Expression data from cyclohexamide treated Human Foreskin Fibroblasts (HFFs) infected with type I Toxoplasma gondii and stimulated with interferon gamma.
ABSTRACT: Pre-infection with Toxoplasma parasites inhibits induction of an interferon gamma responsive gene expression program. In this study we aimed to determine what role host negative regulatory proteins that are induced by Toxoplasma infection, such as SOCS-1, have in this inhibition. We used Affymetrix microarrays to analyze host cell transcription after pre-treatment with the protein synthesis inhibitor cyclohexamide (CHX), Toxoplasma infection, and subsequent interferon-gamma stimulation. Human foreskin fibroblasts (HFFs) were left untreated or treated with CHX for 40min. Cells were infected, or not, with type I (RH) Toxoplasma parasites, for one hour, and subsequently stimulated with human IFN-γ for one hour. Total RNA was isolated and hybridized to Affymetrix Human U133A 2.0 gene chips.
Project description:Toxoplasma strains are known to inhibit the expression of several interferon-gamma induced genes, and a type II strain was shown to dysregulate genome-wide responses to interferon-gamma in human fibroblasts (Kim et al., 2007, J Immunol.). In this study we aimed to determine the effect of infection with three clonal lineages of Toxoplasma, type I, II, and III strains on genome-wide interferon-gamma induced transcription in murine macrophages. We also assessed the effect of the two main Toxoplasma modulators of mouse macrophage transcription, ROP16 and GRA15 (Jensen et al., 2011, Cell Host Microbe). We used Affymetrix microarrays to analyze host cell transcription after Toxoplasma infection and interferon-gamma stimulation. RAW264.7 murine macrophages were left uninfected or infected with type I (RH), type I ∆rop16 (RH ∆rop16), type II (Pru), type II ∆gra15 (Pru ∆gra15), or type II (CEP) parasites at an MOI ~5 for 18 hours and subsequently stimulated with murine IFN-γ for six hours. Plaque assays were done to assess parasite viability. Total RNA was isolated and hybridized to Affymetrix Mouse 430A 2.0 gene chips.
Project description:An early hallmark of Toxoplasma infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of Toxoplasma-targeting activities in immune and non-immune cells, but can also contribute to host immune pathology. Toxoplasma has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ-stimulated genes. We now have identified TgIST as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the NuRD transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1(+) inflammatory monocytes thus highlighting the protective role of TgIST against IFN-γ-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how Toxoplasma has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism. HFF, 2fTGH (STAT1+/+) and U3A (STAT1-/-) human cells were left uninfected or infected for 24 hours with 76KGFP and 76KGFPΔTgIST Toxoplasma strains and stimulated with 100 U/ml IFN-γ for 6 hours before gene expression was measured. Three independent experiments were performed for each condition.
Project description:Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites’ replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors. Comparison of 4 different RNA pools with a 2-Color-Loop Design including 10 microarrays:  T. gondii infected and IFN-gamma treated,  T. gondii infected and untreated,  Non-infected and IFN-gamma treated, and  Non-infected and untreated.
Project description:Toxoplasma gondii is a ubiquitous protozoan pathogen able to infect both mammalian and avian hosts. Surprisingly, just three strains appear to account for the majority of isolates from Europe and N. America. To test the hypothesis that strain divergence might be driven by differences between mammalian and avian response to infection, we examine in vitro strain-dependent host responses in a representative avian host, the chicken. To identify parasite drivers of strain-dependent host response, QTL mapping was used; analysis revealed a locus on Toxoplasma chromosome VIIb. To determine whether this was the parasite gene ROP16, array analysis was performed on chicken embryonic fibroblasts infected with Type I parasites and ROP16-KO parasites (of a Type I background). Chicken embryonic fibroblasts were cultivated in vitro and infected with either Type I (RH) parasites or Type I ROP16-KO parasites; ROP16-dependent host transcriptional responses were then analyzed at 5 hours post-infection.
Project description:Toxoplasma strains have been shown to modulate host cell transcription. We have found a type II Toxoplasma gene, GRA15, which activates the nuclear translocation of the NF-kappaB p65 transcription factor. We used microarrays to determine how GRA15II modulates host cell transcription. HFFs were infected with type I (RH), type I GRA15II (RH GRA15II), type II (Pru), type II GRA15KO (Pru GRA15KO), type III (cep), or type III GRA15II (cep GRA15II) parasites for 18-24 hours. Some samples were also stimulated with TNF-alpha. Total RNA was isolated and hybridized to Affymetrix GeneChip Human Genome U133A 2.0 arrays.
