Assessing Toll-like receptor 3/4 signalling as a phytocannabinoid target in immune cells

Abstract

Toll-like receptors (TLRs) are the sensors of pathogen associated molecules that trigger tailored innate immune intracellular signalling responses. TLRs are expressed on cells of the immune system and play an important role in immune cell activation and inflammatory responses. Indeed, TLRs have been implicated in many diseases, with data from human and animal studies identifying TLRs as players in the pathogenesis of multiple sclerosis (MS). Uncontrolled and atypical activation of TLR signalling can result in neuroinflammation, and in macrophages, innate immune responses to bacterial (via TLR4) and viral (via TLR3) signalling is key in mediating cellular inflammation. Targeting TLR3/4 signalling with novel therapeutics may represent an important avenue on the road to developing improved therapies for MS, and possibly other neuroinflammatory conditions. Cannabinoids are biologically active compounds extracted from the hemp plant Cannabis sativa L. (C. sativa), commonly known as phytocannabinoids, synthesised in our bodies (endogenous cannabinoids) or are artificially created (synthetic cannabinoids). Cannabinoids can reduce the symptoms associated with experimental autoimmune encephalomyelitis (EAE), the murine model of MS, and clinical studies have demonstrated the therapeutic potential of phytocannabinoids in people with (pw)MS. Indeed, Sativex is an oromucosal spray containing cannabidiol (CBD) and Delta9-tetrahydrocannabinol (THC) as its most abundant phytocannabinoid components, in addition to other phytocannabinoids and non-phytocannabinoid components, and has been shown to palliate symptoms associated with MS. The full cellular mechanism of action of the components of Sativex in human cells is unclear. A growing body of literature indicates that cannabinoids can modulate TLR-induced inflammatory signalling events. Indeed, evidence suggests that TLR3 signalling via TIR-domain-containing adaptor-inducing IFN-Beta; (TRIF)-dependent activation of interferon regulatory factor 3 (IRF3), in addition to TLR4 signalling via myeloid differentiation primary response 88 (MyD88) to nuclear factor (NF)-KappaB, may be targeted by cannabinoids to regulate cellular inflammation. The data presented herein characterised TLR3 and TLR4 signalling in the human THP-1 monocyte cell line, THP-1-derived macrophages and primary human peripheral blood mononuclear cells (PBMCs) in terms of inflammatory cytokine (TNFα) chemokine (CCL5, CXCL10) and type I interferon (IFN)-β; expression. This study demonstrates that THP-1 monocytes are a poor model for investigating TLR3-induced signalling mechanisms and that TLR3 and TLR4 signalling is operative in THP-1-derived macrophages. Importantly, CBD and THC differentially targeted MyD88-dependent and independent signalling mechanisms via TLR3 and TLR4 in THP-1-derived macrophages, in terms of IFN-β; and CXCL10 expression. Data also suggests that THC and CBD do not target TLR signalling via the cannabinoid receptors (CB1 and CB2) and the PPARγ; receptor in THP-1 macrophages. In addition, the effects of THC and CBD were examined in PBMCs isolated from pwMS and healthy control (HC) donors. PBMC data identify THC and CBD as potential novel regulators of TLR3/4 signalling in primary immune cells, and highlights possible mechanisms to be targeted in the development of new cannabinoid therapeutics for the treatment of disorders such as MS. Finally, the effect of a range of novel highly purified botanically-derived cannabinoids on the viability of THP-1 monocytes, THP-1 macrophages, and PBMCs from HC donors and pwMS was determined. Findings here suggest that phytocannabinoids are generally well tolerated by immune cells, however the effect was dependent on the cannabinoid used, the concentration administered, and the immune cell examined. Overall this study offers mechanistic insight on the role of phytocannabinoids in modulating cellular inflammatory signalling events controlled by TLR3/4.