The metastasis-associated protein-1 gene encodes a host permissive factor for schistosomiasis, a leading global cause of inflammation and cancer

Authors

  • Sujit S. Nair,

    1. Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC
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  • Anitha Bommana,

    1. Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC
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  • Jeffrey M. Bethony,

    1. Department of Microbiology, Immunology and Tropical Medicine, George Washington University Medical Center, Washington, DC
    2. Human Hookworm Vaccine Initiative, Laboratório de Imunologia Celular e Molecular, Belo Horizonte–Minas Gerais, Brazil
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  • Amanda J. Lyon,

    1. Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC
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  • Kazufumi Ohshiro,

    1. Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC
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  • Suresh B. Pakala,

    1. Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC
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  • Gabriel Rinaldi,

    1. Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC
    2. Department of Microbiology, Immunology and Tropical Medicine, George Washington University Medical Center, Washington, DC
    3. Departamento de Genética, Facultad de Medicina 11800, Universidad de la República (UDELAR), Montevideo, Uruguay
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  • Brian Keegan,

    1. Department of Microbiology, Immunology and Tropical Medicine, George Washington University Medical Center, Washington, DC
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  • Sutas Suttiprapa,

    1. Department of Microbiology, Immunology and Tropical Medicine, George Washington University Medical Center, Washington, DC
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  • Maria V. Periago,

    1. Human Hookworm Vaccine Initiative, Laboratório de Imunologia Celular e Molecular, Belo Horizonte–Minas Gerais, Brazil
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  • Peter J. Hotez,

    1. Department of Microbiology, Immunology and Tropical Medicine, George Washington University Medical Center, Washington, DC
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  • Paul J. Brindley,

    Corresponding author
    1. Department of Microbiology, Immunology and Tropical Medicine, George Washington University Medical Center, Washington, DC
    • George Washington University Medical Center, 2300 I Street, Washington, DC 20037
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    • fax: 202-994-8771

  • Rakesh Kumar

    Corresponding author
    1. Department of Biochemistry and Molecular Biology, George Washington University Medical Center, Washington, DC
    • George Washington University Medical Center, 2300 I Street, Washington, DC 20037
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    • fax: 202-994-8771


  • Potential conflict of interest: Nothing to report.

  • Grant support from the National Center for Research Resources S10RR025565 was provided to the institutional imaging core facility for imaging studies. This work was supported in part by National Institutes of Health Grants CA98823 and CA98823-S1 and institutional new program funds (to R.K.).

Abstract

Schistosoma haematobium is responsible for two-thirds of the world's 200 million to 400 million cases of human schistosomiasis. It is a group 1 carcinogen and a leading cause of bladder cancer that occurs after years of chronic inflammation, fibrosis, and hyperproliferation in the host liver. The coevolution of blood flukes of the genus Schistosoma and their human hosts is paradigmatic of long-term parasite development, survival, and maintenance in mammals. However, the contribution of host genes, especially those discrete from the immune system, necessary for parasite establishment and development remains poorly understood. This study investigated the role of metastasis-associated protein-1 gene (Mta1) product in the survival of S. haematobium and productive infection in the host. Using a Mta−1 null mouse model, here we provide genetic evidence to suggest that MTA1 expression positively influences survival and/or maturation of schistosomes in the host to patency, as we reproducibly recovered significantly fewer S. haematobium worms and eggs from Mta1−/− mice than wild−type mice. In addition, we found a distinct loss of cytokine interdependence and aberrant Th1 and Th2 cytokine responses in the Mta1−/− mice compared to age-matched wild-type mice. Thus, utilizing this Mta1-null mouse model, we identified a distinct contribution of the mammalian MTA1 in establishing a productive host–parasite interaction and thus revealed a host factor critical for the optimal survival of schistosomes and successful parasitism. Moreover, MTA1 appears to play a significant role in driving inflammatory responses to schistosome egg–induced hepatic granulomata reactions, and thus offers a survival cue for parasitism as well as an obligatory contribution of liver in schistosomiasis. Conclusion: These findings raise the possibility to develop intervention strategies targeting MTA1 to reduce the global burden of schistosomiasis, inflammation, and neoplasia. (HEPATOLOGY 2011;)

