Limit Necroptosis through Cleavage of Rip1 Kinase Scott McComb, Bojan Shutinoski, Susan Thurston, Erin Cessford, Kriti Kumar and Subash Sad This information is current as of October 2, 2021. J Immunol published online 5 May 2014 http://www.jimmunol.org/content/early/2014/05/03/jimmun ol.1303380 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2014/05/03/jimmunol.130338 Material 0.DCSupplemental

Why The JI? Submit online. http://www.jimmunol.org/

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 5, 2014, doi:10.4049/jimmunol.1303380 The Journal of Immunology

Cathepsins Limit Macrophage Necroptosis through Cleavage of Rip1 Kinase

Scott McComb,1 Bojan Shutinoski,1 Susan Thurston, Erin Cessford, Kriti Kumar, and Subash Sad

It has recently been shown that programmed necrosis, necroptosis, may play a key role in the development of inflammation. Deci- phering the regulation of this pathway within immune cells may therefore have implications in pathology associated with inflam- matory diseases. We show that treatment of with the pan inhibitor (zVAD-FMK) results in both increased phosphorylation and decreased cleavage of receptor interacting kinase-1 (Rip1), leading to necroptosis that is dependent on autocrine TNF signaling. Stimulation of cells with TLR agonists such as LPS in the presence of zVAD-FMK also induced Rip1- phosphorylation via a TNFR-independent mechanism. Further examination of Rip1 expression under these stimulatory conditions revealed a regulatory cleavage of Rip1 in macrophages that is not apparently attributable to caspase-8. Instead, we provide novel Downloaded from evidence that cysteine family cathepsins, which are highly abundant in myeloid cells, can also cleave Rip1 kinase. Using small in- terfering RNA knockdown, specific inhibitors, and cell-free cleavage assays, we demonstrate that cysteine cathepsins B and S can directly cleave Rip1. Finally, we demonstrate that only through combined inhibition of cathepsins and caspase-8 could a potent induction of macrophage necroptosis be achieved. These data reveal a novel mechanism of regulation of necroptosis by cathepsins within macrophage cells. The Journal of Immunology, 2014, 192: 000–000.

mmune cells must strike a balance between the need for cell Necroptosis was initially coined to describe cell death that was http://www.jimmunol.org/ death and cell survival to effectively eliminate pathogens (1). induced by treatment of cells with TNF-a and the pan caspase I Too little cell death may allow infected cells to escape re- inhibitor zVAD-FMK (zVAD) (12). Interestingly, it was found that moval or raise inadequate inflammatory response, as is exploited the key mediator of apoptosis, caspase-8, acts to limit necroptotic by certain viral pathogens (2). Conversely, too much cell death can cell death by cleaving the receptor-interacting protein kinases-1 prevent cells from performing vital immune functions or drive and -3 (Rip1 and Rip3) (13). Despite clear evidence implicating pathological inflammation, as can occur in Salmonella typhimu- caspase-8 in the regulation of necroptosis (14), specific inhibition rium infection (3) and inflammatory shock (4, 5). The most well- of caspase-8 has also been shown to be inadequate to induce nec-

studied form of cell death, apoptosis, is known to be orchestrated roptosis (15). Active dampening of necroptosis by various mech- by guest on October 2, 2021 by intrinsic or extrinsic signals that drive the activation of caspase anisms may be of particular importance in macrophages, be- (6). Recently, another form of programmed cell death cause these cells express increased levels of cytotoxic mediators that proceeds via necrosis has been identified; this form of cell that might predispose them to necrotic cell death, such as reactive death is known as necroptosis (7). The yin and yang of cell death oxygen species (16). Although inhibition of is necessary is readily apparent in necroptosis. On one hand, necroptotic cell for induction of necroptosis, generally a higher concentration of death has been shown to aid in activating an immune response to zVAD is used for induction of necroptosis (12) than is required to some viruses (8, 9). On the other hand, we have previously shown inhibit caspases alone (17, 18). We therefore considered the pos- that increased necrotic cell death in cellular inhibitor of apoptosis– sibility that additional proteases inhibited by zVAD may be in- deficient macrophages results in impaired control of bacteria (10). volved in regulating necroptosis. In this article, we present novel In addition, necroptotic cell death has been shown to perpetuate evidence that cysteine family cathepsins actively cleave Rip1 ki- various inflammatory pathologies (4, 11). Despite the clear im- nase and are inhibited by zVAD treatment. In addition, we show portance of necrotic cell death to immune function, much of the that a specific inhibitor of caspase-8 was capable of inducing basic mechanisms that regulate necroptosis remain unclear. significant cell death only when combined with inhibitors target- ing cathepsins. Thus, we provide novel insight into the proteolytic control of the key necroptotic kinase, Rip1. Given the ability of Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada macrophages to modulate expression and localization of cathep- 1S.M. and B.S. contributed equally to this work. sins in response to inflammatory stimuli (19, 20), this may rep- Received for publication December 18, 2013. Accepted for publication April 7, 2014. resent an important means of controlling necrotic cell death during an immune response. This work was supported by a grant from the Canadian Institutes of Health Research. Address correspondence and reprint requests to Dr. Subash Sad, Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Materials and Methods Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M, Canada. E-mail address: Subash. [email protected] Mice The online version of this article contains supplemental material. C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All knockout strains were also on the C57BL6 background. TNFR1/ Abbreviations used in this article: C8I, caspase-8 inhibitor; Rip1, receptor-interacting 2/2 protein kinase-1; Rip3, receptor-interacting protein kinase-3; siRNA, small interfer- 2 mice were obtained from Jackson Laboratory. Rip3-deficient mice ing RNA; WT, wild type; zIETD, z-IETD-FMK; zVAD, zVAD-FMK. were kindly provided by Dr. Vishva Dixit (Genentech). All animal ex- periments in this work were carried out as per Canadian Council on An- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 imal Care guidelines.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303380 2 CATHEPSINS PROTECT MACROPHAGES FROM NECROPTOSIS

