The gut mucosal immune response to Toxoplasma gondii.

As an orally acquired pathogen, the immune response to Toxoplasma gondii unfolds in the small intestinal mucosa. There, an array of regulatory and effector immune cells are elicited to combat the parasite through secretion of inflammatory mediators, normally resulting in host protection and pathogen control. Recent studies largely in mice have found that a productive immune response requires the combined recognition of parasite- and commensal-derived antigens by mucosal leucocytes. However, despite the fine-tuned regulatory mechanisms in place to prevent immunopathology, dysregulated responses can occur in genetically susceptible subjects, leading to lethal pro-inflammatory-mediated intestinal damage. Here, we describe the current understanding of the inflammatory players involved in orchestrating immunity or immunopathology in the intestine during the mucosal response to Toxoplasma infection.

Toxoplasma gondii possesses a superantigen activity that selectively expands murine T cell receptor V beta 5-bearing CD8+ lymphocytes.

To investigate early immune responses to the intracellular parasite Toxoplasma gondii, we examined the capacity of nonimmune splenocytes to respond in vitro to intact tachyzoites and soluble tachyzoite antigen (Ag). Both types of stimuli induced high levels of proliferation as well as interferon gamma (IFN-gamma) secretion. Based on several key criteria, the response appeared to be driven by a superantigen present in the parasite. Thus, stimulation of C57BL/6 spleen cells with T. gondii resulted in a preferential threefold expansion of a T cell population expressing the V beta 5 chain of the T cell receptor, and a survey of different inbred mouse strains revealed an inverse correlation between Ag-induced proliferation and genetic deletion of V beta 5. Moreover, proliferation was induced using irradiated Ag-pulsed and infected splenic adherent cells, and was blocked by a major histocompatibility complex class II-specific monoclonal antibody. Furthermore, paraformaldehyde-fixed IAb-, IAk-, and IEk-transfected fibroblast lines were able to specifically bind T. gondii Ag and drive proliferation of T lymphocytes, demonstrating that the response can be mediated by allogeneic class II molecules, and that it does not require cellular Ag processing. It is interesting to note that after 1 wk of culture with Ag, up to 70% of the expanded V beta 5-expressing cells were CD8+. These results provide the first description of a superantigen activity in a protozoan pathogen. In the case of T. gondii, superantigen-driven expansion of IFN-gamma-secreting CD8+ lymphocytes may play a role in the development of the dominant IFN-gamma-dependent, cell-mediated immunity characteristic of infection with this parasite.

Emergence of NK1.1+ cells as effectors of IFN-gamma dependent immunity to Toxoplasma gondii in MHC class I-deficient mice.

CD8+ T lymphocytes have been reported to play a major role in the protective immune response against acute infection with Toxoplasma gondii. In order to further assess the role of CD8+ cells in resistance against this protozoan we examined the ability of beta 2m-deficient mice, which fail to express MHC class I molecules and peripheral CD8+ lymphocytes, to survive tachyzoite challenge following vaccination with an attenuated parasite mutant. Surprisingly, vaccination of beta 2m-deficient mice induced strong resistance to lethal challenge, with > 50% surviving beyond 3 months. Vaccinated beta 2m-deficient mice, but not control heterozygotes, showed a five- to six-fold expansion in spleen cell number and approximately 40% of the splenocytes were found to express the NK markers NK1.1 and asialo GM1. Spleen cells from the vaccinated beta 2m-deficient animals failed to kill either infected host cells or the NK target YAC-1. However, high levels of IFN-gamma were secreted when the cells were cultured in vitro with soluble T. gondii lysate, and this response was abolished by NK1.1+ but not CD4+ and CD8+ lymphocyte depletion, implicating the NK1.1+ population as the major source of IFN-gamma. More importantly, vaccine-induced immunity in beta 2m-deficient mice was completely abrogated by in vivo administration of antibody to NK1.1, asialo GM1, or IFN-gamma. Together, the data suggest that in class I-deficient mice vaccinated against T. gondii, the absence of CD8+ effector cells is compensated for by the emergence of a population of NK1.1+ and asialo GM1+ cells which lack cytolytic activity, and that the protective action of these cells against the parasite is attributable to IFN-gamma production. The induction of this novel NK population may provide an approach for controlling opportunistic infections in immunocompromised hosts.

A role for CD4+ NK1.1+ T lymphocytes as major histocompatibility complex class II independent helper cells in the generation of CD8+ effector function against intracellular infection.