Schistosomiasis is one of the world's most devastating waterborne infectious diseases with 300 million or more people currently infected1 and resulting in almost 300,000 deaths annually in Africa alone.2 Approximately two-thirds of the world's schistosomiasis cases and many of the deaths result from Schistosoma haematobium infection,2 an oncogenic schistosome that is associated with bladder cancer.3Schistosoma haematobium is considered a group 1 carcinogen, and individuals are exposed to schistosome infection when they come in contact with water contaminated by cercariae.4-6 Cercariae penetrate the skin, after which they transform into schistosomula, which are equipped with an antigenic repertoire that enables evasion of host immune response. From infection of subcutaneous tissues, schistosomula enter the circulation and travel to the lungs and then to the liver, where they achieve sexual maturity before entering into the portal venous system or the vesical venous plexus. Eggs released from the paired adults travel to the liver, intestines, and/or bladder, lodging in the tissues and producing granulomatous inflammation, which can lead to fibrosis and, in the case of S. haematobium, neoplastic transformation.4, 5 It can be predicted that schistosomes, similar to most helminth parasites, use diverse host factors to positively stimulate and contribute to their development and sexual maturation.4

Previous studies addressing the host–parasite interactions during schistosomiasis have focused on a subset of the immune response genes used to mount a T helper 1/T helper 2 (Th1/Th2) response during infection. However, the numbers of adult worms in schistosome-infected knockout mouse models of critical immune-response genes such as interleukin-4 (IL-4), IL-6, and IL-10, which control the Th1/Th2 response, remain unaffected.7-9 One interpretation of these observations is that there is an additional, yet-to-be-determined, regulatory pathway (or pathways) that accommodates host permissiveness to schistosome establishment and productive schistosome infection and parasitism.

In both humans and mice, schistosomes elicit chronic inflammatory responses in the host.7-9 In S. haematobium infection, the prolonged inflammatory response is thought to contribute to the development of squamous cell carcinoma.10 Interestingly, granulomatous inflammation due to chronic schistosomiasis has a similar dynamic and cellular manifestation in human and murine models of infection, making mice a physiologically relevant model in which to study this infection.4, 5, 11, 12 Furthermore, the process of dependency of schistosomes on the host factors for a successful infection is evolutionarily conserved among all species of human schistosomes, including S. haematobium and S. mansoni,13 both of which use common host mechanisms to infect its host and immune signals that appear to facilitate parasite development.13

Recent findings from this laboratory have established a novel role of the metastasis-associated protein-1 gene (Mta1) product MTA1, a chromatin-bound component of the NuRD (nucleosome remodeling and deacetylase) complex, in mediating the host inflammatory responses to the components of virus and bacterial infectious products, by regulating the transcription of host immune-responsive genes.14-18 Given that S. haematobium is the only member of group 1 carcinogenic helminths that develops to sexual maturity and can reproduce in mice,3, 19 and the key role we previously identified for overexpressed MTA1 in oncogenesis,20-22 and because physiologic levels of MTA1 participate in the inflammatory response,17 we hypothesized that there may be a link between S. haematobium parasitism and this unique regulator. Furthermore, an exploration of the role of MTA1 in murine S. haematobium infections could help to unlock the links between parasitism and host inflammation. Guided and prompted by our recent findings that demonstrated a key role for MTA1 in modulating the host inflammatory response here, we investigated the role of this protein as a host cofactor for infection by S. haematobium, using schistosome infections in Mta1−/− and wild-type (WT) mice as a model system.

Abbreviations

BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HG, hepatic granulomatous; IgG, immunoglobulin G; IL, interleukin; INF, interferon; MCP-1, monocyte chemoattractant protein-1; mRNA, messenger RNA; Mta1, metastasis-associated protein-1; p.i., postinfection; PBS, phosphate-buffered saline; RT, room temperature; SEA, soluble egg antigen; SWAP, soluble adult worm antigenic preparation; Th1, T helper 1; Th2, T helper 2; TNF-α, tumor necrosis factor-α; WT, wild type.