Generation of bone marrow–derived macrophages struct. Primers used for the inverse PCR to generate the constructs are listed in Supplemental Table 1. For the internal deletion of amino acids, the plasmid Macrophages were derived from bone marrow as previously described (10). encoding for GST-Rip1334-656 was used as a template. The GST-RIP pro- In brief, femurs, tibias, pelvises, and humeri were removed from wild type 2/2 2/2 tein constructs were purified with GE Healthcare GSTrap FF (17-5130-01) (WT), Rip3 , or TNFR1/2 mice as required. Bones were then briefly according to manufacturer recommendations and eluted at pH 8.0. soaked in ethanol and the muscle was removed by agitation with a paper towel. Bare bones were then crushed in a solution of RPMI 1640 con- Immunoprecipitation taining 8% serum (media). M-CSF was then spread into empty petri dishes and allowed to adhere to the dishes for a short amount of time. Bone Coimmunoprecipitations were performed as follows: macrophages were marrow cells were then added to the petri dishes at ∼5 3 106 cells/dish in treated with inhibitors as described in the text and lysed with RIPA buffer 10 ml media with 50 mg/ml gentamicin. Cells were then allowed to dif- containing a inhibitor mixture (04693132001; Roche Applied ferentiate into macrophages over 7–10 d. The expression of F4/80/CD11b Science, Laval, QC, Canada) and phosphatase inhibitor mixture (Sigma- was assessed via flow cytometry to confirm purity (.90% purity). Aldrich P5726). Lysates were then precleared using unconjugated pro- tein G-Sepharose beads (51-3478-CO-EG; GE Healthcare, Waukesha, WI). Cell death induction using various inhibitors Specific Abs targeting Rip1 kinase (BD 610458) or cathepsin S (Santa Cruz sc-29941) were conjugated to beads for 1 h on ice. Ab-conjugated beads Inhibitors were obtained from various commercial sources: zVAD (EMD were then incubated overnight with lysate samples. Finally, immunopre- Millipore 627610), LPS (Sigma L2630), necrostatin-1 (EMD Millipore cipitates were collected by centrifugation and washed with PBS several 480065), E-64-D (Santa Cruz sc-201280), Ca-074-Me (EMD Millipore times. Immunoprecipitates were then denatured by boiling in SDS for 5 min 205531), or calpeptin (Tocris 0448). Inhibitors were dissolved in appropriate before analysis by Western blot (as described earlier). solvents (DMSO or PBS, according to manufacturer’s suggestion). Cell death was induced by various combinations of these inhibitors as described in the Measurement of cathepsin activity text. Generally, macrophages were plated and left for 4–6 h to adhere before cells were treated with inhibitors and incubated at 37˚C for an additional Assessment of cathepsin S activity from primary macrophage lysates was 24 h. Viability or cell death was examined by MTT or fluorescence mi- performed using the Cathepsin S Activity Fluorometric Assay Kit form Downloaded from croscopy as described later. BioVision (K144-100) with minimal modification as follows. After 5 h of treatment with LPS and zVADor control treatment, cells were serum starved Cell death/viability assays with PBS pH 7.4 for 5 min at 37˚C, after which the cells were lysed using four freeze/thaw cycles in liquid nitrogen. Lysates were then examined Cell viability was assessed using MTT (Sigma M5655), which is converted according to manufacturer’s instructions. For cell-free experiments, from yellow to blue by mitochondrial dehydrogenase in living cells. In recombinant cathepsin S (0.2 ng/ml; BioVision) was used to cleave the brief, MTT was added to cells, and cells were incubated at 37˚C for 1–2 h. substrate provided by the kit for 2 h, where no cell lysates were used. An equal volume of isopropanol with 0.02M HCl was added to lyse the http://www.jimmunol.org/ crystals in cells, using vigorous pipetting. Cell survival was then assessed Cell-free cleavage assay by measuring the absorbance at 570 nm on an Emax plate reader (Mo- lecular Devices) because absorbance is directly proportional to the number Reactions were set up with eluted GST-RIP constructs (6 mg) and mouse of cells in culture. cathepsin S (50769-M08H; Sino Biological), mouse (965-CY; Cell death was also measured using propidium iodide (BD 550825) R&D Systems), or (1515-CY-010; R&D Systems) as recom- staining to identify cells that have lost plasma membrane integrity. Hoechst mended by the manufacturers under the following conditions: cathepsin S staining was also used to clearly stain all cells present in the culture. After and cathepsin L reactions were set for 2 or 4 h at 37˚C, pH 7.4; the ca- staining for 15–20 min, cells were examined on an Olympus IX81 fluo- thepsin B reaction for 4 h at 37˚C, pH 6.3. At the end of each reaction, rescent microscope (Olympus, Richmond Hill, ON, Canada). The per- samples were handled for SDS-PAGE. centage of cell death was then determined using Image Pro Software Statistics by guest on October 2, 2021 (Media Cybernetics) for automated counting of the number of propidium iodide–positive cells divided by the number of Hoechst-positive cells. All error bars show SEM, where shown in the text. Student t tests were used to determine the significance of results. All statistical analyses were per- Western blots formed using GraphPad Prism software. Abs targeting the of interest were obtained from various com- mercial sources as follows: mouse anti-Rip1 (BD 610458), mouse anti-Rip3 Results (ProSci 2283), goat anti–cathepsin S (Santa Cruz sc-6505), rabbit anti– zVAD induces TNF- and Rip3-dependent necroptosis in cathepsin B (Santa Cruz sc-6490), rabbit anti-His (Santa Cruz sc-803), and mouse anti-actin (BD 612656). Western blots were performed using macrophage a standard protocol. In brief, protein lysates were run on SDS-PAGE gels Necroptotic cell death is typically induced when cells are treated and transferred to polyvinylidene fluoride membranes. Membranes were then blocked using 5% milk in TBS with 0.05% Tween 20. Abs were with zVAD and TNF-a (7). Within macrophages, zVAD treatment incubated overnight with membranes and detection was performed using alone has also been shown to induce cell death (23, 24), but HRP-conjugated secondary Abs. Results were visualized using the ultra- whether this occurs through necroptosis has not been clear. We sensitive West Femto ECL Kit (Thermo Scientific 34094). observed a dose-dependent loss of macrophage viability upon RNA interference treatment with zVAD for 24 h as determined by MTT assay of mitochondrial activity (Fig. 1A). Consistent with necroptosis, we Small interfering RNA (siRNA) targeted against specific was obtained observed that zVAD treatment results in a dose-dependent in- from various commercial sources as follows: scrambled control (Santa Cruz sc-37007), cathepsin S (Santa Cruz sc-29941), cathepsin B OnTarget crease in a slower migrating form of Rip1 (Fig. 1B), which we SMARTpool (Thermo Scientific L-044712-00-0005), TRIF (Santa Cruz sc- have previously shown to be phosphorylated Rip1 (10). Induction 106845). J774 macrophages were transfected with siRNA using Dharmafect of cell death by zVAD alone occurs only when cells are treated 4 (Thermo Scientific T-2004-02) according to manufacturer’s instruction. with zVAD the same day, underlining the importance of cell stress Day 7 bone marrow–derived macrophages were transfected using the Lonza electroporation kit for bone marrow macrophages (Lonza VPA-1009) in as a variable in the necroptotic response. Next, we observed that accordance with manufacturer’s protocol. After 24 h, transfected cells were zVAD-induced death of primary macrophages was abrogated by treated with various inhibitors, incubated for an additional 24 h, and assessed the addition of the Rip1 kinase inhibitor, necrostatin (Fig. 1C and for protein expression or viability as described in the text. 1D). We also confirmed that Rip32/2 macrophages were highly Plasmid constructs and protein purification resistant to zVAD treatment (Fig. 1E and 1F). Previous work has shown that zVAD-induced necroptosis is dependent upon auto- Bacterial strain Escherichia coli DH5a harboring plasmid encoding for crine TNF-a signaling (15). Similarly, zVAD-induced necroptosis mouse RIPK1 with N-terminal GST tag was obtained from Addgene, plas- mid 11972 (21). By the use of restriction-free cloning strategy (22), plasmids in bone marrow macrophages was dependent on expression of encoding the shorter versions of mouse RIPK1 also with N-terminal GST tag TNFR1 and TNFR2 (Fig. 1G). Thus, these data demonstrate that were generated, and E. coli Rosetta DE3 was transformed with each con- zVAD treatment induces necroptosis in macrophages that is de- The Journal of Immunology 3 Downloaded from

