Development and approbation of a quantitative PCR system for studying the expression of endosomal receptors and cytosolic nucleic acid sensors in mice

Abstract

BACKGROUND: The innate immune response plays a crucial role in protecting the organism against viral pathogens, important part of which are pattern recognition receptors, such as Toll-like and RIG-I-like receptors. It is known that viral invasion, including influenza virus infection, leads to the activation of intracellular pattern recognition receptors such as TLR3, TLR7, TLR8, and TLR9, which are localized in the endoplasmic reticulum, endosomes, and lysosomes, as well as MDA5 and RIG-I, which are cytosolic sensors of viral RNA not associated with cell membranes. The expression of these genes, their proper functioning, and regulation are of critical importance for ensuring an adequate immune response and the establishment of antiviral protection.

AIM: The aim of this study is to develop and validate a quantitative PCR system for assessing the expression of TLR3, TLR7, TLR8, TLR9, MDA5, and RIGI genes in mouse tissues and organs.

METHODS: Gene expression levels were analyzed using reverse transcription polymerase chain reaction with specially developed panels of primers and fluorescent probes. For approbation were selected female inbred BALB/c albino mice aged 8–10 weeks, infected with influenza A/PR8/34 (H1N1) virus.

RESULTS: In this study, a test system based on multiplex polymerase chain reaction was developed for assessing the expression of endosomal receptor genes TLR3, TLR7, TLR8, and TLR9, as well as cytosolic sensors MDA5 and RIG-I. The amplification efficiency was 99 for TLR3, 106 for TLR7, and 107% for the remaining genes. This test system was used to study the expression levels of TLRs and RLRs in the lung and spleen tissues of BALB/c mice infected with influenza A/PR8/34 (H1N1) virus. According to the obtained results, 24 hours post-infection, a significant change in mRNA levels of TLR3, TLR7, TLR8, TLR9, and MDA5 was observed in the lungs but not in the spleens of infected animals.

CONCLUSION: The developed test system can be used for analyzing the expression of certain intracellular PRRs, providing opportunities for a deeper investigation of the pathophysiological mechanisms underlying the immune response.

Keywords

influenza / multiplex polymerase chain reaction / pattern recognition receptors / Toll-like receptors / RIG-I-like receptors / endosomal receptors / cytosolic sensors

References

[1] Sellge G, Kufer TA. PRR-signaling pathways: learning from microbial tactics. Semin Immunol. 2015;27(2):75–84. doi: 10.1016/j.smim.2015.03.009

[2] Sellge G., Kufer T.A. PRR-signaling pathways: learning from microbial tactics // Semin Immunol. 2015. Vol. 27, N 2. P. 75–84. doi: 10.1016/j.smim.2015.03.009

[3] Kouwaki, T., Nishimura, T., Wang, G., & Oshiumi, H. RIG-I-like receptor-mediated recognition of viral genomic RNA of severe acute respiratory syndrome coronavirus-2 and viral escape from the host innate immune responses // Frontiers in immunology. 2021. Vol. 12, P. 700926. doi: 10.3389/fimmu.2021.700926

[4] Hayden MS, Ghosh S. NF-κB in immunobiology. Cell Res. 2011;21(2):223–244. doi: 10.1038/cr.2011.13

[5] Hayden M.S., Ghosh S. NF-κB in immunobiology // Cell Res. 2011. Vol. 21, N 2. P. 223–244. doi: 10.1038/cr.2011.13

[6] Kayesh MEH, Kohara M, Tsukiyama-Kohara K. Recent insights into the molecular mechanisms of the toll-like receptor response to influenza virus infection. Int J Mol Sci. 2024;25(11):5909. doi: 10.3390/ijms25115909

[7] Kayesh M.E.H., Kohara M., Tsukiyama-Kohara K. Recent insights into the molecular mechanisms of the toll-like receptor response to influenza virus infection // Int J Mol Sci. 2024. Vol. 25, N 11. P. 5909. doi: 10.3390/ijms25115909

[8] Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010;11(5):373–384. doi: 10.1038/ni.1863

[9] Kawai T., Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors // Nat Immunol. 2010. Vol. 11, N 5. P. 373–384. doi: 10.1038/ni.1863

