Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of primary human astrocytes from brain treated by stathmin, poly IC and ultrapure LPS to examine the effects of different Toll-like receptor agonists

ABSTRACT: Goal of the experiment was to examine the effects of different Toll-like receptor agonists on the human astrocyte response, in termsof levels of transcripts encoding cytokines, chemokines and theirreceptors. Stathmin and poly IC are considered as agonist for TLR3, LPSas an agonist for TLR4. The manuscript we are about to submit to Journal of Immunology contains these data, and identifies stathmin, aprotein, as a novel TLR3 agonist. We all know that poly IC is, and bycomparing the transcript responses in astrocytes to either poly IC andstathmin, and observing the similarity in these responses, the experiment adds to the evidence that stathmin indeed activates a verysimilar cellular response as poly IC. The LPS-induced response,mediated by TLR4, is included as a reference, to illustrate thatanother TLR-mediated reaction produces quite something different.

ORGANISM(S): Homo sapiens

SUBMITTER: Hans van Noort

PROVIDER: E-TABM-851 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Although viral infection is thought to be associated with subacute thyroiditis and probably with autoimmune thyroid disease, possible changes in thyroid function during the prodromal period of infection or subclinical infection remain largely unknown. Recently, it was shown that pathogen-associated molecular patterns stimulate Toll-like receptors (TLR) and activate innate immune responses by producing type I interferons (IFN). Using a human thyroid follicle culture system, in which de novo synthesized thyroid hormones are released into the culture medium under physiological concentrations of hTSH, we studied the effects of polyinosinic-polycytidylic acid (Poly(I:C)), a chemical analog of viral double-stranded RNA (dsRNA), on TSH-induced thyroid function. Thyrocytes expressed ligands for dsRNA (TLR 3, CD14, and RIG-1) comparable to the TSH receptor. DNA microarray and real-time PCR analyses revealed that dsRNA increased the expression of mRNA for TLR3, IFN-g, interferon-regulating factors, proinflammatory cytokines, and class I MHC, whereas genes associated with thyroid hormonogenesis (NIS, peroxidase, deiodinases) were suppressed. In accordance to these data, Poly(I:C) suppressed TSH-induced 125I uptake and hormone synthesis dose-dependently, accompanied by a decrease in the ratio of 125I-T3/125I-T4 released into the culture medium, whereas peptidoglycan, lipopolysaccharides, or unmethylated CpG DNA, ligands for TLR2, TLR4, and TLR9, respectively, had no significant effect. These inhibitory effects of Poly(I:C) were not blocked by a neutralizing antibody against TLR3 and an anti-IFN alpha/beta receptor antibody. These in vitro findings suggest that when thyrocytes are infected with certain viruses, dsRNA formed intracellularly in thyrocytes may be a cause for thyroid dysfunction, leading to development of autoimmune thyroiditis. This data was published in Endocrinology, vol.148, 3226-3235, 2007. Experiment Overall Design: Two conditioned experimets, control vs. double-stranded RNA (polyI-C), cultured for 6

Project description:Background: Toll-like family of receptors recognizes pathogen-associated molecular patterns (PAMPs) from different organisms. TLR4 is the receptor for bacterial lipopolysaccharide (LPS), dsRNA viral mimic poly(I:C) binds to TLR3, and bacterial CpG DNA signals through TLR9. TLR4 signaling is mediated by adaptor molecules Myd88 and TRIF while TLR3 pathway involves only the TRIF adaptor and TLR9 signals solely through Myd88. Methods: To identify genes other than those in TLR pathways that mediate macrophage response to different PAMPs, RAW264.7 cells were stimulated with LPS, poly(I:C), or CpG DNA, and RNA was profiled on microarrays 6 hrs and 24 hrs post-treatment. Gene expression data were analyzed to determine genes, pathways and transcriptional networks that are in common and unique to each of the three TLR stimuli. Potentially novel candidates revealed by this analysis were tested for their role in innate immunity using RNA interference. Results: Many genes are differentially regulated by LPS and poly(I:C) at both 6 hrs and 24 hrs following treatment, while CpG DNA elicits a much less pronounced transcriptional response. By analyzing gene expression data for networks and pathways, we prioritized differentially expressed genes that are in common to all three PAMPs as well as those shared by LPS and poly(I:C). Knockdown by RNA interference of two genes, Plec1 and TPST1, inhibited production of IL-6 in response to LPS in cultured macrophages. Conclusions: We have identified novel innate immunity genes that may be important in macrophage response to LPS, poly(I:C), and CpG DNA stimuli. Our results provide potential biomarkers and therapeutic targets that should be further investigated in mice and human populations. Keywords: time course For each treatment (Sigma LPS, LIST LPS, and media only), three biological replicates (separate macrophage cultures and RNA isolations) were profiled. Each sample was labeled with Cy3 and Cy5 and co-hybridized with Stratagene Universal Mouse Reference (dye flip techical replicates). Expression at 2 timepoints (6 and 24 hours post-treatment) was assessed.

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Transcription profiling of primary human astrocytes from brain treated by stathmin, poly IC and ultrapure LPS to examine the effects of different Toll-like receptor agonists

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