Toll-Like Receptor Ligands and Interferon-γ Synergize for Induction of Antitumor M1 Macrophages

Abstract

Tumor-associated macrophages may either promote or suppress tumor growth depending on their activation status. Interferon-γ (IFN-γ) has been identified as a key factor for inducing tumoricidal M1 phenotype in macrophages. However, it remains unclear whether IFN-γ is sufficient or if additional stimuli are required. Here, we tested IFN-γ and a panel of toll-like receptor (TLR) agonists for the ability to activate murine macrophages toward a tumoricidal M1 phenotype. The following TLR ligands were used: TLR1/TLR2 agonist Pam3CSK4, TLR2/TLR6 agonist lipotechoic acid, TLR3 agonist poly(I:C), TLR4 agonist lipopolysaccharide (LPS), TLR5 agonist flagellin, TLR7 agonist CL264, and TLR9 agonist CpG. We used an in vitro growth inhibition assay to measure both cytotoxic and cytostatic activity of mouse macrophages against Lewis lung carcinoma (LLC) and MOPC315 plasmacytoma tumor cells. Production of nitric oxide (NO) and cytokines by activated macrophages was quantified. We found that IFN-γ alone was not able to render macrophages tumoricidal. Similarly, macrophage activation with single TLR agonists was inefficient. In sharp contrast, IFN-γ was shown to synergize with TLR agonists for induction of macrophage tumoricidal activity and production of both NO and pro-inflammatory cytokines (TNF-α, IL-12p40, and IL-12p70). Furthermore, IFN-γ was shown to suppress macrophage IL-10 secretion induced by TLR agonists. NO production was necessary for macrophage tumoricidal activity. We conclude that two signals from the microenvironment are required for optimal induction of antitumor M1 macrophage phenotype. Combination treatment with IFN-γ and TLR agonists may offer new avenues for macrophage-based cancer immunotherapy.

Bone Marrow-Derived and Elicited Peritoneal Macrophages Are Not Created Equal: The Questions Asked Dictate the Cell Type Used

“…Polarization was done on adherent macrophages (1 × 10 6 ) with 100 ng/mL IFN-γ or IL4 + IL13 (25 ng/ml each) for 24 h. Cells thus polarized (and with a non-treated control) were used for flow cytometry, respiratory burst, quantitative polymerase chain reaction (qPCR), and cytometric bead arrays. While LPS + IFN-γ is often used together for M1 polarization (10), and synergize in cytotoxicity assays (37,38), we found that a 24 h incubation with IFN-γ was sufficient to M1 polarize (Tables 2-4), with the addition of LPS not significantly enhancing the IFN-γ responses for either cytokine release nor phagocytic rate (Supplemental Figure 1). As cytotoxicity assays are done on the order of days, it may be that feedback and/or gene regulation that occurs in that timeframe contributes to synergy.…”

Poly(I:C) stimulation is superior than Imiquimod to induce the antitumoral functional profile of tumor‐conditioned macrophages

“…As most studies looked at APCs, especially DCs, and as gene modulation appears to be cell‐dependent, it is possible that purified macrophages behave differently. The cytokine network is also important; in fact Muller et al reported that the response to various TLR ligands differed if specific additional cytokines, primarily, IFNγ was available to macrophages .…”

“…As most studies looked at APCs, especially DCs, and as gene modulation appears to be cell-dependent, it is possible that purified macrophages behave differently. The cytokine network is also important; in fact Muller et al reported that the response to various TLR ligands differed if specific additional cytokines, primarily, IFNγ was available to macrophages [41]. Previous studies reported that the potential antitumoral effects of TLR agonists were mediated through activation of innate immunity, as well as by increased recruitment of T-cells within tumors.…”

Malignant transformation of oral leukoplakia is associated with macrophage polarization

“…The exact mechanism of action of Imiquimod is not yet understood. However, there is evidence, that TLR activation in combination with a second pro-inflammatory signal can transform macrophages towards the anti-tumoral M1 type [75].…”