Pam2CSK4 and Pam3CSK4 induce iNOS expression via TBK1 and MyD88 molecules in mouse macrophage cell line RAW264.7
Panthong Kulsantiwong1, Matsayapan Pudla2, Chanya Srisaowakarn1
1Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Objective:
The aim of this study was to investigate the involvement of TLR adaptor molecules, such as TRIF, MyD88, and TBK1 in the induction of iNOS and nitric oxide (NO) production in Pam2CSK4 and Pam3CSK4-treated mouse macrophages.
Method:
Mouse macrophage cell line (RAW264.7) was transfected with trif, myd88, and tbk1 siRNAs before stimulated with Pam2CSK4 and Pam3CSK4. The iNOS gene and protein expression were determined by RT-PCR and immunoblotting, respectively. The NO production was determined by Griess reaction assay.
Results:
The results showed that the induction of iNOS expression and NO production by Pam2CSK4 and Pam3CSK4 were diminished in tbk1 and myd88-depleted mouse macrophages but not trif-depleted cells.
Conclusion:
These results suggested that the TBK1 and MyD88 molecules were essential for the induction of iNOS expression and NO production by both Pam2CSK4 and Pam3CSK4 via TLR2 signaling.
Insights
Toll-like receptor 2 (TLR2) signaling activates inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. TBK1 and MyD88 adaptor molecules are essential for this TLR2-mediated immune response in macrophages.
Area of Science:
- Immunology
- Molecular Biology
Background:
- Toll-like receptors (TLRs) are crucial for innate immunity.
- TLR2 recognizes bacterial lipoproteins like Pam2CSK4 and Pam3CSK4.
- iNOS and NO play significant roles in immune responses.
Purpose of the Study:
- To investigate the role of TLR adaptor molecules TRIF, MyD88, and TBK1.
- To understand their involvement in iNOS and NO production.
- To elucidate the mechanism of Pam2CSK4 and Pam3CSK4-induced responses in macrophages.
Main Methods:
- Used RAW264.7 mouse macrophage cell line.
- Transfected cells with siRNAs targeting trif, myd88, and tbk1.
- Stimulated cells with Pam2CSK4 and Pam3CSK4.
- Assessed iNOS expression (RT-PCR, immunoblotting) and NO production (Griess assay).
Main Results:
- Pam2CSK4 and Pam3CSK4 induced iNOS expression and NO production.
- Depletion of TBK1 or MyD88 significantly reduced this induction.
- Depletion of TRIF did not affect iNOS expression or NO production.
Conclusions:
- TBK1 and MyD88 are critical for TLR2-mediated iNOS induction.
- These adaptor molecules are essential for NO production stimulated by Pam2CSK4 and Pam3CSK4.
- The findings highlight the specific roles of TLR adaptors in TLR2 signaling pathways.
