• Phenotypic features of human neutrophils after ingestion of CA-MRSA contrast with those of phagocytosis-induced apoptosis.

  • Lysis of human neutrophils fed CA-MRSA requires active RIPK-3 but is independent of tumor necrosis factor α, active RIPK-1, and MLKL.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes infections associated with extensive tissue damage and necrosis. In vitro, human neutrophils fed CA-MRSA lyse by an unknown mechanism that is inhibited by necrostatin-1, an allosteric inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK-1). RIPK-1 figures prominently in necroptosis, a specific form of programmed cell death dependent on RIPK-1, RIPK-3, and the mixed-lineage kinase-like protein (MLKL). We previously reported that necrostatin-1 inhibits lysis of human neutrophils fed CA-MRSA and attributed the process to necroptosis. We now extend our studies to examine additional components in the programmed cell death pathway to test the hypothesis that neutrophils fed CA-MRSA undergo necroptosis. Lysis of neutrophils fed CA-MRSA was independent of tumor necrosis factor α, active RIPK-1, and MLKL, but dependent on active RIPK-3. Human neutrophils fed CA-MRSA lacked phosphorylated RIPK-1, as well as phosphorylated or oligomerized MLKL. Neutrophils fed CA-MRSA possessed cytoplasmic complexes that included inactive caspase 8, RIPK-1, and RIPK-3, and the composition of the complex remained stable over time. Together, these data suggest that neutrophils fed CA-MRSA underwent a novel form of lytic programmed cell death via a mechanism that required RIPK-3 activity, but not active RIPK-1 or MLKL, and therefore was distinct from necroptosis. Targeting the molecular pathways that culminate in lysis of neutrophils during CA-MRSA infection may serve as a novel therapeutic intervention to limit the associated tissue damage.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) commonly causes severe infection frequently associated with significant tissue necrosis, even in apparently healthy individuals (reviewed in DeLeo et al1 ). Rapidly recruited to sites of nascent infection, circulating polymorphonuclear neutrophils (PMN) provide critical defense against invading microbes, including staphylococci.2  The frequent and severe staphylococcal infections experienced by individuals with defects in PMN microbicidal action, as seen in patients with chronic granulomatous disease,3  illustrate the intimate link between normal PMN function and optimal innate immune defense against S aureus.

Despite their prominence in human host response to staphylococcal infection, normal PMN fail to kill the entire ingested inoculum of S aureus. In experimental systems, 10% to 50% of ingested organisms remain viable within PMN phagosomes, consequently providing a reservoir of organisms that can support persistent infection.4-6  Furthermore, PMN harboring viable S aureus (PMN-SA), particularly CA-MRSA, lyse,7  thereby serving as a potential source of danger-associated molecular patterns that could fuel additional inflammation and tissue damage.8-10  The recognition that PMN lysis could contribute to the exuberant inflammation that frequently accompanies infection with CA-MRSA11-13  provides the rationale to investigate the host-mediated mechanism of PMN cell death after phagocytosis of CA-MRSA.

Work to date has identified several features of the lysis of human PMN harboring viable SA. The process is somewhat strain-specific with respect to SA, with CA-MRSA associated with the most robust response; requires ingestion of viable SA; and can be interrupted by pharmacologic inhibition of PMN gene transcription or translation, but only when inhibitors are added within the first hour after ingestion of CA-MRSA.7  Inhibition of neither caspase activity nor cathepsin D reduces PMN lysis caused by CA-MRSA.5,7  We previously reported that necrostatin-1 (Nec-1), an allosteric inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK-1),14  blocks lysis of PMN fed CA-MRSA.5  Because RIPK-1 has been implicated in a form of programmed cell death called necroptosis, we concluded that human PMN underwent necroptosis after phagocytosis of CA-MRSA. However, concerns that the high concentration of Nec-1 required to block PMN lysis may have off-target effects and influence signaling systems unrelated to RIPK-1 prompted our expanded study of PMN fate.

The most extensively studied experimental setting for necroptosis has been tumor necrosis factor α (TNFα)-induced lysis of tumor cells,15  which provided the conceptual framework for our studies of human PMN. Unexpectedly, lysis of PMN-SA contrasted with TNFα-induced necroptosis of tumor cells in several notable ways. Lysis of PMN-SA occurred independent of TNFα, active RIPK-1, and the executioner protein mixed lineage kinase-like protein (MLKL). Among the currently recognized components in the necroptotic pathway, only active RIPK-3 was required for lysis of PMN-SA. Taken together, these data suggest that after phagocytosis of CA-MRSA, human PMN undergo a novel form of lytic cell death that differs from necroptosis. Elucidation of critical steps in this cell death pathway may provide novel therapeutic strategies targeting the deleterious and unchecked inflammation associated with CA-MRSA infection.