Materials and Methods

Schistosoma haematobium and Infection of Mice.

Cercariae of Schistosoma haematobium released from experimentally infected Bulinus truncatus truncatus snails were obtained from Dr. Fred A. Lewis, Biomedical Research Institute, Rockville, MD, under National Institutes of Health–National Institute of Allergy and Infectious Diseases contract HHSN272201000005I. This strain of S. haematobium is maintained in the laboratory using hamsters and B. t. truncatus snails.23 Mta1−/− mice and WT mice were bred in our laboratory, as described.17, 24 Mice were infected with cercariae using the percutaneous route, by immersion of the tail for 1 hour in 5 mL water containing ∼700 cercariae. At intervals after infection of 2, 5, 12, and/or 15 weeks, mice were euthanized by overdose of Euthasol (Virbac, Fort Worth, TX). The portal vein was severed, and worms were recovered from the portal circulatory system by perfusion with 150 mM sodium chloride and 15 mM sodium citrate (pH 7.0)25 and were counted. The livers were removed from the carcass, cut into several pieces, one of which was weighed, then subjected to digestion in 4% KOH for 15 hours.26 Subsequently, the numbers of S. haematobium eggs per gram of liver were counted using a 40× objective fitted to a microscope. At necropsy, in addition to the liver, the cardiac blood, spleen, lungs, bladder, and small intestines were collected; part of each was fixed in buffered formalin and the rest were snap-frozen in liquid nitrogen. Studies with schistosome-infected mice were approved by the Institutional Animal Care and Use Committee of The George Washington University Medical Center.

Experimental Design.

We carried out two separate infections of mice with S. haematobium. Within each experiment, we included six to nine mice of each of the Mta1 genotypes: WT (+/+) and knockout (−/−). In the first, we euthanized two or three of each genotype at 2, 5, and 12 weeks after infection. In the second, all mice were euthanized at ∼15 weeks after infection. In mice, adult S. haematobium worms predominantly inhabit the portal venous system, and mature and release eggs by 10 weeks after infection.26, 27

Indirect Enzyme-Linked Immunosorbent Assay.

An indirect enzyme-linked immunosorbent assay (ELISA) was used to measure levels of immunoglobulin G (IgG) to the S. haematobium soluble adult worm antigenic preparation (SWAP) and to the soluble egg antigen (SEA), prepared as described for S. mansoni.28 A pool of positive control sera was derived from equal portions of sera each genotype at 12 weeks after infection. A pool of negative control sera was sourced from uninfected age- and sex-matched mice. PolySorp (Nalge, Nunc International, Rochester, NY) 96-well microtiter plates were coated with 100 μL/well of either 5 μg/mL of SWAP or SEA in carbonate–bicarbonate buffer (pH 9.6), sealed, and incubated overnight at 4°C. Plates were washed three times with phosphate-buffered saline (PBS; pH 7.2) and blocked with 100 μL/well of 3% bovine serum albumin (BSA) (Sigma) diluted in PBS. Control and experimental sera were diluted 1:4000 in PBS, and 100 μL was added to each well of the microtiter plate in duplicate. The plates were sealed and incubated overnight at 4°C and then washed three times with PBS with 0.05% Tween 20 (PBST) at pH 7.2. A biotinylated goat anti-mouse IgG antibody (Vector Laboratories Inc.) was used at 1:5000 in 3% BSA and PBS and applied 100 μL/well and incubated for 90 minutes at room temperature (RT). Thereafter, plates were washed and incubated with a 1:1000 dilution of horseradish peroxidase–conjugated streptavidin (GE Healthcare) in 3% BSA and PBS for 60 minutes at RT in the dark. The plates were incubated for 30 minutes with o-phenylenediamine dihydrochloride in the dark for 30 minutes at RT. Sulfuric acid (50 μL) was added to each well to stop the reaction, and the optical density at 492 nm was determined (SpectraMax 340 PC reader, SOFTmax Pro software; Molecular Devices, Sunnyvale, CA).

Cytokine Profiling: Cytometric Bead Arrays.