FIGURE 1. zVAD treatment induces TNF-a– and Rip3-dependent necroptosis in macrophages. Bone marrow–derived macrophages were cultured for 7–12 d as described in Materials and Methods.(A) Cells were treated with various concentrations of zVAD (as shown) for 24 h, and viability was assessed

using the MTT mitochondrial activity assay. Graph shows cell viability relative to untreated cells. (B) Macrophages were treated with various concentrations http://www.jimmunol.org/ of zVAD for 1 h and examined via Western blot for Rip1 protein expression. (C) Macrophages were treated with 100 mM zVAD with or without the addition of necrostatin for 24 h and stained with Hoechst. Dead cells were identified using propidium iodide staining. (D) The % dead cells were enumerated by automated counting of propidium iodide+ cells versus Hoechst+ cells. An MTT assay was also performed in parallel to confirm inverse correlation of cell death and viability results. (E) Bone marrow macrophages were derived from WT and Rip32/2 mice. Cells were treated with 100 mM zVAD for 24 h and cell death was assessed via MTT assay. (F) The % dead cells was enumerated by automated counting of propidium iodide+ cells versus Hoechst+ cells. An MTT assay was also performed in parallel to confirm inverse correlation of cell death and viability results. (G) Bone marrow macrophages were derived from WT and TNFR1/22/2 mice. Cells were treated with 100 mM zVAD for 24 h, and viability was assessed via MTT assay. All experiments were performed a minimum of three times in duplicate. ***p , 0.0001, **p , 0.005. by guest on October 2, 2021 pendent on autocrine TNF-a signaling, as well as downstream examination of the specific effects of zVAD on Rip1 and Rip3 activation of Rip3 expression. expression. Cells were treated with various combinations of inhibitors and LPS for 1 h, and the expression of Rip1 and Rip3 LPS treatment drives Rip1 phosphorylation independently of was examined. We noted several lower m.w. bands in Rip1 and TNFR signaling Rip3 blots, which we hypothesized to be the cleavage products of LPS is a potent activator of macrophages; thus, we tested the effects Rip1 and Rip3 (Fig. 3A, lanes 1 and 2). Supporting this conclu- LPS might have on macrophage necroptosis. Treatment of cells sion, we observed a consistent loss of these cleavage bands after with LPS resulted in an increased sensitivity to cell death induced treatment with zVAD (Fig. 3A, lane 3). It is noteworthy that the Rip1 by zVAD (Fig. 2A). Importantly, cell death that was induced by kinase Ab we have used targets the C-terminal portion of the protein LPS+zVAD was still fully abrogated by necrostatin, confirming (385-650), and thus the cleavage products visualized are C-terminal necroptosis as the mechanism of cell death (Fig. 2A). We also fragments of Rip1 kinase protein. In the case of Rip3, although observed similar effects with polyinosinic:polycytidylic acid we observed several lower m.w. bands in our gels, only one band (Poly I:C) treatment, suggesting that this effect was not re- (∼20 kDa) was abrogated completely by the application of zVAD. stricted to TLR4 signaling (Fig. 2B). Using siRNA knockdown of Using a specific inhibitor for caspase-8 (z-IETD-FMK [zIETD], TRIF in J774 macrophage cells (Fig. 2C), we confirmed that the 100 mM), there appeared to be little abrogation of Rip1 cleavage LPS-induced increase in sensitivity to zVAD is dependent on the in macrophages. One faint band at ∼42 kDa was absent with TLR4-TRIF pathway (Fig. 2D). TNFR1- and TNFR2-deficient caspase-8 inhibition (Fig. 3A, lane 4). In addition to its ability to macrophages were resistant to cell death in response to zVAD alone, potently inhibit caspases, zVAD has also been shown to inhibit but zVAD+LPS–induced necroptosis was TNFR1 and TNFR2 in- cysteine family cathepsins, such as cathepsins B and S, particu- dependent (Fig. 2E). These results add to previous work showing larly at concentration ranges necessary to induce necroptosis that when combined with zVAD treatment, TLR stimulation can (∼50–100 mM) (17). Thus, we hypothesized that cathepsins may lead to necroptotic cell death in a TNF-independent manner (25). also cleave Rip1 and Rip3. Application of a cell-permeable ca- thepsin inhibitor, E64-D, was able to completely abrogate the Cathepsins cleave Rip1 in macrophages cleavage bands of Rip1 kinase, similarly to zVAD treatment We next considered the possibility that inhibition of other proteases (Fig. 3A, lane 5). Finally, inhibition of both caspase-8 and cathep- by zVAD may regulate necroptosis. Rationale for this stemmed sins resulted in elevated Rip1 phosphorylation and expression along from the observations that high concentrations of zVAD were with a decrease in cleavage bands (Fig. 3A, lane 6). Thus, these data needed that were well above the concentrations required for in- imply that the inhibition of cathepsins by zVAD may potentially hibition of caspases (18). We therefore performed an in-depth play a role in the induction of necroptosis. 4 CATHEPSINS PROTECT MACROPHAGES FROM NECROPTOSIS Downloaded from