Reagents, antibodies, and cells

All reagents were purchased from Fisher Scientific (Pittsburgh, PA), unless otherwise indicated. Heparin was purchased from Fresenius Kabi USA LLC (Lake Zurich, IL), clinical grade dextran T500 from Pharmacosmos (Holbaek, Denmark), Ficoll-Hypaque PLUS from GE Healthcare (Piscataway, NJ), and sterile endotoxin-free water and 0.9% sterile endotoxin-free sodium chloride from Baxter (Deerfield, IL). Human serum albumin (25%) was purchased from USP (Grifols, Canada). Tryptic Soy Broth was purchased from Becton Dickinson (Sparks, MD). Necrostatin-1 was purchased from Sigma Aldrich (St. Louis, MO). Pierce Restore Western Blot Stripping Buffer and Pierce Protein Assay Reagents A and B were purchased from Thermo Fisher Scientific (Waltham, MA). Annexin-V-FITC Apoptosis Kit and necrostatin-1s were purchased from BioVision (Milpitas, CA). Cytotoxicity Detection Kit PLUS (LDH) was purchased from Roche (Indianapolis, IN). GSK’963 (RIPK-1 inhibitor), GSK’962 (inactive RIPK-1 inhibitor), GSK’872 and GSK’843 (RIPK-3 inhibitors) were kindly provided by GlaxoSmithKline (Collegeville, PA). zVAD-fmk and recombinant TNFα were purchased from R&D Systems (Minneapolis, MN), and cycloheximide (CHX) and necrosulfonamide (NSA) were purchased from EMD Millipore (Temecula, CA). Microcolumns and μMACS protein G Microbeads were purchased from Miltenyi Biotec (San Diego, CA). Anti-human RIPK-1 antibody was purchased from BD Biosciences (San Jose, CA), and anti-human caspase 8 (1C12), anti-human phospho-RIPK-1 (D1L3S), and anti-TNFα neutralizing (D1B4) antibodies were purchased from Cell Signaling (Danvers, MA). Anti-human RIPK-3 (B-2) and goat anti-human caspase 8 (C-20) were purchased for Santa Cruz (Dallas, TX), and anti-human Fas-associated protein with death domain (FADD) (IF7) was purchased from Enzo Life Sciences (Farmingdale, NY). Anti-human MLKL and phospho-MLKL (EPR9514) antibodies were purchased from Abcam (Cambridge, MA). Antibodies against β-actin were purchased from BioLegend (San Diego, CA). McCoy’s 5A media-supplemented l-glutamine, Hanks balanced salt solution, and phosphate-buffered saline were purchased from Mediatech (Manassas, VA). The HT-29 human colorectal adenocarcinoma cell line was obtained from ATCC (Manassas, VA).

S aureus culture

Experiments were performed with the USA300 LAC wild-type strain.6 S aureus USA300 LAC (referred to throughout as SA) was cultured overnight in tryptic soy broth at 37°C, with agitation at 180 rpm. Bacteria were diluted to an OD550 of 0.05 in tryptic soy broth and incubated at 37°C with agitation at 180 rpm for 2 hours and 30 minutes until bacteria reached midlogarithmic growth (OD550 between 0.6 and 0.9). Unless otherwise indicated, bacteria were opsonized in HBSS containing 10% pooled human serum, 20 mM HEPES, and 1% HSA for 20 minutes while tumbling at 37°C.

Human PMN isolation and challenge with SA

PMN were isolated from venous blood collected from healthy volunteers (as described in Nauseef16 ). Written consent was obtained from each volunteer in accordance with a protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa. Briefly, heparinized blood was collected and PMN were isolated using dextran sedimentation followed by density gradient separation on Ficoll-Paque PLUS. After hypotonic lysis of erythrocytes, PMN were suspended in HBSS without divalent cations, counted, and adjusted to 20 × 106 cells/mL. The purity of PMN preparations was assessed by HEMA-3 staining and was routinely above 96%. Replicate experiments used PMN from independent donors.

PMN-SA were prepared as follows. PMN were left in buffer or fed opsonized SA (5:1 MOI) and tumbled for 10 min at 37°C. After 10 min phagocytosis, any extracellular bacteria were removed by centrifugation at 300 × g for 5 minutes. PMN-SA were resuspended in HBSS containing 20 mM HEPES and 1% HSA and tumbled for an indicated time.

