Immunometabolic Modulatory Role of Naltrexone in BV-2 Microglia Cells

Abstract

Naltrexone is an opioid receptor antagonist commonly used to treat opioid and alcohol dependence. The use of low dose naltrexone (LDN) was found to have anti-inflammatory properties for treatment of diseases such as fibromyalgia, Crohn’s disease, multiple sclerosis and regional pain syndromes. Related to its anti-neuroinflammatory properties, the mechanism of action is possibly mediated via Toll-like receptor 4 antagonism, which is widely expressed on microglial cells. The aim of the present study was to assess the immunometabolic effects of naltrexone on microglia cells in in vitro conditions. Methods: All experiments were performed in the BV-2 microglial cell line. The cells were treated with naltrexone at 100 μM concentrations corresponding to low dose for 24 h. Cell viability was assessed for every drug dose. To induce additional activation, the cells were pretreated with LPS and IFN-γ. Immunofluorescence was used to analyse the classical microglial activation markers iNOS and CD206, while Seahorse was used for real-time cellular metabolic assessments. mTOR activity measured over the expression of a major direct downstream target S6K was assessed using western blot. Results: LDN induced a shift from highly activated pro-inflammatory phenotype (iNOShighCD206low) to quiescent anti-inflammatory M2 phenotype (iNOSlowCD206high) in BV-2 microglia cells. Changes in the inflammatory profile were accompanied by cellular metabolic switching based on the transition from high glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). LDN-treated cells were able to maintain a metabolically suppressive phenotype by supporting OXPHOS with high oxygen consumption, and also maintain a lower energetic state due to lower lactate production. The metabolic shift induced by transition from glycolysis to mitochondrial oxidative metabolism was more prominent in cells pretreated with immunometabolic modulators such as LPS and IFN-γ. In a dose-dependent manner, naltrexone also modulated mTOR/S6K expression, which underlies the cell metabolic phenotype regulating microglia immune properties and adaptation. Conclusion: By modulating the phenotypic features by metabolic switching of activated microglia, naltrexone was found to be an effective and powerful tool for immunometabolic reprogramming and could be a promising novel treatment for various neuroinflammatory conditions.

Therapeutic trials for long COVID-19: A call to action from the interventions taskforce of the RECOVER initiative

“…Low-dose Naltrexone (LDN) is commonly used off-label for people with ME/CFS, a condition characterized by debilitating fatigue and exertional intolerance that has significant clinical overlap with long COVID ( 78 ). From a mechanistic perspective, one model of LDN’s efficacy is through suppressive effects on microglia cells of the central nervous system and attenuation of proinflammatory cytokines ( 80 ). Microglia are resident macrophages in the brain and spinal cord and normally exist in a resting state but once activated produce proinflammatory factors that interact with neurons to cause hyperalgesia and “sickness response symptoms” such as fatigue, malaise, hypoactivity, sleep changes, etc., which have been demonstrated in animal models ( 80 , 81 ); there is intense interest in the role that these and other macrophage-derived cells might play in long COVID, particularly in neurocognitive symptoms ( 82 ).…”

“…From a mechanistic perspective, one model of LDN’s efficacy is through suppressive effects on microglia cells of the central nervous system and attenuation of proinflammatory cytokines ( 80 ). Microglia are resident macrophages in the brain and spinal cord and normally exist in a resting state but once activated produce proinflammatory factors that interact with neurons to cause hyperalgesia and “sickness response symptoms” such as fatigue, malaise, hypoactivity, sleep changes, etc., which have been demonstrated in animal models ( 80 , 81 ); there is intense interest in the role that these and other macrophage-derived cells might play in long COVID, particularly in neurocognitive symptoms ( 82 ). Additionally, data suggest that the benefits of LDN treatment in ME/CFS are mediated by its regulatory role involving the Transient Receptor Potential Channel Melastatin 3 ( 83 ).…”

Neuroprotection by Skimmianine in Lipopolysaccharide-Activated BV-2 Microglia

“…In this study, the inhibition of neuroinflammation by skimmianine was established following pre-treatment with the compound for 30 min, followed by stimulation with LPS for 24 h. However, a study reported by Kučić et al [ 39 ], showed that the inhibition of neuroinflammation was in fact possible by stimulating BV-2 microglia prior to treatment with low concentrations of naltrexone. It would therefore be interesting if future studies could determine whether reversing the sequence of skimmianine treatment and LPS stimulation would affect the anti-inflammatory activity of the compound.…”

Identification of an immune-related gene prognostic index for predicting prognosis, immunotherapeutic efficacy, and candidate drugs in amyotrophic lateral sclerosis

“…Among them, Astemizole has been reported that immunosuppressive potential of Astemizole against T-cell proliferation and cytokine secretion in macrophages by modulating mitogen-activated protein kinase (MAPK) signaling pathway ( Jakhar et al, 2018 ). As a result of low-dose Naltrexone administration, BV-2 microglia cells acquired a quiescent anti-inflammatory M2 phenotype from highly activated pro-inflammatory phenotypes ( Kučić et al, 2021 ). Furthermore, low-dose naltrexone has been shown to modulate Toll-like receptor four signaling as well as transiently blockend endogenous opioid receptors to reduce glial inflammation ( Toljan and Vrooman, 2018 ).…”