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Introduction: Toxoplasma gondii induces a strong CD8 T cell response characterized by the secretion of IFNγ that promotes host survival during infection. The initiation of CD8 T cell IFNγ responses in vitro differs widely between clonal lineage strains of T. gondii, in which type I strains are low inducers, while types II and III strains are high inducers. We hypothesized this phenotype is due to a polymorphic "Regulator Of CD8 T cell Response" (ROCTR). Methods: Therefore, we screened F1 progeny from genetic crosses between the clonal lineage strains to identify ROCTR. Naïve antigen-specific CD8 T cells (T57) isolated from transnuclear mice, which are specific for the endogenous and vacuolar TGD057 antigen, were measured for their ability to become activated, transcribe Ifng and produce IFNγ in response to T. gondii infected macrophages. Results: Genetic mapping returned four non-interacting quantitative trait loci (QTL) with small effect on T. gondii chromosomes (chr) VIIb-VIII, X and XII. These loci encompass multiple gene candidates highlighted by ROP16 (chrVIIb-VIII), GRA35 (chrX), TgNSM (chrX), and a pair of uncharacterized NTPases (chrXII), whose locus we report to be significantly truncated in the type I RH background. Although none of the chromosome X and XII candidates bore evidence for regulating CD8 T cell IFNγ responses, type I variants of ROP16 lowered Ifng transcription early after T cell activation. During our search for ROCTR, we also noted the parasitophorous vacuole membrane (PVM) targeting factor for dense granules (GRAs), GRA43, repressed the response suggesting PVM-associated GRAs are important for CD8 T cell activation. Furthermore, RIPK3 expression in macrophages was an absolute requirement for CD8 T cell IFNγ differentiation implicating the necroptosis pathway in T cell immunity to T. gondii. Discussion: Collectively, our data suggest that while CD8 T cell IFNγ production to T. gondii strains vary dramatically, it is not controlled by a single polymorphism with strong effect. However, early in the differentiation process, polymorphisms in ROP16 can regulate commitment of responding CD8 T cells to IFNγ production which may have bearing on immunity to T. gondii.
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Toxoplasma , Animales , Ratones , Sitios de Carácter Cuantitativo , Proteínas Protozoarias/metabolismo , Interferón gamma/metabolismo , Linfocitos T CD8-positivos , Diferenciación CelularRESUMEN
Unlike in infection and cancer, T cell exhaustion in autoimmune disease has not been clearly defined. Here we set out to understand inhibitory protein (PD-1, Tim3, CTLA4, Lag3) expression in CXCR5- and CXCR5+ CD8 and CD4 T cells in systemic lupus erythematosus. CXCR5+ CD8 and CD4 T cells express PD-1 and engage B cells in germinal center reactions, leading to autoantibody formation in autoimmunity. We hypothesized that CXCR5+ CD8 T cells develop an exhausted phenotype as SLE autoimmunity expands from initial to chronic, self-perpetuating disease due to chronic self-antigen exposure. Our results indicate that there is no exhaustion frequency differences between sexes, although disease kinetics vary by sex. CXCR5+ CD8 T cells express primarily IFNγ, known to promote autoimmune disease development, whereas CXCR5-CD8 T cells express TNFα and IFNγ as disease progresses from 2-6 months. Tim3 is the highest expressed inhibitory marker for all CD4 and CD8 T cell populations demonstrating potential for terminally exhausted populations. CTLA4 expression on CD4 T cells suggests potential tolerance induction in these cells. We identified exhaustion phenotypes within autoimmune disease that progress with increasing lupus erythematosus severity and possibly provide a feedback mechanism for immunological tolerance. Highlights: CXCR5- and CXCR5+ CD8 T cells expand with rate of disease in SLE mouse model.CXCR5+ CD8 T cells are low contributors to TNFα disease progression unlike CXCR5-CD8 T cells but may increase disease mechanisms through high IFNγ production.Inhibitory markers upregulate in frequency with the highest amounts seen in Tim3+ populations. Tim3+Lag3+ expression may be an indicator of terminal differentiation for all populations.Inhibitory marker expression frequency was unrelated to sex.