Major histocompatibility complex (MHC) class II (A beta) knockout mice were vaccinated with ts-4, an attenuated mutant strain of Toxoplasma gondii, which in normal animals induces strong T cell immunity mediated by interferon gamma (IFN-gamma). After challenge with the lethal parasite strain RH, the knockout mice displayed decreased resistance consistent with absence of CD4+ effectors. Nevertheless, these animals generated CD8+ lymphocyte effectors capable of mediating partial protection through IFN-gamma secretion. Moreover, in vivo neutralization experiments indicated that the development of resistance in knockout mice depends on CD4+ cells as well as interleukin 2 (IL-2). The identity of the IL-2-producing protective cell population was further characterized as CD4+, NK1.1+ by in vitro depletion studies and reverse transcriptase-PCR analysis of fluorescence-activated cell sorter (FACS)-purified CD4+ NK1.1+ T lymphocytes. These results demonstrate that in the absence of conventional MHC class II-restricted CD4+ T lymphocytes, CD8 priming persists and mediates partial protective immunity to T. gondii. Moreover, the data argue that CD4+, NK1.1+ cells, previously implicated in the initiation of T helper cell 2 (Th2) responses through their production of IL-4, can also play a role as alternative IL-2-secreting helper cells in Th1-mediated host resistance to infection.

Functional aspects of Toll-like receptor/MyD88 signalling during protozoan infection: focus on Toxoplasma gondii.

Toll-like receptor (TLR)/MyD88 signalling has emerged as a major pathway of pathogen recognition in the innate immune system. Here, we review recent data that begin to show how this pathway controls the immune response to protozoan infection, with particular emphasis on the opportunistic pathogen Toxoplasma gondii. The various ways that the parasite activates and suppresses TLR/MyD88 signalling defines several key principals that illuminate the complexities of the host-pathogen interaction. We also speculate how TLR/MyD88 signalling might be exploited to provide protection against Toxoplasma, as well as other protozoa and infection in general.

Dysregulation of macrophage signal transduction by Toxoplasma gondii: past progress and recent advances.

The opportunistic protozoan parasite Toxoplasma gondii is well known as a strong inducer of cell-mediated immunity, largely as a result of proinflammatory cytokine induction during in vivo infection. Yet, during intracellular infection the parasite suppresses signal transduction pathways leading to these proinflammatory responses. The opposing responses are likely to reflect the parasite's need to stimulate immunity allowing host survival and parasite persistence, and at the same time avoiding excessive responses that could result in parasite elimination and host immunopathology. This Review summarizes past and present investigations into the effects of Toxoplasma on host cell signal transduction. These studies reveal insight into the profound suppression of proinflammatory cytokine responses that occurs when the parasite infects macrophages and other cells of innate immunity.

T lymphocyte-dependent effector mechanisms of immunity to Toxoplasma gondii.

Immunity to the opportunistic pathogen, Toxoplasma gondii, is highly dependent upon the effector activity of IFN-gamma-producing T lymphocytes. While IFN-gamma is required to survive infection, an understanding of its function remains incomplete. During infection, T. gondii simultaneously induces downregulatory antiinflammatory cytokines, thereby avoiding major host pathology mediated by proinflammatory cytokines such as IFN-gamma. The ability to induce the correct balance between these two opposing host responses likely accounts for the success of this organism as a parasite.

Characterization of Trichinella spiralis antigens sharing an immunodominant, carbohydrate-associated determinant distinct from phosphorylcholine.

The biochemical and immunochemical characteristics of T. spiralis molecules (group II antigens) sharing an immunodominant epitope were examined. Six major proteins, ranging from 43-68 kDa, and from pI 5.0-6.3, express the determinant. Together, they account for at least 3% by weight of the total protein in L1 larval homogenate. The antigens are glycosylated. Following periodate oxidation, they reacted with biotin aminocaproyl hydrazide, and treatment with trifluoromethanesulfonic acid decreased their Mr. Deglycosylated group II antigens lost immunoreactivity with a monoclonal antibody specific for the determinant, and oligosaccharides released by treatment with mild base blocked binding of the monoclonal antibody to native antigens. The determinant on one of the group II antigens (43 kDa) was removed by N-glycanase. Neither phosphorylcholine nor antibody to phosphorylcholine interfered with monoclonal antibody binding to native group II antigens. Together, these results suggest that the immunodominant group II antigen epitope is associated with N- and O-linked oligosaccharides, and that it is not phosphorylcholine.

Differentiation of Toxoplasma gondii from closely related coccidia by riboprint analysis and a surface antigen gene polymerase chain reaction.

