Search

Fibroproliferative diseases are driven by dysregulated tissue repair responses and are a major cause of morbidity and mortality because they affect nearly every organ system. Type 2 cytokine responses are critically involved in tissue repair; however, the mechanisms that regulate beneficial regeneration versus pathological fibrosis are not well understood. Here, we have shown that the type 2 effector cytokine interleukin-13 simultaneously, yet independently, directed hepatic fibrosis and the compensatory proliferation of hepatocytes and biliary cells in progressive models of liver disease induced by interleukin-13 overexpression or after infection with Schistosoma mansoni. Using transgenic mice with interleukin-13 signaling genetically disrupted in hepatocytes, cholangiocytes, or resident tissue fibroblasts, we have revealed direct and distinct roles for interleukin-13 in fibrosis, steatosis, cholestasis, and ductular reaction. Together, these studies show that these mechanisms are simultaneously controlled but distinctly regulated by interleukin-13 signaling. Thus, it may be possible to promote interleukin-13-dependent hepatobiliary expansion without generating pathological fibrosis. VIDEO ABSTRACT., (Published by Elsevier Inc.)

Persistent or dysregulated IL-13 responses are key drivers of fibrosis in multiple organ systems, and this identifies this cytokine as an important therapeutic target. Nevertheless, the mechanisms by which IL-13 blockade leads to the amelioration of fibrosis remain unclear. Because IFN-γ exhibits potent anti-fibrotic activity, and IL-4Rα signalling antagonizes IFN-γ effector function, compensatory increases in IFN-γ activity following IL-13/IL-4Rα blockade might contribute to the reduction in fibrosis. To investigate the role of IFN-γ, we developed novel IL-13(-/-) /IFN-γ(-/-) double cytokine-deficient mice and examined disease progression in models of type 2-driven fibrosis. As predicted, we showed that fibrosis in the lung and liver are both highly dependent on IL-13. We also observed increased IFN-γ production and inflammatory activity in the tissues of IL-13-deficient mice. Surprisingly, however, an even greater reduction in fibrosis was observed in IL-13/IFN-γ double deficient mice, most notably in the livers of mice chronically infected with Schistosoma mansoni. The increased protection was associated with marked decreases in Tgfb1, Mmp12, and Timp1 mRNA expression in the tissues; reduced inflammation; and decreased expression of important pro-inflammatory mediators such as TNF-α. Experiments conducted with neutralizing monoclonal antibodies to IL-13 and IFN-γ validated the findings with the genetically deficient mice. Together, these studies demonstrate that the reduction in fibrosis observed when IL-13 signalling is suppressed is not dependent on increased IFN-γ activity. Instead, by reducing compensatory increases in type 1-associated inflammation, therapeutic strategies that block IFN-γ and IL-13 activity simultaneously can confer greater protection from progressive fibrosis than IL-13 blockade alone. Published 2016. This article is a U.S. Government work and is in the public domain in the USA., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Mice expressing a Cre recombinase from the lysozyme M-encoding locus (Lyz2) have been widely used to dissect gene function in macrophages and neutrophils. Here, we show that while naïve resident tissue macrophages from IL-4Rαf(lox/delta)LysM(Cre) mice almost completely lose IL-4Rα function, a large fraction of macrophages elicited by sterile inflammatory stimuli, Schistosoma mansoni eggs, or S. mansoni infection, fail to excise Il4rα. These F4/80(hi)CD11b(hi) macrophages, in contrast to resident tissue macrophages, express lower levels of Lyz2 explaining why this population resists LysM(Cre)-mediated deletion. We show that in response to IL-4 and IL-13, Lyz2(lo)IL-4Rα(+) macrophages differentiate into an arginase 1-expressing alternatively-activated macrophage (AAM) population, which slows the development of lethal fibrosis in schistosomiasis. In contrast, we identified Lyz2(hi)IL-4Rα(+) macrophages as the key subset of AAMs mediating the downmodulation of granulomatous inflammation in chronic schistosomiasis. Our observations reveal a limitation on using a LysMCre mouse model to study gene function in inflammatory settings, but we utilize this limitation as a means to demonstrate that distinct populations of alternatively activated macrophages control inflammation and fibrosis in chronic schistosomiasis.

