The innate immune response to bacterial and viral molecules involves the coordinated production of cytokines, chemokines, and type I interferons (IFNs), which is orchestrated by toll-like receptors (TLRs). TLRs, and their intracellular signalling intermediates, are closely associated with multiple sclerosis (MS) pathogenesis. Recent data from our laboratory reported that the plant-derived cannabinoids, Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD), regulate viral and bacterial inflammatory signalling pathways controlled by TLR3 and TLR4 in macrophages. The aim of this study was to assess the impact of THC and CBD, when delivered in isolation and in combination (1:1), on TLR3- and TLR4-dependent signalling in peripheral blood mononuclear cells (PBMCs) from people with MS (pwMS; n = 21) and healthy controls (HCs; n = 26). We employed the use of poly(I:C) and lipopolysaccharide (LPS) to induce viral TLR3 and bacterial TLR4 signalling, and PBMCs were pre-exposed to plant-derived highly purified THC (10 μM), CBD (10 μM), or a combination of both phytocannabinoids (1:1 ratio, 10:10 μM), prior to LPS/poly(I:C) exposure. TLR3 stimulation promoted the protein expression of the chemokine CXCL10 and the type I IFN-β in PBMCs from both cohorts. THC and CBD (delivered in 1:1 combination at 10 μM) attenuated TLR3-induced CXCL10 and IFN-β protein expression in PBMCs from pwMS and HCs, and this effect was not seen consistently when THC and CBD were delivered alone. In terms of LPS, TLR4 activation promoted TNF-α expression in PBMCs from both cohorts, and, interestingly, CBD when delivered alone at 10 μM, and in combination with THC (in 1:1 combination at 10 μM), exacerbated TLR4-induced TNF-α protein expression in PBMCs from pwMS and HCs. THC and CBD displayed no evidence of toxicity in primary PBMCs. No significant alteration in the relative expression of TLR3 and TLR4 mRNA, or components of the endocannabinoid system, including the cannabinoid receptor CB₁ (encoded by CNR1 gene) and CB₂ (encoded by CNR2 gene), and endocannabinoid metabolising enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGLL), was determined in PBMCs from pwMS versus HCs. Given their role in inflammation, TLRs are clinical targets, and data herein identify CBD and THC as TLR3 and TLR4 modulating drugs in primary immune cells in vitro. This offers insight on the cellular target(s) of phytocannabinoids in targeting inflammation in the context of MS.

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