Recognition of Cytoplasmic RNA Results in -Dependent Inflammasome Activation and Apoptosis in Human Macrophages This information is current as of September 28, 2021. Johanna Rintahaka, Niina Lietzén, Tiina Öhman, Tuula A. Nyman and Sampsa Matikainen J Immunol 2011; 186:3085-3092; Prepublished online 21 January 2011; doi: 10.4049/jimmunol.1002051 Downloaded from http://www.jimmunol.org/content/186/5/3085

Supplementary http://www.jimmunol.org/content/suppl/2011/01/24/jimmunol.100205 http://www.jimmunol.org/ Material 1.DC1 References This article cites 57 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/186/5/3085.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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Recognition of Cytoplasmic RNA Results in Cathepsin-Dependent Inflammasome Activation and Apoptosis in Human Macrophages

Johanna Rintahaka,* Niina Lietze´n,† Tiina O¨ hman,† Tuula A. Nyman,† and Sampsa Matikainen* dsRNA is an important pathogen-associated molecular pattern that is primarily recognized by cytosolic pattern-recognition receptors of the innate-immune system during virus infection. This recognition results in the activation of inflammasome- associated -1 and apoptosis of infected cells. In this study, we used high-throughput proteomics to identify secretome, the global pattern of secreted , in human primary macrophages that had been activated through the cytoplasmic dsRNA-

recognition pathway. The secretome analysis revealed cytoplasmic dsRNA-recognition pathway-induced secretion of several Downloaded from exosome-associated proteins, as well as basal and dsRNA-activated secretion of lysosomal and pro- tease inhibitors (cystatins). Inflammasome activation was almost completely abolished by cathepsin inhibitors in response to dsRNA stimulation, as well as encephalomyocarditis virus and vesicular stomatitis virus infections. Interestingly, Western blot analysis showed that the mature form of cathepsin D, but not , was secreted simultaneously with IL-18 and inflam- masome components ASC and caspase-1 in cytoplasmic dsRNA-stimulated cells. Furthermore, small interfering RNA-mediated silencing experiments confirmed that cathepsin D has a role in inflammasome activation. Caspase-1 activation was followed by http://www.jimmunol.org/ proteolytic processing of caspase-3, indicating that inflammasome activation precedes apoptosis in macrophages that had recog- nized cytoplasmic RNA. Like inflammasome activation, apoptosis triggered by dsRNA stimulation and virus infection was effectively blocked by cathepsin inhibition. In conclusion, our results emphasize the importance of cathepsins in the innate immune response to virus infection. The Journal of Immunology, 2011, 186: 3085–3092.