A BD Cytometric Bead Array (CBA) Mouse Inflammation Kit and a Mouse Th1/Th2 Cytokine Kit (BD Biosciences) were used. In brief, to detect concentrations of IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, monocyte chemoattractant protein-1 (MCP-1), interferon-γ (INF-γ), and tumor necrosis factor-α (TNF-α) in the serum of S. haematobium–infected mice and positive and negative controls, a standard reference curve (Mouse Inflammation Standard or Mouse Th1/Th2 Cytokine Standards) provided in the kit was used to interpolate levels of each cytokine, expressed as picograms per microliter. Nine-fold serial dilutions were performed with the standard to obtain a standard curve within a range of 20-5000 pg/mL. Each serum sample was diluted 1:2 in Roswell Park Memorial Institute medium for a final volume of 25 μL. In parallel, Roswell Park Memorial Institute medium was also used as a negative control. A cocktail of the beads from each cytokine was prepared with 3 μL of each bead per sample. Cytokine capture bead cocktail (15 μL) was added to samples, standards, and controls. After vortexing for 10 seconds, 18 μL of the Mouse Inflammation Phycoerythrin Detection Reagent or Mouse Th1/Th2 Phycoerythrin Detection Reagent (BD Biosciences) was added. Tubes were incubated at RT in the dark for 2 hours. Samples were washed with 500 μL of washing buffer and centrifuged for 7 minutes at 1000g. After aspirating the supernatants to leave ∼200 μL, samples were analyzed using a FACScan flow cytometer and the CBA Software (BD Biosciences). Results are expressed in picograms per milliliter.

Immunohistochemistry.

Immunohistochemistry was performed as described.29 Five-μm-thick liver sections were cut, warmed to 60°C, deparaffinized in xylene, and rehydrated with graded ethanol. Antigen exposure took place for 20 minutes in antigen retrieval solution, after which endogenous peroxide activity was inactivated with 0.3% H2O2 in methanol. The sections were blocked for 20 minutes in normal goat serum in PBS and incubated with primary antibodies against cytokeratin-19 (CK19) for 3 hours. Samples were rinsed five times in washing buffer and incubated in secondary antibody for 1 hour. Samples were rinsed three times in wash buffer and incubated in horseradish peroxidase–labeled second antibody solution for 15 minutes. Samples were rinsed three times in wash buffer and incubated in diaminobenzidine for 5 minutes. Samples were rinsed three times in wash buffer and counterstained in hematoxylin for 2 minutes. Tissue slides were scored in a blinded fashion as coded unknown specimens by three investigators. For eosinophil staining, tissue sections were processed and stained using the EOPROBE eosinophil staining kit (SurModics In Vitro Diagnostic Products, Eden Prairie, MN). Slides were viewed with a Zeiss 710 confocal laser scanning microscope and scored in a blinded fashion by two investigators.

Quantitative Real-Time Polymerase Chain Reaction.

Quantitative real-time polymerase chain reaction (PCR) was performed as described.17 Sequences of primers are available on request.

Statistical Analysis.

Differences among groups were compared by analysis of variance and Student t test. P values ≤ 0.05 were considered significant.

Results

MTA1 Is a Permissive Host Cofactor for Schistosoma haematobium Survival.

In mice, adult S. haematobium worms predominantly inhabit the portal venous system, where they mature and begin to release eggs 10 weeks after infection. We reproducibly recovered significantly fewer worms from the Mta1−/− mice as compared with age-matched Mta1+/+ WT mice (Fig. 1A; Supporting Fig. 1). Consistent with these results, far fewer eggs also were recovered from livers of S. haematobium–infected Mta1−/− mice than age-matched WT mice (Fig. 1A). Because previous studies have established that schistosome infection is accompanied by an early IgG response to SWAP,4, 5 we evaluated the serum antibody response of schistosome-infected age-matched WT and Mta1−/− animals to SWAP at 2, 5, and 12 weeks postinfection (p.i.) using an indirect ELISA for IgG against SWAP.4, 5 Although levels of IgG were detected in both groups at all three time points, there was a positive correlation between the levels of IgG against SWAP and time postinfection, that is, IgG to SWAP increased progressively from 2 weeks through 5 weeks to 12 weeks p.i. (Fig. 2A). The median optical density at 492 nm for age-matched WT and Mta1−/− mice was 0.365 and 1.052, respectively, at 5 weeks p.i., and the ratio for the age-matched WT group rose to 2.053 at 12 weeks p.i. The similarity in the level of IgG against SWAP suggests a comparable infectivity for both Mta1−/− and WT mice. Hence, the failure of S. haematobium to develop to patent infection in Mta1−/− mice was not due to an inability to establish a successful infection, but to inherent differences of worm maturation and egg deposition in the absence of MTA1 (Fig. 1B).

