Helminth infection reactivates latent γ-herpesvirus via cytokine competition at a viral promoter.

Mammals are coinfected by multiple pathogens that interact through unknown mechanisms. We found that helminth infection, characterized by the induction of the cytokine interleukin-4 (IL-4) and the activation of the transcription factor Stat6, reactivated murine γ-herpesvirus infection in vivo. IL-4 promoted viral replication and blocked the antiviral effects of interferon-γ (IFNγ) by inducing Stat6 binding to the promoter for an important viral transcriptional transactivator. IL-4 also reactivated human Kaposi's sarcoma-associated herpesvirus from latency in cultured cells. Exogenous IL-4 plus blockade of IFNγ reactivated latent murine γ-herpesvirus infection in vivo, suggesting a "two-signal" model for viral reactivation. Thus, chronic herpesvirus infection, a component of the mammalian virome, is regulated by the counterpoised actions of multiple cytokines on viral promoters that have evolved to sense host immune status.

Imaging trematode and nematode parasites.

Recent advances in molecular genetics and in imaging mean that it is now increasingly feasible to image biological processes within helminth parasites and to visualize interactions between worms and their hosts. Moreover, other innovative imaging approaches that are not dependent on transgenic parasites have been applied to, and or developed for, the study of helminth parasites and have provided novel and important insights into the biology of these important pathogens.

IL-4(-/-) mice with lethal Mesocestoides corti infections--reduced Th2 cytokines and alternatively activated macrophages.

Protection against Mesocestoides corti, a cestode that invades vital organs, is dependent on the production of IL-4, as IL-4(-/-) mice were found to have higher parasite burdens when compared with wild-type mice. The goal of this study was to investigate the role of IL-4 in immunity to M. corti, focusing on the immunological profile and on potential mediators of pathology. IL-4(-/-) mice infected with M. corti showed 100% mortality by 32 days, whereas wild-type mice survived for approximately 1 year. Parasite burdens were significantly increased in the liver, peritoneal, and thoracic cavities of IL-4(-/-) mice, associated with impaired recruitment of inflammatory cells and a reduction in monocytes and macrophages. IL-5 production by splenocytes and expression in liver tissue was decreased in infected IL-4(-/-) mice compared with wild-type mice. In contrast, IL-4(-/-) mice produced increased amounts of IFNgamma and TNFalpha. Alternatively activated macrophages were a major feature of liver granulomas in wild-type mice evidenced by Arginase I expression, while livers from infected IL-4(-/-) mice showed impaired alternative macrophage activation without increased classical macrophage activation. Thus, lethality during M. corti infection of IL-4(-/-) mice is associated with decreased Th2 cytokines, increased Th1 cytokines and impairment of alternatively activated macrophages.

Reverse genetics and the study of the immune response to schistosomes.

The sequencing of the schistosome genome and the establishment of techniques for RNAi and transient transfection in these parasites have opened the door for a reverse genetics approach to studying schistosomes. One of the most intriguing aspects of schistosome biology is the interaction of these parasites with the immune system. The immune response underlies the ability of the host to survive while infected and to eventually develop resistance to further infection. However, it is also instrumental in the development of disease due to its role orchestrating granuloma formation around tissue-trapped parasite eggs. While schistosomes have clearly evolved mechanisms for evading host immune responses, their normal development is, paradoxically, also dependent upon the presence of a normal immune system. This article will review recent advances in the development of tools for studying gene function in schistosomes, and discuss how these new tools may be exploited to investigate issues of key importance in the interaction of schistosomes with the host immune system.

Priming of the immune response by schistosome eggs.

Schistosomiasis mansoni is a chronic disease caused by infection with helminths of the genus Schistosoma mansoni. Adult schistosomes live intravascularly, and for transmission of this infection it is necessary for parasite eggs to traverse the endothelium, and migrate to the intestinal lumen, from where they can exit the body to continue the lifecycle. This process is dependent on an intact host CD4 T helper (Th) cell response to egg antigens. Perhaps because of this, eggs have evolved to be highly immunogenic and capable of inducing potent Th responses. The egg-induced Th response is unusual in that it is highly Th2-polarized. The selective pressure on the host to mount a Th2 response against eggs is apparent in the fact that Th2 response-defective mice develop acutely lethal disease when infected with schistosomes. In this review I will focus on the underlying basis for the Th2 bias in the immune response to egg antigens.

Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism.

Type 2 cytokines regulate fibrotic liver pathology in mice infected with Schistosoma mansoni. Switching the immune response to a type 1-dominant reaction has proven highly effective at reducing the pathologic response. Activation of NOS-2 is critical, because type 1-deviated/NO synthase 2 (NOS-2)-deficient mice completely fail to control their response. Here, we demonstrate the differential regulation of NOS-2 and arginase type 1 (Arg-1) by type 1/type 2 cytokines in vivo and for the first time show a critical role for arginase in the pathogenesis of schistosomiasis. Using cytokine-deficient mice and two granuloma models, we show that induction of Arg-1 is type 2 cytokine dependent. Schistosome eggs induce Arg-1, while Mycobacterium avium-infected mice develop a dominant NOS-2 response. IFN-gamma suppresses Arg-1 activity, because type 1 polarized IL-4/IL-10-deficient, IL-4/IL-13-deficient, and egg/IL-12-sensitized animals fail to up-regulate Arg-1 following egg exposure. Notably, granuloma size decreases in these type-1-deviated/Arg-1-unresponsive mice, suggesting an important regulatory role for Arg-1 in schistosome egg-induced pathology. To test this hypothesis, we administered difluoromethylornithine to block ornithine-aminodecarboxylase, which uses the product of arginine metabolism, L-ornithine, to generate polyamines. Strikingly, granuloma size and hepatic fibrosis increased in the ornithine-aminodecarboxylase-inhibited mice. Furthermore, we show that type 2 cytokine-stimulated macrophages produce proline under strict arginase control. Together, these data reveal an important regulatory role for the arginase biosynthetic pathway in the regulation of inflammation and demonstrate that differential activation of Arg-1/NOS-2 is a critical determinant in the pathogenesis of granuloma formation.

Role of gamma interferon in the pathogenesis of severe schistosomiasis in interleukin-4-deficient mice.

In the absence of interleukin-4 (IL-4), infection with Schistosoma mansoni leads to a severe fatal disease rather than the chronic survivable condition that occurs in wild-type (WT) mice. Because the sustained production of NO most closely correlates to weight loss and fatality in infected IL-4(-/-) mice and because gamma interferon (IFN-gamma) is an important inducer of inducible NO synthase, infected IL-4(-/-) mice were treated with anti-IFN-gamma antibodies to determine the role of IFN-gamma during schistosomiasis in WT and IL-4(-/-) animals. When IFN-gamma was neutralized, Th2 responses were enhanced and NO production was reduced in both WT and IL-4(-/-) mice. The decreased NO production correlated with a rescue of proliferation in splenocytes from infected IL-4(-/-) mice. Furthermore, the neutralization of IFN-gamma in vivo improved the gross appearance of the liver and led to a reduction in granuloma size in infected IL-4(-/-) but not WT mice. However, the neutralization of IFN-gamma in vivo did not affect the development of severe disease in infected IL-4(-/-) mice. These results suggest that while the increased production of IFN-gamma does lead to some of the pathology observed in infected IL-4(-/-) mice, it is not ultimately responsible for cachexia and death.

Profound effect of the absence of IL-4 on T cell responses during infection with Schistosoma mansoni.

T cell responses of interleukin (IL)-4(-/-) and wild-type (WT) mice infected with the helper T cell 2 (Th2) response-inducing pathogen Schistosoma mansoni were compared. As expected, given the important role of IL-4 in Th2 response induction, the absence of IL-4 resulted in diminished Th2 responses, apparent as reduced production of IL-4, -5, and -10 by CD4(+) cells isolated from the spleens of infected IL-4(-/-) mice. Surprisingly, these cells produced significantly less interferon (IFN)-gamma and proliferated less than did those from infected WT mice after T cell receptor ligation. CD8(+) cells isolated from infected IL-4(-/-) mice also produced less IFN-gamma than WT CD8 cells, although there was no difference in the proliferative responses of these cell populations. After infection, spleens of infected IL-4(-/-) mice did not enlarge to the same extent as those of WT mice, and attrition of the CD8(+) cell population within this lymphoid organ was noted. Taken together, the data indicate that in addition to inhibiting Th2 response development, the lack of IL-4 during schistosomiasis significantly affects additional aspects of T cell responses.