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Forward genetic approaches have been widely used in parasitology and have proven their power to reveal the complexities of host-parasite interactions in an unbiased fashion. Many aspects of the parasite's biology, including the identification of virulence factors, replication determinants, antibiotic resistance genes, and other factors required for parasitic life, have been discovered using such strategies. Forward genetic approaches have also been employed to understand host resistance mechanisms to parasitic infection. Here, we will introduce and review all forward genetic approaches that have been used to identify host factors involved with Apicomplexa infections, which include classical genetic screens and QTL mapping, GWAS, ENU mutagenesis, overexpression, RNAi and CRISPR-Cas9 library screens. Collectively, these screens have improved our understanding of host resistance mechanisms, immune regulation, vaccine and drug designs for Apicomplexa parasites. We will also discuss how recent advances in molecular genetics give present opportunities to further explore host-parasite relationships.
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Apicomplexa , Pruebas Genéticas , Apicomplexa/genética , Biología , Interacciones Huésped-Parásitos/genética , MutagénesisRESUMEN
Protective immunity to parasitic infections has been difficult to elicit by vaccines. Among parasites that evade vaccine-induced immunity is Toxoplasma gondii, which causes lethal secondary infections in chronically infected mice. Here we report that unlike susceptible C57BL/6J mice, A/J mice were highly resistant to secondary infection. To identify correlates of immunity, we utilized forward genetics to identify Nfkbid, a nuclear regulator of NF-κB that is required for B cell activation and B-1 cell development. Nfkbid-null mice ("bumble") did not generate parasite-specific IgM and lacked robust parasite-specific IgG, which correlated with defects in B-2 cell maturation and class-switch recombination. Though high-affinity antibodies were B-2 derived, transfer of B-1 cells partially rescued the immunity defects observed in bumble mice and were required for 100% vaccine efficacy in bone marrow chimeric mice. Immunity in resistant mice correlated with robust isotype class-switching in both B cell lineages, which can be fine-tuned by Nfkbid gene expression. We propose a model whereby humoral immunity to T. gondii is regulated by Nfkbid and requires B-1 and B-2 cells for full protection.
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Susceptibilidad a Enfermedades/inmunología , Proteínas I-kappa B/inmunología , Inmunidad Humoral/inmunología , Toxoplasmosis Animal/inmunología , Animales , Linfocitos B/inmunología , Ratones , ToxoplasmaRESUMEN
IL-2Rα, in part, comprises the high affinity receptor for IL-2, a cytokine important in immune proliferation, activation, and regulation. IL-2Rα deficient mice (IL-2Rα-KO) develop systemic autoimmune disease and die from severe anemia between 18 and 80 days of age. These mice develop kinetically distinct autoimmune progression, with approximately a quarter dying by 21 days of age and half dying after 30 days. This research aims to define immune parameters and cytokine signaling that distinguish cohorts of IL-2Rα-KO mice that develop early- versus late-stage autoimmune disease. To investigate these differences, we evaluated complete blood counts (CBC), antibody binding of RBCs, T cell numbers and activation, hematopoietic progenitor changes, and signaling kinetics, during autoimmune hemolytic anemia (AIHA) and bone marrow failure. We identified several alterations that, when combined, correlate to disease kinetics. Early onset disease correlates with anti-RBC antibodies, lower hematocrit, and reduced IL-7 signaling. CD8 regulatory T cells (Tregs) have enhanced apoptosis in early disease. Further, early and late end stage disease, while largely similar, had several differences suggesting distinct mechanisms drive autoimmune disease kinetics. Therefore, IL-2Rα-KO disease pathology rates, driven by T cell signaling, promote effector T cell activation and expansion and Treg dysfunction.
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Subunidad alfa del Receptor de Interleucina-2/deficiencia , Transducción de Señal , Linfocitos T Reguladores/inmunología , Animales , Apoptosis , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Eritrocitos/metabolismo , Memoria Inmunológica , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Cinética , Activación de Linfocitos/inmunología , Ratones Endogámicos BALB C , Ratones Noqueados , Timo/crecimiento & desarrolloRESUMEN
Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite's protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.