nnate immune reaction against viral infection begins with endolysosomal compartments of human macrophages (4). In ad- recognition of the virus by specific pattern-recognition re- dition to TLRs, viral RNA is detected by cytoplasmic RIG-I–like I ceptors (PRRs) (1). Macrophages are the central players of receptors (RLRs), such as RIG-I and MDA-5 (5, 6). RIG-I detects by guest on September 28, 2021 innate immunity, and their PRRs detect the presence of pathogen- ssRNA bearing 59 triphosphate and/or short dsRNA molecules associated molecular patterns (PAMPs) and damage-associated mo- with certain sequence or nucleotide preferences (7–9), whereas lecular patterns (DAMPs) (2, 3). This recognition results in pro- MDA-5 is specialized to detect long dsRNA strands (.1kbin duction of antiviral and proinflammatory cytokines, recruitment of length) (10, 11). In macrophages, activation of RIG-I and MDA-5 other inflammatory cells, and, often, apoptosis of the virus-infected induces strong antiviral cytokine response, and recent data also cell. A successful innate-immune reaction also provides a firm connect RLRs to the activation of apoptosis of virus-infected cells basis for the activation of adaptive-immune responses. (12, 13). Several cytoplasmic and membrane-bound PRRs participate in IL-1b and IL-18 are important proinflammatory cytokines that innate-immune recognition of virus infection. TLR8 and TLR3 are produced during virus infection (13–15). IL-1b and IL-18 are detect endocytosed ssRNA or dsRNA molecules, respectively, in synthesized as immature forms: pro–IL-1b and pro–IL-18. Cys- teine protease caspase-1 cleaves these proforms to biologically *Unit of Excellence for Immunotoxicology, Finnish Institute of Occupational Health, active equivalents that are secreted via unconventional - 00250 Helsinki, Finland; and †Protein Chemistry Research Group, Institute of Bio- secretion pathways (16, 17). In parallel, caspase-1 is activated technology, University of Helsinki, 00014 Helsinki, Finland by cytosolic PRRs in a protein complex called inflammasome Received for publication June 21, 2010. Accepted for publication December 15, (18). NLRP3-inflammasome is the most studied caspase-1–acti- 2010. vating molecular platform; it is activated by various heterogeneous This work was supported by grants from the Research Council for Biosciences and DAMPs and PAMPs. The DAMPs include, for example, extra- Environment of the Academy of Finland, the Sigrid Juselius Foundation, and the Helsinki Graduate Program in Biotechnology and Molecular Biology. cellular ATP and crystalline monosodium urate (19, 20). In ad- Address correspondence and reprint requests to Dr. Sampsa Matikainen, Unit of Excel- dition to these endogenous stimuli, NLRP3-inflammasome is acti- lence for Immunotoxicology, Finnish Institute of Occupational Health, Topeliuksenkatu vated by exogenous stimuli, including asbestos, silica, aluminum 41 a A, 00250 Helsinki, Finland. E-mail address: sampsa.matikainen@ttl.fi adjuvant (21, 22), fungal toxins (23), b-glucans (24), and viral The online version of this article contains supplemental material. infections (25–27). Because these activators are chemically and Abbreviations used in this article: CatIII, cathepsin inhibitor III; DAMP, damage- structurally different, they probably are indirectly recognized by associated molecular pattern; EMCV, encephalomyocarditis virus; MOI, multiplicity of infection; PAMP, pathogen-associated molecular pattern; poly-IC, polyinosinic- the NLRP3-inflammasome. It is more likely that they activate in- polycytidylic acid; poly-IC-t, transfected polyinosinic-polycytidylic acid; PRR, flammasome by inducing changes in endogenous molecules that pattern-recognition receptor; RLR, RIG-I–like receptor; t-Bid, truncated form of are then recognized as danger signals (28). Potassium efflux and Bid; VSV, vesicular stomatitis virus. production of reactive oxygen species are common features as- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 sociated with NLRP3-inflammasome activation, and it was re- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002051 3086 CATHEPSIN-DEPENDENT ACTIVATION OF INFLAMMASOME, APOPTOSIS cently shown that thioredoxin-interacting protein links oxidative Amicon Ultra-15 centrifugal filter devices, with a cut-off of 10,000 nom- stress to inflammasome activation (29, 30). inal m.w. limit (Millipore, Bedford, MA). Equal volumes of the concen- Cathepsins are lysosomal that have many immuno- trated cell-culture media were separated on 15% SDS-PAGE. Western blot analysis for total-cell lysates and secreted proteins was carried out as logical activities. Cathepsin B and D are functionally closely re- described earlier (13). Primary Abs were purchased as follows: ASC lated and are the most abundant in this (31–33). polyclonal Ab from Millipore; caspase p20 from Sigma-Aldrich; caspase-3 Maturation of these proteases proceeds through several steps. p19/17 and Bid from Cell Signaling Technology (Danvers, MA); caspase-1 Mature cathepsin B is found as a single-chain form (29 kDa) and p10 (C-20) and cathepsin B from Calbiochem, and cathepsin D (C-20) from Santa Cruz Biotechnology (Santa Cruz, CA). To confirm equal as a two-chain form (25 and 4 kDa) of active (34). Active loading and transfer onto membranes between the samples, membranes cathepsin D is produced through two successive cleavages of were stripped in 0.2 M NaOH for 5 min, washed in PBS-Tween 0.05% preproenzyme, resulting in formation of a two-chain mature en- three times, and stained with ready-to-use SYPRO Ruby Protein Blot zyme (14-kDa L chain and 34-kDa H chain) in (33). Stain, according to the manufacturer’s instructions (Sigma-Aldrich). The Recently, cytoplasmic leakage of cathepsin B was associated with major basally expressed protein band is shown as a loading control. inflammasome activation (29). Secretome characterization We studied the role of cathepsins in the innate-immune re- For secretome characterization, cell-culture supernatants were first col- sponse to cytoplasmic dsRNA in human macrophages. We provide lected and concentrated with Amicon Ultra-15 centrifugal filter devices evidence that cathepsins have a central role in the activation (Millipore), as above, followed by protein purification using a 2-D Clean-Up of inflammasome and apoptosis in response to the cytoplasmic- Kit (GE Healthcare, Pittsburgh, PA). Next, proteins were separated using recognition pathway. SDS-PAGE and visualized with silver staining. For protein identification, whole gel lanes were cut into 30 pieces of equal size, proteins were in-gel digested with trypsin, and the resulting were analyzed by nanoLC- Downloaded from MS/MS, as described previously (35). The liquid chromatography-tandem Materials and Methods Human macrophages, cell stimulations, and viruses Human macrophages were differentiated from isolated blood monocytes of healthy blood donors obtained from the Finnish Red Cross Blood Transfusion Service (Helsinki, Finland), as described previously (14). http://www.jimmunol.org/ Macrophages were transfected with a mimetic of dsRNA, polyinosinic- polycytidylic acid (poly-IC), at 10 mg/ml (Sigma-Aldrich, St. Louis, MO) using Lipofectamine 2000 transfection reagent (Invitrogen Life Techno- logies, Paisley, U.K.), according to the manufacturer’s instructions. Vesic- ular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) were used at a multiplicity of infection (MOI) of 1, and were described pre- viously (35). Cathepsin B inhibitor Ca-074 Me ([L-3-trans-(propylcarba- moyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester), cathepsin inhibitor III (CatIII; Z-Phe-Gly-NHO-Bz-pOMe), and inhibitor Est [(2S,3S)-trans-epoxysuccinyl-L-laucylamodo-3-methylbutane ethyl ester] (also known as E-64d) were purchased from Calbiochem (San by guest on September 28, 2021 Diego, CA). Each sample was obtained and pooled from three separate blood donors, and the results are representatives of three independently and similarly done experiments.