CD8- dendritic cell activation status plays an integral role in influencing Th2 response development.

Whether dendritic cells (DC) play a passive or active role in Th2 response induction is poorly understood. In this study, we show that CD8- DC pulsed with Th2-polarizing Ag (soluble egg Ag (SEA)) from Schistosoma mansoni potently stimulate Th2 responses in vivo and in vitro while failing to undergo a conventional maturation process. Thus, in contrast to DC pulsed with the Th1 response inducing Ag Propionebacterium acnes, SEA-exposed DC exhibit a phenotype that is most similar to that of immature DC, failing to up-regulate expression of CD40, CD54, CD80, CD86, or OX40L; producing no detectable IL-4, IL-10, or IL-12; and displaying only a minor increase in MHC class II expression. Importantly, in vitro derived DC exposed to SEA were phenotypically similar to CD8- DC isolated from active S. mansoni infection. By discriminating between different types of pathogen and responding appropriately, CD8- DC play a major role in the decision process to mount either a Th1 or Th2 response.

Human transforming growth factor-beta activates a receptor serine/threonine kinase from the intravascular parasite Schistosoma mansoni.

The biology of the helminth parasite Schistosoma mansoni is closely integrated with that of its mammalian host. SmRK1, a divergent type I transforming growth factor-beta (TGF-beta) receptor of unknown ligand specificity, was previously identified as a candidate for a receptor that allows schistosomes to respond to host-derived growth factors. The TGF-beta family includes activin, bone morphogenetic proteins (BMPs), and TGF-beta, all of which can play crucial roles in metazoan development. The downstream signaling protein of receptors that respond to TGF-beta and activin is Smad2, whereas the receptors that respond to BMPs signal via Smad1. When a constitutively active mutant of SmRK1 was overexpressed with either schistosome Smad1 (SmSmad1) or SmSmad2, a receptor-dependent modulation of SmSmad phosphorylation and luciferase reporter activity occurred only with SmSmad2. To evaluate potential ligand activators of SmRK1, a chimeric receptor containing the extracellular domain of SmRK1 joined to the intracellular domain of the human type I TGF-beta receptor was used. The chimeric receptor bound radiolabeled TGF-beta and could activate a luciferase reporter gene in response to both TGF-beta 1 and TGF-beta 3 but not BMP7. Confirmatory results were obtained using full-length SmRK1. These experiments implicate TGF-beta as a ligand for SmRK1 and as a potential host-derived regulator of parasite growth and development.

Conserved role for 14-3-3epsilon downstream of type I TGFbeta receptors.

Schistosoma mansoni receptor kinase-1 (SmRK1) is a divergent type I transforming growth factor beta (TGFbeta) receptor on the surface of adult parasites. Using the intracellular domain of SmRK1 as bait in a yeast two-hybrid screen we identified an interaction with S. mansoni 14-3-3epsilon. The interaction which is phosphorylation-dependent is not specific to schistosomes since 14-3-3epsilon also binds to TbetaRI, the human type I TGFbeta receptor. 14-3-3epsilon enhances TGFbeta-mediated signaling by TbetaRI and is the first TbetaRI-interacting non-Smad protein identified that positively regulates this receptor. The interaction of 14-3-3epsilon with schistosome and human TbetaRI suggests a conserved, but previously unappreciated, role for this protein in TGFbeta signaling pathways.

Interleukin-12 promotes pathologic liver changes and death in mice coinfected with Schistosoma mansoni and Toxoplasma gondii.