Figure 1.

MTA1 is a requisite cellular cofactor for productive Schistosomahaematobium infection. Representative images of adult S. haematobium worms (top left panel) and eggs (bottom left panel) recovered from age-matched WT Mta1+/+ mice by portal perfusion and KOH digestion of the liver. (Right panel): Bar plots for total numbers of (A) adult worms and (B) eggs recovered at 12 weeks after infection. KO, knockout.

Figure 2.

ELISA for Schistosoma haematobium (S.h) SWAP and SEA reflect equivalent initial parasite burdens in both genotypes of the host mice but higher levels of mature infections in WT Mta1+/+ mice. (A) Bar plot showing results from ELISA against S. haematobium worm antigen using sera collected from Mta1 WT and −/− knockout (KO) mice at increasing times after infection. (B) Bar plot showing the results from ELISA against S. haematobium egg antigen (SEA) using sera collected from Mta1 WT and KO at various time points during the course of infection. Data are presented as mean ± standard deviation. OD 492nm, optical density at 492 nm wavelength.

In the mouse, the course of schistosome infection progresses through two distinct phases.4, 5, 7-9 The first is an acute phase, which occurs 3-8 weeks after infection when the host is exposed to the migrating immature parasites; this phase is dominated by the T helper (Th1) cellular response.4, 5 As the schistosomes parasites mature in the liver and begin releasing eggs, the Th1 response is gradually diminished by the emergence of a strong Th2 response to the eggs embedded in the liver and other sites, as represented by the immune response to SEA. As such, we measured the levels of IgG against SEA of S. haematobium, using an indirect ELISA as described above. Here, we observed significantly higher levels of IgG in age-matched WT mice compared to Mta1−/− mice (Fig. 2B), indicating the presence of eggs in the portal system of the age-matched WT mice. These results are consistent with the finding of far fewer worms in the portal system in the Mta1−/− mice compared to age-matched WT mice. Collectively, these findings indicate that although the absence of Mta1 does not compromise the susceptibility to S. haematobium infection, expression of MTA1 positively influences survival and/or maturation of schistosomes in the host, and perhaps egg release and deposition as well. These observations suggest that MTA1 may represent a requisite host factor for optimal permissiveness of schistosome infection, culminating in egg production by the adult parasites.

Pivotal Role of MTA1 in Immune Response to S. haematobium Infection.

Because Th1 and Th2 responses are accompanied by the activation of specific cytokines IL-2, IL-12, and IFN-γ for Th1, and TNF-α, IL-4, IL-5, and IL-10 for Th2,4, 5, 7-9, 30-32 we examined the Th1 and Th2 cytokine profiles in the context of a defective S. haematobium infection in the Mta1−/− mice. In our studies, we examined systemic changes in the host immune response by specifically measuring the circulating cytokine concentrations in the sera of age-matched WT and Mta1−/− mice at several time points. As expected, serum Th1 cytokine levels peaked at 5 weeks p.i., whereas serum Th2 cytokine levels peaked at 12 weeks p.i. (Fig. 3). Serum levels of chemokine MCP-1 were similar in age-matched WT and Mta1−/− mice throughout the course of infection (Fig. 3A). MCP-1 is a critical chemokine that controls parasite burden and proinflammatory responses that are predominant in the acute phase of parasite infection.9 Because levels of this mediator remained unaffected in the sera of age-matched WT and Mta1−/− mice, we can assume that there was an equal parasite burden in both genotypes as a result of infection with cercariae. Infection in age-matched WT mice produced a balanced Th1 and Th2 response, which is evident by the lower concentrations of IFN-γ and IL-10 in sera at 12 weeks p.i. compared to Mta1−/− mice (Fig. 3B,F). The levels of the proinflammatory cytokine TNF-α were elevated in age-matched WT mice 5 weeks p.i. compared to uninfected Mta1−/− mice (Fig. 3E). These findings are consistent with the notion that a higher level of this cytokine is reflective of the acute phases of schistosome infection in murine models.4, 5, 7-9, 30-32

Figure 3.

