Recognition of cytoplasmic RNA results in cathepsin-dependent inflammasome activation and apoptosis in human macrophages.
ABSTRACT 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 caspase-1 and apoptosis of infected cells. In this study, we used high-throughput proteomics to identify secretome, the global pattern of secreted proteins, 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 exosome-associated proteins, as well as basal and dsRNA-activated secretion of lysosomal protease cathepsins and cysteine protease 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 cathepsin B, was secreted simultaneously with IL-18 and inflammasome 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 proteolytic processing of caspase-3, indicating that inflammasome activation precedes apoptosis in macrophages that had recognized 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.
- SourceAvailable from: Courtney Wilkins[show abstract] [hide abstract]
ABSTRACT: The immune response to virus infection is initiated when pathogen recognition receptors (PRRs) of the host cell recognize specific nonself-motifs within viral products (known as a pathogen-associated molecular pattern or PAMP) to trigger intracellular signaling events that induce innate immunity, the front line of defense against microbial infection. The replication program of all viruses includes a cytosolic phase of genome amplification and/or mRNA metabolism and viral protein expression. Cytosolic recognition of viral infection by specific PRRs takes advantage of the dependence of viruses on the cytosolic component of their replication programs. Such PRR-PAMP interactions lead to PRR-dependent nonself-recognition and the downstream induction of type I interferons and proinflammatory cytokines. These factors serve to induce innate immune programs and drive the maturation of adaptive immunity and inflammation for the control of infection. Recent studies have focused on identifying the particular viral ligands recognized as nonself by cytosolic PRRs, and on defining the nature of the PRRs and their signaling pathways involved in immunity. The RIG-I-like receptors, RIG-I and MDA5, have been defined as essential PRRs for host detection of a variety of RNA viruses. Novel PRRs and their signaling pathways involved in detecting DNA viruses through nonself-recognition of viral DNA are also being elucidated. Moreover, studies to identify the PRRs and signaling factors of the host cell that mediate inflammatory signaling through inflammasome activation following virus infection are currently underway and have already revealed specific NOD-like receptors (NLRs) as inflammatory triggers. This review summarizes recent progress and current areas of focus in pathogen recognition and immune triggering by cytosolic PRRs.Current opinion in immunology 02/2010; 22(1):41-7. · 10.88 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Infection of cells by microorganisms activates the inflammatory response. The initial sensing of infection is mediated by innate pattern recognition receptors (PRRs), which include Toll-like receptors, RIG-I-like receptors, NOD-like receptors, and C-type lectin receptors. The intracellular signaling cascades triggered by these PRRs lead to transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. However, aberrant activation of this system leads to immunodeficiency, septic shock, or induction of autoimmunity. In this Review, we discuss the role of PRRs, their signaling pathways, and how they control inflammatory responses.Cell 03/2010; 140(6):805-20. · 31.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: When a cell dies in vivo, the event does not go unnoticed. The host has evolved mechanisms to detect the death of cells and rapidly investigate the nature of their demise. If cell death is a result of natural causes - that is, it is part of normal physiological processes - then there is little threat to the organism. In this situation, little else is done other than to remove the corpse. However, if cells have died as the consequence of some violence or disease, then both defence and repair mechanisms are mobilized in the host. The importance of these processes to host defence and disease pathogenesis has only been appreciated relatively recently. This article reviews our current knowledge of these processes.Nature Reviews Immunology 05/2008; 8(4):279-89. · 32.25 Impact Factor
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 caspase-1 and apoptosis of infected cells. In this study, we used high-throughput proteomics to identify secretome,
the global pattern of secreted proteins, 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
exosome-associated proteins, as well as basal and dsRNA-activated secretion of lysosomal protease cathepsins and cysteine 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 cathepsin B, 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
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.
innate immunity, and their PRRs detect the presence of pathogen-
associated molecular patterns (PAMPs) and damage-associated mo-
lecular patterns (DAMPs) (2, 3). This recognition results in pro-
duction of antiviral and proinflammatory cytokines, recruitment of
other inflammatory cells, and, often, apoptosis of the virus-infected
cell. A successful innate-immune reaction also provides a firm
basis for the activation of adaptive-immune responses.