We previously demonstrated that mice concurrently infected with Schistosoma mansoni and Toxoplasma gondii undergo accelerated mortality which is preceded by severe liver damage. Abnormally high levels of serum tumor necrosis factor alpha (TNF-alpha) in the dually infected mice suggested a role for this and related proinflammatory mediators in the pathologic alterations. In order to evaluate the factors involved in increased inflammatory-mediator production and mortality, interleukin-12(-/-) (IL-12(-/-)) mice were coinfected with S. mansoni and T. gondii, and survival and immune responses were monitored. These IL-12(-/-) mice displayed decreased liver damage and prolonged time to death relative to wild-type animals also coinfected with these parasites. Relative to the response of cells from the coinfected wild-type animals, levels of TNF-alpha, gamma interferon, and NO produced by splenocytes from coinfected IL-12(-/-) mice were reduced, and levels of IL-5 and IL-10 were increased, with the net result that the immune response of the dually infected IL-12(-/-) mice was similar to that of the wild-type mice infected with S. mansoni alone. While dually infected wild-type animals succumb in the absence of overt parasitemia, the delayed death in the absence of IL-12 is associated with relatively uncontrolled T. gondii replication. These data support the view that S. mansoni-infected mice are acutely sensitive to infection with T. gondii as a result of their increased hepatic sensitivity to high levels of proinflammatory cytokines; IL-12 and TNF-alpha are implicated in this process.

IL-4 plays a crucial role in regulating oxidative damage in the liver during schistosomiasis.

Liver enlargement and hepatocyte proliferation, normal responses in wild-type (WT) mice infected with the parasitic helminth Schistosoma mansoni, were found to be severely impaired in infected IL-4(-/-) mice. Compared with WT mice, increased levels of O(2)(-), NO, and the more highly reactive ONOO(-) were detected in the liver and produced by lesional cells isolated from liver granulomas of infected IL-4(-/-) mice. Concurrently, antioxidant defenses in the liver, specifically catalase levels, diminished dramatically during the course of infection in these animals. This contrasted to the situation in infected WT mice, where catalase levels remained as high as those in normal mice. Actual levels of reactive oxygen and nitrogen intermediates in the livers of infected IL-4(-/-) animals are thus likely to be considerably higher than those in the livers of infected WT mice. To determine whether these changes contributed to the development of the more severe disease that characterizes infection in the IL-4(-/-) animals, we treated infected IL-4(-/-) mice with uric acid, a potent scavenger of ONOO(-). This resulted in significantly increased hepatocyte proliferation, decreased morbidity, and prolonged survival. Taken together, these data indicate that IL-4 is playing a protective role during schistosomiasis by controlling the tight regulation of the generation of reactive oxygen and nitrogen intermediates in the liver.

Severe schistosomiasis in the absence of interleukin-4 (IL-4) is IL-12 independent.

An interleukin-4 (IL-4)-dependent Th2 response allows wild-type mice to survive infection with the parasite Schistosoma mansoni. In the absence of IL-4, infected mice mount a Th1-like proinflammatory response, develop severe disease, and succumb. Neither the Th1 response nor morbidity is IL-12 dependent in this system.

Functional conservation of Schistosoma mansoni Smads in TGF-beta signaling.

To begin to understand the molecular basis of communication between the parasite Schistosoma mansoni and its mammalian host, we are studying the signaling pathway downstream of S. mansoni receptor kinase-1 (SmRK1), a divergent type I transforming growth factor-beta (TGF-beta) receptor found on the tegumental surface of the parasite. In this study, we have used a homology based PCR approach to clone two S. mansoni Smad (SmSmad) genes; Smads play a pivotal role in the most well understood signaling pathways initiated by the TGF-beta family of ligands in other organisms. Comparison of the amino acid sequences with those of other Smads reveals that the conserved MH1 and MH2 domains of SmSmads show a high degree of identity to homologues in Drosophila. Transcripts for both SmSmads are detected in the same developmental stages as SmRK1, and both are capable of interacting with the intracellular domain of the receptor in vitro. Functional characterization using the human type I TGF-beta receptor further confirms the highly conserved nature of these proteins, as both SmSmads show TGF-beta dependent enhancement of luciferase activity and nuclear translocation in mammalian cells. These data are the first to show a TGF-beta-like receptor/Smad signaling pathway in parasitic helminths and by analogy with other systems, is likely important in regulating schistosome development.