Background: The pathogenic mechanisms underlying pulmonary arterial hypertension resulting from schistosomiasis, one of the most common causes of pulmonary hypertension worldwide, remain unknown. We hypothesized that transforming growth factor-β (TGF-β) signaling as a consequence of Th2 inflammation is critical for the pathogenesis of this disease., Methods and Results: Mice sensitized and subsequently challenged with Schistosoma mansoni eggs developed pulmonary hypertension associated with an increase in right ventricular systolic pressure, thickening of the pulmonary artery media, and right ventricular hypertrophy. Rho-kinase-dependent vasoconstriction accounted for ≈60% of the increase in right ventricular systolic pressure. The pulmonary vascular remodeling and pulmonary hypertension were dependent on increased TGF-β signaling, as pharmacological blockade of the TGF-β ligand and receptor, and mice lacking Smad3 were significantly protected from Schistosoma-induced pulmonary hypertension. Blockade of TGF-β signaling also led to a decrease in interleukin-4 and interleukin-13 concentrations, which drive the Th2 responses characteristic of schistosomiasis lung pathology. Lungs of patients with schistosomiasis-associated pulmonary arterial hypertension have evidence of TGF-β signaling in their remodeled pulmonary arteries., Conclusion: Experimental S mansoni-induced pulmonary vascular disease relies on canonical TGF-β signaling.

A diverse suite of effector immune responses provide protection against various pathogens. However, the array of effector responses must be immunologically regulated to limit pathogen- and immune-associated damage. CD4(+)Foxp3(+) regulatory T cells (Treg) calibrate immune responses; however, how Treg cells adapt to control different effector responses is unclear. To investigate the molecular mechanism of Treg diversity we used whole genome expression profiling and next generation small RNA sequencing of Treg cells isolated from type-1 or type-2 inflamed tissue following Leishmania major or Schistosoma mansoni infection, respectively. In-silico analyses identified two miRNA "regulatory hubs" miR-10a and miR-182 as critical miRNAs in Th1- or Th2-associated Treg cells, respectively. Functionally and mechanistically, in-vitro and in-vivo systems identified that an IL-12/IFNγ axis regulated miR-10a and its putative transcription factor, Creb. Importantly, reduced miR-10a in Th1-associated Treg cells was critical for Treg function and controlled a suite of genes preventing IFNγ production. In contrast, IL-4 regulated miR-182 and cMaf in Th2-associed Treg cells, which mitigated IL-2 secretion, in part through repression of IL2-promoting genes. Together, this study indicates that CD4(+)Foxp3(+) cells can be shaped by local environmental factors, which orchestrate distinct miRNA pathways preserving Treg stability and suppressor function.

Despite effective chemotherapy to treat schistosome infections, re-infection rates are extremely high. Resistance to reinfection can develop, however it typically takes several years following numerous rounds of treatment and re-infection, and often develops in only a small cohort of individuals. Using a well-established and highly permissive mouse model, we investigated whether immunoregulatory mechanisms influence the development of resistance. Following Praziquantel (PZQ) treatment of S. mansoni infected mice we observed a significant and mixed anti-worm response, characterized by Th1, Th2 and Th17 responses. Despite the elevated anti-worm response in PBMC's, liver, spleen and mesenteric lymph nodes, this did not confer any protection from a secondary challenge infection. Because a significant increase in IL-10-producing CD4(+)CD44(+)CD25(+)GITR(+) lymphocytes was observed, we hypothesised that IL-10 was obstructing the development of resistance. Blockade of IL-10 combined with PZQ treatment afforded a greater than 50% reduction in parasite establishment during reinfection, compared to PZQ treatment alone, indicating that IL-10 obstructs the development of acquired resistance. Markedly enhanced Th1, Th2 and Th17 responses, worm-specific IgG1, IgG2b and IgE and circulating eosinophils characterized the protection. This study demonstrates that blocking IL-10 signalling during PZQ treatment can facilitate the development of protective immunity and provide a highly effective strategy to protect against reinfection with S. mansoni.

