Cutting Edge: Blockade of Inhibitor of Proteins Sensitizes Neutrophils to TNF- but Not -Mediated Cell Death and IL-1 β Secretion This information is current as of September 25, 2021. Kaiwen W. Chen, Kate E. Lawlor, Jessica B. von Pein, Dave Boucher, Motti Gerlic, Ben A. Croker, Jelena S. Bezbradica, James E. Vince and Kate Schroder J Immunol 2018; 200:3341-3346; Prepublished online 16 April 2018; Downloaded from doi: 10.4049/jimmunol.1701620 http://www.jimmunol.org/content/200/10/3341 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2018/04/14/jimmunol.170162 Material 0.DCSupplemental References This article cites 26 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/200/10/3341.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology

Cutting Edge: Blockade of Inhibitor of Apoptosis Proteins Sensitizes Neutrophils to TNF- but Not Lipopolysaccharide-Mediated Cell Death and IL-1b Secretion †,‡,1 ,1 Kaiwen W. Chen,*x Kate E. Lawlor,{ Jessica B. von Pein,* Dave Boucher,* Motti Gerlic, Ben A. Croker, Jelena S. Bezbradica,* James E. Vince,†,‡ and Kate Schroder* The mammalian inhibitor of apoptosis proteins (IAPs) (2, 3). IAPs are degraded and/or inhibited upon exposure to are key regulators of cell death and inflammation. A genotoxic stress, proapoptotic stimuli (4), TNF superfamily major function of IAPs is to block the formation of a ligation (5, 6), microbial infection (7), or the ap- Downloaded from cell death–inducing complex, termed the ripoptosome, plication of IAP antagonist compounds (8). Hereditary mu- which can trigger -8–dependent apoptosis or tation can also reduce IAP expression, and in some cases, the caspase-independent necroptosis. Recent studies report clinical manifestation in humans resembles that of cryopyrin- that upon TLR4 or TNF receptor 1 (TNFR1) signaling associated periodic fever syndromes, which are driven by IL-1b (9). Consistent with this, LPS-TLR4 signaling in in macrophages, the ripoptosome can also induce http://www.jimmunol.org/ NLRP3 inflammasome formation and IL-1b matura- IAP-depleted macrophages induces ripoptosome-dependent tion. Whether neutrophils have the capacity to assemble caspase-8 activation, caspase-8–dependent IL-1b matura- a ripoptosome to induce cell death and inflammasome tion, and NLRP3 inflammasome signaling (6, 10, 11). Sim- activation during TLR4 and TNFR1 signaling is un- ilarly, TNF receptor 1 (TNFR1) ligation in XIAP-deficient clear. In this study, we demonstrate that murine dendritic cells activates caspase-8–dependent IL-1b matura- neutrophils can signal via TNFR1-driven ripoptosome tion and NLRP3 activation (12). Notably, TLR4-induced assembly to induce both cell death and IL-1b mat- NLRP3 activation upon IAP loss requires signaling by the TLR3/4 adaptor TRIF, which recruits RIPK3 and RIPK1 uration. However, unlike macrophages, neutrophils by guest on September 25, 2021 suppress TLR4-dependent cell death and NLRP3 inflam- through RIP homotypic interaction motif (RHIM) interac- masome activation during IAP inhibition via deficiencies tions to activate the ripoptosome (6, 10). When caspase-8 in the CD14/TRIF arm of TLR4 signaling. The Journal activity is suppressed, RIPK3 in turn activates the mixed of Immunology, 2018, 200: 3341–3346. lineage kinase domain–like protein (MLKL) to trigger nec- roptosis and drive NLRP3 inflammasome assembly (13). Neutrophils often exhibit specialized innate immune sig- he mammalian inhibitor of apoptosis proteins (IAPs), naling pathways to prolong their otherwise short lifespan (14, cellular IAP (cIAP)1, cIAP2, and X-linked IAP (XIAP), 15). In this study, we investigated whether the ripoptosome T are E3 ubiquitin ligases that inhibit , apoptosis, induces neutrophils to undergo TNFR1- or TLR4-dependent and necroptosis. However, it is increasingly clear that IAPs are apoptosis, necroptosis, or NLRP3 inflammasome assembly. also critical regulators of proinflammatory signaling pathways (1). We demonstrate that deficiencies in the CD14/TRIF arm of Two studies reported that a loss of IAPs induces the for- the TLR4 signaling pathway enable LPS-stimulated neutro- mation of the ripoptosome death-inducing complex, com- phils to resist acute cell death and inflammasome activation prising the core components RIPK1, FADD, and caspase-8 upon IAP depletion. However, at later time points, IAP

