Interleukin-35 (IL-35) belongs to the IL-12 family of heterodimeric cytokines but has a distinct functional profile. IL-35 suppresses T cell proliferation and converts naïve T cells into IL-35-producing iTr35. Here we show that IL-35 signals through a unique IL-12Rβ2:gp130 heterodimer or via homodimers. Conventional T cells are sensitive to IL-35-mediated suppression in the absence of one but not both receptor chains, whereas signaling through both chains is required for IL-35 expression and iTr35 conversion. IL35R signaling requires the transcription factors STAT1 and STAT4, which form a unique heterodimer that binds to distinct sites within the Il12a and Ebi3 promoters. This unconventional mode of signaling, which is distinct from other members of the IL-12 family, may broaden the spectrum and specificity of IL-35-mediated suppression.

Interleukin(IL)-27 is a member of the IL-6 and IL-12 family composed of the IL-27p28 and Epstein Barr-induced virus 1 subunits. While IL-27 was originally identified as a pro-inflammatory factor, subsequent studies have revealed the pleotropic nature of this cytokine. This review discusses recent work that explores the effect of IL-27 on CD4+ T cell subsets, including T regulatory type 1 cells, T follicular helper cells, and Foxp3+ T regulatory cells. Additionally, we highlight studies that identify a role for the IL-27p28 subunit as a cytokine receptor antagonist. Much of the recent work on IL-27 has been relevant to human disease states characterized by inappropriate or excessive inflammation, and this review will discuss potential opportunities to use IL-27 as a therapeutic.

The ability of cytokines to direct the immune response to vaccination, infection and tumors has motivated their use in therapy to augment or shape immunity. To avoid toxic side effects associated with systemic cytokine administration, several approaches have been developed using particle-encapsulated cytokines to deliver this cargo to specific cell types and tissues. Initial work used cytokine-loaded particles to deliver proinflammatory cytokines to phagocytes to enhance antimicrobial and antitumor responses. These particles have also been used to create a cytokine depot at a local site to supplement prophylactic or antitumor vaccines or injected directly into solid tumors to activate immune cells to eliminate established tumors. Finally, recent advances have revealed that paracrine delivery of cytokines directly to T cells has the potential to enhance T-cell mediated therapies. The studies reviewed here highlight the progress in the last 30 years that has established the potential of particle-mediated cytokine immunotherapy.

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. To date, however, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated and it is not known if IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4+ and CD8+ T cells. Importantly, we demonstrate that IFN-γ producing CD4+ T cells, but not innate or CD8+ T cells, can promote the development of ECM in normally resistant IFN-γ−/− mice infected with P. berghei ANKA. Adoptively transferred wild-type (WT) CD4+ T cells accumulate within the spleen, lung and brain of IFN-γ−/− mice and induce ECM through active IFN-γ secretion, which increases accumulation of endogenous IFN-γ−/− CD8+ T cells within the brain. Depletion of endogenous IFN-γ−/− CD8+ T cells abrogated the ability of WT CD4+ T cells to promote ECM. Finally we show that IFN-γ production specifically by CD4+ T cells is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8+ T cell accumulation. These observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4+ T cells promote the development of ECM during P. berghei ANKA infection.

Vitiligo is an autoimmune disease of the skin causing disfiguring patchy depigmentation of the epidermis and, less commonly, hair. Therapeutic options for vitiligo are limited, reflecting in part limited knowledge of disease pathogenesis. Existing mouse models of vitiligo consist of hair depigmentation but lack prominent epidermal involvement, which is the hallmark of human disease. They are thus unable to provide a platform to fully investigate disease mechanisms and treatment. CD8+ T cells have been implicated in the pathogenesis of vitiligo and expression of interferon-gamma (IFN-γ) is increased in the lesional skin of patients, however it is currently unknown what role IFN-γ plays in disease. Here, we have developed an adoptive transfer mouse model of vitiligo using melanocyte-specific CD8+ T cells, which recapitulates the human condition by inducing epidermal depigmentation while sparing the hair. Like active lesions in human vitiligo, histology of depigmenting skin reveals a patchy mononuclear infiltrate and single-cell infiltration of the epidermis. Depigmentation is accompanied by accumulation of autoreactive CD8+ T cells in the skin, quantifiable loss of tyrosinase transcript, and local IFN-γ production. Neutralization of IFN-γ with antibody prevents CD8+ T cell accumulation and depigmentation, suggesting a therapeutic potential for this approach.