FIGURE 2. LPS stimulation induces TNFR–independent, TRIF-dependent necroptosis. (A) Macrophages were treated with LPS (10 ng/ml) in com- bination with various concentrations of zVAD as shown, with or without the addition of necrostatin. Cell viability was assessed after 24 h by MTT assay.(B)

Macrophages were treated with either 10 ng/ml of LPS or Poly I:C at the same time as varying concentrations of zVAD as shown. After 24 h, viability was http://www.jimmunol.org/ measured by MTT assay. (C) J774 macrophage cells were transfected with TRIF, TLR4, or untargeted control siRNA for 24 h, after which a sample of lysates was examined for TRIF knockdown. (D) siRNA-transfected cells were then treated with various concentrations of zVAD and LPS, and incubated for an additional 24 h. Cell viability was then measured using MTT assay. (E) Macrophages were derived from WT or TNFR1/22/2 mice and treated with LPS and various concentrations of zVAD as shown. After 24 h, cell viability was examined via the MTT assay. Graphs show cell viability relative to controls without zVAD. All experiments were performed a minimum of three times in duplicate. ***p , 0.0001, **p , 0.005, *p , 0.05.

To further confirm the role of cathepsins in the cleavage of Rip1, cleavage assay. GST-fused Rip1280–656 was expressed, which con-

we used siRNA knockdown approach to target two key cysteine tained a putative cleavage site(s) for cathepsins based on the Rip1 by guest on October 2, 2021 family cathepsins, which are highly expressed within macrophages cleavage fragments in experiments described earlier (Supplemental (19). Consistent with a role in the cleavage of Rip1, knockdown of Fig. 2A). We observed a clear dose-dependent cleavage of Rip1 either cathepsin S or cathepsin B resulted in less cleavage of Rip1 kinase by mouse cathepsin S (Fig. 5A). Cleavage of recombinant kinase (Fig. 3B). Knockdown of cathepsin S or cathepsin B also Rip1 by cathepsin S resulted in an ∼35- to 40-kDa cleavage resulted in a small but significant increase in the sensitivity of product, consistent with smaller forms of cleaved Rip1 observed macrophages to zVAD-induced cell death (Fig. 3C). Importantly, in macrophages. We also performed a similar cell-free cleavage of siRNA knockdown increased sensitivity to zVAD at concen- Rip1 using cathepsin B, where we observed a number of bands trations that are below the optimal levels required for inhibiting including some slightly heavier ones (∼40–45 kDa) consistent with cathepsins. In a cell-free assay, zVAD directly inhibited the ca- those observed in LPS-treated macrophages (Fig. 5B). We also thepsin S activity (Supplemental Fig. 1). Furthermore, zVAD examined whether cathepsin L can cleave Rip1 kinase, where we inhibited cathepsin S activity in macrophage lysates using a fluo- observed some Rip1 cleavage, albeit more inefficient than that rogenic cathepsin S activity assay (Fig. 3D). These data suggest observed by cathepsins B or S (Fig. 5C). These data indicate that that cysteine family cathepsins act redundantly to cleave Rip1 cathepsins can directly cleave Rip1 kinase. We also confirmed kinase and limit necroptosis within macrophages, and are inhib- that the addition of zVAD to our cell-free cleavage assay inhib- ited by treatment with zVAD. ited the ability of cathepsin S to cleave Rip1 to form a ∼36-kDa To confirm that Rip1 is associating with cathepsin S within band (Fig. 5D). macrophages, we performed coimmunoprecipitation. Upon immu- To map the exact site where Rip1 is cleaved by cathepsins, we noprecipitation with anti–cathepsin S Ab, we noted the associa- generated various truncation mutants of Rip1, which revealed that tion of both cleaved and full-length Rip1 (Fig. 4A, lanes 5 and 6). cleavage of Rip1 by cathepsin S must occur between residues 334 We were not able to reverse immunoprecipitate cathepsin S with and 350 of Rip1 (Fig. 5E). We further narrowed the exact cleav- Rip1 pull-down, perhaps because of a fairly small proportion of age site to 3 aa in the intermediate domain of Rip1 (347–349), Rip1 interacting with cathepsin S. We also confirmed by immu- because cleavage was normal in a deletion mutant lacking residues nofluorescence staining that cathepsins S and B colocalized with 342–347, but absent in the mutant lacking aa 342–349 or Rip1 (Fig. 4B and 4C). These data indicate that cathepsins interact GST-Rip1349–656 (Fig. 5F). These data show that cathepsin S can with Rip1 within macrophages. directly cleave Rip1 kinase at position 348 (Supplemental Fig. 2B). Cathepsins S and B directly cleave Rip1 kinase Cathepsins and caspase-8 cooperate to limit necroptosis Given our surprising finding that cathepsins may act to regulate We finally wanted to assess the role that cathepsin-mediated Rip1 necroptosis, we wanted to directly confirm that cathepsins are cleavage might play in regulating necroptotic cell death of mac- capable of cleaving Rip1 kinase. Thus, we performed a cell-free rophages by applying cathepsin inhibitors in combination with The Journal of Immunology 5

