Cadmium-induced neuroinflammatory cytokines impairs BBB permeability by altering tight junction morphological localization

Cadmium (Cd) is a widespread environmental pollutant found in cigarette smoke, traffic and industrial fumes, as well as paints, batteries, and pesticides. It is toxic to many organs including the central nervous system (CNS) by crossing the blood-brain barrier (BBB), leading to inflammation in the brain parenchyma affecting the glial compartment [1]. As previously reported, Cd-induced glial activation leads to cytokines secretion, such as TNF-α, INF-γ, IL-6, and IL-6, thus contributing to CNS homeostasis perturbance [2]. The present research aimed to study the effect of microglial-released cytokines on endothelial cell compartments at differ- ent time points in order to better understand the role of pro-inflammatory molecules in BBB permeability. For this purpose, different concentrations of TNF-α (1-100 ng/ml) at different time points (3-48 hours) were used on rat brain endothelial cell line (RBE4). Immunofluo- rescence staining, western blotting analysis, and Trans- Endothelial Electrical Resistance (TEER, IVTech, Pisa, Italy), were performed. The results clearly demonstrated that TNF-α 10ng/ml induces the NF-κB nuclear translo- cation after 3h of treatment, thus underlining the tran- scription factor activation pathway. Furthermore, the cytokine treatment alters the zonula occludens 1 (ZO1) distribution on the peripheral edge of RBE4 as early as 10ng/ml after 24-h treatment, showing a more perinuclear localization without a significative decrease of protein expression and no F-actin altered localization. This morphological alteration indicates a putative BBB permeability, confirmed by a TEER decrease with no cell viability alterations in comparison to control, untreated cells. Our findings highlight the deleterious role of environmental pollutants on the CNS directly affecting BBB permeability and indirectly inducing cytokine release from glial compartment, leading to sustained brain endothelial cells permeabilization. This work was supported by #NEXTGENERA- TIONEU (NGEU) and funded by the Ministry of Uni- versity and Research (MUR), National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006) – A Multiscale integrated approach to the study of the nervous system in health and disease (DR. 1553 11.10.2022).