Chemokines play a central role in regulating processes essential to the immune function of T cells1-3, such as their migration within lymphoid tissues and targeting of pathogens in sites of inflammation. Here we track T cells using multi-photon microscopy to demonstrate that the chemokine CXCL10 enhances the ability of CD8+ T cells to control the pathogen T. gondii in the brains of chronically infected mice. This chemokine boosts T cell function in two different ways: it maintains the effector T cell population in the brain and speeds up the average migration speed without changing the nature of the walk statistics. Remarkably, these statistics are not Brownian; rather, CD8+ T cell motility in the brain is well described by a generalized Lévy walk. According to our model, this surprising feature enables T cells to find rare targets with more than an order of magnitude more efficiency than Brownian random walkers. Thus, CD8+ T cell behavior is similar to Lévy strategies reported in organisms ranging from mussels to marine predators and monkeys4-10, and CXCL10 aids T cells in shortening the average time to find rare targets.

During acute infection in human and animal hosts, the obligate intracellular protozoan Toxoplasma gondii infects a variety of cell types, including leukocytes. Poised to respond to invading pathogens, dendritic cells (DC) may also be exploited by T. gondii for spread in the infected host. Here, we report that human and mouse myeloid DC possess functional γ-aminobutyric acid (GABA) receptors and the machinery for GABA biosynthesis and secretion. Shortly after T. gondii infection (genotypes I, II and III), DC responded with enhanced GABA secretion in vitro. We demonstrate that GABA activates GABAA receptor-mediated currents in T. gondii-infected DC, which exhibit a hypermigratory phenotype. Inhibition of GABA synthesis, transportation or GABAA receptor blockade in T. gondii-infected DC resulted in impaired transmigration capacity, motility and chemotactic response to CCL19 in vitro. Moreover, exogenous GABA or supernatant from infected DC restored the migration of infected DC in vitro. In a mouse model of toxoplasmosis, adoptive transfer of infected DC pre-treated with GABAergic inhibitors reduced parasite dissemination and parasite loads in target organs, e.g. the central nervous system. Altogether, we provide evidence that GABAergic signaling modulates the migratory properties of DC and that T. gondii likely makes use of this pathway for dissemination. The findings unveil that GABA, the principal inhibitory neurotransmitter in the brain, has activation functions in the immune system that may be hijacked by intracellular pathogens.

Author Summary

Toxoplasma gondii is an obligate intracellular protozoan parasite and an important food- and water-borne human and veterinary pathogen. Toxoplasmosis is normally self-limiting but severe manifestations occur upon congenital transmission to the developing fetus or during infection in immune-compromised individuals. Toxoplasma invades a variety of cell types and mounting evidence shows that certain white blood cells, e.g. dendritic cells, can shuttle parasites in the infected host by a Trojan horse type of mechanism. Dendritic cells are considered the gatekeepers of the immune system but can, paradoxically, also mediate dissemination of the parasite. Previous work has shown that Toxoplasma induces a hypermigratory state in dendritic cells when they become infected. Here, we show that, shortly after infection by the parasite, dendritic cells start secreting γ-aminobutyric acid (GABA), also known as the major inhibitory neurotransmitter in the brain. We show that dendritic cells express GABA receptors, as well as the machinery to synthesize and transport GABA. When GABA synthesis, transport or receptor function was inhibited, the migration of infected dendritic cells was impaired. In a mouse model of toxoplasmosis, treatment of infected dendritic cells with GABA inhibitors resulted in reduced propagation of the parasite. This study establishes that GABAergic signaling modulates the migratory properties of dendritic cells and that the intracellular pathogen Toxoplasma gondii sequesters the GABAergic signaling of dendritic cells to assure propagation.

Summary

The innate and adaptive immune responses that confer resistance to the intracellular pathogen Toxoplasma gondii critically depend on IL-12 production, which drives interferon-γ (IFN-γ) expression. Certain cytokines can activate STAT3 and limit IL-12 production to prevent infection-associated immune pathology but T.gondii also directly activates STAT3 to evade host immunity. We show that Suppressor of Cytokine Signaling molecule 3 (SOCS3), a target of STAT3 which limits signaling by the pleiotropic cytokine IL-6, is upregulated in response to infection but is dispensable for the immune-inhibitory effects of T. gondii. Unexpectedly, mice with targeted deletion of SOCS3 in macrophages and neutrophils have reduced IL-12 responses and succumb to toxoplasmosis. Anti–IL-6 administration or IL-12 treatment blocked disease susceptibility suggesting that in the absence of SOCS3, macrophages are hypersensitive to the anti-inflammatory properties of IL-6. Thus, SOCS3 has a critical role in suppressing IL-6 signals and promoting immune responses to control T. gondii infection.