Several cytoplasmic and membrane-bound PRRs participate in
innate-immune recognition of virus infection. TLR8 and TLR3
detect endocytosed ssRNA or dsRNA molecules, respectively, in
nnate immune reaction against viral infection begins with
recognition of the virus by specific pattern-recognition re-
ceptors (PRRs) (1). Macrophages are the central players of
endolysosomal compartments of human macrophages (4). In ad-
dition to TLRs, viral RNA is detected by cytoplasmic RIG-I–like
receptors (RLRs), such as RIG-I and MDA-5 (5, 6). RIG-I detects
ssRNA bearing 59 triphosphate and/or short dsRNA molecules
with certain sequence or nucleotide preferences (7–9), whereas
MDA-5 is specialized to detect long dsRNA strands (.1 kb in
length) (10, 11). In macrophages, activation of RIG-I and MDA-5
induces strong antiviral cytokine response, and recent data also
connect RLRs to the activation of apoptosis of virus-infected cells
IL-1b and IL-18 are important proinflammatory cytokines that
are produced during virus infection (13–15). IL-1b and IL-18 are
synthesized as immature forms: pro–IL-1b and pro–IL-18. Cys-
teine protease caspase-1 cleaves these proforms to biologically
active equivalents that are secreted via unconventional protein-
secretion pathways (16, 17). In parallel, caspase-1 is activated
by cytosolic PRRs in a protein complex called inflammasome
(18). NLRP3-inflammasome is the most studied caspase-1–acti-
vating molecular platform; it is activated by various heterogeneous
DAMPs and PAMPs. The DAMPs include, for example, extra-
cellular ATP and crystalline monosodium urate (19, 20). In ad-
dition to these endogenous stimuli, NLRP3-inflammasome is acti-
vated by exogenous stimuli, including asbestos, silica, aluminum
adjuvant (21, 22), fungal toxins (23), b-glucans (24), and viral
infections (25–27). Because these activators are chemically and
structurally different, they probably are indirectly recognized by
the NLRP3-inflammasome. It is more likely that they activate in-
flammasome by inducing changes in endogenous molecules that
are then recognized as danger signals (28). Potassium efflux and
production of reactive oxygen species are common features as-
sociated with NLRP3-inflammasome activation, and it was re-
*Unit of Excellence for Immunotoxicology, Finnish Institute of Occupational Health,
00250 Helsinki, Finland; and†Protein Chemistry Research Group, Institute of Bio-
technology, University of Helsinki, 00014 Helsinki, Finland
Received for publication June 21, 2010. Accepted for publication December 15,
This work was supported by grants from the Research Council for Biosciences and
Environment of the Academy of Finland, the Sigrid Juselius Foundation, and the
Helsinki Graduate Program in Biotechnology and Molecular Biology.
Address correspondence and reprint requests to Dr. Sampsa Matikainen, Unit of Excel-
lence for Immunotoxicology, Finnish Institute of Occupational Health, Topeliuksenkatu
41 a A, 00250 Helsinki, Finland. E-mail address: email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this article: CatIII, cathepsin inhibitor III; DAMP, damage-
associated molecular pattern; EMCV, encephalomyocarditis virus; MOI, multiplicity
of infection; PAMP, pathogen-associated molecular pattern; poly-IC, polyinosinic-
polycytidylic acid; poly-IC-t, transfected polyinosinic-polycytidylic acid; PRR,
pattern-recognition receptor; RLR, RIG-I–like receptor; t-Bid, truncated form of
Bid; VSV, vesicular stomatitis virus.
cently shown that thioredoxin-interacting protein links oxidative
stress to inflammasome activation (29, 30).
Cathepsins are lysosomal proteases that have many immuno-
logical activities. Cathepsin B and D are functionally closely re-
lated and are the most abundant in this protein family (31–33).
Maturation of these proteases proceeds through several steps.
Mature cathepsin B is found as a single-chain form (29 kDa) and
as a two-chain form (25 and 4 kDa) of active enzyme (34). Active
cathepsin D is produced through two successive cleavages of
preproenzyme, resulting in formation of a two-chain mature en-
zyme (14-kDa L chain and 34-kDa H chain) in lysosomes (33).
Recently, cytoplasmic leakage of cathepsin B was associated with
inflammasome activation (29).
