The effects of dimethyl fumarate on nlrp3 inflammasome activation in microglial cells

Inflammasome activation is a developing process differently from classical inflammatory responses. It has been known that inflammasome activation occurs by both microbial agents and the effects of some metabolic stress products (Adenosine triphosphate (ATP) and external agents like asbestos, Inflammasome activation is a process that is initiated by Interleukin-1beta (IL-1beta), Interleukin-18 (IL-18) cytokines production from activated cells and terminated by caspase-1 activation. In this process, inflammasome activation may cause cell death via pyroptotic pathway. Therefore, inflammasome activation is needed to be inhibited. Dimethyl fumarate (DMF) is anti-inflammatory molecule that has pro-metabolic effects. DMF, known as also methyl ester of fumaric acid, has been shown that it provided improvement for multiple sclerosis in clinical parameters and decreased frequency of attacks. And also, it has been established that DMF inhibited inflammasome activation in THP-1 monocyte cells which came from same ancestor of microglial cells. In our work, observing effects of DMF to inflammasome activation in N9 microglial cells has been aimed. In this regard, NOD-like receptor family, pyrin domain containing protein 3 (NLRP3) inflammasome activation in N9 microglial cells was initiated with lipopolysaccharide (LPS) and ATP model. Firstly, the effective and non-toxic dose of DMF was determined. After that, inflammasome activation parameters were observed with real-time PCR for mRNA level, ELISA and Western-blotting for protein level. Additionally, mitochondrial Reactive Oxygen Species (ROS) production and mitochondrial membrane integrity were analyzed via flow cytometry, immunofluorescence (IF) and fluorometric analyses. Consequently, DMF significantly suppressed LPS + ATP induced NLRP3 inflammation, decreased Caspase-1 activity, secretion of IL-1beta and IL-18 cytokines, and reduced the rate of pyroptotic cell death resulting from inflammasome activation. Furthermore, the level of mitochondrial ROS was declined and mitochondrial membrane integrity was restored. This study is the first to demonstrate that DMF is effective to suppress the NLRP3 inflammasome activation in microglial cells.