ARYA Atherosclerosis Journal

Current Issue

By Issue

By Author

By Subject

Author Index

Keyword Index

About Journal

Aims and Scope

Editorial Board

Publication Ethics

Indexing and Abstracting

Related Links

FAQ

Peer Review Process

Journal Metrics

News

Contribution of toll-like receptor 2 and nicotinamide adenine dinucleotide phosphate oxidase to the trimethylamine N-oxide-induced inflammatory reactions in U937-derived macrophages

Document Type : Original Article(s)

Authors

1 Assistant Professor, Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran

2 Assistant Professor, Liver and Digestive Research Center, Research Institute for Health Development AND Department of Clinical Biochemistry, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran

3 Assistant Professor, Liver and Digestive Research Center, Research Institute for Health Development AND Department of Medical Physiology and Pharmacology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran

4 PhD Candidate, Department of Molecular Medicine and Genetics, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran

5 Medical Student, Department of Clinical Biochemistry, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran

Abstract

BACKGROUND: Trimethylamine N-oxide (TMAO) is emerging as a new generation of metabolites related to the activation of inflammatory reactions in the macrophages during atherosclerosis. Stress-activation of cell surface toll-like receptors (TLRs) as well as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) is also assumed to be involved in TMAO-induced inflammatory reaction in the macrophages. To elucidate the possible contribution of TLRs and NOX to the mentioned signaling pathway, we aimed to simultaneously evaluate the expression level of TLR2, TLR6, and NOX2 in TMAO-treated macrophages.METHODS: 2.5 × 106 cells of U937-derived macrophages were treated in triplicates with different concentrations (37.5, 75, 150, and 300 μM) of TMAO for 24 hours. The cells were also treated with tunicamycin (TUN), as a positive control of stress. Normal control group (CTR) cells received no treatment. The viability of treated cells was checked by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was also used to evaluate the relative expression (fold change) of TLR2, TLR6, and NOX2 at messenger ribonucleic acid (mRNA) levels. One-way analysis of variance (ANOVA) with post-hoc Dunnett’s test was performed to compare every mean with that of the control.RESULTS: No cell death occurred because of treatments. Dose of 300 μM of TMAO significantly increased the relative expression of both TLR2 and NOX2 compared to the CTR cells (P < 0.001 for both). The elevation of TLR6 was not statistically significant in all groups of TMAO-treated cells (P > 0.050).CONCLUSION: Our results provide documentation supporting contribution of TLR2 and NOX2 to previously described inflammatory reactions induced by TMAO in macrophages. In addition, they may clarify the proatherogenic role of TMAO in foam cell formation as well as abnormal activation of macrophages during atherosclerosis.

