Synergism of TLR3 and TLR4 agonists during macrophage reprogramming into an antitumor state

Abstract

Introduction. Solid tumor tissue usually contains many macrophages. These cells actively support the progressive growth of malignant cells, stimulate angiogenesis, promote the growth of the fibrotic scaffold the tumor by activating divisions of fibroblasts and their transformation into myofibroblasts, immune responses inhibition with immunosuppressive substances such as IL-10, TGFβ, IDO, PGE2, and an induction of epithelial-mesenchymal transformation of malignant cells, which allows malignant cells to move freely within tissues (invasive tumor growth) and around the host organism (distant metastases). Macrophages’ plasticity, i.e. their ability for transition from one state to another, is well documented by previous studies. Particularly, tumor-promoting macrophages can be reprogrammed into anti-tumor M1 macrophages. External infection-derived signals acting via pathogen recognition receptors (TLR, NOD, etc.), as well as internal damage-derived signals acting via pro-inflammatory cytokine and other receptors, transfer macrophages into M1 state.

The aim of this study is to analyze the synergism of TLR3- and TLR4-agonists during macrophages reprogramming into their M1 antitumor state.

Material and methods. Macrophages were obtained from the mouse bone marrow using in vitro differentiation in the presence of GM-CSF. Cells were activated with synthetic poly I:C (TLR3 agonist) or acidic peptidoglycan of the plant origin (TLR4 agonist, active component of Immunomax, hereinafter IMM). The activation of TLR3- and TLR4-related downstream signaling pathways was measured according to TBK1/IKKε, ERK1 and ERK2 protein kinases, as well as NF-kB transcription factor phosphorylation intensities. Flow cytometry and Western blotting with specific monoclonal antibodies were used to measure phosphorylation degree of the signaling proteins. The expression intensity of Nos2 and Ifnbl mRNAs was investigated by a quantitative RT-PCR. The cytotoxic activity of macrophages was estimated in their co-culture with 4T1 breast carcinoma cells according to the reduction of the tumor cells number.

Results. A transformation of macrophages into an antitumor state can be induced with a combination of TLR3- and TLR4-agonists, more efficiently than with the same agonists used alone. Both agonists induced significant and rapid activation of TBK1/IKKε, ERK1/2 and NF-kB. Compared to the single agonists, the combined action of TLR3 and TLR4 agonists increased the kinases phosphorylation level by only 15-20 %. Even though the activated genes transcription rate was enhanced synergistically. The combined action of TLR3 and TLR4 agonists increased the level of Nos2 and Ifnbl mRNAs transcription by 2.6-3.5 times as compared to the transcription rate of the same genes in response to each of these agonists used alone. TLR3- and TLR4-agonists also synergistically activated the antitumor properties of macrophages in a cytotoxic test against 4T1 carcinoma cells.

Conclusion. The synergism of TLR3 and TLR4 agonists during reprogramming of macrophages into the M1 state has been described for the 1st time. The synergistic action of agonists leads to a multiple increase in the expression of genes encoding antitumor effector proteins, in particular, iNOS and IFN-p, as well as to a significant increase in the antitumor cytotoxic effect of macrophages. At the level of phosphorylation of TBK1/IKKε and ERK1/2 protein kinases, as well as the NF-kB transcription factor, no synergistic effect of TLR3 and TLR4 agonists was found, which indicates the presence of cooperative interaction between other components of these 2 signaling pathways.

Keywords:Toll-like receptors; agonists; synergism; reprogramming of macrophages; signaling; iNOS; IFN-β; antitumor effect

For citation: Bagaev A.V., Rybinets A.S., Fedorova A.A., Ushakova E.I., Lebedeva E.S., Pichugin A.V., Ataullakhanov R.I. Synergism of TLR3 and TLR4 agonists during reprogramming of macrophages to antitumor state. Immunologiya. 2021; 42 (6): 615-30. DOI: https://doi.org/10.33029/0206-4952-2021-42-6-615-630 (in Russian)

Funding. This study was supported by a grant from the RSF No. 20-15-00391.

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

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