The mechanisms underlying schistosomiasis-induced pulmonary hypertension (PH), one of the most common causes of PH worldwide, remain unclear. We sought to determine whether Schistosoma mansoni causes experimental PH associated with pulmonary vascular remodeling in an interleukin (IL)-13-dependent manner. IL-13Ralpha1 is the canonical IL-13 signaling receptor, whereas IL-13Ralpha2 is a competitive nonsignaling decoy receptor. Wild-type, IL-13Ralpha1(-/-), and IL-13Ralpha2(-/-) C57BL/6J mice were percutaneously infected with S. mansoni cercariae, followed by i.v. injection of eggs. We assessed PH with right ventricular catheterization, histological evaluation of pulmonary vascular remodeling, and detection of IL-13 and transforming growth factor-beta signaling. Infected mice developed pulmonary peri-egg granulomas and arterial remodeling involving predominantly the vascular media. In addition, gain-of-function IL-13Ralpha2(-/-) mice had exacerbated vascular remodeling and PH. Mice with loss of IL-13Ralpha1 function did not develop PH and had reduced pulmonary vascular remodeling. Moreover, the expression of resistin-like molecule-alpha, a target of IL-13 signaling, was increased in infected wild-type and IL-13Ralpha2(-/-) but not IL-13Ralpha1(-/-) mice. Phosphorylated Smad2/3, a target of transforming growth factor-beta signaling, was increased in both infected mice and humans with the disease. Our data indicate that experimental schistosomiasis causes PH and potentially relies on up-regulated IL-13 signaling.

Schistosoma blood flukes, which infect over 200 million people globally, co-opt CD4+ T cell-dependent mechanisms to facilitate parasite development and egg excretion. The latter requires Th2 responses, while the mechanism underpinning the former has remained obscure. Using mice that are either defective in T cell receptor (TCR) signaling or that lack TCRs that can respond to schistosomes, we show that naïve CD4+ T cells facilitate schistosome development in the absence of T cell receptor signaling. Concurrently, the presence of naïve CD4+ T cells correlates with both steady-state changes in the expression of genes that are critical for the development of monocytes and macrophages and with significant changes in the composition of peripheral mononuclear phagocyte populations. Finally, we show that direct stimulation of the mononuclear phagocyte system restores blood fluke development in the absence of CD4+ T cells. Thus we conclude that schistosomes co-opt innate immune signals to facilitate their development and that the role of CD4+ T cells in this process may be limited to the provision of non-cognate help for mononuclear phagocyte function. Our findings have significance for understanding interactions between schistosomiasis and other co-infections, such as bacterial infections and human immunodeficiency virus infection, which potently stimulate innate responses or interfere with T cell help, respectively. An understanding of immunological factors that either promote or inhibit schistosome development may be valuable in guiding the development of efficacious new therapies and vaccines for schistosomiasis.

Schistosome helminths constitute a major health risk for the human population in many tropical areas. We demonstrate for the first time that several developmental stages of the human parasite Schistosoma mansoni express arginase, which is responsible for the hydrolysis of l-arginine to l-ornithine and urea. Arginase activity by alternatively activated macrophages is an essential component of the mammalian host response in schistosomiasis. However, it has not been previously shown that the parasite also expresses arginase when it is in contact with the mammalian host. After cloning and sequencing the cDNA encoding the parasite enzyme, we found that many structural features of human arginase are well conserved in the parasite ortholog. Subsequently, we discovered that S. mansoni arginase shares many similar molecular, biochemical and functional properties with both human arginase isoforms. Nevertheless, our data also reveal striking differences between human and schistosome arginase. Particularly, we found evidence that schistosome arginase activity depends upon disulphide bonds by cysteines, in contrast to human arginase. In conclusion, we report that S. mansoni arginase is well adapted to the physiological conditions that exist in the human host.

We explore the controversial issue of the role of eosinophils in host defense against helminthic parasites using the established Schistosoma mansoni infection model in 2 novel mouse models of eosinophil lineage ablation (DeltadblGATA and TgPHIL). No eosinophils were detected in bone marrow of infected DeltadblGATA or TgPHIL mice, despite the fact that serum IL-5 levels in these infected mice exceeded those in infected wild type by approximately 4-fold. Liver granulomata from infected DeltadblGATA and TgPHIL mice were likewise depleted of eosinophils compared with those from their respective wild types. No eosinophil-dependent differences in granuloma number, size, or fibrosis were detected at weeks 8 or 12 of infection, and differential accumulation of mast cells was observed among the DeltadblGATA mice only at week 12. Likewise, serum levels of liver transaminases, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) increased in all mice in response to S mansoni infection, with no eosinophil-dependent differences in hepatocellular damage observed. Finally, eosinophil ablation had no effect on worm burden or on egg deposition. Overall, our data indicate that eosinophil ablation has no impact on traditional measures of disease in the S mansoni infection model in mice. However, eosinophils may have unexplored immunomodulatory contributions to this disease process.