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Linfocitos T CD8-positivos/inmunología , Inflamasomas/inmunología , Interferón gamma/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Animales , Linfocitos T CD8-positivos/parasitología , Femenino , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteínas Protozoarias/genética , Toxoplasmosis Animal/parasitología , Vacuolas/inmunología , Vacuolas/metabolismo , Vacuolas/parasitología , Virulencia/inmunologíaRESUMEN
Over the last century, the alarming surge in allergy and autoimmune disease has led to the hypothesis that decreasing exposure to microbes, which has accompanied industrialization and modern life in the Western world, has fundamentally altered the immune response. In its current iteration, the "hygiene hypothesis" suggests that reduced microbial exposures during early life restricts the production and differentiation of immune cells suited for immune regulation. Although it is now well-appreciated that the increase in hypersensitivity disorders represents a "perfect storm" of many contributing factors, we argue here that two important considerations have rarely been explored. First, the window of microbial exposure that impacts immune development is not limited to early childhood, but likely extends into the womb. Second, restricted microbial interactions by an expectant mother will bias the fetal immune system toward hypersensitivity. Here, we extend this discussion to hypothesize that the cell types sensing microbial exposures include fetal hematopoietic stem cells, which drive long-lasting changes to immunity.
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Feto/inmunología , Hipótesis de la Higiene , Hipersensibilidad/inmunología , Sistema Inmunológico/inmunología , Adulto , Niño , Femenino , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/microbiología , Interacciones Microbiota-Huesped/inmunología , Humanos , Recién Nacido , Inflamación/inmunología , Interacciones Microbianas/inmunología , Embarazo , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
T cell exhaustion is a state of hyporesponsiveness that develops during many chronic infections and cancer. Neutralization of inhibitory receptors, or "checkpoint blockade," can reverse T cell exhaustion and lead to beneficial prognoses in experimental and clinical settings. Whether checkpoint blockade can resolve lethal acute infections is less understood but may be beneficial in vaccination protocols that fail to elicit sterilizing immunity. Since a fully protective vaccine for any human parasite has yet to be developed, we explored the efficacy of checkpoint inhibitors in a mouse model of Toxoplasma gondii reinfection. Mice chronically infected with an avirulent type III strain survive reinfection with the type I RH strain but not the MAS, GUY-DOS, and GT1 parasite strains. We report here that mouse susceptibility to secondary infection correlates with the initial parasite burden and that protection against the RH strain is dependent on CD8 but not CD4 T cells in this model. When given a lethal secondary infection, CD8 and CD4 T cells upregulate several coinhibitory receptors, including PD-1, TIM-3, 4-1bb, and CTLA-4. Moreover, the gamma interferon (IFN-γ) response of CD8 but not CD4 T cells is significantly reduced during secondary infection with virulent strains, suggesting that checkpoint blockade may reduce disease severity. However, single and combination therapies targeting TIM-3, CTLA-4, and/or PD-L1 failed to reverse susceptibility to secondary infection. These results suggest that additional host responses, which are refractory to checkpoint blockade, are likely required for immunity to this pathogen.
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Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Toxoplasma/patogenicidad , Toxoplasmosis Animal/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
For parasites that sequester themselves within a vacuole, new rules governing antigen presentation are coming into focus. Components of the host's autophagy machinery and the parasite's membranous nanotubular network within the parasitophorous vacuole play a major role in determining antigenicity of Toxoplasma proteins. As such, both parasite and vaccinologist may exploit these pathways to regulate the ever important CD8 T cell response to apicomplexan parasites.
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Antígenos/inmunología , Apicomplexa/inmunología , Vacunas Antiprotozoos , Animales , Autofagia , Linfocitos T CD8-positivos/inmunología , HumanosRESUMEN
UNLABELLED: The intracellular parasite Toxoplasma gondii infects a wide variety of vertebrate species globally. Infection in most hosts causes a lifelong chronic infection and generates immunological memory responses that protect the host against new infections. In regions where the organism is endemic, multiple exposures to T. gondii likely occur with great frequency, yet little is known about the interaction between a chronically infected host and the parasite strains from these areas. A widely used model to explore secondary infection entails challenge of chronically infected or vaccinated mice with the highly virulent type I RH strain. Here, we show that although vaccinated or chronically infected C57BL/6 mice are protected against the type I RH strain, they are not protected against challenge with most strains prevalent in South America or another type I strain, GT1. Genetic and genomic analyses implicated the parasite-secreted rhoptry effectors ROP5 and ROP18, which antagonize the host's gamma interferon-induced immunity-regulated GTPases (IRGs), as primary requirements for virulence during secondary infection. ROP5 and ROP18 promoted parasite superinfection in the brains of challenged survivors. We hypothesize that superinfection may be an important mechanism to generate T. gondii strain diversity, simply because two parasite strains would be present in a single meal consumed by the feline definitive host. Superinfection may drive the genetic diversity of Toxoplasma strains in South America, where most isolates are IRG resistant, compared to North America, where most strains are IRG susceptible and are derived from a few clonal lineages. In summary, ROP5 and ROP18 promote Toxoplasma virulence during reinfection. IMPORTANCE: Toxoplasma gondii is a widespread parasite of warm-blooded animals and currently infects one-third of the human population. A long-standing assumption in the field is that prior exposure to this parasite protects the host from subsequent reexposure, due to the generation of protective immunological memory. However, this assumption is based on clinical data and mouse models that analyze infections with strains common to Europe infections with strains common to Europe and North America. In contrast, we found that the majority of strains sampled from around the world, in particular those from South America, were able to kill or reinfect the brains of hosts previously exposed to T. gondii. The T. gondii virulence factors ROP5 and ROP18, which inhibit key host effectors that mediate parasite killing, were required for these phenotypes. We speculate that these results underpin clinical observations that pregnant women previously exposed to Toxoplasma can develop congenital infection upon reexposure to South American strains.