Small interfering RNA experiments After 5 d of cell culture in 12-well plates, macrophages were transfected with 100 nM nontargeting control siRNA (AllStars Negative Control siRNA, Qiagen, Hilden, Germany) and with 50 nM each of two cathepsin D siRNAs (final concentration = 100 nM) (Qiagen) by using HiPerFect Transfection Reagent (Qiagen), according to the manufacturer’s instruction. After 4 h of siRNA treatment, fresh macrophage media were added to the cells. On the following day, the cells were left unstimulated or were transfected with poly-IC for 8 h, after which the cell-culture supernatants were collected, and total proteins were isolated for ELISA and Western blot analyses, respectively.

Quantitative real-time PCR Total RNAwas isolated using an RNeasy Plus Mini Kit (Qiagen) and reverse transcribed with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA), according to the manufacturers’ instructions. Quantitative real-time PCR was performed with an ABI PRISM 7500 Sequence Detection System applying TaqMan chemistry and Predeveloped TaqMan assay primers and probes (Applied Biosystems) and PerfeCTa qPCR FastMix (Quanta Biosciences, Gaithersburg, MD). The Sequence detector system version 1.4 software was used to develop the real-time PCR data (Applied Biosystems). The relative -expression differences were calculated with the comparative ΔΔCt method, as described earlier (36). The results are expressed as relative units, which is a fold change in FIGURE 1. Inflammasome activation precedes apoptosis in human that is normalized to an endogenous reference gene (18S macrophages activated through the cytoplasmic dsRNA-recognition path- rRNA) and is relative to nontemplate control containing molecular-grade way. Human primary macrophages were stimulated with Lipofectamine water instead of a cDNA sample. alone or in complex with poly-IC-t at 10 mg/ml for the indicated times, after which cell-culture media were collected for IL-18 ELISA measurements (A), Western blot analysis and total-cell lysates (B) and concentrated cell-culture media (C) were pre- Total-cell lysates were prepared as previously described (13). To analyze pared for Western blot analysis with Abs against IL-18, caspase-1 p10 and secreted proteins, cell-culture media were collected and concentrated with p20, ASC, and caspase-3 p19/17. The Journal of Immunology 3087

