Short and long-term changes in gene expression mediated by the activation of TLR9

CpG DNA binds to Toll-like receptor 9 to stimulate a strong innate immune response. Despite extensive studies of TLR9 mediated signal transduction, the scope of CpG-induced changes in gene expression is incompletely understood. In particular, the prolonged effects of CpG ODN (oligodeoxynucleotide) on gene activation have not been investigated despite evidence that a single dose of CpG ODN alters immune reactivity for several weeks. This study used gene expression analysis to monitor changes in mRNA levels for 14 days, and identified the genes, pathways, functional groups and regulatory networks triggered in vivo following CpG ODN administration. Two discrete peaks of gene activation (at 3 hr and 5 days) were observed after CpG administration. Both the regulation and function of genes activated during the second peak differed from those triggered shortly after CpG administration. Initial gene up-regulation corresponded to a period when TLR9 ligation stimulated genes functionally associated with the generation of innate and adaptive immune responses (e.g. the NF-kB and B-cell receptor pathways). The second peak reflected processes associated with cell division (e.g., cell cycle and DNA replication & repair). Whereas TNF and IFNG played central roles regulating the first peak of activation, E2F1, E2F2, BRCA1, and HRAS had major impact on the networks controlling the second peak. The complex bimodal pattern of gene expression elicited by CpG ODN administration provides novel insights into the long term effects of CpG DNA on genes associated with immunity and cell proliferation. Data from 4 independent biological replicates/time point (9 time points) and 6 untreated controls were used for all statistical analyses. A reference design was used. Reference cDNA vs. sample cDNA were hybridized to same array.