We studied the role of cathepsins in the innate-immune re-
sponse to cytoplasmic dsRNA in human macrophages. We provide
evidence that cathepsins have a central role in the activation
of inflammasome and apoptosis in response to the cytoplasmic-
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).
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 cysteine protease
inhibitor Est [(2S,3S)-trans-epoxysuccinyl-L-laucylamodo-3-methylbutane
ethyl ester] (also known as E-64d) were purchased from Calbiochem (San
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
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
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 gene-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
gene expression that is normalized to an endogenous reference gene (18S
rRNA) and is relative to nontemplate control containing molecular-grade
water instead of a cDNA sample.
Western blot analysis
Total-cell lysates were prepared as previously described (13). To analyze
secreted proteins, cell-culture media were collected and concentrated with
Amicon Ultra-15 centrifugal filter devices, with a cut-off of 10,000 nom-
inal m.w. limit (Millipore, Bedford, MA). Equal volumes of the concen-
trated cell-culture media were separated on 15% SDS-PAGE. Western blot
analysis for total-cell lysates and secreted proteins was carried out as
described earlier (13). Primary Abs were purchased as follows: ASC
polyclonal Ab from Millipore; caspase p20 from Sigma-Aldrich; caspase-3
p19/17 and Bid from Cell Signaling Technology (Danvers, MA); caspase-1
p10 (C-20) and cathepsin B from Calbiochem, and cathepsin D (C-20)
from Santa Cruz Biotechnology (Santa Cruz, CA). To confirm equal
loading and transfer onto membranes between the samples, membranes
were stripped in 0.2 M NaOH for 5 min, washed in PBS-Tween 0.05%
three times, and stained with ready-to-use SYPRO Ruby Protein Blot
Stain, according to the manufacturer’s instructions (Sigma-Aldrich). The
major basally expressed protein band is shown as a loading control.
For secretome characterization, cell-culture supernatants were first col-
lected and concentrated with Amicon Ultra-15 centrifugal filter devices
Kit (GE Healthcare, Pittsburgh, PA). Next, proteins were separated using
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 peptides were analyzed by nanoLC-
MS/MS, as described previously (35). The liquid chromatography-tandem
macrophages activated through the cytoplasmic dsRNA-recognition path-
way. Human primary macrophages were stimulated with Lipofectamine
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),
and total-cell lysates (B) and concentrated cell-culture media (C) were pre-
pared for Western blot analysis with Abs against IL-18, caspase-1 p10 and
p20, ASC, and caspase-3 p19/17.
Inflammasome activation precedes apoptosis in human
3086CATHEPSIN-DEPENDENT ACTIVATION OF INFLAMMASOME, APOPTOSIS
mass spectrometry data were searched with in-house Mascot version 2.2
through ProteinPilot 3.0 interface against the SwissProt database (version
56.12). The search criteria for Mascot searches were human-specific tax-
onomy, trypsin digestion with one missed cleavage allowed, carbamido-
methyl modification of cysteine as a fixed modification, and oxidation of
methionine as a variable modification. All of the reported protein identi-
fications were statistically significant (p , 0.05).
The percentage of apoptotic cells was assayed with APOPercentage Ap-
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
22.214.171.124) and DP Manager (126.96.36.199) (Center Valley, PA). The stained
and unstained cells were manually counted, and the percentage of apo-
ptotic cells was calculated.
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).
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
(IL-18 p18) and inactive (IL-18 p15/16) forms of this cytokine,
respectively. To analyze the kinetics of inflammasome activation,
poly-IC, a mimetic of dsRNA, was transfected into cytoplasm of
human macrophages, and IL-18 concentration was measured from
cell-culture media with ELISA. Cytoplasmic dsRNA induced
IL-18 secretion at 3 h after transfection, and the greatest levels of
IL-18 were seen at 12 and 18 h after stimulation (Fig. 1A). In
accordance with ELISA data, Western blot analysis of intracel-
lular proteins showed a progressive decrease in constitutively pro-
duced pro–IL-18, as well as accumulation of biologically active
forms of 18-kDa IL-18 and caspase-1 p10 beginning at 3 h after
cytoplasmic dsRNA stimulation (Fig. 1B). Obvious intracellular
formation of activated caspase-3, caspase-3 p19/17, and its
cleavage product IL-18 p16/15 was seen at 6 h after stimulation
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,
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).