FIGURE 2. Secretome characterization of macro- phages activated through the cytoplasmic dsRNA- recognition pathway. A, Human macrophages were stimulated with Lipofectamine alone or in complex with poly-IC-t for 6 and 18 h. Then, cell-culture supernatants were concentrated, and proteins were precipitated, separated using SDS-PAGE, and visual- ized with silver staining. For protein identification, whole gel lanes were cut into 30 pieces of equal size, proteins were in-gel digested with trypsin, and the resulting peptides were analyzed by nanoLC-MS/MS. After 6 h of stimulation, we identified 219 and 291 proteins from control and poly-IC-t samples, respec- tively; after 18 h stimulation, we identified 167 and 424 proteins from control and poly-IC-t samples, Downloaded from respectively. The Venn diagrams show the overlap between protein identification in control and dsRNA- stimulated samples, as well as the overlap in dsRNA- stimulated samples with different time points. The identified proteins included several Ras-related pro- teins (B), as well as cathepsins and cystatins (C). http://www.jimmunol.org/

mass spectrometry data were searched with in-house Mascot version 2.2 IL-18 secretion at 3 h after transfection, and the greatest levels of through ProteinPilot 3.0 interface against the SwissProt database (version IL-18 were seen at 12 and 18 h after stimulation (Fig. 1A). In 56.12). The search criteria for Mascot searches were human-specific tax- accordance with ELISA data, Western blot analysis of intracel- by guest on September 28, 2021 onomy, trypsin digestion with one missed cleavage allowed, carbamido- methyl modification of cysteine as a fixed modification, and oxidation of lular proteins showed a progressive decrease in constitutively pro- methionine as a variable modification. All of the reported protein identi- duced pro–IL-18, as well as accumulation of biologically active fications were statistically significant (p , 0.05). forms of 18-kDa IL-18 and caspase-1 p10 beginning at 3 h after cytoplasmic dsRNA stimulation (Fig. 1B). Obvious intracellular Apoptosis assay formation of activated caspase-3, caspase-3 p19/17, and its The percentage of apoptotic cells was assayed with APOPercentage Ap- cleavage product IL-18 p16/15 was seen at 6 h after stimulation optosis Assay, according to the manufacturer’s guidelines (Biocolor Life Science Assays, County Antrim, U.K.). Photographs were taken with an Olympus DP70 Digital microscope camera, connected to an Olympus IX71 light microscope, and using software DP Controller (version 2.2.1.227) and DP Manager (2.2.1.195) (Center Valley, PA). The stained and unstained cells were manually counted, and the percentage of apo- ptotic cells was calculated.

ELISA measurements The concentrations of secreted IL-18 were analyzed by ELISA, according to the manufacturers’ instructions (Human Medical and Biological Labora- tories, Nagoya, Japan; Bender MedSystems, Vienna, Austria).

Results Inflammasome activation precedes apoptosis in human macrophages during innate-immune recognition of dsRNA We previously showed that cytoplasmic dsRNA recognition results in activation of inflammasome and apoptosis in human macro- phages (13). These events are associated with caspase-1– and caspase-3–mediated processing of pro–IL-18 to biologically active FIGURE 3. Secretion of cathepsin B and D in response to dsRNA (IL-18 p18) and inactive (IL-18 p15/16) forms of this cytokine, transfection. Human macrophages were stimulated with Lipofectamine respectively. To analyze the kinetics of inflammasome activation, alone or in complex with cytoplasmic dsRNA (poly-IC-t) at 10 mg/ml for poly-IC, a mimetic of dsRNA, was transfected into cytoplasm of 1, 3, 6, 12, or 18 h, after which cell lysates (A) and concentrated cell human macrophages, and IL-18 concentration was measured from culture media (B) were prepared for Western blot analysis with Abs against cell-culture media with ELISA. Cytoplasmic dsRNA induced cathepsin B and D. 3088 CATHEPSIN-DEPENDENT ACTIVATION OF INFLAMMASOME, APOPTOSIS with dsRNA (Fig. 1B). Two isoforms of ASC, the central com- Secretome characterization of macrophages activated through ponent of inflammasomes, were constitutively present in human cytoplasmic dsRNA-recognition pathway macrophages, and the expression of both isoforms was slightly Most proteins are secreted through a conventional protein-secre- enhanced in response to dsRNA transfection (Fig. 1B). tion pathway. These proteins contain N-terminal leader signal pep- In addition to processing of proinflammatory cytokines, inflam- tides that direct their transport to the plasma membrane through masome activation is associated with the secretion of its central the endoplasmic reticulum–Golgi pathway (37). Our current and components. To characterize dsRNA-induced inflammasome ac- previous data showed that inflammasome-associated caspase-1 is tivation further, we collected cell-culture supernatants, concen- activated in response to the cytosolic dsRNA-recognition pathway trated them, and performed Western blot analysis of extracellular in human macrophages. Keller et al. (38) showed that active proteins. In accordance with the presented data (Fig. 1A,1B), cyto- caspase-1 is a regulator of unconventional protein secretion of plasmic dsRNA elicited secretion of IL-18 p18, caspase-1 p20, leaderless proteins. To get a global overview of dsRNA-induced and ASC 6 h after stimulation (Fig. 1C). In conclusion, our re- protein secretion in macrophages, we used a combination of pro- sults showed that cytoplasmic dsRNA-induced inflammasome tein separation by SDS-PAGE followed by high-throughput pro- activation precedes apoptosis: caspase-1 activation and IL-18 se- tein identification using liquid chromatography-tandem mass cretion occurred earlier than did activation of caspase-3 and for- spectrometry. At 6 h, we identified 219 and 291 proteins from cell- mation of biologically inactive IL-18 fragments. culture supernatants of control and dsRNA-stimulated macro- Downloaded from http://www.jimmunol.org/