FIGURE 3. Cathepsins cleave Rip1 in macrophages. (A) Bone marrow–derived macrophages were treated with various combinations of LPS, zVAD, C8I, and/or cathepsin inhibitor as shown. After 1 h of treatment, cells were lysed and examined for expression of Rip1 and Rip3 via Western blot. Short (1-min) and long (15-min) exposures were used to identify Rip1 expression by Western blots. Actin expression was used as a loading control. (B) Bone marrow macrophages were transfected with untargeted, cathepsin S (CTSS), or cathepsin B (CTSB) targeting siRNA via electroporation for 24 h. Cells were then treated with LPS for 2 h and examined via Western blot for Rip1, CTSS, and CTSB expression. Actin was used as a loading control. (C) Macrophages trans- Downloaded from fected for 24 h with untargeted control, CTSS or CTSB targeted siRNA were treated with LPS and varying concentra- tion of zVAD as shown. After an addi- tional 24 h, cells were tested for viability using MTT assay. (D) Macrophages were treated with varying concentrations of http://www.jimmunol.org/ LPS and zVAD as shown for 5 h. Lysates were then examined for CTSS activity using a fluorometric kit as described in Materials and Methods.Similarexperi- ments were repeated at least three times. by guest on October 2, 2021 varying concentrations of zVAD+LPS. Cathepsin inhibition alone did Previous work has clearly shown that regulation of Rip1-induced not result in any significant cell death of macrophages, in contrast cell death is a key function of caspase-8 (11). Thus, to confirm the with zVAD, which induced significant cell death. Addition of ca- relative role of caspase-8 and cathepsins in the regulation of thepsin inhibitors E64D, Ca-074-Me, or calpeptin resulted in a small necroptosis, we treated macrophages with LPS and a specific but significant increase in sensitivity to zVAD-induced death (Fig. 6A caspase-8 inhibitor (C8I), zIETD. Although treatment with C8I and Supplemental Fig. 3A–D). The small effect of cathepsin and LPS alone resulted in minimal cell death, when combined inhibitors may be due to the fact that zVAD on its own can inhibit with a cathepsin inhibitor, we observed a significant induction of cathepsins directly. In contrast with the effects of inhibitors of the macrophage necroptosis (Fig. 6B and 6C). Similar results were cathepsins, an inhibitor of the aspartyl protease, obtained with a number of different cathepsin inhibitors in com- cathepsin D (Pepstatin A), did not affect the sensitivity of macro- bination with C8I (Supplemental Fig. 3C and 3D). Consistent with phages to necroptosis (Supplemental Fig. 3A). These results indicate necroptosis, combined caspase-8/cathepsin inhibition–induced that within macrophages, the inhibition of cathepsins leads to an cell death was blocked with the addition of necrostatin (Fig. 6B). increase in necroptosis of macrophages. In addition, these results We also confirmed that caspase-8/cathepsin inhibition was inef- also refute a view that cathepsin activity is necessary for necroptosis. fective at inducing cell death with Rip3 knockout macrophages

FIGURE 4. Cathepsins associate with Rip1 in macrophages. (A) Bone marrow–derived macrophages were treated with LPS or LPS/zVAD for 1 h at 37˚C before lysis with RIPA buffer. Immunoprecipitation was performed overnight using Abs bound to protein G-Sepharose beads. Immunoprecipitated lysates were then denatured and examined via Western blot for Rip1 or cathepsin S (CTSS). HC and LC indicate nonspecific bands corresponding to the H and L chains of the immunoprecipitation Abs, respectively. (B) Macrophages were fixed and stained for CTSS and Rip1, then examined via confocal microscopy. (C) Macrophages were treated with LPS for 1 h, and then were fixed and stained for cathepsin B (CTSB) and Rip1. Cells were examined via confocal microscopy. 6 CATHEPSINS PROTECT MACROPHAGES FROM NECROPTOSIS Downloaded from

FIGURE 5. Cathepsins cleave Rip1 kinase directly. (A) Cathepsin S (CTSS) was mixed with recombinant GST-RIP1280–656 in a cell-free assay as described in Materials and Methods. The reactions were set with decreasing CTSS concentration in subsequent 10-fold dilutions; the highest concentration of CTSS http://www.jimmunol.org/ used was 20 ng/ml. CTSS was developed with anti-His Ab. (B) Recombinant cathepsin B (CTSB) was mixed at varying concentrations with recombinant GST- RIP1280–656 in cell-free assay as described in Materials and Methods. Similar to (A), highest concentration used was 20 ng/ml with subsequent 1:10 dilutions. (C) Recombinant cathepsin L was mixed at varying concentrations with recombinant GST-RIP1280–656 in cell-free assay as described in Materials and Methods and similar to (A)and(B). (D) Recombinant GST-Rip1 was cleaved with recombinant CTSS (2 ng/ml) in the presence of zVADat 0, 100, 50, or 25 mM as shown for 4 h. (E) GST alone, GST-RIP1 constructs, and complete RIP1 with GST tag (see Materials and Methods) were assayed for CTSS cleavage. For each construct, 1.7 ng/ml CTSS was added for 4 h before stopping the reaction. (F) GST-RIP1 constructs with internal deletion of several amino acids or other truncated GST-RIP1 constructs were assayed for CTSS cleavage. For each construct, 5 ng/ml CTSS was added for 2 h before stopping the reaction.

(data not shown). Finally, we examined the role of caspase-8 spe- cleavage of Rip1 kinase as the chief mechanism (7, 11, 13, 28). by guest on October 2, 2021 cifically, using inhibitors targeting other apoptotic caspases (caspase- As a novel addition to this, we have found that cysteine family 9, -3) in combination with the cathepsin inhibitor calpeptin. cathepsins also cleave Rip1 within macrophages. Furthermore, we Combined inhibition of cathepsins and caspase-9 or -3 inhibitors find that combined inhibition of cathepsins and caspase-8 leads to resulted in no significant increase in necroptosis (Supplemental potent induction of necroptosis, indicating that cathepsins and Fig. 3E). Together, these results point to the novel conclusion that caspase-8 cooperate to limit necroptosis. cathepsins and caspase-8 cooperate to regulate necroptosis in The process of zVAD-induced necroptosis has also been ob- macrophages, and inhibition of both is necessary for the induc- served in a number of cell lines, usually requiring exogenous (29) tion of this form of cell death. or autocrine TNF-a signaling (15). In macrophages specifically, zVAD has been shown to induce cell death in the RAW264.1 cell Discussion line, although the mechanism of death was not clear at the time Innate immune cells, such as macrophages, must strike a careful (24). In bone marrow macrophages, we have observed that zVAD balance between the need for cell death to prevent propagation of alone induced necroptosis, which was dependent on autocrine an infectious agent and the need for survival to promote immune TNF-a signaling. The stimulation of macrophages with TLR function. Although apoptosis has long been considered a key ligands LPS or Poly I:C lead to a potent induction of necroptosis mechanism of cell death in the immune system, recently a form of when combined with zVAD stimulation. These data add to a re- programmed necrosis (necroptosis) has also been described (26). cent report showing that TLR4 signaling can lead to necroptosis Control of necroptosis has important implications for the immune in a TNF-independent manner via the TLR4-TRIF pathway (25). system, because this would lead to the release of potent intracel- Interestingly, although zVAD was able to induce necroptosis, lular danger signals to the external environment (7). Viruses have we found that a C8I, zIETD, did not result in cell death. A similar been demonstrated to inhibit Rip1 activation of necroptosis to observation in L929 cells was previously ascribed to less stimulation avoid this type of inflammatory induction (27). In contrast, over- of autocrine TNF-a production with C8I (zIETD) relative to pan- active necroptosis can result in misdirected and damaging in- caspase inhibitor (zVAD) (15). Inconsistent with this, the addition flammatory pathology. For example, we have previously shown of LPS, which can drive necroptosis independently of TNF-a, still that overactive necroptosis of macrophages can be exploited by does not lead to zIETD-induced cell death. Thus, we show that in intracellular bacteria to escape immune response (3, 10). Thus, we combination with signals from TNF-a or LPS, zVAD induces nec- have undertaken a careful investigation of cell death as induced roptosis of macrophages but appears to involve mechanisms in ad- by the pan-caspase inhibitor, which was used in the initial dis- dition to caspase-8 inhibition. covery of necroptosis, zVAD (12). Currently, the prevailing model In examining the expression of Rip1 during necroptosis, we of dampening necroptotic signaling focuses on caspase-8–mediated consistently observed smaller bands that reacted with our anti-Rip1 The Journal of Immunology 7 Downloaded from

