Transcriptional activity of TLR/RLR receptor genes in macrophage-like cells under the influence of drugs based on acridoneacetic acid

Activation of Toll-like receptors (TLRs) is one of the earliest indicators of functional activation of the innate immune system. Therefore, the development of drugs that stimulate the transcription of TLR/RLR genes and at the same time are “multi-target” drugs is an important task of modern immunopharmacology. In this regard, antiviral drugs that combine the properties of interferonogens and immunomodulators, which also include Cycloferon® and its analogues, are of great interest. The purpose of this study was to assess the expression of genes that determine the TLR/RLR signalling reactions of the innate immune system under the influence of immunomodulatory antiviral drugs based on acridoneacetic acid (Cycloferon® and Cycloferon L). The study was conducted using a model of immunocompetent cells: THP-1, differentiated by phorbol ester into macrophage-like cells. Gene expression analysis was performed using real-time polymerase chain reaction. The expression level of genes encoding TLR2, TLR3, TLR4, TLR7, TLR8, TLR9 and RIG-I was studied under the influence of the drugs Cycloferon® and Cycloferon L in three concentrations (156 μg/mL, 312 μg/mL and 625 μg/mL) on 1 hour, 4 hours and 24 hours. It was shown that the drug Cycloferon® at concentrations of 156, 312 and 625 μg/mL at 24 hours of exposure dose-dependently stimulated the expression of TLR2, TLR3, TLR4, TLR7, TLR8 receptor genes. A stable stimulation of the expression of RIG1 receptor genes was found upon exposure 4 hours to the drug in all studied concentrations. For the first time, it was revealed that the drug Cycloferon L stimulated a stable increase in the expression of TLR2, TLR3, TLR4, TLR7, TLR8 genes at an exposure period of 24 hours. Hereby, it was shown that the drugs Cycloferon® and Cycloferon L stimulated the expression of the TLR2, TLR3, TLR4, TLR7, TLR8 genes (and RIG1 for the drug Cycloferon), which are responsible for the synthesis of innate immune receptors.

1. Ershov F.I., Kiselev O.I.Interferons and their inducers (from molecules to drugs). Moscow: GEOTAR-Media, 2005. 368 p.

2. Sokolova T.M., Kosobkova E.N., Shuvalov A.N., Shapoval I.M., Kosorukov V.S., Ershov F.I. of Interferon system gene activity in colon adenocarcinoma cells HCT-116: regulation by interferon-alpha-2B from bacteria or plants. Rossiyskiy bioterapevticheskiy zhurnal = Russian Journal of Biotherapy, 2013, no. 3, pp. 39-44. (In Russ.)

3. Sokolova T.M., Poloskov V.V., Burova O.S., Shuvalov A.N., Sokolova Z.A., Inshakov A.N., Shishkin Yu.V., Ershov F.I. Action interferons and IFN-inductors on TLR/RLRs genes expression and differentiation of tumor cell lines THP-1 and HCT-116. Rossiyskiy bioterapevticheskiy zhurnal = Russian Journal of Biotherapy, 2016, Vol. 15, no. 3, pp. 28-33. (In Russ.)

4. Sokolova T.M., Shuvalov A.N., Poloskov V.V., Ershov F.I. Stimulation of signal transduction genes with drugs Ridostin, Cycloferon and Ingavirin. Tsitokiny i vospaleniye = Cytokines and Inflammation, 2015, no. 2, pp. 26-34. (In Russ.)

5. Sokolova T.M., Shuvalov A.N., Shapoval I.M., Sokolova Z.A., Ershov F.I. Activation of genes controlling the immune signaling pathways: differential individual sensitivity of human blood cells for interferon preparations and IFN inducers. Meditsinskaya immunologiya = Medical Immunology (Russia), 2015, Vol. 17, no. 1, pp. 7-18. (In Russ.) doi: 10.15789/1563-0625-2015-1-7-18.

6. Sukhanov, D.S., Romantsov M.G., Smagina F.I., Kovalenko A.L., Lokteva O.M. Dose-dependent interferon-inducing activity and pharmacokinetics of cycloferon in healthy individuals. Eksperimental’naya i klinicheskaya farmakologiya = Experimental and Сlinical Pharmacology, 2012, no. 75, pp. 23-26. (In Russ.)

7. Assay Guidance Manuel. Available at: http://www/ncgc.nih.gov/guidance/section2.html.

8. El-Zayat S.R., Sibaii H., Mannaa F.A. Toll-like receptors activation, signaling, and targeting: an overview. Bull. Natl Res. Cent., 2019, Vol. 43, 187. doi: 10.1186/s42269-019-0227-2.

9. Fitzgerald K.A., Kagan J.C. Toll-like Receptors and the Control of Immunity. Cell, 2020, Vol. 180, no. 6, pp. 1044-1066.

10. Kawai T., Akira S. Signaling to NF-kappaB by toll-like receptors. Trends Mol. Med., 2007, Vol. 13, no. 11, pp. 460-569.

11. Kramer M.J., Cleeland R., Grunberg E. Antiviral Activity of 10-Carboxymethyl-9-Acridanone. Antimicrob. Agents Chemother., 1976, Vol. 9, no. 2, pp. 233-238.

12. Mancino A., Lawrence T. Nuclear factor-κB and tumor-associated macrophages. Clin. Cancer Res., 2010, Vol. 16, pp. 784-789.

13. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 1983, Vol. 65, no. 1-2, pp. 55-63.

14. Plotnikova M.A., Klotchenko S.A., Kiselev A.A., Gorshkov A.N., Shurygina A-P.S., Vasilyev K.A., Uciechowska-Kaczmarzyk U., Samsonov S.A., Kovalenko A.L., Vasin A.V. Meglumine acridone acetate, the ionic salt of CMA and N-methylglucamine, induces apoptosis in human PBMCs via the mitochondrial pathway. Sci. Rep., 2019, Vol. 9, 18240. doi: 10.1038/s41598-019-54208-9.

15. Tartey S., Takeuchi O. Pathogen recognition and Toll-like receptor targeted therapeutics in innate immune cells. Int. Rev. Immunol., 2017, Vol. 6, pp. 1-17.