A study of the metabolic immune responses produced by some natural products

Macrophages play a crucial role in inflammatory conditions by producing a range of pro-inflammatory molecules, including cytokines, nitric oxide and reactive oxygen species (ROS). Several studies have demonstrated the physiological and inflammatory responses of macrophages to different stimuli, such as lipopolysaccharide (LPS), the Gram-negative-derived bacterial cell wall component. However, research is lacking on the metabolic responses of macrophages to these stimuli. Recently, the involvement of metabolic pathways in the regulation of LPS signalling and macrophage activation has become a focus of research in inflammation. The aim of the present study was to assess and characterise cytokine production and the metabolic profiles of THP-1 monocyte-derived macrophage cells following stimulation with LPS, alone or in combination with different natural products. Some of these natural products, such as melittin and eicosanoids, were considered to act as vaccine adjuvants and to enhance the LPS-stimulated release of cytokines and inflammatory metabolite signalling, while others, such as propolis and gypenoside (Gyp), were suggested to antagonise LPS action, thereby modulating immune responses. Macrophages were therefore viewed as having the potential to undergo different reprogrammed pathways once activated by LPS. In this study, THP-1 cells were differentiated using phorbol 12-myristate 13-acetate (PMA) for 48 hours and then rested for 24 hours by replacing the PMA-containing culture medium with normal medium. The cells were then treated with LPS alone, a natural product alone or a combination of the natural product and LPS for another 24 hours. The THP-1 macrophages were then evaluated for the release of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1Î2 (IL-1Î2), IL-6 and IL-10 using enzyme-linked immunosorbent assay (ELISA) methods. The cells were extracted and their metabolites were characterised by liquid chromatography-mass spectrometry (LC-MS). LPS treatment of THP-1 cells mainly increased glycolysis and the pentose phosphate pathway and decreased the TCA cycle activity. Several biomarker metabolites were altered significantly by LPS, including NADPH, glutathione, oxidised glutathione, L-citrulline, L-arginine, L-ornithine, hypoxanthine, xanthine and urate. The addition of melittin enhanced the LPS effects on the cellular metabolome and cytokine secretion. Several fatty acids, including arachidonic acid, were substantially affected by melittin. Similar responses were found for eicosenoid compounds, which were able to stimulate the immune response and enhance the LPS inflammatory activities. By contrast, propolis (samples obtained from different geographic regions) and Gyp were able to trigger anti-inflammatory actions and inhibit the LPS-induced cytokine release, while also antagonising LPS effects on the cellular metabolome. These findings point to a need to evaluate the possible anti-inflammatory actions of some reported natural products as a strategy for identifying new therapies and confirming the targeted pathways and/or metabolites affected by inflammatory conditions. Understanding the metabolic pathways in reprogrammed macrophages is critical for planning further investigations of immune adjuvants for vaccines. The immunomodulatory effects observed in THP-1 cells highlight the potential use of natural products in clinical therapies.