FIGURE 4. Cathepsins are essential for inflamma- some activation in response to cytoplasmic dsRNA stimulation. Human macrophages were left untreated or were treated with Ca-074 Me (25 mM), Est (50 or by guest on September 28, 2021 150 mM), or CatIII (50 or 150 mM) for 0.5 h before poly-IC-t stimulation for 8 h, after which cell-culture media and total cell lysates were collected. A, C, and D, IL-18 secretion was analyzed with ELISA. B, Concentrated cell-culture media were prepared for Western blot analysis with caspase-1 p20- and ASC- specific Abs. E and F, The cells were preincubated with or without Ca-074 Me (20 mM) for 0.5 h before cytoplasmic dsRNA stimulation. Total RNA was extracted at 8 h, and the expression of IFN-b and IL-29 was analyzed with quantitative RT-PCR, as described in Materials and Methods. G and H, Human primary macrophages were subjected to control and cathepsin D specific-siRNAs (100 nM) for 24 h, after which the cells were stimulated or not with cytoplasmic dsRNA for 8 h. Subsequently, cell-culture media were col- lected, and total protein lysates were prepared for IL-18 ELISA measurement and Western blot analysis with cathepsin B- and cathepsin D-specific Abs, re- spectively. The Journal of Immunology 3089 phages, respectively (Fig. 2A, Supplemental Tables I, II). At 18 h dramatically prevented secretion of biologically active forms of after dsRNA transfection, we detected 424 proteins from cell- IL-18 (Fig. 4A), ASC, and caspase-1 p20 in response to dsRNA culture supernatants compared with 167 proteins from control-cell stimulation (Fig. 4B). Similarly, Est and CatIII clearly decreased supernatants (Fig. 2A, Supplemental Tables III, IV). This shows dsRNA-induced IL-18 secretion in human macrophages (Fig. 4C, that activation of the cytoplasmic RNA-recognition pathway 4D). To study whether the antiviral cytokine response is regulated clearly induces protein secretion by 6 h, with more robust protein by cathepsins, macrophages were transfected with dsRNA in the secretion at 18 h. Our data showed that dsRNA stimulation of absence or presence of Ca-074 Me for 8 h, after which RNA was macrophages activates conventional and unconventional protein- extracted for quantitative RT-PCR analysis. Ca-074 Me had little secretion pathways. The unconventionally secreted proteins detec- effect on IFN-b and IL-29 mRNA expression in response to ted at 18 h after dsRNA stimulation included cystatin A, galectin-3, dsRNA stimulation (Fig. 4E,4F). IL-1R antagonist, macrophage migration inhibitory factor, and We used the siRNA technique to study the specific role of ca- thioredoxin (38, 39). Interestingly, dsRNA stimulation of macro- thepsin D in inflammasome activation. The human primary mac- phages also activated secretion of several exosome-associated rophages were treated with control siRNA and cathepsin D-specific proteins, including actin, ADP ribosylation factor 4, clathrin, heat siRNAs for 24 h, after which the cells were left unstimulated or shock proteins, histones, , Ras-related proteins, tubulin, were stimulated with cytoplasmic dsRNA for 8 h. Silencing of and 14-3-3 proteins (40). Ras-related proteins secreted in response cathepsin D reduced cytoplasmic dsRNA-induced IL-18 secretion to dsRNA stimulation included Rab-1A, Rab-7a, Rab-10, Rab11B, (Fig. 4G). Western blot analysis confirmed that cathepsin D pro- Rab13, and Rap-1b (Fig. 2B). Furthermore, the identified proteins tein expression was strongly decreased in cathepsin D siRNA- contained several lysosomal proteins, including cathepsin B, D, S, treated macrophages, whereas cathepsin B protein expression was Downloaded from and Z (Fig. 2C). Cathepsin B was identified at 18 h after dsRNA not affected (Fig. 4H). In conclusion, our results suggested that stimulation, whereas cathepsin Z was identified only from the con- cathepsins are crucial for efficient inflammasome activation, but trol samples. Cathepsins D and