The tachyzoite of the human pathogen Toxoplasma gondii is morphologically indistinguishable from the proliferative stages of some other zoonotic coccidia, including Sarcocystis. To determine the identity of such coccidia obtained from human tissues and other sources, we compared riboprints (through restriction enzyme analysis of the polymerase chain reaction [PCR]-amplified small subunit rRNA gene) of the following protozoa: the RH and ts-4 strains of T. gondii, lines OH3 and S11, which are two recently isolated T. gondii-like parasites from Brazil, Neospora caninum, Sarcocystis species, and the malarial parasite Plasmodium berghei. In addition, the protozoan genomes were examined by PCR for homologs of surface antigen genes of T. gondii, and by Southern hybridization to the heterologous rRNA gene probe pSM 389. Strains OH3, S11, ts-4, and RH shared identical riboprints, and OH3, S11, and ts-4 have p22 and p30 surface antigen gene structures similar to RH. In contrast, riboprints for N. caninum and T. gondii differ with respect to Dde 1 sites, and moreover, their genomes vary significantly from one another at both the p22 and p30 gene loci. The riboprints of Sarcocystis and P. berghei differ markedly from T. gondii and N. caninum and from each other. Bam HI pSM 389 restriction fragment length polymorphisms differentiate ts-4 from RH, OH3, and S11. Our results confirm that OH3 and S11 are indeed T. gondii, but that N. caninum and T. gondii are likely to be separate species, thereby resolving previous uncertainties concerning the identity of these parasites. Together, the variation in riboprints and surface antigen gene structure reflects the phylogenetic diversity among these coccidia, and in addition, confirms the value of riboprinting in the identification of apicomplexan parasites such as T. gondii.

Border maneuvers: deployment of mucosal immune defenses against Toxoplasma gondii.

Toxoplasma gondii is a highly prevalent protozoan pathogen that is transmitted through oral ingestion of infectious cysts. As such, mucosal immune defenses in the intestine constitute the first and arguably most important line of resistance against the parasite. The response to infection is now understood to involve complex three-way interactions between Toxoplasma, the mucosal immune system, and the host intestinal microbiota. Productive outcome of these interactions ensures resolution of infection in the intestinal mucosa. Nonsuccessful outcome may result in emergence of proinflammatory damage that can spell death for the host. Here, we discuss new advances in our understanding of the mechanisms underpinning these disparate outcomes, with particular reference to initiators, effectors, and regulators of mucosal immunity stimulated by Toxoplasma in the intestine.

Synergy between intraepithelial lymphocytes and lamina propria T cells drives intestinal inflammation during infection.

Oral infection of C57BL/6 mice with Toxoplasma gondii triggers severe necrosis in the ileum within 7-10 days of infection. Lesion development is mediated by Th-1 cytokines, CD4+ T cells, and subepithelial bacterial translocation. As such, these features share similarity to Crohn's disease. Recently, we uncovered a role for intraepithelial lymphocytes (IELs) in mediating pathology after Toxoplasma infection. We show here that αß and not γδ T-cell IELs mediate intestinal damage. By adoptive transfer of mucosal T cells into naive Rag1⁻/⁻ mice, we demonstrate that IELs do not function alone to cause inflammatory lesions, but act with CD4+ T lymphocytes from the lamina propria (LP). Furthermore, recipient mice pretreated with broad-spectrum antibiotics to eliminate intestinal flora resisted intestinal disease after transfer of IELs and LP lymphocytes. Our data provide valuable new insights into the mechanisms of intestinal inflammation, findings that have important implications for understanding human inflammatory bowel disease.

CCR2-dependent intraepithelial lymphocytes mediate inflammatory gut pathology during Toxoplasma gondii infection.

Mice of the C57BL/6 strain develop acute ileal inflammation after infection with the protozoan parasite Toxoplasma gondii. This pathology resembles many key features of human Crohn's disease, including a Th1 cytokine profile with high levels of interferon gamma (IFN-gamma), interleukin 12 (IL)-12, and tumor necrosis factor alpha (TNF)-alpha, presence of pathogenic CD4(+) T cells, and infiltration of gut flora into inflammed tissue. Using CCR2(-/-) mice, we identify a role for this chemokine receptor in the pathogenesis of inflammatory pathology during T. gondii infection. Lack of chemokine (C-C motif) receptor 2 (CCR2) was associated with low levels of CD103(+) T lymphocytes in the intraepithelial compartment, Peyer's patch, and lamina propria relative to wild-type animals. Adoptive transfer of wild-type, but not IFN-gamma(-/-), intraepithelial T lymphocytes converted CCR2 knockout mice from a resistant to susceptible phenotype with respect to parasite-triggered inflammatory gut pathology. These results for the first time show a role for intraepithelial T lymphocytes in pathogenesis of ileitis triggered by a microbial pathogen.