Secretome characterization of macro-
transfection. Human macrophages were stimulated with Lipofectamine
alone or in complex with cytoplasmic dsRNA (poly-IC-t) at 10 mg/ml for
1, 3, 6, 12, or 18 h, after which cell lysates (A) and concentrated cell
culture media (B) were prepared for Western blot analysis with Abs against
cathepsin B and D.
Secretion of cathepsin B and D in response to dsRNA
The Journal of Immunology3087
with dsRNA (Fig. 1B). Two isoforms of ASC, the central com-
ponent of inflammasomes, were constitutively present in human
macrophages, and the expression of both isoforms was slightly
enhanced in response to dsRNA transfection (Fig. 1B).
In addition to processing of proinflammatory cytokines, inflam-
masome activation is associated with the secretion of its central
components. To characterize dsRNA-induced inflammasome ac-
tivation further, we collected cell-culture supernatants, concen-
trated them, and performed Western blot analysis of extracellular
proteins. In accordance with the presented data (Fig. 1A, 1B), cyto-
plasmic dsRNA elicited secretion of IL-18 p18, caspase-1 p20,
and ASC 6 h after stimulation (Fig. 1C). In conclusion, our re-
sults showed that cytoplasmic dsRNA-induced inflammasome
activation precedes apoptosis: caspase-1 activation and IL-18 se-
cretion occurred earlier than did activation of caspase-3 and for-
mation of biologically inactive IL-18 fragments.
Secretome characterization of macrophages activated through
cytoplasmic dsRNA-recognition pathway
Most proteins are secreted through a conventional protein-secre-
tion pathway. These proteins contain N-terminal leader signal pep-
tides that direct their transport to the plasma membrane through
the endoplasmic reticulum–Golgi pathway (37). Our current and
previous data showed that inflammasome-associated caspase-1 is
activated in response to the cytosolic dsRNA-recognition pathway
in human macrophages. Keller et al. (38) showed that active
caspase-1 is a regulator of unconventional protein secretion of
leaderless proteins. To get a global overview of dsRNA-induced
protein secretion in macrophages, we used a combination of pro-
tein separation by SDS-PAGE followed by high-throughput pro-
tein identification using liquid chromatography-tandem mass
spectrometry. At 6 h, we identified 219 and 291 proteins from cell-
culture supernatants of control and dsRNA-stimulated macro-
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
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-
Cathepsins are essential for inflamma-
3088 CATHEPSIN-DEPENDENT ACTIVATION OF INFLAMMASOME, APOPTOSIS
phages, respectively (Fig. 2A, Supplemental Tables I, II). At 18 h
after dsRNA transfection, we detected 424 proteins from cell-
culture supernatants compared with 167 proteins from control-cell
supernatants (Fig. 2A, Supplemental Tables III, IV). This shows
that activation of the cytoplasmic RNA-recognition pathway
clearly induces protein secretion by 6 h, with more robust protein
secretion at 18 h. Our data showed that dsRNA stimulation of
macrophages activates conventional and unconventional protein-
secretion pathways. The unconventionally secreted proteins detec-
ted at 18 h after dsRNA stimulation included cystatin A, galectin-3,
IL-1R antagonist, macrophage migration inhibitory factor, and
thioredoxin (38, 39). Interestingly, dsRNA stimulation of macro-
phages also activated secretion of several exosome-associated
proteins, including actin, ADP ribosylation factor 4, clathrin, heat
shock proteins, histones, integrins, Ras-related proteins, tubulin,
and 14-3-3 proteins (40). Ras-related proteins secreted in response
to dsRNA stimulation included Rab-1A, Rab-7a, Rab-10, Rab11B,
Rab13, and Rap-1b (Fig. 2B). Furthermore, the identified proteins
contained several lysosomal proteins, including cathepsin B, D, S,
and Z (Fig. 2C). Cathepsin B was identified at 18 h after dsRNA
stimulation, whereas cathepsin Z was identified only from the con-
trol samples. Cathepsins D and S were identified from cell-culture
supernatants collected from control and dsRNA-stimulated mac-
rophages. In addition to cathepsins, secretion of cystatins, a family
of cysteine protease inhibitors (41), was seen (Fig. 2C). Secretion
of cystatin A and B was detected at 6 and 18 h after dsRNA stim-
ulation. In contrast to cystatins A and B, cystatin C was also found
in the cell-culture supernatants of control cells.