As an intracellular protozoan parasite, Toxoplasma gondii is likely to exploit proteases for host cell invasion, acquisition of nutrients, avoidance of host protective responses, escape from the parasitophorous vacuole, differentiation, and other activities. T. gondii serine protease inhibitor 1 (TgPI1) is the most abundantly expressed protease inhibitor in parasite tachyzoites. We show here that alternative splicing produces two TgPI1 isoforms, both of which are secreted via dense granules into the parasitophorous vacuole shortly after invasion, become progressively more abundant over the course of the infectious cycle, and can be detected in the infected host cell cytoplasm. To investigate TgPI1 function, the endogenous genomic locus was disrupted in the RH strain background. ΔTgPI1 parasites replicate normally as tachyzoites but exhibit increased bradyzoite gene transcription and labeling of vacuoles with Dolichos biflorus lectin under conditions promoting in vitro differentiation. The differentiation phenotype can be partially complemented by either TgPI1 isoform. Mice infected with the ΔTgPI1 mutant display ∼3-fold-increased parasite burden in the spleen and liver, and this in vivo phenotype is also complemented by either TgPI1 isoform. These results demonstrate that TgPI1 influences both parasite virulence and bradyzoite differentiation, presumably by inhibiting parasite and/or host serine proteases.

Chordoma and chondrosarcoma are malignant bone tumors characterized by the abundant production of extracellular matrix. The resistance of these tumors to conventional therapeutic modalities has prompted us to delineate the gene expression profile of these two tumor types, with the expectation to identify potential molecular therapeutic targets. Furthermore the transcriptional profile of chordomas and chrondrosarcomas was compared to a wide variety of sarcomas as well as to that of normal tissues of similar lineage, to determine whether they express unique gene signatures among other tumors of mesenchymal origin, and to identify changes associated with malignant transformation. A HG-U133A Affymetrix Chip platform was used to determine the gene expression signature in 6 chordoma and 14 chondrosarcoma lesions. Validation of selected genes was performed by qPCR and immunohistochemistry (IHC) on an extended subset of tumors. By unsupervised clustering, chordoma and chondrosarcoma tumors grouped together in a genomic cluster distinct from that of other sarcoma types. They shared overexpression of many extracellular matrix genes including aggrecan, type II & X collagen, fibronectin, matrillin 3, high molecular weight-melanoma associated antigen (HMW-MAA), matrix metalloproteinase MMP-9, and MMP-19. In contrast, T Brachyury and CD24 were selectively expressed in chordomas, as were Keratin 8,13,15,18 and 19. Chondrosarcomas are distinguished by high expression of type IX and XI collagen. Because of its potential usefulness as a target for immunotherapy, the expression of HMW-MAA was analyzed by IHC and was detected in 62% of chordomas and 48% of chondrosarcomas, respectively. Furthermore, western blotting analysis showed that HMW-MAA synthesized by chordoma cell lines has a structure similar to that of the antigen synthesized by melanoma cells. In conclusion, chordomas and chondrosarcomas share a similar gene expression profile of up-regulated extracellular matrix genes. HMW-MAA represents a potential useful target to apply immunotherapy to these tumors.

Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, an additional member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with other Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4+ and CD8+ T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in murine T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore, Foxp3+ T regulatory cells develop normally in Foxp4 deficient animals and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii and acute lymphocytic choriomeningitis virus infections, effector cytokine production during antigen specific recall responses are reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal T cell cytokine recall responses to antigen following pathogenic infection.

Follicular Helper T cells (TFH) are critical for germinal center (GC) formation. The processes that drive their generation and effector potential remain unclear. Here, we define requirements for MHCII antigen presenting cells (APCs) in murine TFH formation by either transiently ablating or restricting antigen presentation to dendritic cells (DCs). We find that cognate interactions with DCs are necessary and sufficient to prime CD4+ T cells towards a CXCR5+ICOS+Bcl6+ TFH intermediate. However, in the absence of additional APCs, these TFH fail to produce IL-21. Furthermore, in vitro priming of naïve T cells by B cells engenders optimal production of IL-21, which induces a GC B cell transcriptional profile. These results support a multi-step model for effector TFH priming and GC initiation, in which DCs are necessary and sufficient to induce a TFH intermediate that requires additional interactions with distinct APCs for full effector function.

Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.

Author Summary

Toxoplasma gondii is an intracellular parasite that infects warm blooded animals, including humans. In these hosts, Toxoplasma establishes a chronic infection in the brain, which the parasite accomplishes in part by injecting effector proteins, which manipulate many cellular processes, into cells it invades. Two recent reports suggested that Toxoplasma may also inject effector proteins into cells it does not invade. To look for these “uninfected-injected” cells, we utilized three different reporter systems that are tied to injection of effector proteins and not to invasion. With these systems, we determined that Toxoplasma injects proteins into cells it does not invade and enough protein is injected to manipulate the uninfected cells in a manner consistent with what occurs in infected cells. Furthermore, by using one of the reporter systems in mice, we verified that these uninfected-injected cells can include systemic immune cells and neurons in the brain. Remarkably, in the brain, the uninfected-injected cells out-number the infected cells by many fold. Together, these results strongly suggest that Toxoplasma manipulates far more cells than previously realized and, given their abundance, these uninfected-injected cells may play a central role in how Toxoplasma engages the host's immune response.

IL-6 and IL-27 are closely related cytokines that play critical, but distinct, roles during infection with Toxoplasma gondii. Thus, IL-6 is required for the development of protective immunity to this pathogen, while IL-27 is required to limit infection-induced pathology. Paradoxically, these factors both signal through glycoprotein 130 (gp130), but little is known about how the signals downstream of gp130 are integrated to coordinate the immune response to infection. To better understand these events, gp130 Y757F mice that have a mutation in gp130 at the binding site for Suppressor of cytokine signaling 3 (SOCS3), a critical negative regulator of gp130 signaling, were infected with T. gondii. These mutant mice were acutely susceptible to this challenge, characterized by an early defect in the production of IL-12 and IFN-γ, and increased parasite burdens. Consistent with the reduced IL-12 levels, IL-6, but not other gp130 cytokines, was a potent antagonist of IL-12 production by gp130 Y757F macrophages and dendritic cells in vitro. Moreover, in gp130 Y757F mice, blocking IL-6 in vivo, or administration of recombinant IL-12, during infection restored IFN-γ production and protective immunity. Collectively, these studies highlight that a failure to abbreviate IL-6-mediated gp130 signaling results in a profound anti-inflammatory signal that blocks the generation of protective immunity to T. gondii.

IL-27 is a cytokine that regulates Th function during autoimmune and pathogen-induced immune responses. Although previous studies have shown that T regulatory cells (Treg) express the IL-27R, and that IL-27 inhibits forkhead box P3 upregulation in vitro, little is known about how IL-27 influences Treg in vivo. The studies presented here show that mice that over-express IL-27 had decreased Treg frequencies and developed spontaneous inflammation. While IL-27 did not cause mature Treg to downregulate forkhead box P3, transgenic over-expression in vivo limited the size of a differentiating Treg population in a bone marrow chimera model, which correlated with reduced production of IL-2, a vital cytokine for Treg maintenance. Together, these data identify an indirect role for IL-27 in shaping the Treg pool.

SUMMARY

European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, II and III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major goal of current research. Here we show that type I and III infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated, while type II infected macrophages are classically activated. The Toxoplasma rhoptry kinase ROP16, which activates STAT6, is responsible for alternative activation. The Toxoplasma dense granule protein GRA15, which activates NF-κB, promotes classical activation by type II parasites. These effectors antagonistically regulate many of the same genes, and mice infected with type II parasites expressing type I ROP16 are protected against Toxoplasma-induced ileitis. Thus, polymorphisms in determinants that modulate macrophage activation influence the ability of Toxoplasma to establish a chronic infection.

Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences—the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The ‘environmental packages’ apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.

The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena), Europe's primary nucleotide sequence resource, captures and presents globally comprehensive nucleic acid sequence and associated information. Covering the spectrum from raw data to assembled and functionally annotated genomes, the ENA has witnessed a dramatic growth resulting from advances in sequencing technology and ever broadening application of the methodology. During 2011, we have continued to operate and extend the broad range of ENA services. In particular, we have released major new functionality in our interactive web submission system, Webin, through developments in template-based submissions for annotated sequences and support for raw next-generation sequence read submissions.