The eosinophil-associated ribonucleases (Ears) are rapidly evolving proteins found in multigene clusters that are unique to each rodent species. Of the 15 independent genes in the Mus musculus cluster, only mEars 1 and 2 are expressed at significant levels at homeostasis. Here we characterize the expression of mEar 6 in the liver and spleen in mice in response to infection with the helminthic parasite, Schistosoma mansoni. Interestingly, expression of mEar 6 is not directly related to the elevated levels of serum IL-5 or tissue eosinophilia characteristic of this disease, as no mEar 6 transcripts were detected in the liver or the spleen from uninfected IL-5-transgenic mice. The coding sequence of mEar 6 has diverged under positive selection pressure (K(a)/K(s) > 1.0) and has a unique unpaired cysteine near the carboxy-terminus of the protein. The high catalytic efficiency of recombinant mEar 6 (k(cat)/K(m) = 0.9 x 10(6)/M/s) is similar to that of the cluster's closest human ortholog, eosinophil-derived neurotoxin (EDN/RNase 2). In summary, we have identified mEar 6 as one of only two RNase A superfamily ribonucleases known to be expressed specifically in response to pathophysiologic stress in vivo.

Plasminogen activator inhibitor-2 (PAI-2) as a potential eosinophil protein was inferred from our gene microarray study of mouse eosinophilopoiesis. Here, we detect 47 kDa intracellular and approximately 60 kDa secretory forms of PAI-2 in purified human eosinophil extracts. PAI-2 is present at variable concentrations in eosinophil lysates, ranging from 30 to 444 ng/10(6) cells, with a mean of 182 ng/10(6) cells from 10 normal donors, which is the highest per-cell concentration among all leukocyte subtypes evaluated. Enzymatic assay confirmed that eosinophil-derived PAI-2 is biologically active and inhibits activation of its preferred substrate, urokinase. Immunohistochemical and immunogold staining demonstrated PAI-2 localization in eosinophil-specific granules. Immunoreactive PAI-2 was detected in extracellular deposits in and around the eosinophil-enriched granuloma tissue encapsulating the parasitic egg in livers of wild-type mice infected with the helminthic parasite Schistosoma mansoni. Among the possibilities, we consider a role for eosinophil-derived PAI-2 in inflammation and remodeling associated with parasitic infection as well as allergic airways disease, respiratory virus infection, and host responses to tumors and metastasis in vivo.

In schistosomiasis mansoni, the chronic egg-induced granulomatous response in the liver and intestines may eventually cause extensive tissue scarring and development of portal hypertension. Indeed, much of the morbidity and mortality associated with this disease is directly attributable to the deposition of connective tissue elements in affected tissues. Elucidating the mechanisms that regulate the severity of schistosomiasis has been a major research objective over the past several years. Research conducted with DNA microarrays as well as investigations with a variety of gene knock-out mice have been particularly helpful in achieving this goal. A notable accomplishment in the past few years was the identification of interleukin-13 (IL-13) and the IL-13 receptor complex as central regulators of disease progression in schistosomiasis. Liver fibrogenesis is severely decreased in infected IL-13-deficient mice as well as in wildtype animals treated with IL-13 antagonists. In contrast, IL-13 effector function increases dramatically in IL-13 receptor alpha2 (IL-13Ralpha2)-deficient mice. These mice develop severe hepatic fibrosis, fail to downregulate granuloma formation in the chronic phase of S. mansoni infection, and succumb to the disease at an accelerated rate; thus, identifying the 'decoy' IL-13 receptor as a critical life sustaining 'off' switch for tissue damaging egg-induced inflammation.

Schistosoma mansoni parasites inhabit three distinct environments including water, intermediate molluscan hosts, and definitive vertebrate hosts. Determining how schistosomes interact with these environments may be one mechanism by which suitable vaccines or novel chemotherapeutic targets will be identified. Towards this end, we describe the identification of a 36-kDa S. mansoni protein that shares extensive sequence similarity to light absorbing rhodopsin guanine protein coupled receptors (GPCRs). This protein, S. mansoni rhodopsin (SmRHO), is the first molecularly characterized GPCR described in schistosomes. Sequence analysis reveals that SmRHO shares extensive phylogenetic conservation among rhodopsins/opsins expressed in water-dwelling invertebrates, possibly indicative of orthology. We demonstrate here that SmRHO is expressed in the free-living, light responsive miracidia and cercaria stages and is down-regulated in the adult, vertebrate residing forms. Moreover, we show that SmRHO is localized to sub-tegumental structures found towards the anterior end of cercariae. As SmRHO may be implicated in schistosome photoreception processes, we have begun a search for additional parasite encoded GPCR super-family members, which may be associated with chemoreception, chemotaxis, and olfaction. Identifying and characterizing new GPCRs may uncover hidden aspects of parasite biology useful towards the development of novel intervention strategies.