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Alelos , Coinfección , Proteínas Protozoarias/genética , Sobreinfección , Toxoplasma/genética , Toxoplasma/patogenicidad , Toxoplasmosis Animal/parasitología , Animales , Ratones Endogámicos C57BL , América del Norte , América del Sur , VirulenciaRESUMEN
The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterise the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice.
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Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Toxoplasmosis Animal/parasitología , Animales , Femenino , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Proteínas Protozoarias/genética , Toxoplasma/química , Toxoplasma/genéticaRESUMEN
Alternative splicing and mRNA editing are known to contribute to transcriptome diversity. Although alternative splicing is pervasive and contributes to a variety of pathologies, including cancer, the genetic context for individual differences in isoform usage is still evolving. Similarly, although mRNA editing is ubiquitous and associated with important biological processes such as intracellular viral replication and cancer development, individual variations in mRNA editing and the genetic transmissibility of mRNA editing are equivocal. Here, we have used linkage analysis to show that both mRNA editing and alternative splicing are regulated by the macrophage genetic background and environmental cues. We show that distinct loci, potentially harboring variable splice factors, regulate the splicing of multiple transcripts. Additionally, we show that individual genetic variability at the Apobec1 locus results in differential rates of C-to-U(T) editing in murine macrophages; with mouse strains expressing mostly a truncated alternative transcript isoform of Apobec1 exhibiting lower rates of editing. As a proof of concept, we have used linkage analysis to identify 36 high-confidence novel edited sites. These results provide a novel and complementary method that can be used to identify C-to-U editing sites in individuals segregating at specific loci and show that, beyond DNA sequence and structural changes, differential isoform usage and mRNA editing can contribute to intra-species genomic and phenotypic diversity.
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Empalme Alternativo , Citidina Desaminasa/genética , Macrófagos/metabolismo , Ratones/genética , Edición de ARN , Desaminasas APOBEC-1 , Animales , Citosina/metabolismo , Ligamiento Genético , Variación Genética , Genoma , Interferón gamma/metabolismo , Macrófagos/parasitología , Ratones Endogámicos C57BL , Sitios de Carácter Cuantitativo , Isoformas de ARN/genética , Toxoplasma/fisiología , Uracilo/metabolismoRESUMEN
Toxoplasma gondii transmission between intermediate hosts is dependent on the ingestion of walled cysts formed during the chronic phase of infection. Immediately following consumption, the parasite must ensure survival of the host by preventing adverse inflammatory responses and/or by limiting its own replication. Since the Toxoplasma secreted effectors rhoptry 16 kinase (ROP16) and dense granule 15 (GRA15) activate the JAK-STAT3/6 and NF-κB signaling pathways, respectively, we explored whether a particular combination of these effectors impacted intestinal inflammation and parasite survival in vivo. Here we report that expression of the STAT-activating version of ROP16 in the type II strain (strain II+ROP16I) promotes host resistance to oral infection only in the context of endogenous GRA15 expression. Protection was characterized by a lower intestinal parasite burden and dampened inflammation. Host resistance to the II+ROP16I strain occurred independently of STAT6 and the T cell coinhibitory receptors B7-DC and B7-H1, two receptors that are upregulated by ROP16. In addition, coexpression of ROP16 and GRA15 enhanced parasite susceptibility within tumor necrosis factor alpha/gamma interferon-stimulated macrophages in a STAT3/6-independent manner. Transcriptional profiling of infected STAT3- and STAT6-deficient macrophages and parasitized Peyer's patches from mice orally challenged with strain II+ROP16I suggested that ROP16 activated STAT5 to modulate host gene expression. Consistent with this supposition, the ROP16 kinase induced the sustained phosphorylation and nuclear localization of STAT5 in Toxoplasma-infected cells. In summary, only the combined expression of both GRA15 and ROP16 promoted host resistance to acute oral infection, and Toxoplasma may possibly target the STAT5 signaling pathway to generate protective immunity in the gut.
