RIPK3 activation leads to cytokine synthesis that continues after loss of cell membrane integrity

Necroptosis is a form of programmed cell death that is defined by activation of the kinase RIPK3, and subsequent cell membrane permeabilization by the effector MLKL. In addition to triggering cell death, RIPK3 activation can promote immune responses through the production of cytokines and chemokines. How active cytokine production is coordinated with the terminal process of necroptosis is unclear. Here, we report that cytokine production continues within necroptotic cells after cells have lost plasma membrane integrity and irreversibly commited to death. The continued production of inflammatory mediators was dependent on mRNA translation, and required maintanence of endoplasmic reticulum integrity, which remained in tact after plasma membrane integrity was lost. The continued translation of cytokines by cellular corpses contributed to necroptotic cell uptake by innate immune cells, as well as priming of adaptive immune responses to antigens associated with necroptotic corpses. These findings imply that necroptosis may represent a program in which cell death and production of inflammatory mediators are coordinated to optimize the immunogenicity of necroptotic cells. Overall design: The NIH-3T3 RIPK3-2xFV cell line has been treated with a chemical dimerizer to induce RIPK3-dependent necroptosis. Experiment A (sample 1-12) is a simple timecourse of dimerizer treatment where cell death increases over time. Experiment B (sample 13-24) takes a snapshot at 4 h, taking a polysome RNA fraction for comparison with total cell RNA. TNF is included as a control that will trigger inflammatory gene expression without cell death. Two biological replicates were analyzed for each of 6 conditions, giving 12 samples per experiment.