FIGURE 6. Cathepsins and caspase-8 cooperate to regulate necroptosis. (A) Bone marrow–derived macrophages were treated with LPS (10 ng/ml), cathepsin inhibitors (10 mg/ml), and varying concentrations of zVAD as shown for 24 h. Cell viability was then assessed by MTT assay. Graph shows cell viability relative to controls without zVAD. (B) Macrophages were treated with LPS, zVAD (50 mM), C8I (100 mM), necrostatin (Nec), and cathepsin inhibitors calpeptin (Cal; 10 mg/ml) or E64D (10 mg/ml) as shown. Cells were incubated for 24 h with inhibitors and assessed for viability via MTT assay. Graph shows cell viability relative to cells treated with LPS alone. (C) Macrophages were treated with C8I (100 mM) or low concentration of zVAD http://www.jimmunol.org/ (25 mM) in the presence or absence of the cathepsin inhibitor E64D (30 mM). Cell death was then assessed using Hoechst and propidium iodide cos- taining. All graphs show viability measurement relative to the untreated control. All experiments were repeated a minimum of three times in duplicate: *p , 0.05, **p , 0.005, ***p , 0.0001.

Ab. Upon treatment with zVAD, we saw a significant reduction in actually results in selective cleavage of few targets (32). Specifi- these bands, leading us to conclude that they were likely cleavage cally, controlled lysosomal breakdown enhances apoptotic cell fragments of Rip1 kinase. In contrast with zVAD, treatment with death via cathepsin-mediated cleavage of Bid into its proapoptotic the C8I zIETD did not affect the cleaved Rip1 forms. In addition to form (33, 34). In the case of necroptosis, cathepsins have been by guest on October 2, 2021 its effects on caspases, zVAD has been demonstrated to also inhibit thought to aid in rapid cellular disintegration (35), but specific data cysteine protease family cathepsins, such as cathepsin B and S (17). are limited. In contrast with this view, we show that inhibition of We reconfirm these findings in this article, showing that at con- cysteine cathepsins actually induced a subtle but significant in- centrations used to induce necroptosis, zVAD inhibits the activity crease in zVAD-induced necroptosis within macrophages. In con- of cathepsin S in macrophage lysates and in cell-free experiments. trast with the subtle effects on zVAD-induced death, cathepsin To test whether cathepsin activity might be driving Rip1 cleavage, inhibitors resulted in significant macrophage necroptosis when we also applied targeted cathepsin inhibitors to macrophages. combined with a specific C8I. Taken together, these data lead to the Although zIETD failed to affect the level of cleaved Rip1, the surprising conclusion that caspase-8 and cathepsins both function to addition of a pan cysteine cathepsin inhibitor, E64D, resulted in actively suppress necroptosis through cleavage of Rip1 kinase. At a total abrogation of cleaved Rip1. We were similarly able to this point it remains unclear whether cleavage of Rip1 occurs in the decrease the cleavage of Rip1 using siRNA knockdown of either cytosolic or lysosomal compartments of the cell. Given that LPS cathepsin S or B. signaling that drives necroptosis is TRIF dependent (36), and Using cell-free cleavage experiments, we clearly demonstrate LPS-induced TRIF signaling has been shown to operate in the that cysteine family cathepsins cleave Rip1, producing fragments endosomal compartment (37), we propose that endosomal cath- of a size (∼36 kDa) consistent with those observed in macro- epsins might function to regulate Rip1 kinase activity in the phages. Based on this and our siRNA experiments, we propose endosomal/phagosomal compartment. that cathepsins S and B likely have redundant function in limiting In our Western blot analysis, Rip1 cleavage products appear to necroptosis. We also observed some cleavage of Rip1 kinase with be predominantly formed by cathepsins, raising the question of how cathepsin L, but it was relatively inefficient in comparison with caspase-8 contributes to regulating necroptosis in macrophages. that observed with cathepsin S or B. Given the size of the Rip1 Caspase-8 is inextricably linked to control of necroptosis in fetal kinase fragments observed, we estimate that the most prominent development (11). Cleavage of Rip1 kinase by caspase-8 was cleavage of Rip1 likely occurs at aa 348. At this time it is unclear identified before the role of Rip1 in nonapoptotic cell death was exactly how cathepsin cleavage of Rip1 affects necroptotic sig- discovered (13), and has since been repeatedly confirmed (38). naling given that the pool of full-length Rip1 remains relatively Given that FADD is thought to recruit caspase-8 to the necrosome unchanged. after activation (14), we hypothesize that caspase-8 may act as a Cathepsins have previously been considered as aggressive prote- more downstream regulator of necroptosis after Rip1 phosphory- ases that once released from the will mediate rapid pro- lation. Consistent with this, caspase-mediated cleavage of Rip1 is teolytic breakdown of the cell (30). Although massive cathepsin generally observed after stimulation of cells with TNF, but not release may result in necrosis (31), slow release of cathepsins under steady-state conditions (13, 28). In contrast with caspase-8, 8 CATHEPSINS PROTECT MACROPHAGES FROM NECROPTOSIS we observe cathepsin mediated cleavage in macrophages under 14. Dillon, C. P., A. Oberst, R. Weinlich, L. J. Janke, T.