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Antígenos de Protozoos/metabolismo , Inflamación/patología , Intestinos/patología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Toxoplasmosis Animal/parasitología , Animales , Antígenos de Protozoos/genética , Citocinas/genética , Citocinas/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Noqueados , Ganglios Linfáticos Agregados/parasitología , Proteínas Tirosina Quinasas/genética , Proteínas Protozoarias/genética , Transducción de Señal , Toxoplasmosis Animal/patologíaRESUMEN
BACKGROUND: Accurate gene model predictions and annotation of alternative splicing events are imperative for genomic studies in organisms that contain genes with multiple exons. Currently most gene models for the intracellular parasite, Toxoplasma gondii, are based on computer model predictions without cDNA sequence verification. Additionally, the nature and extent of alternative splicing in Toxoplasma gondii is unknown. In this study, we used de novo transcript assembly and the published type II (ME49) genomic sequence to quantify the extent of alternative splicing in Toxoplasma and to improve the current Toxoplasma gene annotations. RESULTS: We used high-throughput RNA-sequencing data to assemble full-length transcripts, independently of a reference genome, followed by gene annotation based on the ME49 genome. We assembled 13,533 transcripts overlapping with known ME49 genes in ToxoDB and then used this set to; a) improve the annotation in the untranslated regions of ToxoDB genes, b) identify novel exons within protein-coding ToxoDB genes, and c) report on 50 previously unidentified alternatively spliced transcripts. Additionally, we assembled a set of 2,930 transcripts not overlapping with any known ME49 genes in ToxoDB. From this set, we have identified 118 new ME49 genes, 18 novel Toxoplasma genes, and putative non-coding RNAs. CONCLUSION: RNA-seq data and de novo transcript assembly provide a robust way to update incompletely annotated genomes, like the Toxoplasma genome. We have used RNA-seq to improve the annotation of several Toxoplasma genes, identify alternatively spliced genes, novel genes, novel exons, and putative non-coding RNAs.
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Empalme Alternativo/genética , Anotación de Secuencia Molecular/métodos , ARN Largo no Codificante/genética , Toxoplasma/genética , Transcriptoma/genética , Exones/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Modelos GenéticosRESUMEN
Toxoplasma is a highly successful parasite that establishes a life-long chronic infection. To do this, it must carefully regulate immune activation and host cell effector mechanisms. Here we review the latest developments in our understanding of how Toxoplasma counteracts the immune response of the host, and in some cases provokes it, through the use of specific parasite effector proteins. An emerging theme from these discoveries is that Toxoplasma effectors are master regulators of the pro-inflammatory response, which elicits many of the toxoplasmacidal mechanisms of the host. We speculate that combinations of these effectors present in certain Toxoplasma strains work to maintain an optimal parasite burden in different hosts to ensure parasite transmission.
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Interacciones Huésped-Parásitos/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Humanos , Toxoplasma/patogenicidad , Toxoplasma/fisiología , Toxoplasmosis/parasitología , Virulencia , Factores de Virulencia/inmunologíaRESUMEN
European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, type II, and type 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.
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Intestinos/inmunología , Macrófagos/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Línea Celular , Femenino , Humanos , Intestinos/parasitología , Activación de Macrófagos , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Proteínas Protozoarias/genética , Especificidad de la Especie , Toxoplasma/clasificación , Toxoplasma/genética , Toxoplasmosis/parasitologíaRESUMEN
NF-κB is an integral component of the immune response to Toxoplasma gondii. Although evidence exists that T. gondii can directly modulate the NF-κB pathway, the parasite-derived effectors involved are unknown. We determined that type II strains of T. gondii activate more NF-κB than type I or type III strains, and using forward genetics we found that this difference is a result of the polymorphic protein GRA15, a novel dense granule protein which T. gondii secretes into the host cell upon invasion. A GRA15-deficient type II strain has a severe defect in both NF-κB nuclear translocation and NF-κB-mediated transcription. Furthermore, human cells expressing type II GRA15 also activate NF-κB, demonstrating that GRA15 alone is sufficient for NF-κB activation. Along with the rhoptry protein ROP16, GRA15 is responsible for a large part of the strain differences in the induction of IL-12 secretion by infected mouse macrophages. In vivo bioluminescent imaging showed that a GRA15-deficient type II strain grows faster compared with wild-type, most likely through its reduced induction of IFN-γ. These results show for the first time that a dense granule protein can modulate host signaling pathways, and dense granule proteins can therefore join rhoptry proteins in T. gondii's host cell-modifying arsenal.
