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A transcriptomic and proteomic analysis of the effect of CpG-ODN on human THP-1 monocytic leukemia cells
Cheng-Chin Kuo1, Chu-Wei Kuo, Chi-Ming Liang
1Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan.
Abstract:
The CpG motif of bacterial DNA (CpG-DNA) is a potent immunostimulating agent whose mechanism of action is not yet clear. Here, we used both DNA microarray and proteomic approaches to investigate the effects of oligodeoxynucleotides containing the CpG motif (CpG-ODN) on gene transcription and protein expression profiles of CpG-ODN responsive THP-1 cells. Microarray analysis revealed that 2 h stimulation with CpG-ODN up-regulated 50 genes and down-regulated five genes. These genes were identified as being associated with inflammation, antimicrobial defense, transcriptional regulation, signal transduction, tumor progression, cell differentiation, proteolysis and metabolism. Longer stimulation (8 h) with CpG-ODN enhanced transcriptional expression of 58 genes. Among these 58 genes, none except one, namely WNTI inducible signaling pathway protein 2, was the same as those induced after 2 h stimulation. Proteomic analysis by two-dimensional gel electrophoresis, followed by mass spectrometry identified several proteins up-regulated by CpG-ODN. These proteins included heat shock proteins, modulators of inflammation, metabolic proteins and energy pathway proteins. Comparison of microarray and proteomic expression profiles showed poor correlation. Use of more reliable and sensitive analyses, such as reverse transcriptase polymerase chain reaction, Western blotting and functional assays, on several genes and proteins, nonetheless, confirmed that there is indeed good correlation between mRNA and protein expression after CpG-ODN treatment. This study also revealed that several anti-apoptotic and neuroprotective related proteins, not previously reported, are activated by CpG-DNA. These findings have extended our knowledge on the activation of cells by CpG-DNA and may contribute to further understanding of mechanisms that link innate immunity with acquired immune response(s).
Insights
CpG-DNA activates immune cells by altering gene and protein expression. This study reveals new insights into innate immunity activation and its link to acquired immune responses.
Area of Science:
- Immunology
- Molecular Biology
- Genomics
Background:
- CpG motif of bacterial DNA (CpG-DNA) is a potent immunostimulating agent.
- The precise mechanism of CpG-DNA action remains unclear.
- Understanding CpG-DNA's effects on cellular pathways is crucial for immunology research.
Purpose of the Study:
- To investigate the effects of CpG-DNA on gene transcription and protein expression profiles.
- To explore the impact of CpG-oligodeoxynucleotides (CpG-ODN) on THP-1 cells.
- To elucidate the molecular mechanisms underlying CpG-DNA-mediated immune activation.
Main Methods:
- DNA microarray analysis to assess gene transcription changes.
- Proteomic approaches (2D gel electrophoresis, mass spectrometry) to analyze protein expression.
- Validation using reverse transcriptase polymerase chain reaction, Western blotting, and functional assays.
Main Results:
- CpG-ODN stimulation up-regulated and down-regulated numerous genes related to inflammation, defense, and metabolism.
- Both short-term (2h) and longer-term (8h) CpG-ODN exposure altered distinct sets of genes.
- Proteomic analysis identified up-regulated proteins including heat shock proteins and inflammation modulators.
- Initial poor correlation between microarray and proteomic data was resolved by validation studies, confirming mRNA-protein expression correlation.
- CpG-DNA was found to activate anti-apoptotic and neuroprotective proteins, previously unreported.
Conclusions:
- CpG-DNA significantly impacts cellular gene and protein expression profiles.
- Validated correlations between mRNA and protein levels confirm CpG-ODN's regulatory effects.
- The study identified novel CpG-DNA-activated anti-apoptotic and neuroprotective proteins.
- Findings enhance understanding of CpG-DNA's role in innate immunity and its connection to acquired immunity.
