Cannabidiol (CBD) exerts its anti-inflammatory mechanisms under various inflammatory conditions, including neuroinflammation [1]. Neuroinflammation is a complex concert of immune mediators that range from the innate to adaptive immune system, and CBD has been shown to impact each step of the inflammatory process [1]. For diseases that affect the brain, such as the murine multiple sclerosis (MS) model, experimental autoimmune encephalomyelitis (EAE), treatment with CBD decreased overall clinical symptoms and associated inflammation at the peak of the disease [2], [3], [4], [5], [6], [7]. However, the CBD-associated anti-inflammatory mechanisms that contribute to the attenuation of the EAE remain to be elucidated.

Key innate immune cells of interest are macrophages, as they play a role in disease propagation and attenuation [8]. In pathologies such as MS and its murine model, EAE, macrophages not only make an essential contribution in pro-inflammatory cytokine secretion, such as tumor necrosis factor-alpha (TNF-α) [9], [10], but also phagocytose myelin, resulting in tissue damage [11], [12]. Both microglial cells and infiltrating macrophages contribute to demyelination, although there is evidence that microglial cells and meningeal macrophages specifically are responsible for antigen presentation and T cell activation initially in EAE disease [13].

Given the critical role of macrophages early in EAE disease initiation [13], and the fact that CBD administered for only the first 5 days after EAE disease initiation resulted in attenuation of clinical signs and inflammation [4], it is important to further characterize the effects of CBD on the anti-inflammatory effects of macrophages. It is established that macrophages (and microglial cells) can polarize into at least two phenotypes: the inflammatory phenotype (M1) and an anti-inflammatory, pro-tissue resolving phenotype (M2a) [14]. Thus, the purpose of this study was to examine the effects of CBD on M1/M2 polarization of mouse RAW264.7 macrophages and investigate mechanisms by which CBD was anti-inflammatory. We observed differential effects of CBD on TNF-α, depending on whether it was expressed intracellularly, extracellularly, or secreted, which prompted us to examine the mechanisms by which CBD altered TNF-α secretion. The tumor necrosis factor-α converting enzyme (TACE) is the enzyme responsible for cleaving membrane-bound TNF-α, facilitating its cellular release [15]. Thus, we hypothesized that the mechanism by which CBD suppressed secreted TNF-α is due in part to inappropriate localization of TACE.