[10] Zarember KA, Godowski PJ. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol. 2002;168(2):554–561. doi: 10.4049/jimmunol.168.2.554

[11] Zarember K.A., Godowski P.J. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines // J Immunol. 2002. Vol. 168, N 2. P. 554–561. doi: 10.4049/jimmunol.168.2.554

[12] Masek T, Vopalensky V, Suchomelova P, Pospisek M. Denaturing RNA electrophoresis in TAE agarose gels. Anal Biochem. 2005;336(1):46–50. doi: 10.1016/j.ab.2004.09.010

[13] Masek T., Vopalensky V., Suchomelova P., Pospisek M. Denaturing RNA electrophoresis in TAE agarose gels // Anal Biochem. 2005. Vol. 336, N 1. P. 46–50. doi: 10.1016/j.ab.2004.09.010

[14] Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559–1582. doi: 10.1038/nprot.2006.236

[15] Nolan T., Hands R.E., Bustin S.A. Quantification of mRNA using real-time RT-PCR // Nat Protoc. 2006. Vol. 1, N 3. P. 1559–1582. doi: 10.1038/nprot.2006.236

[16] Mosley YYC, HogenEsch H. Selection of a suitable reference gene for quantitative gene expression in mouse lymph nodes after vaccination. BMC Res notes. 2017;10:1–7. doi: 10.1186/s13104-017-3005-y

[17] Mosley Y.Y.C., HogenEsch H. Selection of a suitable reference gene for quantitative gene expression in mouse lymph nodes after vaccination // BMC Res notes. 2017. Vol. 10. P. 1–7. doi: 10.1186/s13104-017-3005-y

[18] Influenza (Seasonal) [Internet]. WHO. 2023 Oct 3. Available from: https://www.who.int/news-room/fact-sheets/detail/influenza-(seasonal). Accessed: 12 March 2025.

[19] Influenza (Seasonal) [Электронный ресурс] // WHO. 2023 Oct 3. Режим доступа: https://www.who.int/news-room/fact-sheets/detail/influenza-(seasonal). Дата обращения: 12.03.2025.

[20] Thompson WW, Shay DK, Weintraub E, et al. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA. 2003;289(2):179–186. doi: 10.1001/jama.289.2.179

[21] Thompson W.W., Shay D.K., Weintraub E., et al. Mortality associated with influenza and respiratory syncytial virus in the United States // JAMA. 2003. Vol. 289, N 2. P. 179–186. doi: 10.1001/jama.289.2.179

[22] Giri A, Sundar IK. Evaluation of stable reference genes for qPCR normalization in circadian studies related to lung inflammation and injury in mouse model. Sci Rep. 2022;12(1):1764. doi: 10.1038/s41598-022-05836-1

[23] Giri A., Sundar I.K. Evaluation of stable reference genes for qPCR normalization in circadian studies related to lung inflammation and injury in mouse model // Sci Rep. 2022. Vol. 12, N 1. P. 1764. doi: 10.1038/s41598-022-05836-1

[24] Wang JP, Bowen GN, Padden C, et al. Toll-like receptor–mediated activation of neutrophils by influenza A virus. Blood. 2008;112(5):2028–2034. doi: 10.1182/blood-2008-01-132860

[25] Wang J.P., Bowen G.N., Padden C., et al. Toll-like receptor–mediated activation of neutrophils by influenza A virus // Blood. 2008. Vol. 112, N 5. P. 2028–2034. doi: 10.1182/blood-2008-01-132860

[26] Koyama S, Ishii KJ, Kumar H, еt al. Differential role of TLR-and RLR-signaling in the immune responses to influenza A virus infection and vaccination. J Immunol. 2007;179(7):4711–4720. doi: 10.4049/jimmunol.179.7.4711

[27] Koyama S., Ishii K.J., Kumar H., еt al. Differential role of TLR-and RLR-signaling in the immune responses to influenza A virus infection and vaccination // J Immunol. 2007. Vol. 179, N 7. P. 4711–4720. doi: 10.4049/jimmunol.179.7.4711

[28] Hornung V, Barchet W, Schlee M, Hartmann G. RNA recognition via TLR7 and TLR8. Handb Exp Pharmacol. 2008;183:71–86. doi: 10.1007/978-3-540-72167-3_4