S were identified from cell-culture not for the expression of antiviral cytokines, in response to cyto- supernatants collected from control and dsRNA-stimulated mac- plasmic dsRNA stimulation in human macrophages. rophages. In addition to cathepsins, secretion of cystatins, a family Cathepsins are required for the progression of apoptosis of cysteine protease inhibitors (41), was seen (Fig. 2C). Secretion http://www.jimmunol.org/ of cystatin A and B was detected at 6 and 18 h after dsRNA stim- In addition to inflammasome activation, cathepsins are involved ulation. In contrast to cystatins A and B, cystatin C was also found in (apoptosis) (32, 44). We previously in the cell-culture supernatants of control cells. showed that the cytoplasmic dsRNA-recognition pathway acti- vates caspase-3 (13). We used the APOPercentage assay to further Secretion of cathepsin B and D in response to dsRNA demonstrate the activation of apoptosis in dsRNA-treated macro- transfection phages. The assay clearly demonstrated activation of apoptosis in Secretome analysis suggested major changes in secretion of macrophages that were transfected with dsRNA at 18 h (Fig. 5A). cathepsins and their regulators in macrophages activated through the cytoplasmic dsRNA-recognition pathway. To characterize their by guest on September 28, 2021 regulation in response to dsRNA stimulation in more detail, ca- thepsin B and D protein expression and secretion were analyzed in human macrophages by Western blotting. In the intracellular protein fraction, two mature forms of cathepsin B (29 and 25 kDa) were detected in control and cytoplasmic dsRNA-stimulated cells (Fig. 3A). There was a minor decrease in the levels of both forms of cathepsin B beginning at 3 h after cytoplasmic dsRNA stimu- lation. With cathepsin D, a clear decrease in 52-kDa procathepsin D was observed by 3 h after dsRNA transfection, and the intermediate-sized 46-kDa form was seen at 6 and 12 h after dsRNA stimulation. Mature 34-kDa cathepsin D was constantly present in the intracellular fraction, and dsRNA stimulation had no effect on its levels. In the extracellular-protein fraction, proca- thepsin D (52 kDa) was constitutively secreted in human primary macrophages, and dsRNA transfection had little effect on its secretion (Fig. 3B). Secretion of mature cathepsin B and interme- diate-sized 46-kDa cathepsin D occurred at 12 h after dsRNA transfection. Interestingly, secretion of mature 34-kDa cathepsin D into extracellular medium was seen 6 h after dsRNA stimulation (Fig. 3B). Importantly, the secretion of mature 34-kDa cathepsin D was simultaneous with the secretion of inflammasome components FIGURE 5. Cathepsins are required for the progression of apoptosis in ASC and caspase-1 (Fig. 1C). dsRNA-stimulated macrophages. Human macrophages were stimulated in the presence or absence of Ca-074 Me (25 mM) for 0.5 h and then left Cathepsins are essential for inflammasome activation untreated or treated with cytoplasmic dsRNA for 18 h and stained with APOPercentage Apoptosis Assay. A, Representative photographs of control Inflammasome activation has been connected to cytoplasmic (upper panel) and dsRNA-transfected (lower panel) cells (original magni- leakage of cathepsins in response to crystalline structures and fication 3100). B, The apoptotic (purple) and non-apoptotic cells were microbial infections (42, 43). Thus, we next analyzed the possible counted, and the percentage of apoptotic cells was calculated. C and D,The role of cathepsins in cytoplasmic dsRNA-induced inflammasome cells were left unexposed or were exposed to Ca-074 Me and poly-IC-t for 8 activation and antiviral cytokine response with cathepsin inhib- h, as above, and total protein lysates were prepared for Western blot analysis itors Ca-074 Me, a Ca-074–derivate Est, and CatIII. Ca-074 Me with caspase-3 p19/17 and Bid-specific Abs. 3090 CATHEPSIN-DEPENDENT ACTIVATION OF INFLAMMASOME, APOPTOSIS

