FADD ELISA Kit

Fas (CD95) signalling is best known for its role in apoptosis, however, recent reports have shown it to be involved in other cellular responses as well, including inflammation. Fas and its adaptor protein FADD are known to negatively regulate LPS-induced proinflammatory responses, but their role in LPS-induced type I interferon production is unknown. Here, we demonstrate that Fas engagement on macrophages, using an agonistic Fas antibody CH11, augments LPS-induced NF-kappa B responses, causing increased production of TNF alpha, IL-8, IL-6 and IL-12. Conversely, costimulation with both LPS and CH11 causes a significant reduction in the level of interferon-beta (IFN beta) production. This differential effect involves the Fas adaptor FADD because while LPS-induced IL-6 production increased in FADD(-/-) murine embryonic fibroblasts, LPS-induced IFN beta production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD-DD) inhibits LPS-induced IFN beta luciferase but not LPS-induced NF-kappa B luciferase. In contrast, overexpression of full-length FADD inhibited LPS-induced NF-kappa B luciferase activation but was seen to augment LPS-induced IFN beta luciferase. Moreover, FADD-DD inhibits TRIF-, TRAM-, IKK epsilon-, TBK-1- and TRAF3-induced IFN beta luciferase production, with coimmunoprecipitation experiments demonstrating an interaction between FADD and TRIF. These data identify FADD as a novel component of the noncanonical Toll-like receptor 4/IFN beta signalling pathway and demonstrate that both Fas and its adaptor FADD can differentially regulate the production of LPS-induced proinflammatory cytokines and type I interferons.

Heart disease is the leading cause of death worldwide. A key pathogenic factor in the development of lethal heart failure is loss of terminally differentiated cardiomyocytes. However, mechanisms of cardiomyocyte death remain unclear. Here, we discovered and demonstrated that ferroptosis, a programmed iron-dependent cell death, as a mechanism in murine models of doxorubicin (DOX)and ischemia/reperfusion (I/R)-induced cardiomyopathy. In canonical apoptosis and/or necroptosis-defective Ripk3(-/-), Mlkl(-/-), or Fadd(-/-)Mlkl(-/-) mice, DOX-treated cardiomyocytes showed features of typical ferroptotic cell death. Consistently, compared with dexrazoxane, the only FDA-approved drug for treating DOX-induced cardiotoxicity, inhibition of ferroptosis by ferrostatin-1 significantly reduced DOX cardiomyopathy. RNA-sequencing results revealed that heme oxygenase-1 (Hmox1) was significantly up-regulated in DOX-treated murine hearts. Administering DOX to mice induced cardiomyopathy with a rapid, systemic accumulation of nonheme iron via heme degradation by Nrf2-mediated upregulation of Hmox1, which effect was abolished in Nrf2-deficent mice. Conversely, zinc protoporphyrin IX, an Hmox1 antagonist, protected the DOX-treated mice, suggesting free iron released on heme degradation is necessary and sufficient to induce cardiac injury. Given that ferroptosis is driven by damage to lipid membranes, we further investigated and found that excess free iron accumulated in mitochondria and caused lipid peroxidation on its membrane. Mitochondriatargeted antioxidant MitoTEMPO significantly rescued DOX cardiomyopathy, supporting oxidative damage of mitochondria as a major mechanism in ferroptosis-induced heart damage. Importantly, ferrostatin-1 and iron chelation also ameliorated heart failure induced by both acute and chronic I/R in mice. These findings highlight that targeting ferroptosis serves as a cardioprotective strategy for cardiomyopathy prevention.