Secretion of cathepsin B and D in response to dsRNA
Secretome analysis suggested major changes in secretion of
cathepsins and their regulators in macrophages activated through
the cytoplasmic dsRNA-recognition pathway. To characterize their
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
ASC and caspase-1 (Fig. 1C).
Cathepsins are essential for inflammasome activation
Inflammasome activation has been connected to cytoplasmic
leakage of cathepsins in response to crystalline structures and
microbial infections (42, 43). Thus, we next analyzed the possible
role of cathepsins in cytoplasmic dsRNA-induced inflammasome
activation and antiviral cytokine response with cathepsin inhib-
itors Ca-074 Me, a Ca-074–derivate Est, and CatIII. Ca-074 Me
dramatically prevented secretion of biologically active forms of
IL-18 (Fig. 4A), ASC, and caspase-1 p20 in response to dsRNA
stimulation (Fig. 4B). Similarly, Est and CatIII clearly decreased
dsRNA-induced IL-18 secretion in human macrophages (Fig. 4C,
4D). To study whether the antiviral cytokine response is regulated
by cathepsins, macrophages were transfected with dsRNA in the
absence or presence of Ca-074 Me for 8 h, after which RNA was
extracted for quantitative RT-PCR analysis. Ca-074 Me had little
effect on IFN-b and IL-29 mRNA expression in response to
dsRNA stimulation (Fig. 4E, 4F).
We used the siRNA technique to study the specific role of ca-
thepsin D in inflammasome activation. The human primary mac-
rophages were treated with control siRNA and cathepsin D-specific
siRNAs for 24 h, after which the cells were left unstimulated or
were stimulated with cytoplasmic dsRNA for 8 h. Silencing of
cathepsin D reduced cytoplasmic dsRNA-induced IL-18 secretion
(Fig. 4G). Western blot analysis confirmed that cathepsin D pro-
tein expression was strongly decreased in cathepsin D siRNA-
treated macrophages, whereas cathepsin B protein expression was
not affected (Fig. 4H). In conclusion, our results suggested that
cathepsins are crucial for efficient inflammasome activation, but
not for the expression of antiviral cytokines, in response to cyto-
plasmic dsRNA stimulation in human macrophages.
Cathepsins are required for the progression of apoptosis
In addition to inflammasome activation, cathepsins are involved
in programmed cell death (apoptosis) (32, 44). We previously
showed that the cytoplasmic dsRNA-recognition pathway acti-
vates caspase-3 (13). We used the APOPercentage assay to further
demonstrate the activation of apoptosis in dsRNA-treated macro-
phages. The assay clearly demonstrated activation of apoptosis in
macrophages that were transfected with dsRNA at 18 h (Fig. 5A).
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
untreated or treated with cytoplasmic dsRNA for 18 h and stained with
APOPercentage Apoptosis Assay. A, Representative photographs of control
(upper panel) and dsRNA-transfected (lower panel) cells (original magni-
fication 3100). B, The apoptotic (purple) and non-apoptotic cells were
counted, and the percentage of apoptotic cells was calculated. C and D, The
cells were left unexposed or were exposed to Ca-074 Me and poly-IC-t for 8
h, as above, and total protein lysates were prepared for Western blot analysis
with caspase-3 p19/17 and Bid-specific Abs.
Cathepsins are required for the progression of apoptosis in
The Journal of Immunology 3089
Furthermore, Ca-074 Me significantly reduced the percentage
of apoptotic cells (from 45 to 13%) during cytoplasmic dsRNA
stimulation (Fig. 5B). Similarly, Western blot analysis showed
that Ca-074 Me clearly inhibited cytoplasmic dsRNA-induced
caspase-3 activation, the hallmark of apoptosis, as well as com-
pletely inhibited dsRNA-induced formation of the truncated var-
iant of Bid (t-Bid), which promotes apoptosis by permeating the
outer mitochondrial membrane with other proapoptotic proteins,
such as Bax and/or Bad (32) (Fig. 5C, 5D).
