In the present study, we investigate the effects of combined stimulation with IL-6 and extracellular adenosine triphosphate (eATP) – two inflammatory stimuli often increased in neuroinflammatory disorders – on human microglia inflammatory marker expression. We then evaluate the potential of cannabidiol (CBD) treatment to mitigate the resulting microglial activation state in monoculture as well as in coculture with induced pluripotent stem cell (iPSC)-derived cortical neuron-like cells. To characterize IL-6/eATP microglial activation relative to a canonical control, the human microglial cell line HMC3 was activated with either combined 5 ng/mL IL-6 and 1 mM eATP or with combined 100 ng/mL lipopolysaccharide (LPS) and 1 mM eATP.
Proteomic measures of secreted cytokines/chemokines were performed and compared to unstimulated controls. To assess the impact of CBD on IL-6/eATP activated microglia, HMC3 cells were exposed to IL-6/eATP and then treated with varying concentrations of CBD (0.1 µM, 1 µM, and 5 µM) or with 10 µM dexamethasone (Dex) for 6 h and 24 h, after which inflammatory cytokines/chemokines as well as oxidative stress associated enzymes NOS2 and NOS3 were quantified. The CBD concentration identified as most effective in mitigating IL-6/eATP HMC3 activation was then applied to a coculture model of HMC3 microglia and iPSC-derived cortical neuron-like cells.
IL-6/eATP and LPS/eATP differentially activated HMC3 microglia, with LPS/eATP exposure inducing increased IL-8, IL-6, CCL2, VEGF-A secretion and IL-6/eATP stimulation inducing increased IL-1β, IFNγ, CCL2, VEGF-A, and IL-10 secretion relative to unstimulated controls. Application of 1 µM CBD to HMC3 cultures following IL-6/eATP activation resulted in decreased IL-6, VEGF-A, and NOS2 production at 6 h as well as reduced IFNγ, IL-1β, and IL-6 production at 24 h. In HMC3 cocultures with cortical neuron-like cells, IL-6/eATP stimulation increased neuron levels of proinflammatory proteins TNFα, cellular C1q, and NOS2.
It also decreased extracellular levels of inflammatory modulator soluble neuropilin-1 (sNRP-1) and increased neuron levels of the excitatory receptor scaffolding protein PSD-95 relative to the general synaptic marker synaptophysin (SYP). Application of 1 µM CBD for 24 h to IL-6/eATP stimulated cocultures resulted in neuronal TNFα levels, NOS2 levels, and PSD-95/SYP ratios which were statistically indistinguishable from unstimulated cocultures, although sNRP-1 and cell-associated C1q remained at activated levels. Cumulatively, these data indicate that 1 µM CBD partially mitigated IL-6/eATP-associated alterations to HMC3 microglia and cortical neuron-like cells in terms of the assessed inflammatory markers.
These results provide insight into the effects of IL-6/eATP-mediated microglial activation and the potential therapeutic efficacy of CBD in attenuating these effects. The authors would like to thank Dr. Ahmad Arabiyat for beneficial discussions regarding this manuscript.
This research was supported, in part, by the National Institute of Health R03AG067140 and R03AG067970 to M.S.H and by the National Institute of General Medical Sciences T32 Training Grant (Award Number T32GM067545). These authors contributed equally: Daniel J. Department of Biomedical Engineering, Rensselaer Polytechnic Institute (RPI), Troy, NY, USA Daniel J.
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