Aim: Decipher how inflammation drives epilepsy and how NLRP3 targeting impacts epileptic-like activity.
Introduction: Epilepsy is one of the most common neurological diseases in worldwide. Inflammation was linked to the presence of inflammasomes, cytosolic multiprotein complexes, which promote the release of proinflammatory cytokines, namely Il-1β. Although a feedback loop has been described between inflammation and epilepsy, the role of inflammasomes in epilepsy is still unknown. NLRP3 is the most studied inflammasome,1 activated by a two-signal process: 1) a priming signal (as lipopolysaccharides – LPS), which enhances the expression of NLRP3 and pro-IL-1β; and 2) an activating signal (as ATP), which promotes the formation of the complex.
Methods: Organotypic slices were used to assess the interplay between inflammation and epilepsy. Slices were exposed to different concentrations of LPS (5, 10 and 20 ng/mL), either alone or in the presence of ATP (1mM). LPS-induced inflammation was characterized using molecular-based assays, such as ELISA to quantify IL-1β, CBA to measure TNF-α, and western blot to assess the expression of Iba-1, GFAP, NLRP3/ASC, and αII-Spectrin. Field potential recordings were used to evaluate the epileptic-like activity of the slices and the effect of MCC950, a NLRP3 selective inhibitor,2 was assessed.
Results: Results obtained by ELISA showed a significant increase in IL-1β concentration in slices exposed to 10 ng/ml LPS/1mM ATP. TNF-α, assessed by CBA, was also significantly increased in this condition, corroborating the inflammatory phenotype. No changes in NLRP3 expression were observed by immunoblot analysis, but ASC, one component of the inflammasome, showed a decreased expression in LPS/ATP exposed slices, suggestive of its binding to NLRP3 and thus to complex formation.
Furthermore, epileptic-like activity, measured by field potential recordings, was blocked by MCC950 (10μM).
Conclusion: We demonstrate that LPS induces an inflammatory phenotype in organotypic slices. NLRP3 blockade eliminated the epileptic-like activity of the slices.