*Centre for Inflammation and Disease Research, Institute for Molecular Bioscience, The (Fellowship FT130100361 to K.S.). K.W.C. was supported by an ANZ Trustee Medical University of Queensland, St. Lucia, Queensland 4072, Australia; †Division of Inflam- Research Program, K.W.C. and D.B. were supported by The University of Queensland, mation, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria B.A.C. was supported by the American Asthma Foundation and National Institutes of 3052, Australia; ‡Department of Medical Biology, The University of Melbourne, Park- Health Grant 5R01HL124209-04, and M.G. was supported by the Israel Science x ville, Victoria 3050, Australia; Department of Clinical Microbiology and Immunology, Foundation (Grant 1416/15). { Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; and Division Address correspondence and reprint requests to Associate Prof. Kate Schroder, Institute of Hematology/Oncology, Boston Children’s Hospital, Boston, MA 02115 for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, 1K.E.L. and J.B.v.P. contributed equally to this work. Australia. E-mail address: [email protected] ORCIDs: 0000-0003-0471-6842 (K.E.L.); 0000-0002-7233-7177 (J.B.v.P.); 0000- The online version of this article contains supplemental material. 0003-0075-8893 (D.B.); 0000-0001-9518-1833 (M.G.); 0000-0002-0885- Abbreviations used in this article: cIAP, cellular IAP; Cp.A, compound A; FasL, Fas 3599 (B.A.C.); 0000-0001-9261-3805 (K.S.). ; IAP, inhibitor of apoptosis protein; LDH, lactate dehydrogenase; QVD, Q-VD- Received for publication December 4, 2017. Accepted for publication March 19, 2018. Oph; RHIM, RIP homotypic interaction motif; TNFR1, TNF receptor 1; XIAP, X-linked IAP. This work was supported by the National Health and Medical Research Council of Australia (Grant 1101405 and Fellowship 1141466 to J.E.V., Grants 1122240 and Ó 1023297 and Fellowship 1141131 to K.S.) and the Australian Research Council Copyright 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701620 3342 CUTTING EDGE: NEUTROPHILS RESIST TLR4-RIPK3–DEPENDENT CELL DEATH depletion sensitized neutrophils to LPS-induced autocrine 3 h to induce pro–IL-1b and NLRP3 expression, and IAPs TNF signaling, leading to caspase-8–dependent apoptosis and were then inhibited with Cp.A to induce ripoptosome IL-1b activation. If caspase-8 activity is blocked, IFN-g assembly. As expected, LPS stimulation suppressed basal priming rendered neutrophils sensitive to TNFR1-dependent neutrophil apoptosis but, surprisingly, did not enhance Cp.A- necroptosis and subsequent NLRP3 signaling. These findings mediated neutrophil death (Fig. 2A). In contrast, and con- highlight cell type specificity in the regulation of cell death sistent with previous reports (10, 11), LPS stimulation and inflammasome activation. enhanced Cp.A-mediated cell death in macrophages (Fig. 2B). Because LPS/Cp.A treatment did not elicit neutrophil death Materials and Methods (Fig. 2A), we hypothesized that IAP inhibition would be Mice similarly unable to drive caspase-8 and NLRP3 activation in 2 2 2 2 2 2 2 2 C57BL/6, Ice / (16), Tnfr1 / (17), Trif / (18), and Ripk3 / (19) mice LPS-primed neutrophils. We investigated the cleavage status were housed in specific pathogen-free facilities at The University of of caspase-8 and the caspase-8 substrate, Bid, in cells exposed Queensland and the Walter and Eliza Hall Institute. The animal ethics to LPS/Cp.A. Full-length caspase-8 and Bid levels were un- committees of these institutions approved all experimental protocols. affected by neutrophil treatment with Cp.A, regardless of Cell isolation, stimulation, and analysis LPS priming (Fig. 2C), whereas (FasL) triggered Mature bone marrow neutrophils and bone marrow–derived macrophages robust caspase-8 and Bid processing (Fig. 2C). In contrast, in were prepared from murine bone marrow as previously described (10, 15, 20). macrophages, Cp.A readily triggered caspase-8 activation and