Furthermore, Ca-074 Me significantly reduced the percentage their recognition of viruses relies on PRRs. PRRs detect dsRNA, of apoptotic cells (from 45 to 13%) during cytoplasmic dsRNA which is the most important PAMP formed during viral infection. stimulation (Fig. 5B). Similarly, Western blot analysis showed In the present work, we used a high-throughput proteomics ap- that Ca-074 Me clearly inhibited cytoplasmic dsRNA-induced proach to characterize secretome, the global pattern of secreted pro- caspase-3 activation, the hallmark of apoptosis, as well as com- teins, of macrophages activated through the cytoplasmic dsRNA- pletely inhibited dsRNA-induced formation of the truncated var- recognition pathway. The proteomic analysis revealed that dsRNA iant of Bid (t-Bid), which promotes apoptosis by permeating the stimulation of macrophages activates conventional and uncon- outer mitochondrial membrane with other proapoptotic proteins, ventional protein-secretion pathways in human macrophages. such as Bax and/or Bad (32) (Fig. 5C,5D). Especially, exosome-associated proteins were enriched in cell- culture supernatants of macrophages that were transfected with Cathepsins are essential for inflammasome activation and dsRNA. Secretome data also revealed secretion of lysosomal ca- apoptosis triggered by EMCV and VSV infection thepsin proteases and cysteine protease inhibitors (cystatins) in A Picornaviridae EMCV and a Rhabdoviridae VSV are detected macrophages that were activated through the cytoplasmic RNA- by MDA-5 and RIG-I, respectively (7). These RLR-dependent recognition pathway. Finally, we provide evidence that cathe- cytoplasmic RNA-recognition pathways are connected to inflam- psins are essential for inflammasome activation and apoptosis in masome activation and apoptosis (12, 13, 45). To study whether response to dsRNA stimulation and viral infection. cathepsins are important for inflammasome activation and apo- IL-1b and IL-18 are proinflammatory cytokines that are es- ptosis in response to RNA virus infection, macrophages were left sential for the initiation and maintenance of inflammation (16). untreated or were treated with Ca-074 Me for 0.5 h. After this, the IL-1b and IL-18 lack N-terminal leader signal peptides; thus, Downloaded from cells were left uninfected or were infected with EMCV or VSV at they are released from macrophages via an unconventional protein- an MOI of 1 for 8 or 16 h, and IL-18 secretion and activation of secretion pathway (46). Cysteine protease caspase-1 processes apoptosis were studied, respectively. Ca-074 Me inhibited EMCV- their proforms into their biologically active equivalents in a cyto- and VSV-induced IL-18 secretion (Fig. 6A,6B). Similarly, Ca-074 plasmic molecular structure called inflammasome (18). After this Me clearly abolished EMCV- and VSV-induced caspase-3 acti- processing step, the biologically active forms of IL-1b and IL-18

