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Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor-α-induced shock
Andreas Linkermann1, Jan H Bräsen, Federica De Zen
1Division of Nephrology and Hypertension, Christian-Albrechts University, Kiel, Germany.
Abstract:
Tumor necrosis factor receptor (TNFR) signaling may result in survival, apoptosis or programmed necrosis. The latter is called necroptosis if the receptor-interacting protein 1 (RIP1) inhibitor necrostatin-1 (Nec-1) or genetic knockout of RIP3 prevents it. In the lethal mouse model of TNFα-mediated shock, addition of the pan-caspase inhibitor zVAD-fmk (zVAD) accelerates time to death. Here, we demonstrate that RIP3-deficient mice are protected markedly from TNFα-mediated shock in the presence and absence of caspase inhibition. We further show that the fusion protein TAT-crmA, previously demonstrated to inhibit apoptosis, also prevents necroptosis in L929, HT29 and FADD-deficient Jurkat cells. In contrast to RIP3-deficient mice, blocking necroptosis by Nec-1 or TAT-crmA did not protect from TNFα/zVAD-mediated shock, but further accelerated time to death. Even in the absence of caspase inhibition, Nec-1 application led to similar kinetics. Depletion of macrophages, natural killer (NK) cells, granulocytes or genetic deficiency for T lymphocytes did not influence this model. Because RIP3-deficient mice are known to be protected from cerulein-induced pancreatitis (CIP), we applied Nec-1 and TAT-crmA in this model and demonstrated the deterioration of pancreatic damage upon addition of these substances. These data highlight the importance of separating genetic RIP3 deficiency from RIP1 inhibition by Nec-1 application in vivo and challenge the current definition of necroptosis.
Insights
Genetic RIP3 deficiency protects mice from TNFα-mediated shock, unlike RIP1 inhibition. This challenges the definition of necroptosis and highlights differences between genetic knockout and chemical inhibition in vivo.
Area of Science:
- Cellular signaling pathways
- Immunology
- Molecular biology
Background:
- Tumor necrosis factor receptor (TNFR) signaling can lead to cell survival, apoptosis, or programmed necrosis (necroptosis).
- Necroptosis is typically defined by inhibition of receptor-interacting protein 1 (RIP1) with necrostatin-1 (Nec-1) or genetic knockout of RIP3.
- In TNFα-mediated shock models, pan-caspase inhibitors like zVAD-fmk (zVAD) accelerate death.
Purpose of the Study:
- To investigate the protective effects of RIP3 deficiency against TNFα-mediated shock.
- To compare the in vivo effects of genetic RIP3 deficiency with RIP1 inhibition using Nec-1 or TAT-crmA.
- To re-evaluate the definition and mechanisms of necroptosis.
Main Methods:
- Utilized RIP3-deficient mice in a lethal mouse model of TNFα-mediated shock.
- Administered pan-caspase inhibitor zVAD-fmk (zVAD) and RIP1 inhibitors Nec-1 and TAT-crmA.
- Assessed cell death and survival in various cell lines (L929, HT29, FADD-deficient Jurkat) and in vivo models, including cerulein-induced pancreatitis (CIP).
- Depleted specific immune cell populations (macrophages, NK cells, granulocytes) and used T lymphocyte-deficient mice.
Main Results:
- RIP3-deficient mice were significantly protected from TNFα-mediated shock, even with caspase inhibition.
- In contrast, Nec-1 or TAT-crmA treatment accelerated death in TNFα/zVAD-mediated shock and even in the absence of zVAD.
- Nec-1 and TAT-crmA exacerbated pancreatic damage in the cerulein-induced pancreatitis model.
- Depletion or deficiency of immune cells did not affect the TNFα-mediated shock model.
Conclusions:
- Genetic RIP3 deficiency confers distinct protection compared to RIP1 inhibition in vivo.
- The definition of necroptosis needs re-evaluation, particularly distinguishing between genetic and pharmacological inhibition of RIP1.
- RIP1 inhibition can have detrimental effects independent of necroptosis, as observed in shock and pancreatitis models.
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