Cytotoxicity assays

HT-29 (2.5 × 106 cells/well) were treated with or without the following inhibitors: Nec-1, Nec-1s, GSK’963, GSK’962, GSK’872, GSK’843, and NSA for 30 min at 37°C. To induce necroptosis, HT-29 cells were treated with zVAD-fmk (50 μM) and CHX (10 μg/mL) for 30 min at 37°C before addition of TNFα (10 ng/mL). After 22 hours, supernatants were collected and centrifuged at 300 × g for 5 minutes, and clarified supernatants were assayed for LDH. PMN were left in buffer alone or treated with Nec-1, Nec-1s, GSK’963, GSK’962, GSK’872, GSK’843, and NSA for 20 min while tumbling at 37°C. PMN were left in buffer or challenged with SA, as described earlier. After 3 hours, supernatants were collected and centrifuged at 300 × g for 5 minutes, and clarified supernatants were assayed for LDH. To assess for toxicity mediated directly by inhibitors, we measured LDH released by unstimulated PMN; the highest concentration of each inhibitor was also assessed (supplemental Figure 1, available on the Blood website). LDH was measured using the Cytotoxicity Detection Kit PLUS, according the manufacturer’s instructions.

Immunoprecipitation and immunoblot analyses

HT-29 cells were lysed in M2 buffer (20 mM tris[hydroxymethyl]aminomethane at pH 7.4, 0.5% NP40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5 mM PMSF, 20 mM glycerol phosphate, 1 mM sodium orthovanadate, and 1 μg/mL leupeptin), as previously described by Cai et al.17  PMN were collected by centrifugation at individual points, and lysed by sonication (as described by Witko-Sarsat et al.18 ) with the following modifications. PMN were resuspended at 50 × 106 cells/mL in sonication buffer (50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 4 mM phenylmethylsulfonyl fluoride, 0.4 mM leupeptin, 0.4 mM pepstatin A, 1 mM sodium orthovanadate, 3 mM EGTA, 3 mM EDTA, and 20 mM glycerol phosphate), and sonicated on ice for 10 s at an output wattage of 1 in the XL2000 QSonica. Lysates were then spun at 20 000 × g for 5 minutes to remove insoluble material. For immunoprecipitation, 2 μg anti-goat caspase 8 was incubated with 60 million cell equivalents of lysate and 50 μL protein G microbeads for 30 minutes on ice. For immunoblot analyses, supernatants were frozen at −80°C until analysis. The immunoprecipitate was loaded onto microcolumns, and columns were washed as previously described by Witko-Sarsat et al.18  The immunoprecipitate was eluted in 50 μL sample buffer and resolved by polyacrylamide gel electrophoresis on a 4% to 15% sodium dodecyl sulfate gel. For blots of lysates, 20 μg protein was resolved by polyacrylamide gel electrophoresis on a 10% sodium dodecyl sulfate gel for RIPK-1 and a 5% to 15% sodium dodecyl sulfate gradient gel for MLKL and transferred to nitrocellulose for immunoblotting. The following dilutions were used for immunoblots: 1:500 for RIPK-1, 1:1000 for phospho-RIPK-1, 1:500 for RIPK-3, 1:500 for FADD, 1:500 for caspase 8, 1:1000 for MLKL, 1:1000 for phospho-MLKL, 1:1000 for β-actin, 1:20 000 anti-mouse horseradish peroxidase, and 1:20 000 anti-rabbit horseradish peroxidase.

Statistical analysis

Statistical comparisons were performed using 1-way ANOVA followed by Dunnett’s or Tukey’s posttests. All analyses used GraphPad Prism software. P values < .05 were considered statistically significant.

Human subject approval

Written consent was obtained from each volunteer in accordance with a protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa.

TNFα and lysis of PMN-SA

TNFα has a paradoxical effect on the fate of many types of cells, including PMN, prompting cell survival or, alternatively, apoptosis in PMN as a function of the concentration of TNFα. For example, low concentrations of TNFα (0.1-1.0 ng/mL) decrease apoptosis, whereas high concentrations of TNFα (≥ 10 ng/mL) enhance PMN apoptosis.19  In addition, TNFα-driven cell lysis of cultured tumor cells represents the most extensively studied form of necroptosis.15  In established experimental models, necroptosis of HT-29 cells, a human colorectal adenocarcinoma cell line, follows treatment with TNFα, CHX, and the pan-caspase inhibitor zVAD-fmk.20  Because PMN-SA released 2.7 ng/mL ± 0.8 ng/mL TNFα (n = 3) simultaneously with significant PMN lysis, we reasoned that autocrine activation of the TNFα receptor on PMN-SA may drive the lytic process.

To examine the relationship between TNFα and the lysis of PMN-SA, we used the TNFα-neutralizing antibody D1B4 to counteract TNFα stimulation. As proof of principle, we first demonstrated that D1B4 inhibited necroptosis of HT-29 cells in a concentration-dependent fashion (Figure 1A). In contrast to its inhibition of HT-29 cells, D1B4 had no effect on the lysis of PMN-SA (Figure 1B), suggesting engagement of the TNFα receptor in PMN did not promote lysis of PMN-SA.

Figure 1.

