Various studies have implicated neuroinflammatory NLRP3 inflammasome activation in a host of neurodegenerative diseases with neurovascular aspects, though the interaction between the inflammasome and the blood-brain barrier has yet to be deciphered. This project aims to assess the effect of NLRP3 inflammasome activation on the integrity of the neural endothelium, independent of cell death, and investigate the underlying pathways. Furthermore, this project aimed to elucidate the electrophysiological effects of endothelial TLR4 activation by LPS, with potassium efflux being a key regulator of NLRP3 inflammasome activation.
hCMEC/D3 cells were cultured in EGM-2 Basal Medium and SingleQuots supplements 5% foetal bovine serum, 0.4% human fibroblast growth factor-B, 0.04% hydrocortisone and 0.1% ascorbic acid along with 1% Pen-Strep. LPS treatments were conducted over 6 hours, or 5 hours + hour 10µM nigericin with or without an hour of 20µM MCC950 treatment. Permeability assays were conducted on endothelial monolayers cultured on 0.4µm Transwell membranes, followed by ion permeability assessment by a Voltohmeter, and 2000kDa Dextran-Fluorescein flow determined by ID3 Spectramax plate reader. Protein for western blotting was obtained by scraping with RIPA buffer and membranes were read using a LICOR ODESSEY. Immunofluorescence was imaged using a Zeiss AxioObserver7 confocal microscope and analysed by ImageJ.
Whole-cell currents were recorded using a MultiClamp 700B amplifier for data acquisition (n=6-10 cells per condition) and analysis was performed with Clampex 11.2 (Molecular Devices) software. Whole-cell currents were recorded in HEPES buffered standard solution at a perfusion rate of 4ml/min and ∼35ºC.
All graphed data is presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 10 software and used one- or two-way ANOVA followed by Tukey’s multiple comparisons, or unpaired t-tests. Significance was set at *p < 0.05.
Following treatment, Dextran permeability increased by 5.89×10-12±1.513×10-12 mol/cm2×S (p=0.0078) and TEER decreased by 42.68±7.944% (p=0.0003) in comparison to the healthy controls. MCC950 prior to nigericin addition showed a 36.97±10.26 % increase (p=0.013) in ion permeability compared with the LPS + nigericin treated groups (n=3-7 independent experiments). Western blotting confirmed inflammasome activation and immunofluorescence showed inflammasome localisation, a shift in morphology and a reduction of tight and adherens junction localisation to the cell membranes in NLRP3 inflammasome activated cells (3 replicates). Interestingly, LPS treatment reduced whole-cell currents at 50mV by -100.2±31.37pA (p=0.0085, N=6-7) compared with untreated cells and V ¹/² K⁺ by 10.99±4.302mV (p=0.0268, N=6-7).
These findings imply that NLRP3 activation is disruptive to the BBB by altering the morphology and junction protein expression of microvasculature endothelial cells, though further research is needed to decipher the underlying pathways involved. The effect of NLRP3 inflammasome inhibitor MCC950 in reversing some of these effects could show some future therapeutic potential for rescuing inflammatory-associated vascular dysfunction in AD in conjunction with anti-amyloid treatments. The electrophysiology studies show a novel effect of TLR4 activation that may influence K⁺ efflux for inflammasome activation.