The purity of neutrophil isolation was 98–100%. Cells were stimulated with the subsequent cleavage of Bid, similar to FasL stimulation Downloaded from 100 ng/ml ultrapure Escherichia coli K12 LPS (InvivoGen), 100 ng/ml human recombinant TNF (PeproTech), 5 mM nigericin (Sigma-Aldrich), 10 mM (Fig. 2C). Q-VD-Oph (QVD; R&D Systems), 1 mM MCC950, 80 mM dynasore Next, we assessed inflammasome activation by examining (Sigma-Aldrich), 100 ng/ml SuperFasLigand (Enzo Life Sciences), or com- ASC polymerization and caspase-1 and IL-1b cleavage in cells pound A (Cp.A) (0.25–1 mM). Each batch of Cp.A was titrated and used at exposed to LPS/Cp.A. The NLRP3 agonist nigericin stimu- the most potent dose. Cell death was assessed by propidium iodide uptake (10) or lactate dehydrogenase (LDH) release into the culture supernatant lated ASC polymerization, caspase-1 cleavage, and IL-1b

(Promega). Quantitative RT-PCR relative to the reference gene Hprt was maturation in LPS-primed cells, whereas Cp.A did not trigger http://www.jimmunol.org/ performed as described (15). any hallmarks of inflammasome activation in LPS-primed or Statistical analyses -unprimed neutrophils but did so in macrophages (Fig. 2D–F). As anticipated, given the dual pathways (caspase-8 versus Statistical analyses were performed using the nonparametric Mann–Whitney t b test. Data were considered significant when p values were #0.05. caspase-8/NLRP3–dependent caspase-1) mediating IL-1 cleavage downstream of the ripoptosome (10, 11), caspase-1 2 2 Results and Discussion deficiency (Ice / ) only partially suppressed Cp.A-induced IAP inhibition induces neutrophil cell death in the presence of IL-1b secretion from LPS-primed macrophages (Fig. 2E,