TNFα does not contribute to PMN-SA lysis. (A) HT-29 cells were pretreated without or with varied concentrations of TNFα neutralizing antibody (D1B4) and then stimulated with CHX, zVAD-fmk, and TNFα (CZT), as described in “Methods.” After 22 hours, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using repeated measures 1-way ANOVA and Tukey posttest (*P < .05 vs untreated, ***P < .0001 vs untreated, and #P < .05 vs CZT + 0, 10, or 100 ng/ml D1B4). (B) PMN were pretreated without or with varied concentrations of D1B4 and then incubated without or with SA. After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (***P < .0001 vs SA). (C) Reduced caspase 8 immunoprecipitates from sonicates of PMN or PMN-SA at 0, 1, and 3 hours after phagocytosis were probed for the presence of FADD, RIPK-3, RIPK-1, and caspase 8 by immunoblotting. Shown is a representative experiment of 5.

Figure 1.

TNFα does not contribute to PMN-SA lysis. (A) HT-29 cells were pretreated without or with varied concentrations of TNFα neutralizing antibody (D1B4) and then stimulated with CHX, zVAD-fmk, and TNFα (CZT), as described in “Methods.” After 22 hours, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using repeated measures 1-way ANOVA and Tukey posttest (*P < .05 vs untreated, ***P < .0001 vs untreated, and #P < .05 vs CZT + 0, 10, or 100 ng/ml D1B4). (B) PMN were pretreated without or with varied concentrations of D1B4 and then incubated without or with SA. After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (***P < .0001 vs SA). (C) Reduced caspase 8 immunoprecipitates from sonicates of PMN or PMN-SA at 0, 1, and 3 hours after phagocytosis were probed for the presence of FADD, RIPK-3, RIPK-1, and caspase 8 by immunoblotting. Shown is a representative experiment of 5.

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Induction of necroptosis by ligation of death receptors, including TNFα, Fas ligand, and TNF-related apoptosis-inducing ligand, relies on the assembly the ripoptosome complex, which contains FADD, RIPK-3, RIPK-1, and full-length, inactive caspase 8. In contrast, apoptosis relies on a ripoptosome complex with FADD, RIPK-3, RIPK-1, and active caspase 8.21  The cytosol of unstimulated PMN contained ripoptosomes composed of FADD, RIPK-3, RIPK-1, and caspase 8 (PMN 0 h in Figure 1C). Furthermore, the association of FADD, RIPK-3, and RIPK-1 with full-length caspase 8 did not vary over time in either control PMN or PMN-SA (Figure 1C), and the caspase 8 recovered is inactive during lysis of PMN-SA, as we reported previously.5 

Contribution of RIPK-1 to lysis of PMN-SA

Our conclusion that necroptosis causes lysis of PMN-SA5  relies heavily on its inhibition by Nec-1, an allosteric inhibitor of RIPK-1.14  However, Nec-1 inhibits lysis of PMN-SA at a relatively high concentration of the inhibitor. Moreover, Nec-1 has many off-target effects, including inhibition of proximal T cell receptor signaling, p21 protein-activated kinase 1, protein kinase A catalytic α subunit, MAP kinase-interacting serine/threonine kinase 1 (MKNK1), and indoleamine 2,3-dioxygenase.22-24  Nec-1s, an analog of Nec-1 that is ∼10-fold more effective in inhibiting RIPK-1, does not inhibit human indoleamine 2,3-dioxygenase.22  We found that 5 μM Nec-1s inhibited TNFα-treated HT-29 cells and was nearly equipotent to 50 μM Nec-1 (Figure 2A). However, 20 μM Nec-1s did not significantly inhibit PMN-SA (Figure 2B). Therefore, we reasoned that the effect of 200 μM Nec-1 on PMN-SA lysis did not reflect selective inhibition of RIPK-1.

Figure 2.

Necrostatin-1 inhibits lysis in a RIPK-1-independent manner. (A) HT-29 cells were pretreated without or with 1 μM or 5 μM Nec-1s or 50 μM Nec-1, and then stimulated with or without CHX, zVAD-fmk, and TNFα (CZT), as described in “Methods.” After 22 hours, LDH in supernatants was measured. Bars represent the mean of 3 experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (**P < .01; ***P < .001 vs CZT). (B) PMN were pretreated without or with 20 μM Nec-1s or 200 μM Nec-1, and then were either left in buffer alone or challenged with SA, as indicated. After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (**P < .01; ***P < .0001 vs SA). (C) HT-29 cells were pretreated without or with Nec-1, varied concentrations of GSK’963 (active inhibitor) or of GSK’962 (inactive enantiomer), and then stimulated with or without CHX, zVAD-fmk, and TNFα (CZT), as described in “Methods.” After 22 hours, LDH in supernatants was measured. Bars represent the mean of at least 3 experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (*P < .05 vs untreated; #P < .05 vs GSK’962; ^P < .05 vs CZT). (D) PMN were pretreated without or with Nec-1 or varied concentrations of GSK’963. PMN were either left in buffer alone or challenged with SA as indicated. After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (*P < .05 vs untreated; #P < .05 vs SA). (E) Nonreduced lysates from untreated and CZT-treated HT-29, PMN, and PMN-SA were analyzed by immunoblot for RIPK-1, phosphorylated-RIPK-1, and, in the PMN samples, β-actin. Shown is a representative of 3 separate experiments. Samples were separated in lanes on the same gel, but were noncontiguous. Splicing is denoted with a thin vertical line.