Keywords

References
  1. Lin TY, Timasheff SN. Why do some organisms use a urea-methylamine mixture as osmolyte? Thermodynamic compensation of urea and trimethylamine N-oxide interactions with protein. Biochemistry 1994; 33(42): 12695-701.
  2. Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN. Living with water stress: Evolution of osmolyte systems. Science 1982; 217(4566): 1214-22.
  3. Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 2011; 472(7341): 57-63.
  4. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002; 105(9): 1135-43.
  5. Chen YM, Liu Y, Zhou RF, Chen XL, Wang C, Tan XY, et al. Associations of gut-flora-dependent metabolite trimethylamine-N-oxide, betaine and choline with non-alcoholic fatty liver disease in adults. Sci Rep 2016; 6: 19076.
  6. Xu R, Wang Q. Towards understanding brain-gut-microbiome connections in Alzheimer's disease. BMC Syst Biol 2016; 10(Suppl 3): 63.
  7. Al-Obaide MAI, Singh R, Datta P, Rewers-Felkins KA, Salguero MV, Al-Obaidi I, et al. Gut microbiota-dependent trimethylamine-N-oxide and serum biomarkers in patients with T2DM and advanced CKD. J Clin Med 2017; 6(9): 86.
  8. Missailidis C, Hallqvist J, Qureshi AR, Barany P, Heimburger O, Lindholm B, et al. Serum trimethylamine-n-oxide is strongly related to renal function and predicts outcome in chronic kidney disease. PLoS One 2016; 11(1): e0141738.
  9. Delzenne NM, Cani PD. Gut microbiota and the pathogenesis of insulin resistance. Curr Diab Rep 2011; 11(3): 154-9.
  10. Oellgaard J, Winther SA, Hansen TS, Rossing P, von Scholten BJ. Trimethylamine N-oxide (TMAO) as a new potential therapeutic target for insulin resistance and cancer. Curr Pharm Des 2017; 23(25): 3699-712.
  11. Liu X, Liu H, Yuan C, Zhang Y, Wang W, Hu S, et al. Preoperative serum TMAO level is a new prognostic marker for colorectal cancer. Biomark Med 2017; 11(5): 443-7.
  12. Warrier M, Shih DM, Burrows AC, Ferguson D, Gromovsky AD, Brown AL, et al. The TMAO-generating enzyme flavin monooxygenase 3 is a central regulator of cholesterol balance. Cell Rep 2015; 10(3): 326-38.
  13. Chistiakov DA, Bobryshev YV, Kozarov E, Sobenin IA, Orekhov AN. Role of gut microbiota in the modulation of atherosclerosis-associated immune response. Front Microbiol 2015; 6: 671.
  14. Seldin MM, Meng Y, Qi H, Zhu W, Wang Z, Hazen SL, et al. Trimethylamine N-oxide promotes vascular inflammation through signaling of mitogen-activated protein kinase and nuclear factor-kappaB. J Am Heart Assoc 2016; 5(2): e002767.
  15. Geng J, Yang C, Wang B, Zhang X, Hu T, Gu Y, et al. Trimethylamine N-oxide promotes atherosclerosis via CD36-dependent MAPK/JNK pathway. Biomed Pharmacother 2018; 97: 941-7.
  16. Seneviratne AN, Sivagurunathan B, Monaco C. Toll-like receptors and macrophage activation in atherosclerosis. Clin Chim Acta 2012; 413(1-2): 3-14.
  17. Falck-Hansen M, Kassiteridi C, Monaco C. Toll-like receptors in atherosclerosis. Int J Mol Sci 2013; 14(7): 14008-23.
  18. Erridge C. The roles of Toll-like receptors in atherosclerosis. J Innate Immun 2009; 1(4): 340-9.
  19. Yiu JH, Dorweiler B, Woo CW. Interaction between gut microbiota and toll-like receptor: From immunity to metabolism. J Mol Med (Berl) 2017; 95(1): 13-20.
  20. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: Physiology and pathophysiology. Physiol Rev 2007; 87(1): 245-313.
  21. Chen ML, Zhu XH, Ran L, Lang HD, Yi L, Mi MT. Trimethylamine-N-oxide induces vascular inflammation by activating the NLRP3 inflammasome through the SIRT3-SOD2-mtROS signaling pathway. J Am Heart Assoc 2017; 6(9): e006347.
  22. Weber C, Noels H. Atherosclerosis: Current pathogenesis and therapeutic options. Nat Med 2011; 17(11): 1410-22.
  23. Mohammadi A, Gholamhoseyniannajar A, Yaghoobi MM, Jahani Y, Vahabzadeh Z. Expression levels of heat shock protein 60 and glucose-regulated protein 78 in response to trimethylamine-N-oxide treatment in murine macrophage J774A.1 cell line. Cell Mol Biol (Noisy -le -grand) 2015; 61(4): 94-100.
  24. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ, et al. Amplification efficiency: Linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 2009; 37(6): e45.
  25. Suzuki S, Kubo A, Shinano H, Takama K. Inhibition of the electron transport system in Staphylococcus aureus by trimethylamine-N-oxide. Microbios 1992; 71(287): 145-8.
  26. Loscalzo J. Lipid metabolism by gut microbes and atherosclerosis. Circ Res 2011; 109(2): 127-9.
  27. Malhotra JD, Kaufman RJ. The endoplasmic reticulum and the unfolded protein response. Semin Cell Dev Biol 2007; 18(6): 716-31.
  28. Schroder M, Kaufman RJ. ER stress and the unfolded protein response. Mutat Res 2005; 569(1-2): 29-63.
  29. Kennedy D, Samali A, Jager R. Methods for studying ER stress and UPR markers in human cells. Methods Mol Biol 2015; 1292: 3-18.
  30. Mohammadi A, Vahabzadeh Z, Jamalzadeh S, Khalili T. Trimethylamine-N-oxide, as a risk factor for atherosclerosis, induces stress in J774A.1 murine macrophages. Adv Med Sci 2018; 63(1): 57-63.
  31. Xu Q. Role of heat shock proteins in atherosclerosis. Arterioscler Thromb Vasc Biol 2002; 22(10): 1547-59.
  32. Cole JE, Georgiou E, Monaco C. The expression and functions of toll-like receptors in atherosclerosis. Mediators Inflamm 2010; 2010: 393946.
  33. Curtiss LK, Tobias PS. Emerging role of Toll-like receptors in atherosclerosis. J Lipid Res 2009; 50(Suppl): S340-S345.
  34. Curtiss LK, Tobias PS. The toll of Toll-like receptors, especially toll-like receptor 2, on murine atherosclerosis. Curr Drug Targets 2007; 8(12): 1230-8.
  35. Beutler B. Inferences, questions and possibilities in Toll-like receptor signalling. Nature 2004; 430(6996): 257-63.
  36. Tobias P, Curtiss LK. Thematic review series: The immune system and atherogenesis. Paying the price for pathogen protection: Toll receptors in atherogenesis. J Lipid Res 2005; 46(3): 404-11.
  37. Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med 2013; 19(5): 576-85.
  38. Abdi M, Vahabzadeh Z, Ghanivash A, Farhadi L, Hakhamaneshi M S, Andalibi P. Investigation of the effect of trimethylamine-N-oxide on the proinflammatory cytokine genes expression in U937-derived macrophages. Sci J Kurdistan Univ Med Sci 2018; 23(3): 1-9. [In Persian].
ARYA Atherosclerosis Journal
Volume 17, Issue 1
January and February 2021
Pages 1-7
Files
Share
How to cite
Statistics