Geoffrey N. Gobert

Immunopathogenesis of human schistosomiasis

Schistosomiasis continues to be a significant cause of parasitic morbidity and mortality worldwide. This review considers the basic features of the pathology and clinical outcomes of hepatointestinal and genitourinary schistosomiasis, presents an overview of the numerous studies on animal models that have clarified many of the immunopathological features, and provides insight into our current understanding of the immunopathogenesis and genetic control of human schistosomiasis. In murine schistosomiasis, pathology is induced by a CD4+ Th2 driven granulomatous response directed against schistosome eggs lodged in the host liver. The Th2 cytokines IL‐4 and IL‐13 drive this response, whereas IL‐10, IL13Rα2, IFN‐γ and a subset of regulatory T‐cells act to limit schistosome induced pathology. A variety of cell types including hepatic stellate cells, alternatively activated macrophages and regulatory T‐cells have also been implicated in the pathogenesis of schistosomiasis. Current knowledge suggests the immunopathogenic mechanisms underlying human schistosomiasis are likely to be similar. The review also considers the future development of anti‐pathology schistosome vaccines. As fibrosis is an important feature of many other diseases such as Crohns disease and sarcoidosis, a comprehensive understanding of the cellular and molecular mechanisms involved in schistosomiasis may also ultimately contribute to the development an effective disease intervention strategy for other granulofibrotic diseases.

Exposed proteins of the Schistosoma japonicum tegument.

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.

Developmental gene expression profiles of the human pathogen Schistosoma japonicum

BackgroundThe schistosome blood flukes are complex trematodes and cause a chronic parasitic disease of significant public health importance worldwide, schistosomiasis. Their life cycle is characterised by distinct parasitic and free-living phases involving mammalian and snail hosts and freshwater. Microarray analysis was used to profile developmental gene expression in the Asian species, Schistosoma japonicum. Total RNAs were isolated from the three distinct environmental phases of the lifecycle – aquatic/snail (eggs, miracidia, sporocysts, cercariae), juvenile (lung schistosomula and paired but pre-egg laying adults) and adult (paired, mature males and egg-producing females, both examined separately). Advanced analyses including ANOVA, principal component analysis, and hierarchal clustering provided a global synopsis of gene expression relationships among the different developmental stages of the schistosome parasite.ResultsGene expression profiles were linked to the major environmental settings through which the developmental stages of the fluke have to adapt during the course of its life cycle. Gene ontologies of the differentially expressed genes revealed a wide range of functions and processes. In addition, stage-specific, differentially expressed genes were identified that were involved in numerous biological pathways and functions including calcium signalling, sphingolipid metabolism and parasite defence.ConclusionThe findings provide a comprehensive database of gene expression in an important human pathogen, including transcriptional changes in genes involved in evasion of the host immune response, nutrient acquisition, energy production, calcium signalling, sphingolipid metabolism, egg production and tegumental function during development. This resource should help facilitate the identification and prioritization of new anti-schistosome drug and vaccine targets for the control of schistosomiasis.

Temporal Expression of Chemokines Dictates the Hepatic Inflammatory Infiltrate in a Murine Model of Schistosomiasis

Schistosomiasis continues to be an important cause of parasitic morbidity and mortality world-wide. Determining the molecular mechanisms regulating the development of granulomas and fibrosis will be essential for understanding how schistosome antigens interact with the host environment. We report here the first whole genome microarray analysis of the murine liver during the progression of Schistosoma japonicum egg-induced granuloma formation and hepatic fibrosis. Our results reveal a distinct temporal relationship between the expression of chemokine subsets and the recruitment of cells to the infected liver. Genes up-regulated earlier in the response included T- and B-cell chemoattractants, reflecting the early recruitment of these cells illustrated by flow cytometry. The later phases of the response corresponded with peak recruitment of eosinophils, neutrophils, macrophages and myofibroblasts/hepatic stellate cells (HSCs) and the expression of chemokines with activity for these cells including CCL11 (eotaxin 1), members of the Monocyte-chemoattractant protein family (CCL7, CCL8, CCL12) and the Hepatic Stellate Cell/Fibrocyte chemoattractant CXCL1. Peak expression of macrophage chemoattractants (CCL6, CXCL14) and markers of alternatively activated macrophages (e.g. Retnla) during this later phase provides further evidence of a role for these cells in schistosome-induced pathology. Additionally, we demonstrate that CCL7 immunolocalises to the fibrotic zone of granulomas. Furthermore, striking up-regulation of neutrophil markers and the localisation of neutrophils and the neutrophil chemokine S100A8 to fibrotic areas suggest the involvement of neutrophils in S. japonicum-induced hepatic fibrosis. These results further our understanding of the immunopathogenic and, especially, chemokine signalling pathways that regulate the development of S. japonicum-induced granulomas and fibrosis and may provide correlative insight into the pathogenesis of other chronic inflammatory diseases of the liver where fibrosis is a common feature.