Using a Schistosoma mansoni egg-induced granuloma model, we examined the ability of CpG oligodeoxynucleotides (ODN) to suppress Th2-type cytokine expression and to prophylactically immunize against Th2-dependent pulmonary pathology. The mechanism was examined by studying Th2 response regulation in cytokine-deficient mice. Surprisingly, our findings revealed several functions of CpG DNA that were completely IL-12 independent. Most striking was the marked suppression in Th2 cytokine expression and granulomatous inflammation observed in egg/CpG-sensitized IL-12-deficient mice. Immune deviation was not dependent on NK or B cells. However, a role for IL-10, B7.1, and CD40 expression in Th2 response inhibition was suggested. Indeed, CpG ODN up-regulated all three elements in both wild-type and IL-12-deficient mice. The role of IL-10 was demonstrated in mice exhibiting combined deficiencies in IL-12 and IL-10. Here, a marked increase in egg-specific IL-4/IL-5-producing cells confirmed a role for both cytokines in Th2 response inhibition. Nevertheless, the frequency of Th2-producing cells was again reduced by CpG ODN. However, in marked contrast to IL-12-deficient animals, a significant increase in IFN-gamma-producing cells likely explains the reduced Th2 response in IL-10/IL-12-deficient mice. Thus, a novel IL-12-independent type 1-inducing pathway was revealed in the combined absence of IL-12 and IL-10. Together, these data demonstrate 1) that the Th1-promoting activity of CpG DNA is controlled by IL-12 and IL-10, and 2) that Th2 response inhibition by CpG ODN involves IL-12-independent changes in IL-10 and costimulatory molecule expression. These findings illustrate the utility of CpG DNA as adjuvants for vaccines designed to prevent Th2-dependent immunopathology.

A fundamental obstacle to vaccine development in schistosomiasis mansoni is a lack of understanding of what type of an immune response should be invoked. We have addressed this central issue by using the radiation-attenuated cercariae vaccine in mice genetically engineered to exhibit highly polarized type 1 (IL-10/IL-4-deficient) or type 2 (IL-10/IL-12-deficient) cytokine and Ab phenotypes. Our data show that while significant differences in immunity exist after a single vaccination with irradiated cercariae in double cytokine-deficient vs wild-type mice, these differences disappear after two vaccinations. The most important finding of these studies, however, was revealed in vaccinated IL-10-deficient mice. These mice developed a mixed and elevated type 1- and type 2-associated immune response and developed anti-schistosome immunity at levels equal to or better than those in wild-type mice. This immunity in IL-10-deficient mice correlated with higher parasite-specific Ab titers, greater proliferative capacity of lymphocytes, increased frequency of IFN-gamma- and IL-4-secreting cells, elevated perivascular/peribronchial inflammatory responses in the lung, and greater in vitro schistosomulacidal capacity of parasite Ag-elicited cells. These results suggest that optimal vaccine-induced immunity against schistosomes is linked not to the development of a highly polarized response, but, rather, to the induction of both type 1- and type 2-associated immune responses.

Mice with severe combined immunodeficiency (SCID mice) lack functional B and T cells. Egg laying by Schistosoma mansoni and S. japonicum was delayed in SCID mice, but in a matter of weeks worm fecundity was equivalent to that in intact mice. SCID mice formed smaller hepatic granulomas and showed less fibrosis than did intact mice. The reduction in egg-associated pathology in SCID mice correlated with marked reductions in interleukin-4 (IL-4), IL-5, IL-13, and gamma interferon mRNA expression in the liver. S. mansoni infections were frequently lethal for SCID mice infected for more than 9 weeks, while S. japonicum-infected SCID mice died at the same rate as infected intact mice. We were unable to affect hepatic granuloma formation or egg laying by worms in SCID mice by administration of recombinant murine tumor necrosis factor alpha (TNF-alpha). In fact, SCID and BALB/c mice appeared to express nearly equivalent levels of TNF-alpha mRNA in their granulomatous tissues, suggesting that there is little or no deficit in TNF-alpha expression in infected SCID mice. The data indicate that TNF-alpha may be in large part derived from a non-T-cell source. Together, these findings provide little evidence that TNF-alpha alone can reconstitute early fecundity, granuloma formation, or hepatic fibrosis in schistosome-infected SCID mice.

