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Inductive and suppressive networks regulate TLR9-dependent gene expression in vivo.

ABSTRACT: Bacterial DNA expressing unmethylated CpG motifs binds to TLR9, thereby stimulating a broadly protective, innate immune response. Although CpG-mediated signal transduction has been studied, the scope of TLR9-dependent gene expression is incompletely understood. To resolve these issues, mice were treated with immunostimulatory CpG oligonucleotides (ODN) and splenic mRNA levels monitored from 30 min through 3 days by microarray. Through the unique application of bioinformatic analysis to these experimental data, this study is the first to describe the complex regulatory networks responsible for TLR9-mediated gene expression. Current results are the first to establish that CpG-induced stimulation of the innate immune system proceeds in multiple waves over time, and gene up-regulation is mediated by a small number of temporally activated "major inducers" and "minor inducers". An additional study of TNF knockout mice supports the conclusion that the regulatory networks identified by our bioinformatic analysis accurately identified CpG ODN-driven gene-gene interactions in vivo. Equally important, this work identifies the counter-regulatory mechanisms embedded within the signaling cascade that suppresses the proinflammatory response triggered in vivo by CpG DNA stimulation. Identifying these network interactions provides novel and global insights into the regulation of TLR9-mediated gene activation, improves our understanding of TLR-mediated host defense, and facilitates the development of interventions designed to optimize the nature and duration of the ensuing response.

SUBMITTER: Klaschik S

PROVIDER: S-EPMC2669411 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Inductive and suppressive networks regulate TLR9-dependent gene expression in vivo.

Klaschik Sven S Tross Debra D Klinman Dennis M DM

Journal of leukocyte biology 20090129 5

Bacterial DNA expressing unmethylated CpG motifs binds to TLR9, thereby stimulating a broadly protective, innate immune response. Although CpG-mediated signal transduction has been studied, the scope of TLR9-dependent gene expression is incompletely understood. To resolve these issues, mice were treated with immunostimulatory CpG oligonucleotides (ODN) and splenic mRNA levels monitored from 30 min through 3 days by microarray. Through the unique application of bioinformatic analysis to these exp ...[more]

PMID: 19179452

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Project description:CpG DNA interacts with TLR9 to stimulate a broadly protective innate immune response. This study uses bioinformatic network analysis of microarray data to identify the genes and regulatory networks triggered by CpG ODN. CpG treatment induced significant gene up-regulation (p < 0.00001) in the spleen within 30 min, peaking with the activation of >500 genes at 3 hr, and declining progressively thereafter. There were reproducible changes in the pattern of gene expression over time. This was mediated by a small group of “major inducers” (IL1A, IL1B, TNF, IFNG) whose activity was modulated by several “minor inducers” (NFKB1, IL6, IL15, IL18, STAT1, STAT2). The subsequent decline in gene activation was mediated by “suppressors” (MYC, IL1RN, SOCS1, SOCS3, NFKBIA, IL10, FOS) that actively down-regulated gene expression and targeted both major and minor inducers. Thus, the regulation of TLR9 dependent gene activation involves multiple waves of stimulation mediated by a small number of “major” and “minor” inducers followed by the active inhibition of gene expression by “suppressors”. Keywords: time series design Endotoxin-free phosphorothioate ODN were synthesized at the CBER core facility (FDA, Bethesda, MD) as previously described. Mice were injected intraperitoneal (i.p.) with 400 ìg of an equimolar mixture of CpG ODNs 1555 (GCTAGACGTTAGCGT) and 1466 (TCAACGTTGA) or control ODNs 1612 (GCTAGATGTTAGCGT) and 1471 (TCAAGCTTGA). Spleens were surgically removed from mice under sterile conditions after 0.5, 1, 3, 9, 24 hr, and/or 72 hr, diced, and stored at -800 C in RNAlater (Qiagen, Valencia, CA). Data from 4 independent experiments/time point and 4-6 untreated controls were used for all statistical analyses. Reproducibility was established by comparing gene expression profiles among similarly treated mice from independent experiments in mAdb (referenced above). Expression analyses were performed using BRB ArrayTools (Biometric Research Branch, NCI). Data were background corrected, flagged values were removed, spots in which both signals were <100 were filtered out, ratios were log base 2 transformed and lowess intensity dependent normalization was used to adjust for differences in labeling intensities of the Cy3 and Cy5 dyes (Yang et al., 2002). Analysis was restricted to genes present on >70% of the arrays after filtering. The gene expression profile of all treatment groups was compared to that of the control groups.

Dataset Information

Inductive and suppressive networks regulate TLR9-dependent gene expression in vivo.

Publications

Inductive and suppressive networks regulate TLR9-dependent gene expression in vivo.

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