-B. Kang, T. Ben-Moshe, T. W. Mak, D. Wallach, and D. R. Green. 2012. Survival function of the FADD- steady-state conditions, indicating that this may be a more up- CASPASE-8-cFLIP(L) complex. Cell Rep. 1: 401–407. stream mode of regulating Rip1 activity. Given that the cleavage 15. Wu, Y.-T., H.-L. Tan, Q. Huang, X.-J. Sun, X. Zhu, and H.-M. Shen. 2011. zVAD- sites are after the kinase domain and that these cleaved forms induced necroptosis in L929 cells depends on autocrine production of TNFa mediated by the PKC-MAPKs-AP-1 pathway. Cell Death Differ. 18: 26–37. persist within the cells, it seems possible that Rip1-cleaved forms 16. Forman, H. J., and M. Torres. 2002. Reactive oxygen species and cell signaling: could potentially interact with and inhibit the formation of the respiratory burst in macrophage signaling. Am.J.Respir.Crit.CareMed.166: S4–S8. necrosome. Further work will be needed to elucidate how pro- 17. Rozman-Pungercar, J., N. Kopitar-Jerala, M. Bogyo, D. Turk, O. Vasiljeva, I. Stefe, P. Vandenabeele, D. Bro¨mme, V. Puizdar, M. Fonovic´, et al. 2003. In- teolysis by cathepsins and caspase-8 regulates necroptosis. hibition of -like cysteine proteases and legumain by caspase-specific The idea of cathepsins acting as antinecroptotic factors is a sur- inhibitors: when reaction mechanism is more important than specificity. Cell prising finding, yet given an expanding role of cathepsins in the Death Differ. 10: 881–888. 18. Hayashi, H., M. Cuddy, V. C.-W. Shu, K. W. Yip, C. Madiraju, P. Diaz, apoptotic program, it seems to fit well into the paradigm of pro- T. Matsuyama, M. Kaibara, K. Taniyama, S. Vasile, et al. 2009. Versatile assays apoptotic factors acting to inhibit necroptosis. Interestingly, ex- for high throughput screening for activators or inhibitors of intracellular pro- pression of both cathepsin S and B has been well documented at teases and their cellular regulators. PLoS ONE 4: e7655. 19. Schmid, H., R. Sauerbrei, G. Schwarz, E. Weber, H. Kalbacher, and C. Driessen. inflammatory sites in various disease models including pancreatitis 2002. Modulation of the endosomal and lysosomal distribution of cathepsins B, (39), inflammatory bowel disease (40), and atherosclerosis (41). It L and S in human monocytes/macrophages. Biol. Chem. 383: 1277–1283. 20. Beers, C., K. Honey, S. Fink, K. Forbush, and A. Rudensky. 2003. Differential seems plausible that cathepsin-mediated cleavage of Rip1 kinase regulation of cathepsin S and cathepsin L in interferon g-treated macrophages. J. could play a role in perpetuating macrophage survival and function Exp. Med. 197: 169–179. within inflammatory sites. In addition to this, elevated expression of 21. Wu, C.-J., D. B. Conze, T. Li, S. M. Srinivasula, and J. D. Ashwell. 2006. Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF- cathepsins has repeatedly been shown to be a marker for enhanced kappaB activation. [Published erratum appears in 2006 Nat. Cell Biol. 8: 424.] Downloaded from tumorigenicity and poor prognosis (42). Given the novel role we Nat. Cell Biol. 8: 398–406. have identified for cathepsins in cleaving Rip1 kinase and limiting 22. Unger, T., Y. Jacobovitch, A. Dantes, R. Bernheim, and Y. Peleg. 2010. Appli- cations of the Restriction Free (RF) cloning procedure for molecular manipu- macrophage necroptosis, it is an exciting possibility that cathepsins lations and protein expression. J. Struct. Biol. 172: 34–44. may function similarly to enhance resistance to necrotic cell death 23. Wu, Y.-T., H.-L. Tan, Q. Huang, Y.-S. Kim, N. Pan, W.-Y. Ong, Z. G. Liu, within cancer cells. Furthermore, using cathepsin inhibition to target C.-N. Ong, and H.-M. Shen. 2008. Autophagy plays a protective role during zVAD-induced necrotic cell death. Autophagy 4: 457–466. macrophage function may represent a novel treatment modality for 24. Kim, S. O., and J. Han. 2001. Pan-caspase inhibitor zVAD enhances cell death in both cancer and inflammatory diseases. RAW246.7 macrophages. J. Endotoxin Res. 7: 292–296. http://www.jimmunol.org/ 25. He, S., Y. Liang, F. Shao, and X. Wang. 2011. Toll-like receptors activate pro- grammed necrosis in macrophages through a receptor-interacting kinase-3- Acknowledgments mediated pathway. Proc. Natl. Acad. Sci. USA 108: 20054–20059. We thank Renu Dudani for technical contributions. 26. Christofferson, D. E., and J. Yuan. 2010. Necroptosis as an alternative form of programmed cell death. Curr. Opin. Cell Biol. 22: 263–268. 27. Mocarski, E. S., J. W. Upton, and W. J. Kaiser. 2012. Viral infection and the Disclosures evolution of -regulated apoptotic and necrotic death pathways. Nat. Rev. Immunol. 12: 79–88. The authors have no financial conflicts of interest. 28. Declercq, W., T. Vanden Berghe, and P. Vandenabeele. 2009. RIP kinases at the crossroads of cell death and survival. Cell 138: 229–232. 29. Zhang, D.-W., J. Shao, J. Lin, N. Zhang, B.-J. Lu, S.-C. Lin, M.-Q. Dong, and References J. Han. 2009. RIP3, an energy metabolism regulator that switches TNF-induced by guest on October 2, 2021 1. Golstein, P., D. M. Ojcius, and J. D.