Chronic granulomatous disease (CGD) is a genetic disorder of NADPH oxidase in which phagocytes are defective in generating reactive oxidants. CGD patients suffer from recurrent infections and exuberant and persistent tissue granuloma formation. We hypothesized that abnormal granulomata in CGD may result from aberrant T-cell-mediated cytokine responses. To assess Th-1-type cytokine responses and granulomata, we challenged p47(phox-/-) and wild-type mice with avirulent (SmD) or virulent (SmT) variants of Mycobacterium avium 2-151. To assess Th-2-type cytokine responses and granulomata, we used Schistosoma mansoni eggs (SME). Mononuclear cells were harvested, and cytokine responses were determined by enzyme-linked immunosorbent assay or reverse transcriptase PCR. Following SmD or SmT challenge, splenocytes from p47(phox-/-) and wild-type mice generated similar polar Th-1 responses (increased levels of gamma interferon and basal levels of interleukin 4 [IL-4] and IL-5). By 8 weeks after SmT challenge, exuberant splenic granulomata developed in p47(phox-/-) and wild-type mice. After SME challenge, thoracic lymph node mononuclear cells from p47(phox-/-) and wild-type mice generated similar mixed Th-1 and Th-2 cytokine responses to SME antigen and concanavalin A. Peak lung granuloma sizes and rates of regression were similar in p47(phox-/-) and wild-type mice. These results suggest that exuberant granulomatous inflammation in CGD is probably not the result of skewing of T-cell responses toward the Th-1 or Th-2 pole. Appropriate regression of established tissue granulomata in p47(phox-/-) mice challenged with SME suggests that abnormal granuloma formation in CGD is stimulus dependent and is not an invariant feature of the disease.

Mice immunized with radiation-attenuated cercariae of Schistosoma mansoni display resistance to challenge infection, which increases with multiple boosting. Protection in animals receiving a single vaccination is thought to involve a primarily cell-mediated, IFN-gamma-dependent mechanism, while humoral immunity has been shown to contribute to challenge rejection in multiply (three times) immunized mice. To better understand the respective contribution of the B lymphocyte- and IFN-gamma-dependent effector arms in host resistance, we compared vaccine-induced immunity in B cell-deficient (muMT) and IFN-gamma knockout (GKO) animals. Unexpectedly, after a single vaccination, B cell knockout (KO) mice displayed reduced protection against challenge infection, although they developed a normal IFN-gamma-dominated cytokine response. This defect in resistance was equivalent to that displayed by GKO animals. Moreover, whereas two additional vaccinations significantly increased the level of immunity in wild-type mice, the protection in B cell KO animals remained unchanged. In contrast, multiple vaccination resulted in increased but, nevertheless, defective resistance in GKO mice. Since FcR gamma KO mice, which lack functional FcgammaRI, FcgammaRIII, and FcepsilonRI, show no defects in vaccine-induced resistance after immunization either one or three times, the B cell-dependent mechanism of protection involved does not appear to require FcR signaling. Together, these findings indicate that effective vaccination against schistosomes depends on the simultaneous induction of both humoral and cell-mediated immunity, a conclusion that may explain the limited success of most subunit vaccine protocols designed to preferentially induce either B cell- or IFN-gamma-dependent protective mechanisms.

The production of Th1-type cytokines is associated with strong cell-mediated immunity, while Th2-type cytokines typically dominate humoral immune responses. In mice vaccinated a single time with attenuated cercariae of Schistosoma mansoni, the protection induced is associated with Th1 cytokine-dependent, cell-mediated immunity. In contrast, mice vaccinated multiple times display a more Th2-type dominant cytokine response and develop Ab-dependent resistance. We have previously shown that IL-12 enhances cell-mediated immunity in singly vaccinated mice. In the present study, we asked what effects administering IL-12 as an adjuvant would have on the development of a protective humoral response in multiply immunized animals. We found that multiply immunized/IL-12-treated mice displayed a marked increase in resistance to challenge infection, with some animals demonstrating complete protection. The IL-12-vaccinated mice developed strongly polarized Th1 responses but, importantly, also showed significant increases in parasite-specific Ab and, in particular, IgG2a, IgG2b, and IgG1 isotypes. Passive transfer demonstrated an enhanced ability of serum from these animals to protect naive recipients. In addition, animals vaccinated in the presence of IL-12 also developed macrophages with increased nitric oxide-dependent killing activity against the parasites. Together, these data demonstrate that IL-12, initially described as an adjuvant for cell-mediated immunity, may be used to simultaneously to promote both humoral and cell-mediated protective responses against infection.

