Toll-like receptor 9-dependent gene expression in vivo is regulated by inductive and suppressive networks

Introduction

Synthetic oligodeoxynucleotides (ODN) expressing CpG motifs mimic the immunostimulatory activity of bacterial DNA. CpG ODN interact with Toll-like receptor (TLR) 9 to stimulate an innate immune response characterized by the production of Th1 and proinflammatory cytokines, and the functional maturation of immune cells. Changes in gene expression mediated by the in vivo administration of CpG ODN were identified using microarrays.

Objective

We predicted that microarrays could be used to identify reproducible changes in gene expression induced by CpG ODN activation in mice treated in vivo over time, and that network analysis would allow us to identify regulators of gene expression.

Materials

cDNA was generated from total RNA isolated from spleen cells of mice 30 minutes to 3 days after in vivo treatment with 400 μg CpG or control ODN.

Methods

Dual-color hybridizations (Biomicro) were performed on murine genome microarrays (NCI). Analyses were conducted on four independently derived RNA samples for each time point. R² was 0.90 ± 0.04 for all matched samples. Network analysis was performed using Ingenuity Pathway Analysis.

Results

Differential gene activation (P < 0.00001) was observed within 30 minutes of CpG ODN treatment (25 genes), peaked at 3 hours (529 upregulated genes), and fell to near background levels after 72 hours. TNFα, IL-1β, NF-κB and IFNγ played central roles in upregulating the early expression of immune-related genes. Of interest, two distinct patterns of gene expression were observed. One subset of genes was activated shortly after CpG ODN administration and remained upregulated for a prolonged period, while unique subsets of additional genes were activated at specific time points, and were rapidly downregulated. Several genes responsible for this downregulation (MYC, IL1RN, SOCS1) were identified.

Conclusion

This analysis identifies two distinct patterns of gene regulation associated with CpG-induced activation of the innate immune system of mice. A small number of regulatory genes triggers the patterned upregulation of immune related genes from 30 minutes through 72 hours. A separate set of downregulatory genes subsequently dampens what would otherwise be a continuous positive feedback loop.