Figure 2.

Necrostatin-1 inhibits lysis in a RIPK-1-independent manner. (A) HT-29 cells were pretreated without or with 1 μM or 5 μM Nec-1s or 50 μM Nec-1, and then stimulated with or without CHX, zVAD-fmk, and TNFα (CZT), as described in “Methods.” After 22 hours, LDH in supernatants was measured. Bars represent the mean of 3 experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (**P < .01; ***P < .001 vs CZT). (B) PMN were pretreated without or with 20 μM Nec-1s or 200 μM Nec-1, and then were either left in buffer alone or challenged with SA, as indicated. After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (**P < .01; ***P < .0001 vs SA). (C) HT-29 cells were pretreated without or with Nec-1, varied concentrations of GSK’963 (active inhibitor) or of GSK’962 (inactive enantiomer), and then stimulated with or without CHX, zVAD-fmk, and TNFα (CZT), as described in “Methods.” After 22 hours, LDH in supernatants was measured. Bars represent the mean of at least 3 experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (*P < .05 vs untreated; #P < .05 vs GSK’962; ^P < .05 vs CZT). (D) PMN were pretreated without or with Nec-1 or varied concentrations of GSK’963. PMN were either left in buffer alone or challenged with SA as indicated. After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Tukey posttest (*P < .05 vs untreated; #P < .05 vs SA). (E) Nonreduced lysates from untreated and CZT-treated HT-29, PMN, and PMN-SA were analyzed by immunoblot for RIPK-1, phosphorylated-RIPK-1, and, in the PMN samples, β-actin. Shown is a representative of 3 separate experiments. Samples were separated in lanes on the same gel, but were noncontiguous. Splicing is denoted with a thin vertical line.

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GSK’963, a more active and selective inhibitor of RIPK-1 than is Nec-1,25  blocked TNFα-induced necroptosis of HT-29 cells (Figure 2C), as previously reported by Seo et al.,26  with no effect of the inactive enantiomer, GSK’962. However, PMN-SA lysed in the presence of GSK’963 at concentrations that blocked necroptosis of HT-29 cells (Figure 2D), suggesting PMN-SA lysed independent of active RIPK-1. During necroptosis, RIPK-1 undergoes phosphorylation, and we detected phosphorylated RIPK-1 in HT-29 cells that underwent TNFα-stimulated necroptosis (Figure 2E). In contrast, PMN-SA lacked phospho-RIPK-1, a finding that supports our conclusion that lysis occurred independent of RIPK-1 activity.

RIPK-3 and lysis of PMN-SA

Although RIPK-1 phosphorylation of RIPK-3 typically initiates signaling events that culminate in necroptosis, RIPK-3 can drive necroptosis independent of RIPK-1 activity.27-29  Inhibition of RIPK-3 with GSK’872 or GSK’843 reduced necroptosis of HT-29 cells in a concentration-dependent fashion (Figure 3A-B), consistent with earlier reports linking activation of RIPK-3 with necroptosis.30  At a concentration that inhibited necroptosis of HT-29 cells by 50%, GSK’872 or GSK’843 (Figure 3A-B) reduced lysis of PMN-SA 26% and 25%, respectively (Figure 3C), suggesting lysis of PMN-SA depended in part on active RIPK-3.

Figure 3.

PMN-SA lysis is RIPK-3-dependent. HT-29 cells were pretreated without or with varied concentrations of GSK’872 (A) or GSK’843 (B), and then stimulated with or without CHX, zVAD-fmk, and TNFα (CZT) as described in “Methods.” After 22 hours, LDH in supernatants was analyzed. Bars represent the mean of at least 3 experiments ± SEM. P values were determined using a 1-way ANOVA and Dunnett posttest (*P < .05 vs CZT; ***P < .0001 vs CZT). (C) PMN were pretreated with or without 200 μM Nec-1, 50 μM GSK’872 or 50 μM GSK’843. PMN were either left in buffer alone or challenged with SA, as indicated in “Methods.” After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Dunnett posttest (***P < .0001 vs SA).

Figure 3.