exogenous TNF 2F). In all, the failure of LPS/Cp.A treatment to induce cell by guest on September 25, 2021 death, caspase-8 processing, ASC polymerization, and IL-1b To mimic IAP loss during physiological conditions, we treated cleavage in neutrophils indicates that TLR4 ligation cannot mature bone marrow neutrophils with the bivalent SMAC signal ripoptosome-induced cell death or NLRP3 activation mimetic Cp.A to degrade or inhibit cIAP1, cIAP2, and XIAP in these cells. (8). Cp.A triggered rapid cIAP1 degradation in neutrophils at doses as low as 125 nM (Fig. 1A) and induced modest but Neutrophils appear unable to robustly signal via CD14-TRIF significant neutrophil death (Fig. 1B), whereas Cp.A elicited robust macrophage death (Fig. 1C) by promoting autocrine In the absence of IAPs, LPS triggers RIPK3-dependent cell TNF-TNFR1 signaling and the subsequent caspase-8 activa- death and NLRP3 activation in macrophages by using the tion (6, 8). The modest induction of neutrophil death by endosomal TLR4 signaling adaptor TRIF, which recruits and Cp.A suggested that Cp.A induces insufficient autocrine TNF activates RIPK3 through RHIM interactions and so is required signaling for robust cell death induction at this time point. for ripoptosome-dependent cell death induction (10). We thus Indeed, Cp.A failed to significantly induce TNF secretion queried the capacity of neutrophils to induce TRIF signaling. compared with LPS treatment (Fig. 1D), and the addition We first examined levels of the LPS coreceptor CD14, which of exogenous TNF sensitized neutrophils to Cp.A-mediated is required for the internalization of the TLR4-LPS complex killing (Fig. 1E, Supplemental Fig. 1). Thus, although into endosomes to initiate TRIF signaling (21), and resultant Cp.A does not itself induce sufficient TNF production from IFN regulatory factor (IRF)-3 phosphorylation and IFN-b neutrophils to induce robust cell death, IAP depletion licenses production. CD14 was expressed on the surface of murine neutrophil apoptosis to exogenous TNF, demonstrating that macrophages but was undetectable on neutrophils (Fig. 3A). neutrophils can signal cell death via the ripoptosome. Correspondingly, LPS induced IRF3 phosphorylation and IFN-b induction in macrophages but not in neutrophils TLR4 signaling does not trigger caspase-8–dependent cell death and (Fig. 3B, 3C). Despite the lack of TRIF-dependent signaling, NLRP3 activation when IAPs are inhibited in neutrophils neutrophils displayed robust TRIF-independent outputs, in- TLR4 signaling in IAP-depleted macrophages triggers RIPK3- cluding LPS-induced NF-kB p65 phosphorylation (Fig. 3B) dependent ripoptosome-mediated cell death, IL-1b process- and TNF production (Fig. 1D). ing, and NLRP3 activation (6, 10, 11). Having established We next sought to demonstrate that neutrophil CD14 that neutrophils have the capacity to assemble a ripoptosome deficiency blocks LPS/Cp.A–induced cell death and NLRP3 (Fig. 1B, 1E), we next examined whether IAPs modulate inflammasome activation. Because of the technical challenges ripoptosome-dependent cell death and NLRP3 activation in associated with overexpressing CD14 in a short-lived cell LPS-stimulated neutrophils. Cells were primed with LPS for type, such as primary neutrophils, we assessed the impact of The Journal of Immunology 3343

ABCDE

FIGURE 1. TNF signaling and IAP inhibition triggers neutrophil cell death. (A) Neutrophils were stimulated with Cp.A for 2 h, and cIAP depletion was analyzed by immunoblot. Data are representative of two independent experiments. (B and C) Cells were treated with Cp.A for 6 h, and PI uptake was quantified by flow cytometry. (D) Neutrophils were stimulated with 1 mM Cp.A or 100 ng/ml LPS for 6 h, and TNF secretion was determined. (E) Neutrophils were treated with 100 ng/ml recombinant TNF and 1 mM Cp.A for 6 h, and LDH release assay was determined. Data are mean + SEM of data pooled from three independent experiments (B and C) or mean + SD of technical triplicates from one experiment that is representative of two independent experiments (D and E). **p # 0.005, ****p # 0.0001. blocking CD14 internalization on macrophage CD14/TLR4/ points, LPS-induced autocrine TNF engages TNFR1 to TRIF/RIPK3 signaling, cell death, and IL-1b production contribute to cell death and IL-1b production (10). Given using the dynamin inhibitor dynasore (21). In keeping with that LPS induces TNF production from neutrophils TRIF/RIPK3–mediated apoptosis and necroptosis (10), (Fig. 1D), we investigated whether chronic LPS/Cp.A expo- blocking CD14 internalization with dynasore significantly sure elicits neutrophil death and IL-1b production via auto- Downloaded from reduced macrophage apoptosis (LPS/Cp.A), necroptosis crine TNF-TNFR1 signaling. Although acute (5 h) LPS/Cp.A (LPS/Cp.A/pan-caspase inhibitor QVD that blocks caspase- exposure was a poor inducer of neutrophil death and 8), and IL-1b secretion (Fig. 3D, 3E). The lack of CD14 inflammasome activation (Fig. 2), LPS/Cp.A exposure for expression in neutrophils, coupled with the requirement for 16 h triggered substantial (∼50–60% above background) cell CD14 internalization to induce LPS/Cp.A–induced RIPK3- death (Fig. 4A, Supplemental Fig. 2A) and IL-1b secretion dependent cell death and IL-1b activation, indicates that (Fig. 4B) from wild type neutrophils in a TNFR1-dependent http://www.jimmunol.org/ neutrophils suppress LPS-induced cell death induction and manner (Fig. 4A, 4B). Because chronic LPS can upregulate inflammasome assembly via deficiencies in the CD14/TRIF CD14 expression in neutrophils (22), we confirmed that arm of TLR4 signaling. enhanced CD14 signaling was not responsible for increased death, as death was TRIF-independent (Supplemental Fig. Neutrophils undergo apoptosis and necroptosis via TNFR1 2B). We next examined whether inhibiting caspase-8 during In macrophages, LPS/Cp.A induces rapid cell death and IL-1b this treatment regimen switches neutrophil apoptotic cell activation in a TRIF-dependent manner (10), but at later time death to necroptosis by treating cells with the pan-caspase by guest on September 25, 2021

