NLRP3 Inflammasome assembly in microglia-induced reactive astrocytes

Abstract

Event Abstract Back to Event NLRP3 Inflammasome assembly in microglia-induced reactive astrocytes Antónia Fortunato1, 2, Catarina C. Almeida1, 2, Ana M. Sebastião1, 2* and Claudia A. Valente1, 2* 1 Institute of Pharmacology and Neurosciences, Faculty of Medicine, University of Lisbon, Portugal 2 Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Portugal Microglia and astrocytes work together as the immune effectors in the Central Nervous System (CNS). Upon a prolonged insult or injury, these cells acquire a reactive, pro-inflammatory phenotype, resulting in neuronal damage [1]. At the core of this inflammatory response is the NLRP3 inflammasome (NLRP3), a cytosolic multiprotein complex that leads to Interleukin-1beta (IL-1beta) secretion. The canonical mechanism of NLRP3 activation involves: 1) a priming signal, which induces an increased production of pro-IL-1beta and NLRP3 protein, and 2) an activation signal, which mediates the oligomerization of the inflammasome subunits [1]. Although NLRP3 assembly in microglia is established, its presence in astrocytes remains to be confirmed. Furthermore, recent data show that microglia and astrocytes exhibit a reciprocal modulation in CNS [2]. Hence, this work aims to explore the production and assembly of NLRP3 in astrocytes and to clarify the role of microglia in these events. To do so, primary cultures of astrocytes, microglia and co-cultures were prepared from newborn Sprague-Dawley rats. After 17 days in vitro, cells were stimulated with LPS (100ng/ml) for 24h, as the priming stimulus, with or without the oligomerization signal: ATP (1mM). Through immunocytochemistry, morphological alterations and NLRP3 expression in astrocytes were visualized. Upon LPS/ATP stimulation, astrocytes exhibit a more reactive phenotype, confirmed by STAT3 staining [3] and an increased NLRP3 expression in co-cultures, when compared with astrocytes cultured alone. Next, the effect of LPS/ATP stimuli on A1 astrocytes induced by microglia-derived factors, namely IL-1α (3ng/mL), TNFα (30 ng/mL) and C1q (400 ng/mL), for 24h [4] was assessed. NLRP3 assembly in reactive astrocytes was analyzed by immunocytochemistry and immunoprecipitation, while the levels of IL-1beta were assessed by ELISA. Our preliminary results suggest that astrocyte-microglia communication is indeed important for NLRP3 assembly and activation in astrocytes. However, if cell-to-cell contact is necessary remains to be determined.