Impact of MTA1 status on inflammatory and Th1/Th2 cytokines in response to S. haematobium infection. Cytokine responses in age-matched WT and Mta1−/− mice at 2, 5, and 12 weeks post-infection (p.i.): (A) MCP-1, (B) IFN-γ, (C) IL-2, (D) IL-12p70, (E) TNF-α, (F) IL-10, (G) IL-5, and (H) IL-4. Serum samples were used to determine the levels of inflammatory and Th1/Th2 cytokines by cytokine bead array assays. Data are presented as mean ± standard deviation.

Among the other Th1 cytokines assayed in sera, we observed that IL-12p70, the bioactive form of IL-12,4, 5, 33, 34 was significantly higher in the Mta1−/− mice soon after infection and remained elevated during the course of this study (Fig. 3D). Among the Th2 cytokines evaluated, levels of IL-4 and IL-5 were significantly elevated in the Mta1−/− mice at 12 weeks p.i., whereas levels of the immunomodulator IL-10 were elevated early during the course of infection when compared to age-matched WT mice (Fig. 3F-H). Earlier studies have demonstrated a regulatory role for IL-10 in egg-induced pathology from S. mansoni infection, and in some cases, this has been shown to suppress granulomatous inflammations.4, 5, 7-9, 33, 34 IL-12 is a key inducer of Th1-associated inflammatory response, which provides protection against intracellular infections.33, 34 The importance of IL-12 in the pathogenesis of schistosomiasis has been shown in experiments where the inclusion of SEA plus IL-12 resulted in smaller granulomas and less-severe fibrosis.33, 34 Decreased fibrosis was associated with diminished Th2 response and increased Th1 cytokine production.33, 34

MTA1 Is a Host Determinant of Schistosome Egg–Induced Hepatic Granulomatous Inflammation and Hyperproliferation.

In the case of schistosome infection, the infective cercariae are guided by factors contributed by the host that serve as molecular cues for their development and sexual maturation of the adult blood fluke in the liver. In our studies, maturation of adult worms and egg-laying was severely compromised in Mta1−/− mice. Pathology of schistosomiasis is largely determined by egg-induced hepatic granulomatous (HG) response. Immunohistochemical analysis of liver sections from age-matched WT and Mta1−/− mice revealed significant number of granulomatous lesions in age-matched WT mice that were completely absent in Mta1−/− mice (Fig. 4). High levels of TNF-α in tandem with low levels of IFN-γ have also been shown to promote granulomatous lesions in the liver during schistosome infection.7-9, 33, 34 Accordingly, higher levels of TNF-α and lower levels of IFN-γ in age-matched WT mice mice at 5 and 12 weeks p.i. correlated well with an increase in HG inflammation and hyperproliferation, as evident by CK19-positive staining and eosinophil infiltration in the liver tissues of infected mice (Figs. 4 and 5).

Figure 4.

Mta1-WT mice show a higher degree of eosinophil infiltration in liver compared to age-matched Mta1−/− mice 12 weeks p.i. Liver tissue sections from infected WT and Mta1−/− mice were stained for eosinophils using an EO-probe kit. Sections were scanned using Zeiss 710 confocal microscope, and images were recorded using a 40× objective.

Figure 5.