Role of IL-6 in directing the initial immune response to schistosome eggs.

The eggs of Schistosoma mansoni are strong inducers of a Th2 response, and previous work has shown that Ag-specific IL-6 is produced within 24 h after the injection of eggs into mice. Investigations to determine the role of IL-6 in orchestrating the early response to schistosome eggs have revealed that IL-12 is rapidly produced in lymph node cell cultures from egg-injected mice. This "early" IL-12 primes for the production of IL-6 and IFN-gamma, for in IL-12-/- mice egg injection fails to stimulate increased production of either of these cytokines. Furthermore, IL-6 also up-regulates IL-10 production which, together with IL-6, negatively regulates IL-12 and IFN-gamma production. Finally, IL-10 down-regulates the production of its inducer, IL-6. These data indicate that the anti-inflammatory role of IL-6 may be effected through negative regulation of type 1 (IFN-gamma) and type 1-associated (IL-12) cytokines either directly (by IL-6) or indirectly (through the induction of IL-10) and suggest that one mechanism by which eggs may support the development of Th2 responses is through the negative regulation of the type 1 response.

Atypical post-translational modification and targeting of a Schistosoma mansoni surface receptor, a member of the transforming growth factor beta receptor family of cell surface receptors.

The surface membrane of the intravascular parasite Schistosoma mansoni is composed of not one but two closely apposed lipid bilayers which overlie a syncytial cellular layer, known as the tegument or neodermis. To gain insights into how membrane proteins are transported to and displayed on this unusual surface structure, we have investigated the post-translational modification and targeting of SmRK-1, a receptor and type I membrane protein expressed on the parasite surface, using heterologous expression systems. While SmRK-1 enters the secretory pathway in these systems, our data indicate that the SmRK-1 N-terminal signal peptide is either not cleaved by signal peptidase or is only eleven amino acids long or less. Retention of the signal peptide is accompanied by N-linked glycosylation of an asparagine residue within the predicted signal peptide. The SmRK-1 signal peptide is not capable of directing another cytoplasmic protein to the secretory pathway, suggesting that the signal for insertion of the SmRK-1 extracellular domain into the endoplasmic reticulum resides elsewhere in the protein. Further, SmRK-1 is inefficiently transported to the cell surface in mammalian cells, suggesting that the schistosome neodermis possesses specialized systems for receptor targeting and localization.

Caveolae-like structures in the surface membrane of Schistosoma mansoni.

Specialized regions of cellular membranes termed detergent-insoluble glycosphingolipid-enriched membrane domains (DIG) have been identified in mammalian cells and shown to contain signalling molecules, cholesterol, sphingolipids and caveolae. Here we report that the unusual double surface membrane of the tegument of the trematode parasite Schistosoma mansoni possesses biochemically distinct domains analogous to DIG. When subjected to sucrose density gradient centrifugation, a detergent-extracted tegument from adult parasites yielded a low-density fraction consisting of detergent-insoluble complexes (DIC). Several tegument proteins were concentrated in DIC and a subset of these were labelled when adult schistosomes were biotinylated using a membrane-impermeant reactive biotin prior to extraction. The GPI-linked proteins alkaline phosphatase (SmAP), Sm200, the membrane-bound protein Sm23, and a protein recognized by an antibody against human caveolin, co-purified with DIC whereas soluble proteins, such as paramyosin and aldolase, were found at the bottom of the gradient. Antibodies against DIC immunoprecipitated a subset of worm surface proteins and immunolabeled the dorsal tegument of adult worms. Transmission electron microscopy of DIC revealed caveolae-like structures in the double bilayer surface structure. These results suggest that the tegument of adult S. mansoni possesses specialized membrane domains that are resistant to detergent-extraction, contain a subset of total tegument membrane proteins, and bear caveola-like invaginations, and thus are analogous to DIG.