Supplementary Materials1. production is dependent on mRNA translation and requires maintenance of endoplasmic reticulum integrity that remains after plasma membrane integrity is lost. The continued translation of cytokines by cellular corpses contributes to necroptotic cell up-take by innate immune cells and priming of adaptive immune responses to antigens associated with necroptotic corpses. These findings imply that cell death and production of inflammatory mediators are coordinated to optimize the immunogenicity of necroptotic cells. In Brief Necroptotic cell death is associated with cytokine production. Orozco et al. show that necroptotic cell corpses continue to synthesize cytokines after they have lost membrane integrity and committed to cell death. This activity involves continued mRNA translation and requires ER function that continues after plasma membrane rupture. Graphical Abstract INTRODUCTION Programmed cell death can occur via several pathways, and the way a cell dies influences subsequent immune responses (Yatim et al., 2017). Although apoptosis is generally considered immunologically silent, lytic forms of cell death, such as pyroptosis and necroptosis, can occur in response to pathogenic infection and are associated with inflammation and adaptive immunity (Green and Llambi, 2015). It is now appreciated that these cell death programs influence the immune system through the active generation of immunostimulatory signals during cell death. The activating cleavage of interleukin-1 (IL-1) and IL-18 by caspase-1 that accompanies pyroptosis is a well-described example of this paradigm (de Vasconcelos et al., 2016; Vande Walle and Lamkanfi, 2016). Necroptosis is a distinct cell death program, triggered in response to receptor ligation or RWJ-67657 viral infection through formation of a cytosolic complex containing the receptor-interacting protein kinases RIPK1 (Degterev et al., 2008; Lin et al., 2004) and RIPK3 (Cho et al., 2009; He RWJ-67657 et al., 2009; Zhang et al., 2009) and subsequent phosphorylation of the membrane-disrupting pseudokinase MLKL (Chen et al., 2013; Sun et al., 2012; Wu et al., 2013; Zhao et al., 2012). Several recent studies have highlighted additional roles for the RIP kinases in promoting nuclear factor B (NF-B)-dependent transcriptional responses, which in some cases occur simultaneously with necroptotic cell death (Snyder et al., 2019; Yatim et al., 2015). We have previously reported that this transcriptional signaling leads to an increase in cross-priming of T cells responsive to antigens derived from necroptotic cells. However, this finding raises the question of how Rabbit polyclonal to YY2.The YY1 transcription factor, also known as NF-E1 (human) and Delta or UCRBP (mouse) is ofinterest due to its diverse effects on a wide variety of target genes. YY1 is broadly expressed in awide range of cell types and contains four C-terminal zinc finger motifs of the Cys-Cys-His-Histype and an unusual set of structural motifs at its N-terminal. It binds to downstream elements inseveral vertebrate ribosomal protein genes, where it apparently acts positively to stimulatetranscription and can act either negatively or positively in the context of the immunoglobulin k 3enhancer and immunoglobulin heavy-chain E1 site as well as the P5 promoter of theadeno-associated virus. It thus appears that YY1 is a bifunctional protein, capable of functioning asan activator in some transcriptional control elements and a repressor in others. YY2, a ubiquitouslyexpressed homologue of YY1, can bind to and regulate some promoters known to be controlled byYY1. YY2 contains both transcriptional repression and activation functions, but its exact functionsare still unknown a necroptotic cell is able to actively generate immunostimulatory cytokines while RWJ-67657 committing to the terminal process of cell death. Notably, an older report indicated that, although caspase activation associated with apoptosis actively suppresses protein translation by cleaving translation initiation factors, necroptotic cells retain the ability to translate mRNAs up to the point of death, as defined by loss of membrane integrity (Saelens et al., 2005). Here, we report that cells undergoing necroptosis in response to direct RIPK3 activation or viral infection continue synthesis of cytokines and chemokines for several hours after they have lost plasma membrane integrity and irreversibly committed to cell death. This process involves continued mRNA translation in cellular corpses and proceeds via an endoplasmic reticulum (ER)-dependent mechanism that reflects maintenance of ER integrity after MLKL-mediated plasma membrane (PM) permeabilization. This continued cytokine and chemokine synthesis enhances the uptake of necroptotic-cell-derived material and contributes to the immunogenicity of necroptotic cell-derived antigens Together, these findings define an unexpected mechanism by which cells that have irreversibly committed to cell death continue to influence inflammatory and immune responses. RESULTS RIPK3 Activation Leads to Cytokine Synthesis that Continues after Loss of PM Integrity To study the effects of RIPK3 RWJ-67657 activation, we employed a previously described system in which RIPK3 can be activated directly, independent of upstream receptor signaling (Orozco et al., 2014). Briefly, we created a chimeric form of RIPK3, composed of murine RIPK3 fused to tandem copies of the dimerizable domain FKBPF36V. We term the resulting chimeric, activatable RIPK3 construct acRIPK3 (Figure 1A). Consistent with previous reports (Orozco et al., 2014; Yatim et al., 2015), clonal populations of NIH 3T3 cells expressing acRIPK3 underwent rapid and uniform necroptosis upon addition of the small-mole-cule dimerizer drug, as measured by the uptake of the cell-impermeable DNA-binding dye Sytox Green. We observed that 99% of cells were positive for Sytox Green 3 h after addition of dimerizer (Figure 1B). The robust induction of necroptosis by this system was further confirmed by kinetic imaging using an In-cuCyte system, release of lactate dehydrogenase (LDH), and CellTiter-Blue viability assay RWJ-67657 (Figure S1A). Open in a.