Analysis of genes induced by bacterial immunostimulatory CpG-DNA

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Bacterial DNA and immunostimulatory oligodeoxyribonuceotides (ISS-ODN) can induce macrophages, monocytes, and dendritic cells to secrete proinflammatory cytokines such as IL-6, IL-12 and TNF-a, and up-regulate MHC class II, CD40 and CD86 expressions. To further characterize molecular consequences of the recognition of a specific pattern in bacterial DNA, we used a sensitive PCR-based subtraction approach, termed suppression subtractive hybridization (SSH), to isolate genes upregulated in ISS-ODN-stimulated murine spleen cells.Sequence analysis of 87 clones isolated by SSH revealed that 41 clones were found to correspond exactly to known mouse protein in the BLAST program. Six clones were revealed to have 80-90% homology with known human or rat proteins. Thirty-one clones showed homology with known cDNA whose gene products are not allocated to any functions. Remaining 9 clones showed no significant homology with any sequences in the public database.Overexpression of NF-kB p105, IFN regulatory factor-1 (IRF-1), proteasome activator (PA) 28b, IRG2 (IFN-IP), and MyD88, which have not previously been reported as overexpressed in the ISS-ODN-stimulated cells, were confirmed by using RT-PCR.