PMN-SA lysis is RIPK-3-dependent. HT-29 cells were pretreated without or with varied concentrations of GSK’872 (A) or GSK’843 (B), and then stimulated with or without CHX, zVAD-fmk, and TNFα (CZT) as described in “Methods.” After 22 hours, LDH in supernatants was analyzed. Bars represent the mean of at least 3 experiments ± SEM. P values were determined using a 1-way ANOVA and Dunnett posttest (*P < .05 vs CZT; ***P < .0001 vs CZT). (C) PMN were pretreated with or without 200 μM Nec-1, 50 μM GSK’872 or 50 μM GSK’843. PMN were either left in buffer alone or challenged with SA, as indicated in “Methods.” After 3 h, LDH in supernatants was measured. Bars represent the average of at least 3 separate experiments ± SEM. P values were determined using a 1-way ANOVA and Dunnett posttest (***P < .0001 vs SA).

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The intact plasma membrane exhibits lipid asymmetry, whereby phosphatidylserine resides on the inner leaflet, inaccessible to binding with probes such as Annexin V. Compromise of membrane integrity, as occurs during apoptosis and other forms of programmed cell death, causes phosphatidylserine to be exposed on the cell surface, and cells stain positive for Annexin V. Complete loss of membrane integrity renders cells permeable to propidium iodide (PI), and PI staining typically indicates cell death. With these staining properties in mind, we used flow cytometry to determine whether RIPK-3 influenced the changes in plasma membrane integrity of PMN-SA. We measured binding of Annexin V and staining by PI of PMN-SA in the absence or presence of RIPK-3 inhibitors. In the absence of SA, normal PMN were predominantly Annexin V, PI after 3 hours of incubation (Figure 4). However, within 3 hours of ingesting SA, PMN-SA had changed dramatically, with a minority of cells still healthy (Annexin V, PI) and the majority staining with Annexin V, or Annexin V and PI. Inhibition of RIPK-3 with either GSK’872 or GSK’843, neither of which alone caused lysis of PMN in the absence of SA (supplemental Figure 1), increased the fraction of intact PMN-SA with normal membrane asymmetry (Annexin V) and decreased the cell population that lost normal membrane asymmetry (Annexin V+) and barrier function (Annexin V+, PI+) (Figure 4; supplemental Figure 2). Taken together, these data demonstrate that RIPK-3 contributes to early changes in programmed cell death pathways, including Annexin V and PI staining, as well as lysis of PMN-SA (Figure 3C), and that inhibition of RIPK-3 redirected the fate of PMN-SA by preventing lysis, and thereby extending survival of PMN-SA.

Figure 4.

GSK’872 and GSK’843 each inhibits phosphatidylserine exposure in PMN-SA. PMN were pretreated without or with 50 μM GSK’872 or 50 μM GSK’843 and then left in buffer alone or challenged with SA, as indicated in “Methods.” After 3 hours, PMN were stained with Annexin V-FITC and PI to detect surface exposure of phosphatidylserine and to assess membrane integrity, respectively, and analyzed by flow cytometry. Bars represent the mean of 3 separate experiments ± SEM. P values were determined using repeated measures 1-way ANOVA and Dunnett posttest (*P < .05 vs SA).

Figure 4.

GSK’872 and GSK’843 each inhibits phosphatidylserine exposure in PMN-SA. PMN were pretreated without or with 50 μM GSK’872 or 50 μM GSK’843 and then left in buffer alone or challenged with SA, as indicated in “Methods.” After 3 hours, PMN were stained with Annexin V-FITC and PI to detect surface exposure of phosphatidylserine and to assess membrane integrity, respectively, and analyzed by flow cytometry. Bars represent the mean of 3 separate experiments ± SEM. P values were determined using repeated measures 1-way ANOVA and Dunnett posttest (*P < .05 vs SA).

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MLKL and lysis of PMN-SA

The necroptosis pathway terminates with the loss of membrane integrity after insertion of oligomers of MLKL into the plasma membrane.17,31  Phosphorylation of MLKL by RIPK-3 prompts the conformational changes that allow high molecular weight oligomers to form.17  NSA covalently binds to cysteine 86 in human MLKL, thereby blocking changes in its conformation and its capacity to act as the executioner protein in necroptosis.31 

In a concentration-dependent fashion, NSA inhibited necroptosis of TNFα-stimulated HT-29 cells (Figure 5A), as expected and previously reported in Sun et al.31  In contrast, NSA treatment did not alter lysis of PMN-SA (Figure 5B). Furthermore, whereas stimulated HT-29 cells expressed phosphorylated MLKL and high-molecular-weight oligomers of MLKL (∼250 kDa under nonreducing conditions [Figure 5C]), PMN-SA lacked evidence for either phosphorylation or oligomerization of MLKL, despite expressing the ∼54 kDa monomeric MLKL (Figure 5C). Taken together, the lack of inhibition of lysis by NSA and the absence of both phosphorylated and oligomerized MLKL in PMN-SA suggest MLKL did not promote lysis of PMN-SA.

