Dataset Information

Transcriptional Responses in the Murine Spleen after Toxoplasma gondii Infection: Inflammasome and Mucus-Associated Genes.

ABSTRACT: The spleen plays an important role in coordinating both adaptive and innate immune responses. Here, the transcriptional response to T. gondii infection in the murine spleen was characterized concerning inflammasome sensors (two different models: seven days after oral or four weeks after intraperitoneal infection). Additionally, Tff1KO and Tff3KO mice were investigated because TFF genes are often upregulated during inflammation. The expression of the pattern-recognition receptors Nlrp3, Nlrp12, and Nlrp1a was significantly increased after infection. This increase was diminished in Tff1KO and Tff3KO mice pointing towards a positive regulation of the inflammatory response by Tff1 and Tff3. Furthermore, the transcription of Tff1 (encoding a motogenic lectin) and other secretory genes was analyzed, i.e., gastrokines (Gkn), IgG Fc binding protein (Fcgbp), and the mucin Muc2. The corresponding gene products belong to an interactome protecting mucous epithelia. Tff1 was significantly induced after infection, which might increase the motility of immune cells. In contrast, Gkn3, Fcgbp, and Muc2 were downregulated seven days after oral infection; whereas four weeks after i.p. infection only Gkn3 remained downregulated. This might be an indication that Gkn3, Fcgbp, and Muc2 are involved in the transient disruption of the splenic architecture and its reorganization, which is characteristic after T. gondii infection.

SUBMITTER: Znalesniak EB

PROVIDER: S-EPMC5486068 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Transcriptional Responses in the Murine Spleen after Toxoplasma gondii Infection: Inflammasome and Mucus-Associated Genes.

Znalesniak Eva B EB Fu Ting T Salm Franz F Händel Ulrike U Hoffmann Werner W

International journal of molecular sciences 20170610 6

The spleen plays an important role in coordinating both adaptive and innate immune responses. Here, the transcriptional response to T. gondii infection in the murine spleen was characterized concerning inflammasome sensors (two different models: seven days after oral or four weeks after intraperitoneal infection). Additionally, Tff1KO and Tff3KO mice were investigated because TFF genes are often upregulated during inflammation. The expression of the pattern-re ...[more]

PMID: 28604600

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Project description:BackgroundToxoplasma gondii is capable of persisting in the brain, although it is efficiently eliminated by cellular immune responses in most other sites. While Toll-like receptor 2 (TLR2) reportedly plays important roles in protective immunity against the parasite, the relationship between neurological disorders induced by T. gondii infection and TLR2 function in the brain remains controversial with many unknowns. In this study, primary cultured astrocytes, microglia, neurons, and peritoneal macrophages obtained from wild-type and TLR2-deficient mice were exposed to T. gondii tachyzoites. To characterize TLR2-dependent functional pathways activated in response to T. gondii infection, gene expression of different cell types was profiled by RNA sequencing.ResultsDuring T. gondii infection, a total of 611, 777, 385, and 1105 genes were upregulated in astrocytes, microglia, neurons, and macrophages, respectively, while 163, 1207, 158, and 1274 genes were downregulated, respectively, in a TLR2-dependent manner. Overrepresented Gene Ontology (GO) terms for TLR2-dependently upregulated genes were associated with immune and stress responses in astrocytes, immune responses and developmental processes in microglia, metabolic processes and immune responses in neurons, and metabolic processes and gene expression in macrophages. Overrepresented GO terms for downregulated genes included ion transport and behavior in astrocytes, cell cycle and cell division in microglia, metabolic processes in neurons, and response to stimulus, signaling and cell motility in macrophages.ConclusionsTo our knowledge, this is the first transcriptomic study of TLR2 function across different cell types during T. gondii infection. Results of RNA-sequencing demonstrated roles for TLR2 varied by cell type during T. gondii infection. Our findings facilitate understanding of the detailed relationship between TLR2 and T. gondii infection, and elucidate mechanisms underlying neurological changes during infection.

Project description:Induction of immunity that limits Toxoplasma gondii infection in mice is critically dependent on the activation of the innate immune response. In this study, we investigated the role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain (NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3 inflammasome, resulting in the rapid production and cleavage of interleukin-1? (IL-1?), with no measurable cleavage of IL-18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice produced large quantities of IL-18 but had no measurable IL-1? in their serum. Infection of mice deficient in NLRP3, caspase-1/11, IL-1R, or the inflammasome adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly, mice deficient in NLRP1 also displayed increased parasite loads and acute mortality. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis.Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain "sensor" proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the proinflammatory cytokines interleukin-1? (IL-1?) and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. We report here that T. gondii is a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. Using mice deficient in IL-18 and IL-18R, we show that the IL-18 cytokine plays a pivotal role by limiting parasite replication to promote murine survival.

