Antitumor activity of human macrophages activated by NOD1 and TLR4 receptor agonists in vitro

Abstract

Introduction. Tumor-associated macrophages play an important role in tumor development by modulating inflammation, immunosuppression and angiogenesis. Depending on the microenvironment, macrophages can exhibit both pro- and antitumor activity. Agonists of pattern recognition receptors, including Toll-like receptors (TLR) and NOD receptors, promote the induction of antitumor activity of macrophages. It is known that combined stimulation of NOD1 and TLR4 receptors in macrophages and other cell types results in a synergistic increase in the expression and production of pro-inflammatory cytokines. However, the effect of a combination of NOD1 and TLR4 agonists on the antitumor properties of macrophages has not been studied.

The aim of the work was to study the mechanisms of antitumor activity of macrophages stimulated by NOD1, TLR4 receptor agonists and their combination in vitro.

Material and methods. The cells of the erythromyeloid HLA-I-negative K562 line labeled with CFSE were used as targets for macrophages. Target cells were cocultured with human macrophages in the presence of a NOD1 agonist, TLR4 agonist, combination thereof or without agonists. K562 cultures without macrophages served as controls. 72 hours after the start of the experiment, the content of viable target cells in cultures was assessed using flow cytometry. To clarify the contribution of soluble mediators and surface molecules to the antitumor activity of macrophages, the effect of activated macrophage supernatants on K562 cells was studied, and macrophages and K562 cells were separated using semipermeable membranes. Neutralizing antibodies were used to assess the contribution of TNF and IFN-β to the antitumor effect of macrophages. NADPH oxidase was inhibited with apocynin.

Results. Macrophages not activated by NOD1 or TLR4 agonists enhanced the growth of K562 tumor cells compared to the control. Separate NOD1 and TLR4 agonists abolished the protumor effect of macrophages, while stimulation of macrophages with a combination of agonists led to a multiple decrease in the number of K562 cells compared to the control. In the absence of macrophages NOD1 and TLR4 agonists did not affect the number of K562 cells. The antitumor effect of macrophages activated by a combination of NOD1 and TLR4 agonists was partially abolished by anti-TNF antibodies and when macrophages and K562 cells were separated by a semipermeable membrane. It has been shown that one of the components of the antitumor activity of macrophages activated by a combination of NOD1 and TLR4 agonists is an antiproliferative effect.

Discussion contains an analysis of the possible mechanisms of the antitumor effect of macrophages activated by a combination of NOD1 and TLR4 agonists.

Conclusion. The possibility of switching the activity of human macrophages from protumor to antitumor under the action of a combination of NOD1 and TLR4 agonists in vitro has been shown. To implement the antitumor response of macrophages against K562 cells, TNF secretion and direct intercellular interaction between K562 and macrophages are required. The results obtained allow to consider the combination of NOD1 and TLR4 agonists as a potential anticancer drug.

Keywords:macrophages; antitumor immunity; K562 cells; pattern recognition receptors; NOD1; TLR4; tumor necrosis factor

For citation: 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://doi.org/10.33029/0206-4952-2022-43-5-548-557

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. Performing experiments, writing the article – Murugina N.E.; performing experiments – Murugin V.V.; planning and performing experiments, writing and editing the article – Pashenkov M.V.

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