FIGURE 2. LPS/Cp.A treatment does not induce neutrophil cell death or secretion of mature IL-1b. Cells were left untreated, stimulated with recombinant SuperFasLigand (100 ng/ml) for 8 h, or primed for 3 h with 100 ng/ml LPS prior to stimulation with Cp.A (500 nM) or nigericin (5 mM) for 5 h. (A and B) LDH release, (C) Caspase-8 and BID cleavage in cell extracts (XT) and culture supernatants (S/N), and (D and E) IL-1b release were assessed. (F) ASC oligomers in the Triton X-100 insoluble (TX-insol) fraction and cleaved caspase-1 (p20) and mature IL-1b (p17) in the cell supernatants were detected. Data are mean + range of two independent experiments (A and B), mean + SEM of three independent experiments (D and E), or representative of three independent experiments (C and F). 3344 CUTTING EDGE: NEUTROPHILS RESIST TLR4-RIPK3–DEPENDENT CELL DEATH

A B

C D E Downloaded from

FIGURE 3. CD14 internalization is required for RIPK3-dependent cell death and IL-1b production. (A) Surface expression of CD14 in unstimulated cells and http://www.jimmunol.org/ (B) immunoblot analysis of cell extracts of LPS-stimulated cells. Data are representative of three (A) or two (B) independent experiments. (C) Ifnb mRNA induction in LPS-stimulated cells. (D and E) Macrophages were primed for 3 h with 100 ng/ml LPS and then stimulated with Cp.A (1 mM) for 6 h. Dynasore (80 mM) was added 20 min prior to LPS stimulation, and QVD (10 mM) was added in the final 20 min of priming. Data are mean + SD of technical triplicates representative of four independent experiments (C) or mean + SEM of three independent experiments (D and E). *p # 0.05, **p # 0.005, ***p # 0.001. inhibitor QVD prior to Cp.A exposure. Surprisingly, QVD We considered the possibility that neutrophils resist LPS/Cp. blocked chronic LPS/Cp.A–induced neutrophil cell death A/QVD–mediated death because of insufficient TNFR1 sig- (Fig. 4A) and abrogated neutrophil IL-1b secretion (Fig. 4B). naling or expression of necroptotic death machinery. IFN-g by guest on September 25, 2021

