Melatonin Maintains Macrophage M1 Phenotype to Reverse LPS-stimulated Tumor Immune Tolerance

Abstract

Background: Lipopolysaccharide (LPS) is a potent trigger of macrophage-mediated inflammation and its repeated stimulation results in immune tolerance. This study is to explore the cellular mechanisms of LPS-mediated tumor immune tolerance and to investigate whether melatonin can reverse this tolerance. Methods: The effect of melatonin and LPS on macrophages was assessed by cell proliferation, morphological changes, phagocytosis and autophagy in vitro. The tumor-preventing effect of melatonin and LPS were evaluated in the urethane-induced lung carcinoma model and in the H22 liver cancer allograft model. Immunofluorescence, immunohistochemistry and ELISA were used to examine protein expression. The related targets and pathways of melatonin were predicted by comprehensive bioinformatics, and the clinical association of bacterial infections and survival was evaluated in cancer patients by meta-analysis.Results: In vitro，Raw264.7 macrophages were polarized toward the M1 phenotype by single LPS administration but toward the M2 phenotype by repeated LPS administration. Interestingly, combination treatment with repeated LPS and 10 µM melatonin prevented macrophage polarization toward the M2-like phenotype and exerted lasting antitumor efficacy. In the urethane-induced lung carcinoma model, repeated LPS administration stimulated macrophage polarization toward the M2 phenotype and promoted lung carcinogenesis, which was abrogated by macrophage depletion, while melatonin alone or in combination with repeated LPS challenge restored M1-like macrophages and prevented carcinogenesis. In the H22 liver cancer allograft model, melatonin maintained the macrophage phenotype and promoted the tumor-suppressing effect of repeated LPS challenge. Furthermore, we found that macrophages repeatedly stimulated with LPS had a high level of surface lipid rafts that mediated PI3K/AKT and JAK2/STAT3 signaling and prevented both LPS sensitivity and immune response by self-expression of PD-L1 and surface expression of PD-1 receptor on NK cells, whereas melatonin decreased surface lipid rafts and PI3K/AKT and JAK2/STAT3 signaling. Finally, we conducted a comprehensive bioinformatics analysis of melatonin-relevant targets and pathways involved in M2 macrophage polarization and evaluated the clinical associations of bacterial infections and survival in cancer patients. Conclusions: This study suggests a function of melatonin in regulating macrophage polarization to maintain LPS-stimulated tumor immune surveillance.