[29] Hornung V., Barchet W., Schlee M., Hartmann G. RNA recognition via TLR7 and TLR8 // Handb Exp Pharmacol. 2008. Vol. 183. P. 71–86. doi: 10.1007/978-3-540-72167-3_4

[30] Koh YT, Scatizzi JC, Gahan JD, et al. Role of nucleic acid–sensing tlrs in diverse autoantibody specificities and anti-nuclear antibody–producing B cells. J Immunol. 2013;190(10):4982–4990. doi: 10.4049/jimmunol.1202986

[31] Koh Y.T., Scatizzi J.C., Gahan J.D., et al. Role of nucleic acid–sensing tlrs in diverse autoantibody specificities and anti-nuclear antibody–producing B cells // J Immunol. 2013. Vol. 190, N 10. P. 4982–4990. doi: 10.4049/jimmunol.1202986

[32] Goffic RL, Balloy V, Lagranderie M, et al. Detrimental contribution of the Toll-like receptor (TLR) 3 to influenza A virus– induced acute pneumonia. PLoS Pathog. 2006;2(6):e53. doi: 10.1371/journal.ppat.0020053

[33] Goffic R.L., Balloy V., Lagranderie M., et al. Detrimental contribution of the Toll-like receptor (TLR) 3 to influenza A virus-induced acute pneumonia // PLoS Pathog. 2006. Vol. 2, N 6. P. e53. doi: 10.1371/journal.ppat.0020053

[34] Majde JA, Kapás L, Bohnet SG, et al. Attenuation of the influenza virus sickness behavior in mice deficient in Toll-like receptor 3. Brain Behav Immun. 2010;24(2):306–315. doi: 10.1016/j.bbi.2009.10.011

[35] Majde J.A., Kapás L., Bohnet S.G., et al. Attenuation of the influenza virus sickness behavior in mice deficient in Toll-like receptor 3 // Brain Behav Immun. 2010. Vol. 24, N 2. P. 306–315. doi: 10.1016/j.bbi.2009.10.011

[36] Bhargavan B, Woollard SM, Kanmogne GD. Toll-like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication. Cell Signal. 2016;28(2):7–22. doi: 10.1016/j.cellsig.2015.11.005

[37] Bhargavan B., Woollard S.M., Kanmogne G.D. Toll-like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication // Cell Signal. 2016. Vol. 28, N 2. P. 7–22. doi: 10.1016/j.cellsig.2015.11.005

[38] Xie J, Zhang S, Hu Y, et al. Regulatory roles of c-jun in H5N1 influenza virus replication and host inflammation. Biochim Biophys Acta. 2014;1842(12 Pt A):2479–2488. doi: 10.1016/j.bbadis.2014.04.017

[39] Xie J., Zhang S., Hu Y., et al. Regulatory roles of c-jun in H5N1 influenza virus replication and host inflammation // Biochim Biophys Acta. 2014. Vol. 1842, N 12 Pt A. P. 2479–2488. doi: 10.1016/j.bbadis.2014.04.017

[40] Meng D, Huo C, Wang M, et al. Influenza a viruses replicate productively in mouse mastocytoma cells (P815) and trigger pro-inflammatory cytokine and chemokine production through TLR3 signaling pathway. Front Microbiol. 2017;7:2130. doi: 10.3389/fmicb.2016.02130

[41] Meng D., Huo C., Wang M., et al. Influenza a viruses replicate productively in mouse mastocytoma cells (P815) and trigger pro-inflammatory cytokine and chemokine production through TLR3 signaling pathway // Front Microbiol. 2017. Vol. 7. P. 2130. doi: 10.3389/fmicb.2016.02130

[42] Zhang X, Li Z, Peng Q, et al. Epstein-Barr virus suppresses N6-methyladenosine modification of TLR9 to promote immune evasion. J Biol Chem. 2024;300(5):107226. doi: 10.1016/j.jbc.2024.107226

[43] Zhang X., Li Z., Peng Q., et al. Epstein-Barr virus suppresses N6-methyladenosine modification of TLR9 to promote immune evasion // J Biol Chem. 2024. Vol. 300, N 5. P. 107226. doi: 10.1016/j.jbc.2024.107226

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