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Summary: Oxylipins are major immunomodulating mediators, yet studies of inflammation focus mainly on cytokines. Here, using a standardized whole-blood stimulation system, we characterized the oxylipin-driven inflammatory responses to various stimuli and their relationships with cytokine responses. We performed a pilot study in 25 healthy individuals using 6 different stimuli: 2 bacterial stimuli (LPS and live BCG), 2 viral stimuli (vaccine-grade poly I:C and live H1N1 attenuated influenza), an enterotoxin superantigen and a Null control. All stimuli induced a strong production of oxylipins but most importantly, bacterial, viral, and T cell immune responses show distinct oxylipin signatures. Integration of the oxylipin and cytokine responses for each condition revealed new immune networks improving our understanding of inflammation regulation. Finally, the oxylipin responses and oxylipin-cytokine networks were compared in patients with active tuberculosis or with latent infection. This revealed different responses to BCG but not LPS stimulation highlighting new regulatory pathways for further investigations.

Summary: 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. RIPK3 activation can also promote immune responses via 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 even after they have lost cell membrane integrity and irreversibly committed to death. This continued cytokine 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 uptake 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. : 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. Keywords: necroptosis, cell death, phagocytosis, cytokines, RIPK3, MLKL

Systems approaches for the study of immune signaling pathways have been traditionally based on purified cells or cultured lines. However, in vivo responses involve the coordinated action of multiple cell types, which interact to establish an inflammatory microenvironment. We employed standardized whole-blood stimulation systems to test the hypothesis that responses to Toll-like receptor ligands or whole microbes can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, that captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. Furthermore, we used donor variability to identify shared inter-cellular pathways and trace cytokine loops involved in gene expression. This provides strategies for dimension reduction of large datasets and deconvolution of innate immune responses applicable for characterizing immunomodulatory molecules. Moreover, we provide an interactive R-Shiny application with healthy donor reference values for induced inflammatory genes.

Programmed necrosis (or necroptosis) is a form of cell death triggered by the activation of receptor interacting protein kinase-3 (RIPK3). Several reports have implicated mitochondria and mitochondrial reactive oxygen species (ROS) generation as effectors of RIPK3-dependent cell death. Here, we directly test this idea by employing a method for the specific removal of mitochondria via mitophagy. Mitochondria-deficient cells were resistant to the mitochondrial pathway of apoptosis, but efficiently died via tumor necrosis factor (TNF)-induced, RIPK3-dependent programmed necrosis or as a result of direct oligomerization of RIPK3. Although the ROS scavenger butylated hydroxyanisole (BHA) delayed TNF-induced necroptosis, it had no effect on necroptosis induced by RIPK3 oligomerization. Furthermore, although TNF-induced ROS production was dependent on mitochondria, the inhibition of TNF-induced necroptosis by BHA was observed in mitochondria-depleted cells. Our data indicate that mitochondrial ROS production accompanies, but does not cause, RIPK3-dependent necroptotic cell death.

Contraction is a critical phase of immunity whereby the vast majority of effector T cells die by apoptosis, sparing a population of long-lived memory cells. Where, when, and why contraction occurs has been difficult to address directly due in large part to the rapid clearance of apoptotic T cells in vivo. To circumvent this issue, we introduced a genetically encoded reporter for caspase-3 activity into naive T cells to identify cells entering the contraction phase. Using two-photon imaging, we found that caspase-3 activity in T cells was maximal at the peak of the response and was associated with loss of motility followed minutes later by cell death. We demonstrated that contraction is a widespread process occurring uniformly in all organs tested and targeting phenotypically diverse T cells. Importantly, we identified a critical window of time during which antigen encounters act to antagonize T cell apoptosis, supporting a causal link between antigen clearance and T cell contraction. Our results offer insight into a poorly explored phase of immunity and provide a versatile methodology to study apoptosis during the development or function of a variety of immune cells in vivo.