vation and formation of t-Bid (Fig. 6C,6D). In conclusion, our are secreted by poorly defined mechanisms. The molecular com- http://www.jimmunol.org/ results showed that activation of apoptosis during viral infections position of the activated inflammasome varies, depending on the is dependent on cathepsins. virus that infects the cell, but it contains at least caspase-1 and a member of a pyrin domain family protein ASC. Picornaviruses, Discussion such as EMCV, and long-cytoplasmic dsRNA stretches activate Innate immunity against viruses includes production of antiviral caspase-1 and NLRP3-inflammasome via MDA-5, whereas VSV and proinflammatory cytokines and induction of apoptosis. Mac- triggers caspase-1 independently of NLRP3 via RIG-I (45). In all rophages are the central players in the innate-immune system, and cases, the specific mechanisms that activate inflammasomes are by guest on September 28, 2021

FIGURE 6. Cathepsins are essential for in- flammasome activation and apoptosis during EMCV and VSV infection. A and B,Human macrophages were left untreated or were treated with Ca-074 Me (25 mM) for 0.5 h before in- fection with EMCV or VSV at an MOI of 1 for 8 h. After 8 h of infection, cell-culture super- natants were collected, and secretion of IL-18 was measured by ELISA. C and D, The cells were left untreated or were treated with Ca-074 Me (45 mM) for 0.5 h before infection with EMCV or VSV at an MOI of 1 for 16 h. Then total-cell lysates were prepared for Western blot analysis with caspase-3 p19/17 and Bid-specific Abs. The Journal of Immunology 3091 not fully understood. Our data showed that a mimetic of long- cysteine protease inhibitors, including cystatin A, B, and C) (41). cytoplasmic dsRNA, transfected poly-IC (poly-IC-t), activates Our data showed that cystatins are secreted simultaneously with inflammasome, which was observed as the appearance of active cathepsins in macrophages that are activated through the dsRNA- caspase-1 and intracellular accumulation of biologically active IL- recognition pathway. Thus, cystatins may bind to cathepsin B and 18. This all coincided with the onset of IL-18 secretion (Fig. 1). limit its extralysosomal functions. Interestingly, More importantly, caspase-3 was activated with delayed kinetics does not encode aspartate-specific protease inhibitors, which could compared with caspase-1 (Fig. 1B), indicating that inflammasome antagonize functions of cathepsin D (55); moreover, cathepsin D activation preceded the initiation of apoptosis in macrophages is able to inactivate cystatins (56, 57). Importantly, we found that that had been activated through the cytosolic dsRNA-recognition cathepsin D was secreted earlier than cathepsin B in dsRNA- pathway. stimulated cells. Thus, it may be possible that the prior release of Membrane transfer is a common mode of intercellular com- cathepsin D ensures cathepsin B-mediated inflammasome activa- munication between immune cells. Exosome secretion is a form of tion and apoptosis. The balance between the actions of cystatins membrane transfer that is activated by various environmental and lysosomal leakage of cathepsins is likely important for the signals, including TLR activation and/or stress conditions (40). progression of both of these innate-immune reactions. Our secretome analysis revealed secretion of several exosome- In our experiments, inflammasome activation in response to associated proteins, including actin, ADP ribosylation factor 4, dsRNA stimulation, as well as EMCV and VSV infection, was clathrin, heat shock proteins, histones, integrins, Ras-related pro- completely abolished by cathepsin inhibitor Ca-074 Me. Ca-074 teins, tubulin, and 14-3-3 proteins, in response to dsRNA stimu- Me is a cell-permeable and irreversible inhibitor of intracellular lation. Of these Ras-related proteins, Rab-1A, Rab-7a, Rab-10, cathepsins, and its primary target is cathepsin B. In addition to Downloaded from Rab11B, Rab13, and Rap-1b are especially interesting because Ca-074 Me, a Ca-074-derivate Est and cathepsin inhibitor CatIII they are involved in protein transport and secretion (47). In- clearly reduced inflammasome activation. We found that secretion terestingly, secretome analysis showed that Rab-1A, Rab-13, and of the mature form of cathepsin D, but not that of cathepsin B, Rap-1b secretion was already seen at 6 h after dsRNA trans- occurred simultaneously with inflammasome components ASC fection, and it was associated with secretion of inflammasome and caspase-1. Interestingly, silencing of cathepsin D with siRNAs

components. It was recently shown that another member of the reduced dsRNA-induced IL-18 secretion (Fig. 4G), demonstrating http://www.jimmunol.org/ RabGTPase family (Rab39a) binds caspase-1 and is involved in a role for cathepsin D in inflammasome activation. It is likely that caspase-1–dependent secretion of IL-1b in response to LPS cathepsins have redundant functions, and knockdown of a single stimulation (48). Clearly, further studies are needed to clarify the cathepsin does not result in complete inhibition of inflammasome role of Ras-related proteins in IL-1b and IL-18 secretion during activation, as was also shown in our study. In conclusion, our re- viral infections. sults suggest an important role for cathepsins in the innate-immune Lysosomes are acidic organelles in which different metabolic response to virus infection, but further studies are required to end products and contents of late endosomes and phagosomes are show the precise role of different cathepsins in inflammasome degraded. In addition to inflammasome activation, moderate ly- activation and apoptosis. sosomal damage leads to apoptosis. It is possible that the acidic by guest on September 28, 2021 environment and the action of produce apoptotic sig- Acknowledgments nals that induce apoptosis when released into cytoplasm. Cathep- We thank Dr. Jesper Melchjorsen for kindly providing EMCV and VSV. sins are the best-studied class of lysosomal hydrolases. 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