Figure 5.

Lysis of PMN-SA is MLKL-independent. (A) HT-29 cells were pretreated with 0, 0.5, or 5 μM NSA and then stimulated with or without CHX, zVAD-fmk, and TNFα (CZT), as described in “Methods.” After 22 hours, LDH in supernatants was measured. Bars represent the mean of 3 experiments ± SEM. P values were determined using a repeated measures 1-way ANOVA and Tukey posttest (*P < .05 vs CZT and #P < .05 vs 0.5 μM NSA). (B) PMN were pretreated with or without 0, 0.5, or 5 μM NSA, and then left in buffer alone or challenged with SA, as described in “Methods.” After 3 h, LDH in supernatants was analyzed. Bars represent the average of at least 3 separate experiments ± SEM. (C) Nonreduced lysates from treated and untreated HT-29 cells, PMN, and PMN-SA were immunoblotted and probed for MLKL, phosphorylated-MLKL, and, in the PMN samples, β-actin. Shown is a representative of 3 experiments. Samples were separated in lanes on the same gel, but were noncontiguous. Splicing is denoted with a thin vertical line.

Figure 5.

Lysis of PMN-SA is MLKL-independent. (A) HT-29 cells were pretreated with 0, 0.5, or 5 μM NSA and then stimulated with or without CHX, zVAD-fmk, and TNFα (CZT), as described in “Methods.” After 22 hours, LDH in supernatants was measured. Bars represent the mean of 3 experiments ± SEM. P values were determined using a repeated measures 1-way ANOVA and Tukey posttest (*P < .05 vs CZT and #P < .05 vs 0.5 μM NSA). (B) PMN were pretreated with or without 0, 0.5, or 5 μM NSA, and then left in buffer alone or challenged with SA, as described in “Methods.” After 3 h, LDH in supernatants was analyzed. Bars represent the average of at least 3 separate experiments ± SEM. (C) Nonreduced lysates from treated and untreated HT-29 cells, PMN, and PMN-SA were immunoblotted and probed for MLKL, phosphorylated-MLKL, and, in the PMN samples, β-actin. Shown is a representative of 3 experiments. Samples were separated in lanes on the same gel, but were noncontiguous. Splicing is denoted with a thin vertical line.

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To understand the pathogenesis of the tissue necrosis that frequently accompanies severe staphylococcal infections, we probed the molecular mechanisms by which PMN harboring viable CA-MRSA undergo lytic cell death, a terminal event that releases intracellular contents that can act as danger-associated molecular patterns and fuel exuberant inflammation.8-10 

To elucidate necroptosis pathways, many investigators have stimulated HT-29 cells with a cocktail of TNFα in the presence of executioner caspase inhibitors (eg, zVAD-fmk) and inhibitors of apoptosis proteins (eg, CHX). Under these experimental conditions, TNFα induces lysis in a RIPK-1-, RIPK-3-, and MLKL-dependent fashion. Assembly of the TNF receptor complex IIb, also known as the ripoptosome, activates RIPK-1 and RIPK-3, which subsequently phosphorylates MLKL. Phosphorylated MLKL oligomerizes and inserts into the plasma membrane to create pores and compromise cellular integrity.17,31,32  We used TNFα-induced necroptosis of HT-29 cells as a paradigm with which to compare the fate of PMN-SA. In contrast to the effect of a TNFα-neutralizing antibody on necroptosis of HT-29 cells, blocking autocrine TNFα signaling had no effect on PMN-SA lysis. Furthermore, components of the cytosolic TNF receptor complex IIb, a critical step in TNF receptor-mediated RIPK-1-RIPK-3 activation, was unchanged in PMN over time in the presence of SA (Figure 1).

We previously reported that PMN-SA undergoes necroptosis, a conclusion based on inhibition of lysis by Nec-1.5  However, the failure to link TNFα or ripoptosome activation to PMN-SA lysis prompted us to reexamine the importance of RIPK-1 in the fate of PMN-SA. Whereas Nec-1 inhibited lysis of TNFα-treated HT-29 cells and of PMN-SA, a more potent and selective inhibitor of RIPK-1, GSK’963,25  blocked necroptosis of TNFα-treated HT-29 cells, as expected, but had no effect on PMN-SA (Figure 2). Furthermore, phosphorylation of RIPK-1, a prerequisite for catalyzing the downstream events in the prototypical necroptosis cascade33  and detected in TNFα-stimulated HT-29 cells, did not occur in PMN-SA (Figure 2E). Taken together, these data suggest that lysis of PMN-SA was independent of RIPK-1 activity and that the inhibition by Nec-1 we observed earlier reflects its off-target effects, as previously observed in other experimental settings.22,23,34,35 