Influence of MTA1 on the formation of granulomatous inflammatory lesions in liver. Paraffin-embedded liver tissue sections from age-matched WT and Mta1−/− mice at 5 and 12 weeks after exposure to Schistosoma haematobium cercariae were stained with an anti–cytokeratin-19 (Ck-19) antibody followed by hematoxylin and eosin (H&E). Stained slides were scored under phase contrast microscopy for HG regions. Representative liver sections from uninfected, control mice (panel A, WT Mta1; panel B, Mta1−/−), infected mice at 12 weeks (C, D) after infection. Regions showing high infiltration of polymorphonuclear cells were scored as positive for inflammation. Percentage HG-positive and CK19-positive zones from 20 fields were determined and represented as a bar plot (F). (E) shows an adult male S. haematobiumin situ in a portal venule in a WT mouse 12 weeks after exposure to cercariae (H&E stain). Images were captured under a 20× objective.

Intrahepatic Immune Response and Hepatic CD4-Positive T Cell Repertoire Favors Successful Parasitism Involving a Functional MTA1 Pathway.

Because adult schistosomes mature to sexually reproductive forms in the liver, we measured cytokine levels in the liver from age-matched WT or Mta1−/− mice by quantitative real-time RT-PCR. Upon analysis of RNA isolated from liver at 2 and 12 weeks p.i., Mta1−/− mice showed, on average, a 12-fold increase in messenger RNA (mRNA) levels encoding IL-10 and IL-12 within 2 weeks p.i. compared to age-matched WT littermates. Furthermore, mRNA levels of some of the Th1 and Th2 cytokines in both genotypes reflected the cytokine production profile observed in sera (Fig. 6A-E). Remarkably, Mta1 mRNA levels were significantly up-regulated in the liver of age-matched WT mice as the infection progressed (Fig. 7A). Analysis of total hepatic CD4 expression revealed that CD4 expression in Mta1−/− mice was several-fold less in WT mice at 2 and 12 weeks p.i., suggesting involvement of Mta1 in the regulation of CD4-specific T cells that may facilitate infection (Fig. 7B).

Figure 6.

Liver cytokine expression reveals loss of cytokine interdependence in Mta1−/− mice. For analysis of liver cytokine mRNA levels in Mta1-WT (+/+) and Mta1-KO (−/−) mice, liver tissue (50 mg) was used for RNA isolation and complementary DNA synthesis. Expression levels of Th1 and Th2 cytokine was assessed by quantitative real-time PCR. *P < 0.01; **P < 0.001.

Figure 7.

Mta1 is an early host-responsive gene following S. haematobium infection and regulates expression of CD4+ T cell population. Expression levels of (A) Mta1 and (B) CD4 was assessed by quantitative real-time PCR. *P < 0.01; **P < 0.001.

Discussion

MTA1, a member of the NuRD complex, has been widely associated with human cancer.21, 22, 35 The Mta1 gene product is a chromatin-bound coregulator involved in transcriptional regulation of genes associated with multiple cellular pathways.21 Recent investigations from this laboratory have established that in addition to the oncogenic role of MTA1 overexpression,29 physiological levels of MTA1 play an important role in the host inflammatory responses to the products of viral and bacterial infections, including to the HBx core antigen of hepatitis B virus and to lipopolysaccharide, due to its role in the transcriptional regulation of host immune response genes.14-18 These findings underpin the notion, explored here, that master regulators such as MTA1, which is important in cancer, could also be of fundamental importance in establishing productive parasitic infections; that is, MTA1 may have an inherent role in supporting parasitism. We explored this hypothesis here, using murine schistosomiasis haematobia as a model. Whereas a consistent goal in research on schistosomiasis has been to decipher the complexity and diversity of host immune responses to these complex pathogens, the contribution of host genes discrete from those of the adaptive immune system necessary for parasite establishment and development remains little understood. The present findings indicate that MTA1 is a critical factor for survival of S. haematobium (Fig. 1A) and that the absence of MTA1 leads to production of cytokines inimical to successful parasitism by schistosomes. To our knowledge, this report is the first to ascribe a productive relationship between the expression of Mta1 and a successful infection by a pathogen associated with human cancer—in this case, the African blood fluke S. haematobium.