Project description:Toxoplasma gondii is a ubiquitous protozoan pathogen able to infect both mammalian and avian hosts. Surprisingly, just three strains appear to account for the majority of isolates from Europe and N. America. To test the hypothesis that strain divergence might be driven by differences between mammalian and avian response to infection, we examine in vitro strain-dependent host responses in a representative avian host, the chicken. Chicken embryonic fibroblasts were cultivated in vitro and infected with different strains of Toxoplasma gondii (Type II = ME49, Type III = CEP); host transcriptional responses were then analyzed at 24 hours post-infection.
Project description:Identification of genomic anchors across the MHC in untreated MRC5 fibroblasts, and fibroblasts treated with IFN-gamma, using high resolution microarrays. Identification of genomic anchors using MRC5 fibroblasts, and fibroblasts treated with IFN-gamma: Loop associated DNA vs Matrix associated DNA, 2 biological replicates
Project description:Interferon-gamma (IFN-gamma) is approved as a drug to treat chronic granulomatous disease and osteopetrosis and is also used in hyperimmunoglobulin E syndromes. These primary immunodeficiencies involve defects in neutrophils/polymorphonuclear cells. Neutrophils mature in the bone marrow and then enter the blood where they quickly undergo apoptotic cell death with a half-life of 5-10 hours. Therefore we reasoned that IFN-gamma might exert its effects on neutrophils via prolonged exposure to cells undergoing maturation toward a terminally differentiated state in the marrow rather than by its brief exposure to short-lived circulating cells. To explore this possibility we made use of PLB-985 cells which are a myeloblast-like myeloid cell line that can be differentiated into a mature, neutrophil-like state by treatment with various agents including DMSO. In initial studies we investigated transcription and protein expression in PLB-985 cells undergoing DMSO-mediated maturation in the presence or absence of IFN-gamma and identified several classes of immunity related genes that were differentially expressed in the presence of the drug and could potentially explain the immune supportive properties of IFN-gamma. Next we explored if the effect of IFN-gamma on expression of these genes is dependent on whether the cells are undergoing maturation; to do this we compared the effects of IFN-gamma on cells cultured with and without DMSO. For a subset of genes the expression level changes caused by IFN-gamma were much greater in maturing cells than non-maturing cells. These findings indicate that developmental changes associated with cell maturation can modulate the effects of IFN-gamma and supports our hypothesis that the effects of the drug on developing neutrophils in the bone marrow may be very different from its effects on mature cells in the blood. We also compared the effects on gene expression of applying IFN-gamma either during DMSO mediated PLB-985 cell maturation or after maturation i.e. IFN-gamma was applied to already mature cells. For some genes we found that IFN-gamma applied to mature cells caused large changes in expression similar to those seen when IFN-gamma was applied during maturation, however for other genes no effect was seen when drug was applied to mature cells but large effects were seen when drug was applied during DMSO treatment. This data suggests that, in the former class of genes, the effects of IFN-gamma application during maturation are largely the result of the effects of the drug on cells approaching full maturity but in the latter class of genes the effects of IFN-gamma only become apparent when it is present throughout maturation. This further supports our hypothesis that the effects of IFN-gamma are modulated by developmental changes associated with cell maturation.
Project description:IL-27 is a potent antagonist of TH1-mediated inflammation, but the basis for this effect is not fully understood. Recent studies identified a population of T-bet+ CXCR3+ Treg that limit TH1-mediated immune pathology. The studies presented here demonstrate that IL-27-mediated STAT1 activation promotes Treg expression of T-bet and CXCR3. Infection with Toxoplasma gondii induced a similar Treg population that limits T cell responses and this population at mucosal sites is IL-27-dependent. Furthermore, transfer of Treg ameliorated the infection-induced CD4+ T cell-mediated pathology observed in IL-27p28-/- mice. Although IFN-γ promoted a similar population of cells in the periphery, it did not compensate for the absence of IL-27 at mucosal sites and microarray analysis revealed that Treg exposed to either cytokine have distinct transcriptional profiles. These findings suggest that IFN-γ and IL-27 have different roles in Treg biology but define IL-27 as a key cytokine that promotes the development of Treg specialized to control TH1 immunity. Three conditions were analyzed across two timepoints. Inducible regulatory T cells (iTreg) were generated in vitro in the presence of IL-27, IFNg or under 'Neutral' conditions as a control. Samples were collected at 10 hours and 2 days during the culture period. Three biological replicates were used for each condition.
Project description:Toxoplasma strains have been shown to modulate host cell transcription. We have found a type II Toxoplasma gene, GRA15, which activates the nuclear translocation of the NF-kappaB p65 transcription factor. We used microarrays to determine how GRA15II modulates host cell transcription, and whether this transcription is dependent on the p65 transcription factor. WT or p65-/- MEFs were infected with type I (RH), type I GRA15II (RH GRA15II), type II (Pru), or type II GRA15KO (Pru GRA15KO) parasites for 18-24 hours. Total RNA was isolated and hybridized to Affymetrix GeneChip Human Genome U133A 2.0 arrays.