The data presented here clearly demonstrate that IL-12 can act as an adjuvant, suppressing both granuloma formation and fibrosis induced after natural schistosome infection. Recently, we showed that IL-12 can increase protective immunity provided by an attenuated larval schistosome vaccine as well. In both cases the vaccines appear to suppress in large part the parasite-induced Th2 responses. Thus, the use of cytokines as adjuvants offers a rational approach for immunomodulation when the effector mechanism of a particular vaccine is known. Clearly, IL-12 has enormous potential for modulating the outcome of immunization and may have broad application in preventing a variety of different infectious diseases.

To assess the role of IFN-gamma in the in vivo regulation of Th subset differentiation by IL-12, schistosome egg-induced Th2 responses and granuloma formation were studied in IFN-gamma knock-out (gamma KO) mice in which the absence of endogenous IFN-gamma is assured. Rather than suppressing pathology and eosinophilia as observed in wild-type animals, exogenous IL-12 in egg-injected gamma KO mice exacerbated Th2-dependent granuloma formation while failing to reduce peak tissue eosinophilia. Similarly, instead of inhibiting its production, IL-12 caused a dramatic increase in serum IgE levels in gamma KO animals after egg injection. Although the suppressive effects of IL-12 on Th2 responses were blocked in the absence of IFN-gamma, lymphocyte proliferation and IL-2 production were enhanced, a phenomenon which may underlie the observed exacerbation of egg-induced pathology. These findings formally establish that IL-12 inhibits Th2 development indirectly in vivo through the stimulation of IFN-gamma synthesis. In contrast, its promotion of Th1-associated responses seems to be at least partly a result of the direct action of the cytokine.

Cytokine responses of peripheral blood mononuclear cells from humans infected with Schistosoma mansoni were assessed. By ELISA and ELISPOT, persons with acute and hepatosplenic infections produced higher levels of interleukin (IL)-4 and IL-5 and higher frequencies of IL-4-producing cells in response to mitogen than did uninfected persons. In contrast, mitogen-induced production of the Th1 cytokine interferon-gamma (IFN-gamma) did not differ from that of uninfected controls. Upon stimulation with egg antigens, many patients responded with elevated IL-4 mRNA levels but displayed no appreciable increases in Th1 (i.e., IFN-gamma and IL-2) cytokine transcripts. Nevertheless, in cells stimulated with adult worm antigen, a more mixed Th0-type response was observed with production of both Th1 and Th2 cytokines. These results support previous findings in laboratory mice that schistosome infection results in increased production of Th2 cytokines. Unlike mice, infected humans do not display a generalized down-modulation in Th1 responses but instead show a selective deficiency in IFN-gamma and usually IL-2 responses to egg antigens.

In the mouse, infection with the helminth parasite Schistosoma mansoni results in the selective induction of CD4+ T lymphocytes belonging to the Th2 subset. Schistosome ova are responsible for the development of Th2 responses seen in patently infected animals and the injection of eggs s.c. into the footpad leads to the development of elevated Th2 cytokine production by T cells in the draining popliteal lymph node. Using the egg injection model, we have shown that IL-12 suppresses schistosome egg-induced Th2 responses as evidenced by decreased IL-4, IL-5, and IL-10 secretion in vitro while increasing the production of the Th1 cytokine IFN-gamma. Similar responses were obtained using either total lymph node cells or purified CD4+ T cells, indicating that IL-12 acts at the T cell level. When given as a single injection IL-12 was most effective at inhibiting Th2 responses when administered 2 days after egg inoculation, a time when T cells are still in a Th0 phase. The suppression of Th2 responses induced by IL-12 was blocked when the animals were simultaneously injected with neutralizing anti-IFN-gamma mAb, either systemically or systemically plus locally. Anti-IFN-gamma also inhibited the enhancement of IFN-gamma responses induced by IL-12 but only if the mAb was administered systemically plus locally. NK cells are likely to be a major source of the immunoregulatory IFN-gamma, because the effects of IL-12 on Th2 cytokine production were suppressed in mice treated with anti-asialo-GM1 Abs. Together these results suggest that IL-12 may have potential use in preventing or treating parasite-induced pathology resulting from Th2 cytokine production.

