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Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages
Published on: November 3, 2014
Specific siRNA downregulated TLR9 and altered cytokine expression pattern in macrophage after CpG DNA stimulation
Bin Qiao1, Baohua Li, Xiuli Yang
1Department of Immunology and Key Laboratory of Molecular Medicine of the Ministry of Education, Shanghai Medical College of Fudan University, Shanghai 200032, China.
Abstract:
Bacterial CpG DNA or synthetic oligonucleotides (ODNs) that contain unmethylated CpG motifs (CpG ODN) can directly activate antigen-presenting cells (APCs) to secrete various cytokines through the intracellular receptor TLR9. Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial to the subsequent immune responses. To date, cytokine profiles in APCs upon CpG ODN stimulation in vitro are not fully investigated. In the present study, vector-based siRNA was used to downregulate TLR9 expression. Cytokine profiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid upon CpG ODN stimulation. We found that not all the cytokine expressions by the macrophage were decreased while TLR9 was downregulated. IL-12, TNF-alpha, IFN-gamma and IL-1beta expressions were significantly decreased, but IL-6, IFN-beta and IL-10 expressions were not affected. Interestingly, the level of IFN-alpha was even increased. This alteration of cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role of CpG DNA is more complicated in the pathogenesis and prevention of diseases.
Insights
CpG DNA activates immune cells via TLR9. Downregulating Toll-like receptor 9 (TLR9) in macrophages affects specific cytokine production, revealing a complex role for CpG DNA in disease.
Area of Science:
- Immunology
- Molecular Biology
- Cellular Biology
Background:
- Bacterial CpG DNA and synthetic oligonucleotides (ODNs) containing unmethylated CpG motifs activate antigen-presenting cells (APCs) via Toll-like receptor 9 (TLR9).
- The resulting cytokine profiles are critical for adaptive immune responses.
- Understanding these profiles upon CpG ODN stimulation in vitro is incomplete.
Purpose of the Study:
- To investigate the impact of TLR9 downregulation on cytokine profiles in macrophages stimulated with CpG ODN.
- To elucidate the specific roles of TLR9 in mediating CpG DNA-induced cytokine secretion.
Main Methods:
- Utilized vector-based small interfering RNA (siRNA) to downregulate TLR9 expression in the murine macrophage cell line RAW264.7.
- Stimulated transfected macrophages with CpG ODN.
- Analyzed the expression of various cytokines using quantitative methods.
Main Results:
- Downregulation of TLR9 significantly decreased the expression of IL-12, TNF-alpha, IFN-gamma, and IL-1beta.
- IL-6, IFN-beta, and IL-10 expression levels remained unaffected by TLR9 downregulation.
- Interestingly, IFN-alpha expression was observed to increase.
Conclusions:
- TLR9 signaling is essential for the induction of certain pro-inflammatory cytokines (IL-12, TNF-alpha, IFN-gamma, IL-1beta) by CpG ODN.
- The secretion of other cytokines (IL-6, IFN-beta, IL-10) is TLR9-independent.
- CpG DNA's role in disease pathogenesis and prevention is more intricate than previously understood, involving differential regulation of cytokine responses.
