Abstract
BackgroundOligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are known to exert a strong adjuvant effect on Th1 immune responses. Although several genes have been reported, no comprehensive study of the gene expression profiles in human cells after stimulation with CpG ODN has been reported.ResultsThis study was designed to identify a CpG-inducible gene cluster that potentially predicts for the molecular mechanisms of clinical efficacy of CpG ODN, by determining mRNA expression in human PBMC after stimulation with CpG ODN. PBMCs were obtained from the peripheral blood of healthy volunteers and cultured in the presence or absence of CpG ODN 2006 for up to 24 hours. The mRNA expression profile was evaluated using a high-density oligonucleotide probe array, GeneChip®. Using hierarchical clustering-analysis, out of a total of 10,000 genes we identified a cluster containing 77 genes as having been up-regulated by CpG ODN. This cluster was further divided into two sub-clusters by means of time-kinetics. (1) Inflammatory cytokines such as IL-6 and GM-CSF were up-regulated predominantly 3 to 6 hours after stimulation with CpG ODN, presumably through activation of a transcription factor, NF-κB. (2) Interferon (IFN)-inducible anti-viral proteins, including IFIT1, OAS1 and Mx1, and Th1 chemoattractant IP-10, were up-regulated predominantly 6 to 24 hours after stimulation. Blocking with mAb against IFN-α/β receptor strongly inhibited the induction of these IFN-inducible genes by CpG ODN.ConclusionThis study provides new information regarding the possible immunomodulatory effects of CpG ODN in vivo via an IFN-α/β receptor-mediated paracrine pathway.
Highlights
Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are known to exert a strong adjuvant effect on Th1 immune responses
Identification of CpG ODN-inducible genes Using hierarchical clustering-analysis of the gene expression profiles of approximately 10,000 genes, we identified a cluster containing 77 genes which were up-regulated by CpG ODN (Fig. 1)
With respect to the treatment of chronic viral infections, our study demonstrated that CpG ODN induces production of mRNA for anti-viral proteins, including IFN-induced protein with tetratricopeptide repeats 1 (IFIT1), oligoadenylate synthetase 1 (OAS1), myxovirus (influenza virus) resistance 1 (Mx1), IFN-stimulated gene 15 (ISG15) and TRAIL
Summary
Oligodeoxynucleotides Phosphorothioate ODNs were synthesized at Operon Technologies (Alameda, CA). The sequence of the CpG ODN 2006 used in the present study was 5'-TCG TCG TTT TGT CGT TTT GTC GTT-3' [6]. The sequence of the GpC control ODN was 5'-TGC TGC TTT TGT GCT TTT GTG CTT-3', which was modified by substitutions of GpC for the CpG in CpG ODN 2006. (clone: GIR-208, IgG1, Pharmingen, San Diego, CA) or isotype-matched control mAbs (IgG1: SouthernBiotech, Birmingham, AL, IgG2a: eBioscience, San Diego, CA) were added simultaneously with CpG ODN or IFNs throughout the culture period. GeneChip® expression analysis Total RNA was extracted by RNeasy® (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Gene expression was analyzed with the GeneChip® Human Genome U95Av2 probe array (Affymetrix, Santa Clara, CA), which contains the oligonucleotide probe sets for approximately 10,000 genes, according to the manufacturer's protocol (Expression Analysis Technical Manual). Hierarchical-clustering analysis was performed using a minimum distance value of 0.001, a separation ratio of 0.5 and the standard definition of the correlation distance
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