Morbidity in humans infected with Schistosoma mansoni results primarily from the deposition of parasite eggs in portal areas where they induce a granulomatous response. In mice infected with this helminth granuloma formation is a CD4+ T helper (Th) cell-dependent process that is associated with a strong Th2 cytokine response which appears to evolve through a Th0 phase. In this report, we asked whether endogenously synthesized or exogenously induced interferon (IFN)gamma through its suppression of Th2 cell expansion exerts a regulatory role on egg pathology. Depletion of IFN-gamma or natural killer cells resulted in a marked enhancement of granuloma formation around intravenously injected eggs and was associated with increased Th2 and decreased Th1 and interleukin (IL)12 mRNA expression. Similar changes occurred when egg-injected mice were treated with neutralizing monoclonal antibodies specific for IL-12 indicating a role for this cytokine in the regulation of the granulomatous response. In contrast, treatment with exogenous rIL-12 profoundly inhibited primary granuloma formation while increasing IFN-gamma, IL-2, IL-10, and IL-12 pulmonary mRNA levels and suppressing IL-4, IL-5, IL-6, and IL-13 mRNA expression. Cytokine depletion studies indicated that the effects of IL-12 could be attributed primarily to increased IFN-gamma. Importantly, IL-12 also inhibited secondary granuloma formation in mice presensitized with eggs demonstrating a role for the cytokine in reversing established Th2-type responses. Moreover, mice sensitized with eggs in combination with IL-12 to precommit them toward a Th1 response developed only minimal granulomas upon subsequent egg challenge. The latter findings suggest that simultaneous vaccination with antigen plus IL-12 may provide a strategy for the prevention of schistosome egg pathology as well as other diseases stemming from Th2 cytokine production.

Like many pathogens that undergo an intravascular stage of development, larvae of the helminth parasite Schistosoma mansoni migrate through the blood vessels, where they are in close contact with endothelial cells. In vitro exposure of murine endothelial cells to various cytokines (interferon gamma, tumor necrosis factor alpha, and interleukin 1 alpha or 1 beta) resulted in their activation to kill schistosomula through an arginine-dependent mechanism involving production of nitric oxide (NO). Cytokine-treated endothelial cells showed increased expression of mRNA for the inducible form of the NO synthase, and both NO production and larval killing were suppressed by treatment with competitive inhibitors. The effector function of cytokine-treated endothelial cells was similar to that of activated inflammatory tissue macrophages, although activation appeared to be differentially regulated in these two cell types. Activated endothelial cells killed older (18-day) forms of the parasite, such as those currently thought to be a primary target of immune elimination in the lungs of mice previously vaccinated with radiation-attenuated cercariae, as well as newly transformed larvae. In C57BL/6 mice, which become resistant to S. mansoni infection as a result of vaccination with irradiated cercariae, endothelial cell morphology characteristic of activation was observed in the lung by 1-2 weeks after challenge infection. Similar endothelial cell changes were absent in P-strain mice, which do not become resistant as a result of vaccination. Together, these observations indicate that endothelial cells, not traditionally considered to be part of the immune system, may play an important role in immunity to S. mansoni and, by means of NO-dependent killing, could serve as effectors of resistance to other intravascular pathogens.

Granulomas induced by parasite eggs are the primary lesions in mice infected with Schistosoma mansoni. Temporal analysis of cytokine mRNA expression in lung tissue containing synchronized granulomas demonstrated a Th0-like pattern of lymphokine expression. IFN-gamma, IL-1 beta, and IL-6 were the primary cytokines induced by day 1 in developing lung granulomas initiated by i.v. egg injection. These changes were followed by increases in expression of IL-2, IL-4, and IL-10 mRNA on day 3 and TNF-alpha and IL-5 mRNA on day 6. Nearly all cytokine mRNA reached maximal levels by day 6, which preceded the peak in granuloma size seen on day 14. In vivo treatment of egg-injected mice with either anti-IL-2 or anti-IL-4 antibodies significantly diminished the size of circumoval granulomas in the lungs. Both groups of antibody-treated animals displayed a marked reduction in IL-4 as well as IL-5 mRNA expression, although IFN-gamma and IL-2 mRNA levels were unchanged or slightly increased. These findings confirm previous observations suggesting a role for IL-2 in egg-induced pathology via the generation of Th2-associated responses, and also indicate a primary function for IL-4 in granuloma formation. Analysis of responses after injection of eggs into nude mice demonstrated that only the Th2 cytokines IL-4 and IL-5 are exclusively dependent on T cells for their induction. Taken together, these data suggest that Th2 cells producing IL-4 play a major role in egg granuloma formation, and that the induction and ultimate down-modulation of Th2-like responses may be influenced by non-T-cell-derived cytokines.