Abstract
Objective
The aim of this study is to investigate the involvement of TLR9 in the regulation of iNOS expression and nitric oxide (NO) production in Porphyromonas gingivalis LPS-treated mouse macrophages.
Methods
Mouse macrophage cell line (RAW264.7) was transfected with siRNAs against TLR9 and then stimulated with P. gingivalis LPS. At indicated time points, the activated cells were lysed. Gene and protein expression of iNOS were determined by RT-PCR and immunoblotting, respectively. The level of nitric oxide (NO) production in the supernatant of the activated cells was determined by Griess reaction assay.
Results and conclusion
Depletion of TLR9 in mouse macrophages demonstrated the markedly decreased iNOS gene and protein expression by P. gingivalis LPS compared to those of the wild-type or control siRNA transfected cells. In consistent with these results, the level of NO secretion was also significantly diminished in TLR9-depleted cells after challenged with P. gingivalis LPS. These results indicate that TLR9 involves in the regulation of the iNOS expression and the NO secretion in P. gingivalis LPS-treated macrophages.
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Acknowledgements
MP acknowledges support from the Talent Management Program of Mahidol University. This work was supported by Thailand Research Fund Grant Number MRG5980057 for support of MP and Grant Number BRG5980004 for support of PU.
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11_2018_1168_MOESM1_ESM.tif
Supplementary Fig. 1. TLR2 and TLR4 expression in TLR9-depleted mouse macrophages. Mouse macrophages were transfected with siRNAs against TLR9. At indicated time points, the cells were lysed. TLR2 and TLR4 mRNA expression were measured by RT-PCR (TIF 364 KB)
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Pudla, M., Srisatjaluk, R. & Utaisincharoen, P. Induction of inducible nitric oxide synthase (iNOS) in Porphyromonas gingivalis LPS-treated mouse macrophage cell line (RAW264.7) requires Toll-like receptor 9. Inflamm. Res. 67, 723–726 (2018). https://doi.org/10.1007/s00011-018-1168-1
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DOI: https://doi.org/10.1007/s00011-018-1168-1
Keywords
- iNOS
- Nitric oxide
- Porphyromonas gingivalis LPS
- RAW264.7
- TLR9