-E. Young. 1991. Cell death mechanisms and cell death from apoptosis to necrosis. Science 325: 332–336. the immune system. Immunol. Rev. 121: 29–65. 30. Guicciardi, M. E., M. Leist, and G. J. Gores. 2004. Lysosomes in cell death. 2. Upton, J. W., W. J. Kaiser, and E. S. Mocarski. 2010. Virus inhibition of RIP3- Oncogene 23: 2881–2890. dependent necrosis. Cell Host Microbe 7: 302–313. 31. Yamashima, T., and S. Oikawa. 2009. The role of lysosomal rupture in neuronal 3. Robinson, N., S. McComb, R. Mulligan, R. Dudani, L. Krishnan, and S. Sad. death. Prog. Neurobiol. 89: 343–358. 2012. Type I interferon induces necroptosis in macrophages during infection 32. Repnik, U., V. Stoka, V. Turk, and B. Turk. 2012. Lysosomes and lysosomal with Salmonella enterica serovar Typhimurium. Nat. Immunol. 13: 954–962. cathepsins in cell death. Biochim. Biophys. Acta 1824: 22–33. 4. Linkermann, A., J. H. Bra¨sen, F. De Zen, R. Weinlich, R. A. Schwendener, 33. Guicciardi, M. E., J. Deussing, H. Miyoshi, S. F. Bronk, P. A. Svingen, C. Peters, D. R. Green, U. Kunzendorf, and S. Krautwald. 2012. Dichotomy between RIP1- S. H. Kaufmann, and G. J. Gores. 2000. Cathepsin B contributes to TNF-alpha- and RIP3-mediated necroptosis in tumor necrosis factor-a-induced shock. Mol. mediated hepatocyte apoptosis by promoting mitochondrial release of cyto- Med. 18: 577–586. chrome c. J. Clin. Invest. 106: 1127–1137. 5. Duprez, L., N. Takahashi, F. Van Hauwermeiren, B. Vandendriessche, 34. Cirman, T., K. Oresic´, G. D. Mazovec, V. Turk, J. C. Reed, R. M. Myers, V. Goossens, T. Vanden Berghe, W. Declercq, C. Libert, A. Cauwels, and G. S. Salvesen, and B. Turk. 2004. Selective disruption of lysosomes in HeLa P. Vandenabeele. 2011. RIP kinase-dependent necrosis drives lethal systemic cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like inflammatory response syndrome. Immunity 35: 908–918. lysosomal cathepsins. J. Biol. Chem. 279: 3578–3587. 6. MacFarlane, M., and A. C. Williams. 2004. Apoptosis and disease: a life or death 35. Vanlangenakker, N., T. Vanden Berghe, and P. Vandenabeele. 2012. Many decision. EMBO Rep. 5: 674–678. stimuli pull the necrotic trigger, an overview. Cell Death Differ. 19: 75–86. 7. Vandenabeele, P., L. Galluzzi, T. Vanden Berghe, and G. Kroemer. 2010. Mo- 36. Cusson-Hermance, N., S. Khurana, T. H. Lee, K. A. Fitzgerald, and lecular mechanisms of necroptosis: an ordered cellular explosion. Nat. Rev. Mol. M. A. Kelliher. 2005. Rip1 mediates the Trif-dependent toll-like receptor 3- and Cell Biol. 11: 700–714. 4-induced NF-kB activation but does not contribute to interferon regulatory 8.Cho,Y.S.,S.Challa,D.Moquin,R.Genga,T.D.Ray,M.Guildford,and factor 3 activation. J. Biol. Chem. 280: 36560–36566. F. K.-M. Chan. 2009. Phosphorylation-driven assembly of the RIP1-RIP3 complex 37. Kagan, J. C., T. Su, T. Horng, A. Chow, S. Akira, and R. Medzhitov. 2008. regulates programmed necrosis and virus-induced inflammation. Cell 137: 1112–1123. TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon- 9. Rebsamen, M., L. X. Heinz, E. Meylan, M.-C. Michallet, K. Schroder, beta. Nat. Immunol. 9: 361–368. K. Hofmann, J. Vazquez, C. A. Benedict, and J. Tschopp. 2009. DAI/ZBP1 38. Rajput, A., A. Kovalenko, K. Bogdanov, S.-H. Yang, T.-B. Kang, J.-C. Kim, recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate J. Du, and D. Wallach. 2011. RIG-I RNA helicase activation of IRF3 tran- NF-kappaB. EMBO Rep. 10: 916–922. scription factor is negatively regulated by caspase-8-mediated cleavage of the 10. McComb, S., H. H. Cheung, R. G. Korneluk, S. Wang, L. Krishnan, and S. Sad. RIP1 protein. Immunity 34: 340–351. 2012. cIAP1 and cIAP2 limit macrophage necroptosis by inhibiting Rip1 and 39. Lyo, V., F. Cattaruzza, T. N. Kim, A. W. Walker, M. Paulick, D. Cox, J. Cloyd, Rip3 activation. Cell Death Differ. 19: 1791–1801. J. Buxbaum, J. Ostroff, M. Bogyo, et al. 2012. Active cathepsins B, L, and S in 11. Kaiser, W. J., J. W. Upton, A. B. Long, D. Livingston-Rosanoff, L. P. Daley- murine and human pancreatitis. Am. J. Physiol. Gastrointest. Liver Physiol. 303: Bauer, R. Hakem, T. Caspary, and E. S. Mocarski. 2011. RIP3 mediates the G894–G903. embryonic lethality of caspase-8-deficient mice. Nature 471: 368–372. 40. Menzel, K., M. Hausmann, F. Obermeier, K. Schreiter, N. Dunger, F. Bataille, 12. Degterev, A., Z. Huang, M. Boyce, Y. Li, P. Jagtap, N. Mizushima, G. D. Cuny, W. Falk, J. Scholmerich, H. Herfarth, and G. Rogler. 2006. Cathepsins B, L and T. J. Mitchison, M. A. Moskowitz, and J. Yuan. 2005. Chemical inhibitor of D in inflammatory bowel disease macrophages and potential therapeutic effects nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat. of cathepsin inhibition in vivo. Clin. Exp. Immunol. 146: 169–180. Chem. Biol. 1: 112–119. 41. Li, X., Z. Liu, Z. Cheng, and X. Cheng. 2012. Cysteinyl cathepsins: multi- 13. Lin, Y., A. Devin, Y. Rodriguez, and Z. G. Liu. 1999. Cleavage of the death functional in cardiovascular disease. Chonnam Med. J. 48: 77–85. domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev. 42. Mohamed, M. M., and B. F. Sloane. 2006. Cysteine cathepsins: multifunctional 13: 2514–2526. enzymes in cancer. Nat. Rev. Cancer 6: 764–775.