TLR-4 Agonist Induces IFN-gamma Production Selectively in Proinflammatory Human M1 Macrophages through the PI3K-mTOR- and JNK-MAPK-Activated p70S6K Pathway

IFN-gamma, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-gamma production by murine macrophages, IFN-y production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-gamma and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-gamma. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-gamma production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-gamma that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-gamma production, suggesting that differentiation with IFN-gamma plays a key role in IFN-gamma induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-gamma production in M1 macrophages. Our results show that TLR-4-induced IFN-gamma production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-gamma may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.