Project description:BackgroundThe obligate intracellular parasite Toxoplasma gondii establishes a life-long chronic infection within any warm-blooded host. After ingestion of an encysted parasite, T. gondii disseminates throughout the body as a rapidly replicating form during acute infection. Over time and after stimulation of the host immune response, T. gondii differentiates into a slow growing, cyst form that is the hallmark of chronic infection. Global transcriptome analysis of both host and parasite during the establishment of chronic T. gondii infection has not yet been performed. Here, we conducted a dual RNA-seq analysis of T. gondii and its rodent host to better understand host and parasite responses during acute and chronic infection.ResultsWe obtained nearly one billion paired-end RNA sequences from the forebrains of uninfected, acutely and chronically infected mice, then aligned them to the genomic reference files of both T. gondii and Mus musculus. Gene ontology (GO) analysis of the 100 most highly expressed T. gondii genes showed less than half were shared between acute and chronic infection. The majority of the highly expressed genes common in both acute and chronic infection were involved in transcription and translation, underscoring that parasites in both stages are actively synthesizing proteins. Similarly, most of the T. gondii genes highly expressed during chronic infection were involved in metabolic processes, again highlighting the activity of the cyst stage at 28 days post-infection. Comparative analyses of host genes using uninfected forebrain revealed over twice as many immune regulatory genes were more abundant during chronic infection compared to acute. This demonstrates the influence of parasite development on host gene transcription as well as the influence of the host environment on parasite gene transcription.ConclusionsRNA-seq is a valuable tool to simultaneously analyze host and microbe transcriptomes. Our data shows that T. gondii is metabolically active and synthesizing proteins at 28 days post-infection and that a distinct subset of host genes associated with the immune response are more abundant specifically during chronic infection. These data suggest host and pathogen interplay is still present during chronic infection and provides novel T. gondii targets for future drug and vaccine development.

Project description:Tools for tuning endogenous gene expression are key to determining the genetic basis of diverse cellular phenotypes. Although synthetic regulatable promoters are available in Toxoplasma, scalable methods for targeted and combinatorial downregulation of gene expression-like RNA interference-have yet to be developed. To investigate the feasibility of CRISPR-mediated transcriptional regulation, we examined the function of two catalytically inactive Cas9 (dCas9) orthologs, from Streptococcus pyogenes and Streptococcus thermophilus, in Toxoplasma. Following the addition of single-guide RNAs (sgRNAs) targeting the promoter and 5' untranslated region (UTR) of the surface antigen gene SAG1, we profiled changes in protein abundance of targeted genes by flow cytometry for transcriptional reporters and immunoblotting. We found that the dCas9 orthologs generated a range of target gene expression levels, and the degree of repression was durable and stably inherited. Therefore, S. pyogenes and S. thermophilus dCas9 can effectively produce intermediate levels of gene expression in Toxoplasma. The distinct sgRNA scaffold requirements of the two dCas9s permit their orthogonal use for simultaneous examination of two distinct loci through transcriptional modulation, labeling for microscopy-based studies, or other dCas9-based approaches. Taking advantage of newly available genomic transcription start site data, these tools will aid in the development of new loss-of-function screening approaches in Toxoplasma. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular parasite of humans and animals that causes life-threatening disease in immunocompromised patients, fetal abnormalities when contracted during gestation, and recurrent eye lesions in some patients. Despite its health implications, about half of the Toxoplasma genome still lacks functional annotation. A particularly powerful tool for the investigation of an organism's cell biology is the modulation of gene expression, which can produce the subtle phenotypes often required for informing gene function. In Toxoplasma, such tools have limited throughput and versatility. Here, we detail the adaptation of a new set of tools based on CRISPR-Cas9, which allows the targeted downregulation of gene expression in Toxoplasma. With its scalability and adaptability to diverse genomic loci, this approach has the potential to greatly accelerate the functional characterization of the Toxoplasma genome.

Dataset Information

Transcriptional Responses in the Murine Spleen after Toxoplasma gondii Infection: Inflammasome and Mucus-Associated Genes.

Publications

Transcriptional Responses in the Murine Spleen after Toxoplasma gondii Infection: Inflammasome and Mucus-Associated Genes.

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