Transcriptome profiling of lung schistosomula,in vitro cultured schistosomula and adult Schistosoma japonicum

Abstract.The schistosomulum is the main target of vaccine-induced protective immunity; however, most studies have utilized schistosomula produced by mechanical transformation of infective larvae followed by in vitro culture rather than larvae isolated directly from the lungs of infected mammals. Using transmission electron microscopy, we demonstrated that there was little difference in the ultrastructure of Schistosoma japonicum schistosomula obtained by the two methods. However, significant differences in gene expression profiles were apparent when we used an oligonucleotide microarray to compare the gene expression profiles of schistosomula obtained in vivo from lung tissue with those maintained in vitro, and with adult worms of S. japonicum. It is likely that host environmental factors, which cannot be reliably reproduced in vitro, do influence the growth, development and overall biology of schistosomes. Thus caution is urged when using in vitro-cultured schistosomes and mechanically transformed/cultured schistosomula in molecular, biochemical and immunological studies.

Cellular and chemokine-mediated regulation in schistosome-induced hepatic pathology

In hepatic schistosomiasis, pathology arises when schistosome eggs become lodged in the host liver, evoking an interleukin 4 (IL-4)- and IL-13-mediated dominant CD4(+) Th2 immune response. This response leads to the development of granulomas and fibrosis, with eosinophils, neutrophils, macrophages, hepatic stellate cells, and lymphocytes all identified as major cellular contributors to these events. This review outlines the cellular and molecular mechanisms of hepatic schistosomiasis, with an emphasis on the major cellular components and their release of chemokines. The differences between Schistosoma mansoni- and Schistosoma japonicum-induced hepatic granuloma are also discussed. This comprehensive overview of the processes associated with hepatic schistosomiasis may provide new insights into improved treatment for both schistosomiasis and other granulofibrotic diseases.

Transcriptional changes in Schistosoma mansoni during early schistosomula development and in the presence of erythrocytes.

Background Schistosomes cause more mortality and morbidity than any other human helminth, but control primarily relies on a single drug that kills adult worms. The newly transformed schistosomulum stage is susceptible to the immune response and is a target for vaccine development and rational drug design. Methodology/Principal Findings To identify genes which are up-regulated during the maturation of Schistosoma mansoni schistosomula in vitro, we cultured newly transformed parasites for 3 h or 5 days with and without erythrocytes and compared their transcriptional profiles using cDNA microarrays. The most apparent changes were in the up-regulation of genes between 3 h and 5 day schistosomula involved in blood feeding, tegument and cytoskeletal development, cell adhesion, and stress responses. The most highly up-regulated genes included a tegument tetraspanin Sm-tsp-3 (1,600-fold up-regulation), a protein kinase, a novel serine protease and serine protease inhibitor, and intestinal proteases belonging to distinct mechanistic classes. The inclusion of erythrocytes in the culture medium resulted in a general but less pronounced increase in transcriptional activity, with the highest up-regulation of genes involved in iron metabolism, proteolysis, and transport of fatty acids and sugars. Conclusions We have identified the genes that are up-regulated during the first 5 days of schistosomula development in vitro. Using a combination of gene silencing techniques and murine protection studies, some of these highly up-regulated transcripts can be targeted for future development of new vaccines and drugs.