Cathepsins are essential for inflammasome activation and
apoptosis triggered by EMCV and VSV infection
A Picornaviridae EMCV and a Rhabdoviridae VSV are detected
by MDA-5 and RIG-I, respectively (7). These RLR-dependent
cytoplasmic RNA-recognition pathways are connected to inflam-
masome activation and apoptosis (12, 13, 45). To study whether
cathepsins are important for inflammasome activation and apo-
ptosis in response to RNA virus infection, macrophages were left
untreated or were treated with Ca-074 Me for 0.5 h. After this, the
cells were left uninfected or were infected with EMCVor VSVat
an MOI of 1 for 8 or 16 h, and IL-18 secretion and activation of
apoptosis were studied, respectively. Ca-074 Me inhibited EMCV-
and VSV-induced IL-18 secretion (Fig. 6A, 6B). Similarly, Ca-074
Me clearly abolished EMCV- and VSV-induced caspase-3 acti-
vation and formation of t-Bid (Fig. 6C, 6D). In conclusion, our
results showed that activation of apoptosis during viral infections
is dependent on cathepsins.
Innate immunity against viruses includes production of antiviral
and proinflammatory cytokines and induction of apoptosis. Mac-
rophages are the central players in the innate-immune system, and
their recognition of viruses relies on PRRs. PRRs detect dsRNA,
which is the most important PAMP formed during viral infection.
In the present work, we used a high-throughput proteomics ap-
teins, of macrophages activated through the cytoplasmic dsRNA-
recognition pathway. The proteomic analysis revealed that dsRNA
stimulation of macrophages activates conventional and uncon-
ventional protein-secretion pathways in human macrophages.
Especially, exosome-associated proteins were enriched in cell-
culture supernatants of macrophages that were transfected with
dsRNA. Secretome data also revealed secretion of lysosomal ca-
thepsin proteases and cysteine protease inhibitors (cystatins) in
macrophages that were activated through the cytoplasmic RNA-
recognition pathway. Finally, we provide evidence that cathe-
psins are essential for inflammasome activation and apoptosis in
response to dsRNA stimulation and viral infection.
IL-1b and IL-18 are proinflammatory cytokines that are es-
sential for the initiation and maintenance of inflammation (16).
IL-1b and IL-18 lack N-terminal leader signal peptides; thus,
they are released from macrophages via an unconventional protein-
secretion pathway (46). Cysteine protease caspase-1 processes
their proforms into their biologically active equivalents in a cyto-
plasmic molecular structure called inflammasome (18). After this
processing step, the biologically active forms of IL-1b and IL-18
are secreted by poorly defined mechanisms. The molecular com-
position of the activated inflammasome varies, depending on the
virus that infects the cell, but it contains at least caspase-1 and
a member of a pyrin domain family protein ASC. Picornaviruses,
such as EMCV, and long-cytoplasmic dsRNA stretches activate
caspase-1 and NLRP3-inflammasome via MDA-5, whereas VSV
triggers caspase-1 independently of NLRP3 via RIG-I (45). In all
cases, the specific mechanisms that activate inflammasomes are
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 EMCVor 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
Cathepsins are essential for in-
3090CATHEPSIN-DEPENDENT ACTIVATION OF INFLAMMASOME, APOPTOSIS
not fully understood. Our data showed that a mimetic of long-
cytoplasmic dsRNA, transfected poly-IC (poly-IC-t), activates
inflammasome, which was observed as the appearance of active
caspase-1 and intracellular accumulation of biologically active IL-
18. This all coincided with the onset of IL-18 secretion (Fig. 1).
More importantly, caspase-3 was activated with delayed kinetics
compared with caspase-1 (Fig. 1B), indicating that inflammasome
activation preceded the initiation of apoptosis in macrophages
that had been activated through the cytosolic dsRNA-recognition
Membrane transfer is a common mode of intercellular com-
munication between immune cells. Exosome secretion is a form of
membrane transfer that is activated by various environmental
signals, including TLR activation and/or stress conditions (40).