Recent emphasis in studies of programmed cell death have highlighted the prominent roles of RIPK-3 and MLKL in the necroptosis pathway.36,37  The RIPK-3 inhibitors GSK’872 and GSK’843 blocked necroptosis of TNFα-treated HT-29 cells, as expected, as well as lysis of PMN-SA (Figures 3 and 4), thereby suggesting PMN-SA lysis also required catalytically active RIPK-3. However, whereas necroptosis of HT-29 cells under our experimental conditions depended on MLKL, as expected, lysis of PMN-SA was not inhibited by NSA and was not associated with phosphorylation of MLKL (Figure 5C), observations that together suggest that necrosis of human PMN fed CA-MRSA occurred independent of MLKL. Taken together, our data demonstrate that lysis of PMN-SA differs from necroptosis in several ways.

During programmed cell death, RIPK-1 operates both as a kinase, enzymatically phosphorylating RIPK-3, and as a structural component of a cytoplasmic protein complex. In the latter role, inactive RIPK-1 can serve as an adaptor for RIPK-3 to undergo autophosphorylation and promote downstream signaling (reviewed in Moriwaki and Chan38 ). Examples of necroptosis independent of active RIPK-1 in vitro and in vivo have been reported. Toll-like receptor 3 and Toll-like receptor 4 activate RIPK-3 through their association with Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-β, and cytoplasmic DNA sensor DNA-dependent activator of interferon-regulatory factors activates RIPK-3 in response to several viruses, including influenza A and murine cytomegalovirus.28,29,39-41  Whereas RIPK-3 drives MLKL-dependent cell death in these examples, RIPK-3 mediates necroptosis independent of RIPK-1 and MLKL in cardiomyocytes via a pathway dependent on calmodulin-dependent protein kinase II and mitochondria.27 

Our finding that lysis of human PMN fed SA was independent of MLKL contrast with the MLKL-dependent necroptosis observed to occur in murine myeloid cells,42-45  a difference that most likely reflects species-specific features of human and mouse cell death pathways, as previously reported. For example, the relative roles of RIPK-1 and RIPK-3 in acetaminophen-induced necrosis of human and murine hepatocytes differ. Nec-1 protects mice from acetaminophen-induced cell death,46  whereas RIPK-1 inhibition by Nec-1 or knock down has no effect on human hepatocytes,47  which are instead protected by the RIPK-3 inhibitor dabrafenib. Thus, the importance of MLKL in the fate of murine, but not human, PMN represents another of the many differences between murine and human neutrophils, both in general and specifically with respect to responses to S aureus.48-50  Although our studies did not identify the executioner protein that mediates PMN lysis in lieu of MLKL, it is important to recognize that cell death pathways are not only cell- and species-specific but also depend on the inducing agent. For example, a recent report by Wang et al showed that granulocyte-macrophage colony-stimulating factor priming and adhesion molecule ligation results in PMN lysis that is Nec-1-inhibitable, RIPK-3-dependent, and TNFα-independent.51  In contrast to lysis of PMN-SA,7  events described by Wang et al depend on the activity of the phagocyte NADPH oxidase.51 

In summary, the fate of PMN-SA contrasts in several ways with the prototypical necroptosis of HT-29 cells. Human PMN fed CA-MRSA underwent programmed cell death and lysis by a mechanism that required RIPK-3, but was independent of active RIPK-1 and MLKL. The incentives to elucidate the mechanisms of underlying PMN fate during inflammation rest on the promise that delineation of the molecular pathways that culminate in lysis of PMN-SA will provide novel targets for intervention as adjuncts to antibiotic therapy during severe CA-MRSA infection.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

The authors acknowledge Kevin Leidal and Sally McCormick-Hill for their excellent technical assistance. The authors thank Peter Gough, John Bertin, and the team at GlaxoSmithKline for kindly providing RIPK-1 and RIPK-3 inhibitors.

This work was supported by National Institutes of Health, National Institute of Allergy and Infectious Diseases grants AI116546 and AI044642, a Merit Review award from the Veterans Affairs, and use of facilities at the Iowa City Department of Veterans Affairs Medical Center, Iowa City, Iowa.

Contribution: M.C.G.-W. contributed to study design, execution of experiments, analyzing data, and writing of the manuscript; S.K. contributed to execution of experiments, analyzing data, and editing the manuscript; and W.M.N. contributed to study design, analyzing data, and writing of the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: William M. Nauseef, Inflammation Program and Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, D160 MTF, 2501 Crosspark Rd, Coralville, IA 52241; e-mail: william-nauseef@uiowa.edu.

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Author notes

*

M.C.G.-W. and S.K. contributed equally to this study.