A Toxoplasma gondii Superantigen: Biological effects and implications for the host-parasite interaction.

Superantigens exert their biological effects by activating large families of T cells, based on expression of the variable beta chain of the T-cell receptor. As a result, the reactive cells proliferate, secrete high levels of inflammatory cytokines, and ultimately die or become anergic to further stimulation. It is now becoming clear that the intracellular protozoan Toxoplasma gondii has many of these same superantigenic properties. As discussed here by Eric Denkers, this activity may play a key role in the induction of cell-mediated immunity to the parasite, and may prove to be responsible for much of the pathology associated with the clinical manifestations of toxoplasmosis.

Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection.

The intracellular protozoan Toxoplasma gondii is a widespread opportunistic parasite of humans and animals. Normally, T. gondii establishes itself within brain and skeletal muscle tissues, persisting for the life of the host. Initiating and sustaining strong T-cell-mediated immunity is crucial in preventing the emergence of T. gondii as a serious pathogen. The parasite induces high levels of gamma interferon (IFN-gamma) during initial infection as a result of early T-cell as well as natural killer (NK) cell activation. Induction of interleukin-12 by macrophages is a major mechanism driving early IFN-gamma synthesis. The latter cytokine, in addition to promoting the differentiation of Th1 effectors, is important in macrophage activation and acquisition of microbicidal functions, such as nitric oxide release. During chronic infection, parasite-specific T lymphocytes release high levels of IFN-gamma, which is required to prevent cyst reactivation. T-cell-mediated cytolytic activity against infected cells, while easily demonstrable, plays a secondary role to inflammatory cytokine production. While part of the clinical manifestations of toxoplasmosis results from direct tissue destruction by the parasite, inflammatory cytokine-mediated immunopathologic changes may also contribute to disease progression.

IFN-gamma overproduction and high level apoptosis are associated with high but not low virulence Toxoplasma gondii infection.

Toxoplasma gondii is an opportunistic intracellular parasite which induces a highly strong type 1 cytokine response. The present study focuses on defining the factors influencing the outcome of infection with tachyzoites of the type I, highly lethal RH strain, relative to the type II, low virulence strain ME49. Infection with the RH strain led to widespread parasite dissemination and rapid death of mice; in contrast, mice survived low virulence strain ME49 infection, and tachyzoite dissemination was much less extensive. Furthermore, massive apoptosis and disintegration of the splenic architecture was characteristic of RH, but not ME49, infection. In addition, hyperinduction of IFN-gamma and lack of NO production were found during RH, in contrast to ME49 infection. These data demonstrate that Toxoplasma strain characteristics exert a profound effect on the host immune response and that the latter itself is a crucial determinant in parasite virulence.

Toxoplasma gondii triggers granulocyte-dependent cytokine-mediated lethal shock in D-galactosamine-sensitized mice.

To investigate the capacity of Toxoplasma gondii to induce cytokine-mediated toxicity, we employed a murine model of lethal shock in which hypersensitivity to microbial toxins is induced by D-galactosamine (D-Gal). Animals injected with D-Gal and tachyzoite lysate died within 12 to 24 h, whereas administration of D-Gal or lysate alone was nonlethal. Analyses of plasma cytokines revealed peaks of tumor necrosis factor (TNF) alpha and interleukin-12 (IL-12) 1 and 3 to 5 h after injection, respectively, and gradually rising levels of gamma interferon (IFN-gamma) continuing until death. Nitric oxide (NO) levels in serum paralleled IFN-gamma production. Transaminase assays revealed elevated levels of liver-associated enzymes in sera of lethally injected mice, indicating severe hepatic damage. Depletion of IL-12, TNF, IFN-gamma, and NO rescued mice from the lethal effect of antigen (Ag) and D-Gal. T-cell-deficient animals remained sensitive to D-Gal and lysate, suggesting that T lymphocytes do not contribute to the response. Nevertheless, monoclonal antibody (MAb)-mediated granulocyte depletion completely abrogated D-Gal- and Ag-induced mortality and accompanying liver pathology. Finally, mice acutely infected with T. gondii displayed highly elevated NO and liver enzyme levels in serum immediately prior to death, and administration of anti-TNF MAb prolonged survival by approximately 24 h. Our results demonstrate that T. gondii induces lethal inflammatory cytokine shock in D-Gal-sensitized animals and suggest that a similar pathology may contribute to manifestations of acute toxoplasmosis.

Protective role for interleukin-5 during chronic Toxoplasma gondii infection.

