Background

The Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that promotes the expression of antioxidant genes, protecting cells against oxidative stress and it also exerts immunomodulatory properties. The activation of Nrf2 is one of the proposed mechanisms of action of dimethyl fumarate (DMF), an approved drug for Multiple Sclerosis (MS).

DMF is rapidly metabolized into its active metabolite monomethyl fumarate (MMF) in the intestine. However, there is a lack of studies comparing the effects of both compounds. The combination of other Nrf2 activators could be relevant as adjuvants for DMF in neuroinflammation. Cannabidiol (CBD), a cannabinoid that attenuates MS in murine models, is known to have antioxidant properties, although there are no studies on Nrf2 activation by CBD.

Objectives

The aim of this study was to evaluate the in vitro effects of DMF, MMF and CBD on the activation of Nrf2 in neurons and microglia.

Methods

Primary hippocampal neurons and the microglial cell line BV-2 were treated for 4 hours with either vehicle, DMF (1-30 µM), MMF (1-30 µM) or CBD (1-10 μM). Cells were fixed, permeabilized and stained with a Nrf2 antibody. Activation of Nrf2 was considered as nuclear translocation, measured by confocal microscopy as the mean density of nuclear fluorescence. Five fields were taken from each condition in 3 experiments. One-way ANOVA test was used, considering p<0.05 statistically significant.

Results

DMF induced Nrf2 translocation in both neurons and microglia. However, Nrf2 translocation in neurons needed a higher dose (30 µM) than microglia (10 µM), and the induction was lower in neurons (four-fold increase) than in microglia (eight-fold increase). We did not find activation of Nrf2 with MMF in neither neurons nor microglia. CBD induced a dose-dependent Nrf2 activation in neurons, statistically significant at 6 and 7 μM, with a higher increase than that of DMF (8 and 12-fold compared to vehicle, respectively). CBD did not produce any effect on Nrf2 activation in microglia.

Conclusions

Our results support the idea that DMF acts as a neuroprotective and immunomodulatory drug through the activation of the Nrf2 pathway in neurons and microglia. We also demonstrate that DMF and MMF differ in their mechanisms of action, as we did not see Nrf2 activation with MMF. CBD could be relevant in neuroprotection as an adjuvant to DMF, as it induces a higher Nrf2 activation than DMF. CBD’s mechanism of action differs between neurons and microglia.

Background

The circulatory pattern of encephalitogenic cells (ECs) in the adoptively transferred experimental autoimmune encephalomyelitis (at-EAE) is not well known. Multimodal imaging nanoparticles (NPs), that combine more than one imaging moieties to be monitored by different imaging tecniques have gained special attention for cell monitoring.

Objectives

To evaluate the in vitro toxicity and internalization of new chitosan-based multimodal imaging NPs that incorporated IR820 NIR fluorescent probe, and superparamagnetic iron nanoparticles in ECs. To study the distribution of ECs labelled with internalized NPs after the induction of at-EAE.

Methods

Splenocytes and lymph nodes from C57BL/6 mice with actively induced EAE were cultured for 24, 48, 72 and 96 hours with MOG_35-55 (25µg/ml), IL-12 (25ng/ml), IL-18 (25ng/ml) and different dilutions of NPs (1/50 and 1/100) which had different concentrations of Fe (1/10 and 1/100). ECs viability (up to 90%) was assessed by flow cytometry and Alamar blue. NPs internalization was analyzed by confocal microscopy and flow cytometry using 633nm red laser.

After selecting the best cell viability and IR emission culture condition with NPs, 2 x 10⁶ ECs were injected intraperitoneally to induce at-EAE. Mice were sacrificed at different time points (1, 2, 3, 4, 7 and 24 days) and biodistribution of ECs was analyzed by confocal microscopy in heart, lymph nodes, lung, liver, kidney, spleen, brain and spinal cord..

To check whether the internalization of NPs affected the encephalitogenic capacity, clinical assessment was carried out for 24 days in two groups of animals. One of them was treated with ECs cultured with NPs, and the other with ECs without NPs.

Results

Confocal images obtained by fluorescence and reflection,corroborate the internalization of NPs and their persistence inside the cells over time. NPs did not affect the viability nor the encephalitogenic capacity of ECs. The highest fluorescence signal in ECs was obtained with the less diluted NPs (1/50) and the maximum concentration of Fe (1/10). These conditions were chosen for the induction of at-EAE. We have found the presence of ECs at days 1, 2 and 3 post injection in brain, spinal cord, lymph nodes and spleen.

Conclusions

Our findings suggest that activated ECs cross the blood-brain barrier rapidly after inoculation, locating themselves mainly in the central nervous system, where they trigger the inflammatory response that will produce the clinical characteristics of at-EAE. New multimodal imaging NPs allowed successful ECs monitoring due to their good tolerability and low toxicity.