A B C

D

FIGURE 4. Autocrine TNF signaling induces neutrophil apoptotic and necroptotic cell death and IL-1b production. Neutrophils were primed with IFN-g (100 ng/ml) for 2 h, then treated with 100 ng/ml LPS for 3 h prior to Cp.A (1 mM) stimulation for 16 h. QVD (10 mM) and MCC950 (1 mM) were added to cells 20 min prior to Cp.A. (A) PI uptake, (B) IL-1b release, and (C) TNF were quantified. Data are mean + SEM of cells from six independent mice. (D) Schematic of ripoptosome-mediated cell death and IL-1b cleavage in macrophages and neutrophils. *p # 0.05, **p # 0.005, ***p # 0.001, ****p # 0.0001. The Journal of Immunology 3345 enhances TLR4-driven TNF production (23) and induces the trigger a switch to necroptosis unless cells were first stimulated expression of necroptotic drivers, RIPK3, and MLKL in with IFN-g or exogenous TNF, indicating that circulating macrophages (24), and so we investigated whether IFN-g levels of proinflammatory cytokines dictate whether neutro- performs similar functions in neutrophils to sensitize these phils undergo necroptosis in vivo. The finding that cell death cells to necroptosis. IFN-g did not induce expression of Ripk3 pathways are distinctly regulated in neutrophils as compared mRNA (Supplemental Fig. 2C) and moderately upregulated with closely-related myeloid cells highlights the important Mlkl mRNA expression in neutrophils (Supplemental Fig. influence of cell identity on immune signaling pathways. Such 2D), but markedly enhanced LPS-stimulated TNF produc- cell type–specific signaling adaptations have likely evolved to tion from neutrophils (Fig. 4C). IFN-g indeed sensitized provide a coordinated inflammatory response in vivo. 2 2 wild type but not Tnfr1 / neutrophils to LPS/Cp.A/QVD– mediated death and IL-1b secretion (Fig. 4A, 4B), and IFN-g/ LPS/Cp.A–treated wild type neutrophils cleaved caspase-8 Acknowledgments and IL-1b (Supplemental Fig. 2E). IFN-g did not sensitize We thank TetraLogic Pharmaceuticals, Prof. John Silke and Prof. Heinrich neutrophils to TRIF-mediated cell death upon chronic LPS/ Korner for reagents, and Dr. Akshay D’Cruz for technical assistance. We apologize for omitting citations to relevant publications because of space Cp.A exposure (Supplemental Fig. 2B). In all, this indicates constraints. that IFN-g sensitized neutrophils to TNF-mediated nec- roptosis, a conclusion also supported by the observation that exogenous TNF or FasL triggered Ripk3-dependent, nec- Disclosures Downloaded from roptotic death in IFN-g–untreated neutrophils (Supplemental K.S. holds a full-time Associate Professorial Research Fellow appointment at Fig. 2F). Interestingly, IFN-g/LPS/Cp.A–induced IL-1b se- The University of Queensland and conducts research focused on inflammation cretion was largely insensitive to the NLRP3 inhibitor, and inflammasomes. K.S. is a coinventor on patent applications for NLRP3 MCC950 (Fig. 4C), suggesting that neutrophil IL-1b was inhibitors, which have been licensed to Inflazome Ltd. (Dublin, Ireland). Infla- zome is developing drugs that target the NLRP3 inflammasome to address predominantly processed by caspase-8 and not caspase-1 un- unmet clinical needs in inflammatory disease. K.S. served on the Scientific Ad- der these conditions. In macrophages, necroptotic cell death visory Board of Inflazome in 2016–2017. The other authors have no financial http://www.jimmunol.org/ activates the NLRP3 inflammasome and drives IL-1b secre- conflicts of interest. tion in a cell-intrinsic manner (13). IL-1b production from neutrophils exposed to IFN-g/LPS/Cp.A/QVD occurs via the same pathway because MCC950 suppressed IL-1b secretion References from these necroptotic neutrophils (Fig. 4B). Taken together, 1. Silke, J., and J. Vince. 2017. IAPs and cell death. Curr. Top. Microbiol. Immunol. 403: 95–117. our data indicate that when IAPs are blocked, TNFR1 sig- 2. Feoktistova, M., P. Geserick, B. Kellert, D. P. Dimitrova, C. Langlais, M. Hupe, naling triggers apoptosis and IL-1b activation, whereas IFN-g K. Cain, M. MacFarlane, G. Ha¨cker, and M. 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