Transcriptional response of macrophages to combined stimulation of a NOD- and a Toll-like receptor

• Anna M. Masyutina
• Mikhail V. Pashenkov

Abstract

Introduction. Activation of innate immune cells by agonists of two or more pattern-recognition receptors can result in mutual blunting or enhancement of activation signals. Based on expression of selected genes, it is assumed that combined stimulation of NOD- and Toll-like receptors results in synergistic enhancement of cellular response (the effect of agonist combination is greater than sum of effects of individual agonists). However, this assumption has not been tested using whole-transcriptome analysis.

Aim – to conduct whole transcriptome analysis of human macrophages activated by a combination of NOD1 and TLR4 receptor agonists in vitro.

Material and methods. Macrophages were obtained by culturing of healthy donor blood monocytes with granulocyte-macrophage colony-stimulating factors and stimulated by agonists of NOD1 and TLR4 separately or in combination for 1 and 4 h. Transcriptomes were evaluated using high-throughput RNA sequencing (RNA-seq) and real-time PCR (RT-PCR). To identify synergistically inducible genes, potentiation indices were calculated (response to agonist combination divided by sum of responses to separate agonists).

Results. Synergisticall inducible genes comprised no more than 10 % of all genes induced by NOD1 and/or TLR4 agonists separately or in combination. Typical features of synergistically inducible genes were low basal expression and high response to combined stimulation. Despite their relatively low number, this group of genes is of great functional importance, because it includes genes of key pro-inflammatory cytokines. Results of bioinformatic analysis point at the role of NF-κB and AP-1 transcription factor families in the regulation of expression of synergistically inducible genes.

Conclusion. Knowledge of characteristics and mechanisms of synergistic effects will allow to select appropriate targets to suppress excessive cytokine production during cytokine storm, as well as to choose appropriate agonist combinations for therapeutic application in clinical situations demanding enhancement of innate immune response.

Keywords:macrophages; transcriptome; RNA sequencing; NOD1; TLR4; synergy

References

1. Budikhina A.S., Murugina N.E., Maximchik P.V., Dagil Y.A., Nikolaeva A.M., Balyasova L.S., et al. Interplay between NOD1 and TLR4 receptors in macrophages: nonsynergistic activation of signaling pathways results in synergistic induction of proinflammatory gene expression. J Immunol. 2021; 206 (9): 2206–220. DOI: https://www.doi.org/10.4049/JIMMUNOL.2000692

2. Fritz J.H., Girardin S.E., Fitting C., Werts C., Mengin-Lecreulx D., Caroff M., et al. Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2- activating agonists. Eur J Immunol. 2005; 35 (8): 2459–70. DOI: https://www.doi.org/10.1002/eji.200526286

3. Tada H., Aiba S., Shibata K.I., Ohteki T., Takada H. Synergistic effect of Nod1 and Nod2 agonists with toll-like receptor agonists on human dendritic cells to generate interleukin-12 and T helper type 1 cells. Infect Immun. 2005; 73 (12): 7967–76. DOI: https://www.doi.org/10.1128/IAI.73.12.7967-7976.2005

4. Tukhvatulin A., Dzharullaeva A.S., Tukhvatulina N.M., Shcheblyakov D.V., Shmarov M.M., Dolzhikova I.V., et al. Powerful complex immunoadjuvant based on synergistic effect of combined TLR4 and NOD2 activation significantly enhances magnitude of humoral and cellular adaptive immune responses. PLoS One. 2016; 11 (5): e0155650. DOI: https://www.doi.org/10.1371/journal.pone.0155650

5. van Beelen A.J., Zelinkova Z., Taanman-Kueter E.W., Muller F.J., Hommes D.W., Zaat S.A.J., et al. Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells to promote interleukin-17 production in human memory T cells. Immunity. 2007; 27 (4): 660–9. DOI: https://www.doi.org/10.1016/j.immuni.2007.08.013

6. Van Heel D.A., Ghosh S., Hunt K.A., Mathew C.G., Forbes A., Jewell D.P., et al. Synergy between TLR9 and NOD2 innate immune responses is lost in genetic Crohn’s disease. Gut. 2005; 54 (11): 1553–7. DOI: https://www.doi.org/10.1136/gut.2005.065888

