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


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

For citation: Masyutina A.M., Pashenkov M.V. Transcriptional response of macrophages to combined stimulation of a NOD-like and a Toll-like receptor. Immunologiya. 2023; 44 (4): 408–18. DOI: (in Russian)

Funding. The work was supported by the Russian Science Foundation grant No. 21-15-00211.

Conflict of interests. The authors declare no conflict of interests.

Authors’ contribution. Conducting experiments and analysing results – Masyutina A.M.; designing experiments, analysing results and writing the article – Pashenkov M.V.


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Musa R. Khaitov

Corresponding member of Russian Academy of Sciences, MD, Professor, Director of the NRC Institute of Immunology FMBA of Russia