Comparative analysis of cytokine profiles following S. haematobium infection in both genotypes indicated a loss of cytokine interdependence and aberrant cytokine production in the Mta1−/− mice when compared to WT age-matched animals (Fig. 3). Notably, elevated levels of the Th1 cytokine IL-12 did not suppress IL-10 in Mta1−/− mice during the early stages of infection (Fig. 3D,F). Furthermore, elevated levels of Th2 cytokine IL-4 did not suppress levels of Th1 cytokine IL-2 in Mta1−/− at 12 weeks p.i. (Fig. 3C,H). Age-matched WT mice exhibited mixed Th1 and Th2 responses, suggesting a strong interdependence and a balance among the cytokines, which possibly contributed to the success of the infection (Fig. 3A-H). It is noteworthy that although the levels of IL-10 were significantly elevated in the Mta1−/− mice, there was no resulting mortality (Fig. 3F). In light of these findings, it is tempting to speculate that sustained IL-12 expression and an early Th2 response mediated by IL-5 and IL-10, as observed in the infected Mta1−/− mice, played a role in the demise of maturing worms in these mice. These observations also raise the intriguing possibility that MTA1, as a host factor, plays a causative role in S. haematobium development, by influencing expression of cytokines conducive to productive parasitism.

Our studies suggest that a complex interplay between the host factor MTA1 and the schistosome is required for the optimal persistence of the parasite in the host, without causing immune-mediated deleterious effects for the host. We speculate that the blood fluke uses MTA1, a master regulator of at least several well-characterized downstream genes,21 to control the host microenvironment, suggesting that Mta1 might be an early response gene to helminth parasite infection, in similar fashion to exposure to lipopolysaccharide,21 and thus could favor parasitism by providing a key developmental cue (Fig. 7A). MTA1 is expressed in multiple organs and tissues,22, 36 including the skin, lungs, and liver, all sites through which the developing schistosome migrates.

Earlier studies have used diverse mouse models to understand host factors in schistosomiasis. Studies that used S. mansoni infections in Rag1−/− mice deficient for B and T lymphocytes30, 37 are instructive in that they demonstrate a key role for CD4+ hepatic lymphocytes in maturation of adult schistosomes. A recent report concluded that schistosomes adopt innate immune signals for development in the host. Furthermore, the studies demonstrated that the CD4+ T cell population was critical in regulating the mononuclear phagocyte population in liver after schistosome infection.38 Here, we observed far fewer worms in the portal perfusate of Mta1−/− mice, indicating that the lack of MTA1 was a predominant factor in determining parasite migration to the hepatoportal system and maturation in the liver. Detailed analysis of CD4 expression in livers of uninfected age-matched WT and Mta1−/− mice by quantitative real-time PCR revealed no differences in CD4 expression, indicating that CD4 expression did not contribute to the unsuccessful parasitism of the Mta1−/− mice. Intriguingly, CD4 expression was higher in WT mice as infection progressed, suggesting that MTA1 might be involved in regulation and or specific homing of hepatic CD4-positive T cell lymphocytes in response to infection. Considering the aberrant cytokine profile observed in the Mta1−/− mice, we speculate that MTA1 is involved in maturation of a specific subset of CD4-positive T cells. These findings provide additional support to the notion that MTA1 represents a permissive stimulus or cofactor for parasite survival in the immunologically intact host.

These findings highlight a likely role of MTA1 as a master coregulator in the pathogenesis of schistosomiasis. We hypothesize that MTA1 provides a necessary niche for the host–parasite interaction and that MTA1 plays a key role in driving granulomatous-specific inflammatory reactions in the liver. Because schistosomes can survive for many years, it is tempting to speculate that MTA1 is a permissive host regulatory factor that maintains the worms and elevates cancer-causing fibrotic lesions. In addition, because expression of MTA1 is up-regulated during schistosomiasis, as occurs with lipopolysaccharide and HBx,18 and because elevated levels of MTA1 promote cancer,21, 22, 35 the findings establish a rationale for prospective clinical investigations on the links between MTA1, schistosomiasis haematobia, granulomatous-specific inflammation in liver, and incidence of schistosome-associated bladder cancer.10

Acknowledgements

We thank Drs. Fred A. Lewis, Yung-san Liang, and Allen Cheever for helpful discussions and for providing Schistosoma haematobium stages, and Dr. Stephanie Constant for assistance with cytological analysis.

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