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Proteínas Protozoarias/metabolismo , Transducción de Señal , Toxoplasma/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Células Cultivadas , Regulación de la Expresión Génica , Genoma de Protozoos , Humanos , Interleucina-12/biosíntesis , Ratones , Ratones Noqueados , Transporte de Proteínas , Toxoplasma/genética , Factor de Transcripción ReIA/deficiencia , Transcripción GenéticaRESUMEN
The T cell receptor (TCR) and associated CD3gammaepsilon, deltaepsilon, and zetazeta signaling dimers allow T cells to discriminate between different antigens and respond accordingly, but our knowledge of how these parts fit and work together is incomplete. In this study, we provide additional evidence that the CD3 heterodimers congregate on one side of the TCR in both the alphabeta and gammadeltaTCR-CD3 complexes. We also report that the other side of the alphabetaTCR mediates homotypic alphabetaTCR interactions and signaling. Specifically, an erythropoietin receptor-based dimerization assay was used to show that, upon complex assembly, the CD3epsilon chains of two CD3 heterodimers are arranged side-by-side in both the alphabeta and gammadeltaTCR-CD3 complexes. This system was also used to show that alphabetaTCRs can dimerize in the cell membrane and that mutating the unusual outer strands of the Calpha domain impairs this dimerization. Finally, we present data showing that, for CD4 T cells, the mutations that impair alphabetaTCR dimerization also alter ligand-induced calcium mobilization, TCR accumulation at the site of pMHC contact, and polarization toward the site of antigen contact. These data reveal a "functional-sidedness" to the alphabetaTCR constant region, with dimerization occurring on the side of the TCR opposite from where the CD3 heterodimers are located.
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Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Animales , Células Presentadoras de Antígenos/citología , Complejo CD3/metabolismo , Señalización del Calcio , Línea Celular , Membrana Celular/metabolismo , Polaridad Celular , Humanos , Espacio Intracelular/metabolismo , Ratones , Modelos Moleculares , Mutación/genética , Multimerización de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/citologíaRESUMEN
gammadelta Tau cells, together with alphabeta Tau cells, are abundantly present in the epithelial layer of the small intestine (IEL) and are essential for the host's first line of defense. Whether or not gammadelta IELs, like alphabeta IELs, are derived from thymocytes that encounter self-Ags in the thymus is unclear. In this study, we report that a natural population of gammadelta T cells that are specific for the nonclassical MHC class I molecules T10 and T22 are present in the IEL compartment of mice that do not express T10/T22. Furthermore, the small intestinal homing receptor CCR9 is preferentially expressed on gammadelta thymocytes that have yet to encounter a ligand, and gammadelta thymocytes with high affinity for self-ligand are CCR9(low). These observations suggest that the Ag-specific repertoire of gammadelta IELs is not biased toward thymic Ags. Instead, gammadelta IELs appear suited to respond to novel Ags revealed in pathological settings.
Asunto(s)
Antígenos/inmunología , Epitelio/inmunología , Intestino Delgado/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Timo/inmunología , Secuencia de Aminoácidos , Animales , Movimiento Celular/inmunología , Humanos , Intestino Delgado/citología , Células Jurkat , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T gamma-delta/química , Receptores CCR/deficiencia , Receptores CCR/genética , Receptores CCR/inmunología , Receptores CCR/metabolismoRESUMEN
gammadelta T cells contribute uniquely to host immune defense, but how they do so remains unclear. Recent work suggests that thymic selection does little to constrain gammadelta T cell antigen specificities, but instead determines their effector fate. When triggered through the T cell receptor, ligand-experienced cells make IFNgamma, whereas ligand-naïve gammadelta T cells produce IL-17, a major initiator of inflammation. These advances warrant a fresh look at how gammadelta T cells may function in the immune system.