of OVA-induced airway inflammation likely through enhancing IL-10 and down-regulation of IL-5 and IL-17A.

Hepatocyte Proliferation/Growth Arrest Balance in the Liver of Mice during E. multilocularis Infection: A Coordinated 3-Stage Course

Background Alveolar echinococcosis (AE) is characterized by the tumor-like growth of Echinococcus (E.) multilocularis. Very little is known on the influence of helminth parasites which develop in the liver on the proliferation/growth arrest metabolic pathways in the hepatocytes of the infected liver over the various stages of infection. Methodology/Principal Findings Using Western blot analysis, qPCR and immunohistochemistry, we measured the levels of MAPKs activation, Cyclins, PCNA, Gadd45β, Gadd45γ, p53 and p21 expression in the murine AE model, from day 2 to 360 post-infection. Within the early (day 2–60) and middle (day60–180) stages, CyclinB1 and CyclinD1 gene expression increased up to day30 and then returned to control level after day60; Gadd45β, CyclinA and PCNA increased all over the period; ERK1/2 was permanently activated. Meanwhile, p53, p21 and Gadd45γ gene expression, and caspase 3 activation, gradually increased in a time-dependent manner. In the late stage (day180–360), p53, p21 and Gadd45γ gene expression were significantly higher in infected mice; JNK and caspase 3 were activated. TUNEL analysis showed apoptosis of hepatocytes. No significant change in CyclinE, p53 mRNA and p-p38 expression were observed at any time. Conclusions Our data support the concept of a sequential activation of metabolic pathways which 1) would first favor parasitic, liver and immune cell proliferation and survival, and thus promote metacestode fertility and tolerance by the host, and 2) would then favor liver damage/apoptosis, impairment in protein synthesis and xenobiotic metabolism, as well as promote immune deficiency, and thus contribute to the dissemination of the protoscoleces after metacestode fertility has been acquired. These findings give a rational explanation to the clinical observations of hepatomegaly and of unexpected survival of AE patients after major hepatic resections, and of chronic liver injury, necrosis and of hepatic failure at an advanced stage and in experimental animals.

Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4+ CD25+ Treg Effector Molecule, Leads to Improved Control of Echinococcus multilocularis Infection in Mice.

Background The growth potential of the tumor-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune-mediated processes. We previously showed that Fibrinogen-like-protein 2 (FGL2), a novel CD4+CD25+ Treg effector molecule, was over-expressed in the liver of mice experimentally infected with E. multilocularis. However, little is known about its contribution to the control of this chronic helminth infection. Methods/Findings Key parameters for infection outcome in E. multilocularis-infected fgl2-/- (AE-fgl2-/-) and wild type (AE-WT) mice at 1 and 4 month(s) post-infection were (i) parasite load (i. e. wet weight of parasitic metacestode tissue), and (ii) parasite cell proliferation as assessed by determining E. multilocularis 14-3-3 gene expression levels. Serum FGL2 levels were measured by ELISA. Spleen cells cultured with ConA for 48h or with E. multilocularis Vesicle Fluid (VF) for 96h were analyzed ex-vivo and in-vitro. In addition, spleen cells from non-infected WT mice were cultured with rFGL2/anti-FGL2 or rIL-17A/anti-IL-17A for further functional studies. For Treg-immune-suppression-assays, purified CD4+CD25+ Treg suspensions were incubated with CD4+ effector T cells in the presence of ConA and irradiated spleen cells as APCs. Flow cytometry and qRT-PCR were used to assess Treg, Th17-, Th1-, Th2-type immune responses and maturation of dendritic cells. We showed that AE-fgl2-/- mice exhibited (as compared to AE-WT-animals) (a) a significantly lower parasite load with reduced proliferation activity, (b) an increased T cell proliferative response to ConA, (c) reduced Treg numbers and function, and (d) a persistent capacity of Th1 polarization and DC maturation. Conclusions FGL2 appears as one of the key players in immune regulatory processes favoring metacestode survival by promoting Treg cell activity and IL-17A production that contributes to FGL2-regulation. Prospectively, targeting FGL2 could be an option to develop an immunotherapy against AE and other chronic parasitic diseases.

The Potential Role of Th9 Cell Related Cytokine and Transcription Factors in Patients with Hepatic Alveolar Echinococcosis

Human alveolar echinococcosis (AE) is a lethal parasitic infectious disease which may lead to liver failure if left untreated. It is caused by the larval stage of the fox tapeworm Echinococcus multilocularis and usually develops a substantial infiltrative occupation in solid organs. During the infection, T helper subsets are known to play crucial role in crosstalk between the parasite and human host. Th9 cells, a new member of CD4+ T cell family which is characterized by its specific cytokine IL-9 and transcription factors PU.1 and IRF-4, have been known recently to have a critical role in allergic diseases, and cancers as well as the parasitic infection. To assess the potential role of Th9 cells during the infection, the mRNA levels of IL-9, PU.1, and IRF-4 both in peripheral blood mononuclear cells and in liver tissues were, respectively, detected by using real-time PCR. The plasma concentration levels of IL-9 were detected by using enzyme linked immunosorbent assay (ELISA). Th9 related cytokine IL-9 and transcription factors PU.1 and IRF-4 mRNA levels elevated both in PBMCs, and in hepatic lesion and paralesion tissues in AE patients. This may facilitate the infiltrative growth of the parasite and its persistence in human host.

Depletion of FoxP3+ Tregs improves control of larval Echinococcus multilocularis infection by promoting co-stimulation and Th1/17 immunity

The growth potential of the tumor‐like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune‐mediated processes. Previous studies had shown that regulatory T cells (Tregs) become gradually up‐regulated in the course of both chronic human and murine AE. Thus we now tackled the role of FoxP3+ Tregs and FoxP3+‐Treg‐regulated immune response in contributing to the control of this helminthic infection.