Tissue specific profiling of females of Schistosoma japonicum by integrated laser microdissection microscopy and microarray analysis.

Background The functions of many schistosome gene products remain to be characterized. A major step towards elucidating function of these genes would be in defining their sites of expression. This goal is rendered difficult to achieve by the generally small size of the parasites and the lack of a body cavity, which precludes analysis of transcriptional profiles of the tissues in isolation. Methodology/Principal Findings Here, we describe a combined laser microdissection microscopy (LMM) and microarray analysis approach to expedite tissue specific profiling and gene atlasing for tissues of adult female Schistosoma japonicum. This approach helps to solve the gene characterization “bottle-neck” brought about by acoelomy and the size of these parasites. Complementary RNA obtained after isolation from gastrodermis (parasite gut mucosa), vitelline glands and ovary by LMM were subjected to microarray analyses, resulting in identification of 147 genes upregulated in the gastrodermis, 4,149 genes in the ovary and 2,553 in the vitellaria. Conclusions This work will help to shed light on the molecular pathobiology of this debilitating human parasite and aid in the discovery of new targets for the development of anti-schistosome vaccines and drugs.

Cloning and Characterisation of Schistosoma japonicum Insulin Receptors

Background Schistosomes depend for growth and development on host hormonal signals, which may include the insulin signalling pathway. We cloned and assessed the function of two insulin receptors from Schistosoma japonicum in order to shed light on their role in schistosome biology. Methodology/Principal Findings We isolated, from S. japonicum, insulin receptors 1 (SjIR-1) and 2 (SjIR-2) sharing close sequence identity to their S. mansoni homologues (SmIR-1 and SmIR-2). SjIR-1 is located on the tegument basal membrane and the internal epithelium of adult worms, whereas SjIR-2 is located in the parenchyma of males and the vitelline tissue of females. Phylogenetic analysis showed that SjIR-2 and SmIR-2 are close to Echinococcus multilocularis insulin receptor (EmIR), suggesting that SjIR-2, SmIR-2 and EmIR share similar roles in growth and development in the three taxa. Structure homology modelling recovered the conserved structure between the SjIRs and Homo sapiens IR (HIR) implying a common predicted binding mechanism in the ligand domain and the same downstream signal transduction processing in the tyrosine kinase domain as in HIR. Two-hybrid analysis was used to confirm that the ligand domains of SjIR-1 and SjIR-2 contain the insulin binding site. Incubation of adult worms in vitro, both with a specific insulin receptor inhibitor and anti-SjIRs antibodies, resulted in a significant decrease in worm glucose levels, suggesting again the same function for SjIRs in regulating glucose uptake as described for mammalian cells. Conclusions Adult worms of S. japonicum possess insulin receptors that can specifically bind to insulin, indicating that the parasite can utilize host insulin for development and growth by sharing the same pathway as mammalian cells in regulating glucose uptake. A complete understanding of the role of SjIRs in the biology of S. japonicum may result in their use as new targets for drug and vaccine development against schistosomiasis.

The ultrastructural architecture of the adult Schistosoma japonicum tegument

The tegument of the adult blood fluke Schistosoma japonicum is in direct contact with the host blood and immune systems. A comprehensive understanding of the ultrastructure of the tegument is crucial to the understanding of how the parasite maintains itself within the mammalian host. Important functions such as nutritional uptake and immune evasion are suspected functions of the tegument and this review discusses these aspects and presents some insights into some of these crucial functions. Transmission electron microscopy has allowed the identification of ultrastructural features of the adult S. japonicum, some of which differ from the reported features of other schistosome species. Morphological differences within the tegument of the adult S. japonicum are noted between sexes, among different regions of the worms and between aspects along the length of the parasite. Differences included variations in the ultrastructure, size and number of tegumental bodies and mitochondria within the matrix, and differences in the relative area of the apical surface of the tegument. Functions of the various components of the tegument matrix and specialised functions of different regions of the male and female parasites are discussed based on ultrastructural findings and previously reported biochemical and molecular data.