The nuclear factor κB transcription factor c-Rel is exclusively expressed in immune cells and plays a role in numerous cellular functions including proliferation, survival and production of chemokines and cytokines. c-Rel has also been implicated in the regulation of multiple genes involved in innate and adaptive immune responses to the intracellular protozoan parasite Toxoplasma gondii, in particular IL-12. To better understand how this transcription factor controls the CD8+ T-cell response to this organism, wild-type (WT) and c-Rel−/− mice were challenged with a replication-deficient strain of T. gondii that expresses the model antigen ovalbumin (OVA). These studies revealed that c-Rel was required for optimal primary expansion of OVA-specific CD8+ T cells and that immunized c-Rel-deficient mice were susceptible to challenge with a virulent strain of T. gondii. However, when c-Rel−/− cells specific for OVA were adoptively transferred into a WT recipient, or c-Rel−/− mice were treated with IL-12 at the time of immunization, there was no apparent proliferative defect. Surprisingly, upon secondary challenge, antigen-specific CD8+ T cells in c-Rel−/− mice expanded to a much greater degree in terms of frequency as well as numbers when compared with WT mice. Despite this, the cytokine responses of c-Rel−/− mice remained defective, consistent with their susceptibility to secondary challenge. Together, these results indicate that in this infection model, the major influence of c-Rel in generation of CD8+ T-cell responses is through its regulation of the inflammatory environment, rather than playing a substantial T-cell-intrinsic role.

Under normal conditions the immune system has limited access to the brain; however, during toxoplasmic encephalitis (TE), large numbers of T cells and APCs accumulate within this site. A combination of real time imaging, transgenic reporter mice, and recombinant parasites allowed a comprehensive analysis of CD11c+ cells during TE. These studies reveal that the CNS CD11c+ cells consist of a mixture of microglia and dendritic cells (DCs) with distinct behavior associated with their ability to interact with parasites or effector T cells. The CNS DCs upregulated several chemokine receptors during TE, but none of these individual receptors tested was required for migration of DCs into the brain. However, this process was pertussis toxin sensitive and dependent on the integrin LFA-1, suggesting that the synergistic effect of signaling through multiple chemokine receptors, possibly leading to changes in the affinity of LFA-1, is involved in the recruitment/retention of DCs to the CNS and thus provides new insights into how the immune system accesses this unique site.

Author Summary

Toxoplasmic encephalitis (TE), caused by the protozoan parasite Toxoplasma gondii, can be potentially life threatening especially in immuno-compromised individuals. Immune cells including dendritic cells have been shown to accumulate in the brain during chronic toxoplasmosis; however, little is known about their function, their behavior in vivo, and the mechanisms by which they migrate into the brain. In the present studies, we utilize a combination of real time imaging, transgenic reporter mice, and recombinant parasites to reveal the distinct behavior and morphologies of dendritic cells within the brain and their ability to interact with parasites and effector T cells during TE. The CNS DCs were also found to exhibit a unique chemokine receptor expression pattern during infection, and the migration of DCs into the brain was mediated through a pertussis toxin (which blocks signaling downstream of several chemokine receptors) sensitive process and dependent on the integrin LFA-1. There is currently a poor understanding of the events that lead to DC recruitment to the CNS during inflammation in general, and our studies provide new insights into the mechanisms by which antigen-presenting cells gain access to the brain during infection.

The ROP16 kinase of Toxoplasma gondii is injected into the host cell cytosol where it activates signal transducer and activator of transcription (STAT)-3 and STAT6. Here, we generated a ROP16 deletion mutant on a Type I parasite strain background, as well as a control complementation mutant with restored ROP16 expression. We investigated the biological role of the ROP16 molecule during T. gondii infection. Infection of mouse bone marrow-derived macrophages with rop16-deleted (ΔROP16) parasites resulted in increased amounts of IL-12p40 production relative to the ROP16-positive RH parental strain. High level IL-12p40 production in ΔROP16 infection was dependent on the host cell adaptor molecule MyD88, but surprisingly was independent of any previously recognized T. gondii triggered pathway linking to MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1ß and IL-18). In addition, ROP16 was found to mediate the suppressive effects of Toxoplasma on LPS-induced cytokine synthesis in macrophages and on IFN-γ-induced nitric oxide production by astrocytes and microglial cells. Furthermore, ROP16 triggered synthesis of host cell arginase-1 in a STAT6-dependent manner. In fibroblasts and macrophages, failure to induce arginase-1 by ΔROP16 tachyzoites resulted in resistance to starvation conditions of limiting arginine, an essential amino acid for replication and virulence of this parasite. ΔROP16 tachyzoites that failed to induce host cell arginase-1 displayed increased replication and dissemination during in vivo infection. We conclude that encounter between Toxoplasma ROP16 and the host cell STAT signaling cascade has pleiotropic downstream effects that act in multiple and complex ways to direct the course of infection.