To investigate the role of interleukin-5 (IL-5) during Toxoplasma gondii infection, IL-5 knockout (KO) mice and C57BL/6 control mice were infected intraperitoneally with ME49 cysts and the course of infection was monitored. The mortality rate during chronic infection was significantly greater in IL-5-deficient animals, and consistent with this finding, the KO mice harbored a greater number of brain cysts and tachyzoites than did their wild-type counterparts. Although the IL-5 KO animals did not succumb until late during infection, increased susceptibility, as measured by accelerated weight loss, was detectable during the acute stages of infection. The amounts of total immunoglobulin (Ig), IgM, and IgG2b were comparable in both strains, while the amount of IgG1 was much smaller in IL-5 KO mice. Spleen cell production of IL-12 in response to T. gondii antigen was approximately threefold lower in the KO strain, and this decrease correlated with a selective loss of B lymphocytes during culture. A link between the presence of B cells and augmented IL-12 production was established by the finding that after removal of B cells with monoclonal antibody and complement, wild-type- and KO-derived cells produced equivalent levels of IL-12 in response to T. gondii antigen. These results demonstrate a protective role of IL-5 against T. gondii infection and suggest that IL-5 may play a role in the production of IL-12.

Trichinella spiralis: influence of an immunodominant, carbohydrate-associated determinant on the host antibody response repertoire.

Host antibody responses to the G2.1 epitope, a carbohydrate-associated determinant shared by several Trichinella spiralis glycoproteins, were examined by competitive inhibition enzyme-linked immunosorbent assay (ELISA). The G2.1 epitope dominated the AKR/J mouse antibody response whether the antigens were injected or introduced through infection, as determined by the serum blocking ability of a G2.1 epitope-specific monoclonal antibody (mAb). Serum T. spiralis-binding activity from several other infected mouse strains was blocked 22 to 86% by the G2.1 epitope-specific mAb. In addition to mice, the G2.1 epitope evoked powerful antibody responses in four other species. The binding activity of Trichinella-reactive antibodies from infected rats and pigs was inhibited 56 and 34%, respectively, by the mAb. Greater than 48% of the T. spiralis serum-binding activity from 4/5 infected humans was G2.1-specific. Most of the rabbit antibody response induced by injection of a previously characterized 43-kDa antigen was also directed to the G2.1 determinant. The specificities of 10 T. spiralis-reactive mAb were examined, and 7 reacted with the immunodominant epitope. Finally, of nine helminth species examined, only T. spiralis and T. pseudospiralis extracts efficiently blocked G2.1-specific antibody binding to solid-phase antigens. These results suggest that the responses to G2.1 epitope may play an important role during infection.

Host resistance to Toxoplasma gondii: model for studying the selective induction of cell-mediated immunity by intracellular parasites.

Cell-mediated immunity (CMI) provides a major host defense against infectious disease and malignancy. Nevertheless, the mechanisms underlying the induction of CMI remain poorly understood and this lack of knowledge remains an important impasse in the design of rationally based vaccines and immunotherapies. Toxoplasma gondii is an intracellular pathogen that, as part of its normal life cycle, induces a potent CMI response leading to host resistance. Studies on the interaction of this parasite with the immune system therefore provide a unique approach for identifying events that selectively lead to the triggering of host-protective CMI. In addition, this knowledge may provide new strategies for immunotherapy of opportunistic infections in immunodeficient hosts.

Induction and regulation of host cell-mediated immunity by Toxoplasma gondii.

Toxoplasma gondii is a highly infectious intracellular parasite which, if left unchecked by the immune system, rapidly overwhelms its intermediate hosts, as illustrated by the pathogenesis of toxoplasmic encephalitis in patients with AIDS. In order to insure both its host's and consequently its own survival simultaneously, T. gondii induces a potent gamma-interferon (IFN-gamma)-dependent cell-mediated immunity early in infection that controls the replication of the protozoan and facilitates transformation into the dormant cyst stage. The protective IFN-gamma is derived from three sources: natural killer cells; and CD4+ and CD8+ T lymphocytes, which can partially compensate for each other in knockout mice lacking the appropriate major histocompatibility complex-restricting elements. At least two properties of the parasite appear to be responsible for the early induction of these effector cells. The first is a hydrophobic molecule (or group of related molecules) that triggers interleukin 12 (IL-12), tumour necrosis factor alpha and IL-1beta synthesis in macrophages. This response can also promote HIV replication in the same cells. The second is a superantigen activity that drives IFN-gamma-producing Vbeta5+ CD8+ T cells. These potentially lethal responses are later regulated through the triggering of IL-10 and by the induction of anergy in the superantigen-stimulated Vbeta5+ T cell population.