Dissection of TLR4-Induced Necroptosis Using Specific Inhibitors of Endocytosis and P38 MAPK

Abstract

Necroptosis is a pathway of inflammatory cell death that is associated with several pathologies and is induced by ligation of surface TLR or cytokine receptors in macrophages. Many signaling pathways depend on endocytosis, a process mediated by GTPases such as dynamin. We evaluated the role of dynamin-dependent endocytosis in the necroptosis of macrophages using various dynamin inhibitors. Using flow cytometry, we confirmed that during necrosome signaling, various dynamin inhibitors (e.g. Dyngo 4a and Dynasore) blocked the internalization of TLR4, which also resulted in the inhibition of cytokine production. Despite the similar impact of Dynasore and Dyngo 4a on TLR4 endocytosis and cytokine production, only Dyngo 4a prevented TLR4-induced necroptosis of macrophages. Further studies indicated that Dyngo 4a was a potent stimulator of the p38 MAPK pathway, and activation of this pathway by Dyngo 4a was responsible for the inhibition of necroptosis of macrophages following TLR4 signaling. Thus, these studies reveal the previously unknown role of the p38 MAPK pathway in regulating the activation of necrosome signaling.