Author Summary

Toxoplasma gondii is an extremely widespread intracellular protozoan parasite that establishes long-lasting infection in humans and animals. Because Toxoplasma infection is most often asymptomatic, it is evident that this parasite has developed sophisticated ways to manipulate host immunity. Recently, the parasite ROP16 kinase was identified as an important determinant of host cell signaling. During cell invasion, ROP16 is injected into the host cell cytoplasm and subsequently localizes to the nucleus. Here, we report the generation of ROP16 knockout parasites (ΔROP16) as well as ΔROP16 complementation mutants (ΔROP16:1) and we describe the biological effects of deleting and re-inserting this molecule. We find that ROP16 controls the ability to activate multiple host cell signaling pathways and simultaneously suppress macrophage proinflammatory responses. Deletion of ROP16 increases parasite ability to replicate and disseminate during in vivo infection. This increased growth response may arise from ROP16-dependent activation of host arginase-1. Induction of arginase-1 limits availability of arginine, an amino acid that is required for parasite growth and host-inducible nitric oxide production. Our results provide new insight into the complex interactions between an intracellular eukaryotic pathogen and its host cell.

The Toxoplasma gondii population consists of multiple strains, defined by genotype and virulence. Previous studies have established that protective immunity to this organism is mediated by IL-12, which drives T cells to produce IFN-γ. Paradoxically, while type I and type II strains of T. gondii both induce IL-12 and IFN-γ in the mouse, type I parasites are lethal, whereas type II strains establish chronic infection. The cellular basis for these strain-dependent differences remains unclear. To better understand these events, the CD8+ T cell and dendritic cell (DC) responses to transgenic, OVA-expressing type I RH (RH OVA) and type II Prugniuad (Pru OVA) parasites were examined. Pru OVA-infected mice developed a robust DC response at the site of infection and the draining lymph node and generated a population of endogenous OVA-specific CD8+ T cells. In contrast, RH OVA-infected mice had fewer DC and OVA-specific CD8+ T cells. RH OVA-infected mice given pre-activated OVA-specific CD8+ T cells were protected, suggesting that reduced DC-derived signals contributed to the low OVA-specific CD8+ T cell numbers observed during type I infection. Indeed, DC depletion prior to Pru OVA infection resulted in a failure to generate activated OVA-specific CD8+ T cells, and IL-12p70 treatment during RH OVA infection modestly increased the number of Ag-specific cells. Together, these data are consistent with a model of immunity to T. gondii in which strain-dependent DC responses shape the generation of Ag-specific CD8+ T cells and determine the outcome of infection.

Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8+ T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8+ T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8+ T-cell activation was assayed using ‘B3Z’ reporter cells in vitro, or adoptively-transferred OVA-specific ‘OT-I’ CD8+ T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8+ T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8+ T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products.

Toxoplasma gondii is an intracellular protozoan parasite that infects a wide variety of warm-blooded hosts and can have devastating effects in the developing fetus as well as the immunocompromised host. An appreciation of how this organism interacts with the host immune system is crucial to understanding the pathogenesis of this disease. The last decade has been marked by the application of various imaging techniques, such as bioluminescent imaging as well as confocal and multiphoton microscopy to study toxoplasmosis. The ability to manipulate parasites to express fluorescent/bioluminescent markers or model antigens/enzymes combined with the development of reporter mice that allow the detection of distinct immune populations have been crucial to the success of many of these studies. These approaches have permitted the visualization of parasites and immune cells in real-time and provided new insights into the nature of host–pathogen interactions. This article highlights some of the advances in imaging techniques, their strengths and weaknesses, and how these techniques have impacted our understanding of the interaction between parasites and various immune populations during toxoplasmosis.