Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor-α-induced shock

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.

Figure 1

TAT-crmA inhibits both apoptosis and necroptosis in vitro. (A) Jurkat T cells were left untreated or treated with recombinant human TNFα plus CHX, zVAD and TAT-crmA as indicated. After 5 h of incubation at 37°C, cells were stained for phosphatidylserine exposure with annexin V-FITC antibody and analyzed by FACS. Presence of zVAD or TAT-crmA prevented TNFα/CHX-induced apoptotic cell death. (B) L929, HT29 and FADD^−/− Jurkat cells were left untreated or treated with TNFα plus CHX, zVAD, Nec-1 and TAT-crmA as indicated. After 5 h of incubation at 37°C, cells were stained with annexin V-FITC and analyzed by FACS. Necroptosis was induced by TNFα/CHX + zVAD and protection from necroptotic cell death was achieved by the addition of Nec-1 or TAT-crmA. (C) Western blot of cyclophilin A in the supernatant of L929 cells that were left untreated or treated for 7 h with TNFα, zVAD, Nec-1, TAT-crmA or mut.TAT-crmA, as indicated.

Figure 2

Genetic deficiency of RIP3 protects from TNFα-mediated shock in the presence and absence of caspase inhibition. Wild-type mice (open circles) and RIP3-deficient mice (black squares, n = 8 per group) were treated with mouse TNFα in the absence (A) or presence (B) of zVAD. Survival is presented as a Kaplan-Meyer plot. Genetic RIP3-deficiency protects from both TNFα-mediated shock (P < 0.001) and hyperacute TNFα/zVAD-mediated shock (P < 0.01), respectively.

Figure 3

Blocking necroptosis accelerates death and worsens organ damage after TNFα/zVAD-mediated hyperacute shock. (A) Mice were left untreated (open circles), injected intravenously with mouse TNFα alone (black squares) or in addition with zVAD (open triangles), zVAD + Nec-1 (open squares) or zVAD + TAT-crmA (black circles). Survival is presented as a Kaplan-Meyer plot. Whereas TNFα-treated mice die significantly later than TNFα/zVAD-treated animals (P < 0.01), both TNFα/zVAD/Nec-1- and TNFα/zVAD/TAT-crmA-treated mice died significantly earlier compared with TNFα/zVAD-treated mice (P < 0.02, n = 7). (B) Three h after TNFα-injection (basic experimental setting is identical to 3A), mice were euthanized, and kidneys were harvested and prepared for PAS-staining. Representative sections are shown, and a renal damage score was used for quantification. (C) Compared to the TNFα-treated group, significantly elevated renal damage was detected in the TNFα/zVAD-, TNFα/zVAD/Nec-1- and the TNFαzVAD/TAT-crmA-treated animals (n = 3). Scale bar, 20 μm.

Figure 4

Necrostatin-1 accelerates death and worsens organ damage after TNFα-mediated shock in the absence of caspase inhibition. (A) Mice were left untreated (open circles) or treated intravenously with mouse TNFα alone (black squares) or in addition to Nec-1 (open triangles). Survival is presented as a Kaplan-Meyer plot (n = 8). (B, C) Three h after TNFα-injection (basic experimental setting is identical to Figure 3A), mice were euthanized and kidneys were harvested and prepared for PAS-staining. Representative sections are demonstrated and a kidney damage score was used for quantification. (D) In an independent approach (basic experimental setting is identical to Figure 3A), 2.5 h after TNFα-injection, mice were euthanized and jejunum was harvested and HE stained. Representative sections are presented. The arrowhead points to the typical appearance of edema within the tunica muscularis that was exclusively visible in the TNFα/zVAD/Nec-1 group. Scale bars, 20 μm (B) and 50 μm (D).

Figure 5

Blocking necroptosis deteriorates acute experimental pancreatitis. Cerulein-induced pancreatitis mice were left untreated or given i.p. injections of cerulein once every hour for 10 h. In addition to every second injection of cerulein, either vehicle or zVAD, Nec-1 and TAT-crmA were injected as indicated. 14 h after the last injection mice were sacrificed and blood samples were taken. Levels of serum amylase (A) and serum lipase (B) are shown for each group as indicated. In parallel, pancreatic tissue samples were harvested. Representative sections are demonstrated in C, and a pancreatic damage score was used for quantification (D) (n = 5, * P < 0.05, ** P < 0.02). Scale bar, 50 μm.