7. van Heel D.A., Ghosh S., Butler M., Hunt K., Foxwell B.M.J., Mengin-Lecreulx D., et al. Synergistic enhancement of Toll-like receptor responses by NOD1 activation. Eur J Immunol. 2005; 35 (8): 2471–6. DOI: https://www.doi.org/10.1002/eji.200526296

8. Pichugin A.V., Bagaev A.V., Lebedeva E.S., Chulkina M., Ataullakhanov R.I. Synergistic cytokine production by murine dendritic cells in response to their simultaneous activation with pairs of agonists of different innate immune receptors. Immunologiya. 2017; 38 (2): 118–23. DOI: https://www.doi.org/10.18821/0206-4952-2017-38-2-118-123

9. Takada H., Galanos C. Enhancement of endotoxin lethality and generation of anaphylactoid reactions by lipopolysaccharides in muramyl-dipeptide-treated mice. Infect Immun. 1987; 55 (2): 409–13. DOI: https://www.doi.org/10.1128/iai.55.2.409-413.1987

10. Murch O., Abdelrahman M., Kapoor A., Thiemermann C. Muramyl dipeptide enhances the response to endotoxin to cause multiple organ injury in the anesthetized rat. Shock. 2008; 29 (3): 388–94. DOI: https://www.doi.org/10.1097/SHK.0b013e3181453e59

11. Poltorak A., He X., Smirnova I., Liu M.Y., Van Huffel C., Du X. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene. Science. 1998; 282 (5396): 2085–8. DOI: https://www.doi.org/10.1126/science.282.5396.2085

12. Girardin S.E., Travassos L.H., Hervé M., Blanot D., Boneca I.G., Philpott D.J. et al. Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. J Biol Chem. 2003; 278 (43): 41702–8. DOI: https://www.doi.org/10.1074/jbc.M307198200

13. Masyutina A.M., Murugina N.E., Pashchenkova Y.G., Pashenkov M. V. Comparison of transcriptional profiles of human macrophages activated by agonists of NOD1 and TLR4 receptors. Immunologiya. 2023; 44 (1): 16–27. DOI: https://www.doi.org/10.33029/0206-4952-2023-44-1-16-27

14. Patro R., Duggal G., Love M.I., Irizarry R.A., Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017; 14 (4): 417–9. DOI: https://www.doi.org/10.1038/nmeth.4197

15. Benjamini Y., Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B. 1995; 57 (1): 289–300. DOI: https://www.doi.org/10.1111/j.2517-6161.1995.tb02031.x

16. Gearing L.J., Cumming H.E., Chapman R., Finkel A.M., Woodhouse I.B., Luu K., et al. CiIIder: A tool for predicting and analysing transcription factor binding sites. PLoS One. 2019; 14 (9): e0215495. DOI: https://www.doi.org/10.1371/journal.pone.0215495

17. Napolitani G., Rinaldi A., Bertoni F., Sallusto F., Lanzavecchia A. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1 -polarizing program in dendritic cells. Nat. Immunol. 2005; 6 (8): 769–76. DOI: https://www.doi.org/10.1038/ni1223

18. Tukhvatulin A.I., Gitlin I.I., Shcheblyakov D. V., Artemicheva N.M., Burdely L.G., Shmarov M.M., et al. Combined stimulation of Toll-like receptor 5 and Nod1 strongly potentiates activity of NF-κB, resulting in enhanced innate immune reactions and resistance to Salmonella enterica serovar typhimurium infection. Infect Immun. 2013; 81 (10): 3855–64. DOI: https://www.doi.org/10.1128/IAI.00525-13

19. Bagaev A.V., Rybinets A.S., Fedorova A.A., Ushakova E.I., Lebedeva E.S., Pichugin A.V., et al. Synergism of TLR3 and TLR4 agonists during macrophage reprogramming into an antitumor state. Immunologiya. 2021; 42 (6): 615–30. DOI: https://www.doi.org/10.33029/0206-4952-2021-42-6-615-630

20. Murugina N.E., Murugin V.V., Pashenkov M.V. Antitumor activity of human macrophages activated by NOD1 and TLR4 receptor agonists in vitro. Immunologiya. 2022; 43 (5): 548–57. DOI: https://www.doi.org/10.33029/0206-4952-2022-43-5-548-557

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