Our secretome analysis revealed secretion of several exosome-
associated proteins, including actin, ADP ribosylation factor 4,
clathrin, heat shock proteins, histones, integrins, Ras-related pro-
teins, tubulin, and 14-3-3 proteins, in response to dsRNA stimu-
lation. Of these Ras-related proteins, Rab-1A, Rab-7a, Rab-10,
Rab11B, Rab13, and Rap-1b are especially interesting because
they are involved in protein transport and secretion (47). In-
terestingly, secretome analysis showed that Rab-1A, Rab-13, and
Rap-1b secretion was already seen at 6 h after dsRNA trans-
fection, and it was associated with secretion of inflammasome
components. It was recently shown that another member of the
RabGTPase family (Rab39a) binds caspase-1 and is involved in
caspase-1–dependent secretion of IL-1b in response to LPS
stimulation (48). Clearly, further studies are needed to clarify the
role of Ras-related proteins in IL-1b and IL-18 secretion during
Lysosomes are acidic organelles in which different metabolic
end products and contents of late endosomes and phagosomes are
degraded. In addition to inflammasome activation, moderate ly-
sosomal damage leads to apoptosis. It is possible that the acidic
environment and the action of hydrolases produce apoptotic sig-
nals that induce apoptosis when released into cytoplasm. Cathep-
sins are the best-studied class of lysosomal hydrolases. Cathepsin
B and D are closely related cysteine- and aspartate-specific pro-
teases, respectively, and they were shown to promote programmed
cell death (apoptosis) (44, 49). Reactive oxygen species-mediated
translocation of cathepsins from lysosomes to cytoplasm is an
early event in apoptosis; it occurs before loss of mitochondrial
membrane potential, release of cytochrome c, and activation of
the apoptotic caspase cascade (50, 51). Our results showed that in-
hibition of cathepsin activity with Ca-074 Me clearly reduced the
percentage of apoptotic cells in cytoplasmic dsRNA-stimulated
macrophages (Fig. 5B). Furthermore, caspase-3 activation in re-
sponse to dsRNA stimulation and viral infection was significantly
reduced by cathepsin inhibition (Figs. 5C, 6C, 6D). This showed
that apoptosis triggered by the cytoplasmic RNA-recognition path-
way is cathepsin dependent. Furthermore, Ca-074 Me com-
pletely inhibited formation of t-Bid in response to dsRNA mimetic
and viral stimuli (Figs. 5D, 6C, 6D). t-Bid promotes mitochondrial
outer membrane permeabilization and the mitochondrial apoptosis
pathway, as described above (52). Our results suggested that ly-
sosomal cathepsins function upstream of mitochondrial damage
to activate apoptosis during virus infection.
Cathepsin B and D have versatile immunological functions
(i.e., they are involved in MHC class II-mediated Ag presentation
and TLR signaling) (53, 54). Cathepsin research has been com-
plicated by their functional redundancy, as exemplified by ca-
thepsin knockout studies in mice (31, 53). Under normal condi-
tions, lysosomal proteases are scarcely found outside cells, and
their activities are controlled intracellularly (i.e., by endogenous
cysteine protease inhibitors, including cystatin A, B, and C) (41).
Our data showed that cystatins are secreted simultaneously with
cathepsins in macrophages that are activated through the dsRNA-
recognition pathway. Thus, cystatins may bind to cathepsin B and
limit its extralysosomal functions. Interestingly, human genome
does not encode aspartate-specific protease inhibitors, which could
antagonize functions of cathepsin D (55); moreover, cathepsin D
is able to inactivate cystatins (56, 57). Importantly, we found that
cathepsin D was secreted earlier than cathepsin B in dsRNA-
stimulated cells. Thus, it may be possible that the prior release of
cathepsin D ensures cathepsin B-mediated inflammasome activa-
tion and apoptosis. The balance between the actions of cystatins
and lysosomal leakage of cathepsins is likely important for the
progression of both of these innate-immune reactions.
In our experiments, inflammasome activation in response to
dsRNA stimulation, as well as EMCV and VSV infection, was
completely abolished by cathepsin inhibitor Ca-074 Me. Ca-074
Me is a cell-permeable and irreversible inhibitor of intracellular
cathepsins, and its primary target is cathepsin B. In addition to
Ca-074 Me, a Ca-074-derivate Est and cathepsin inhibitor CatIII
clearly reduced inflammasome activation. We found that secretion
of the mature form of cathepsin D, but not that of cathepsin B,
occurred simultaneously with inflammasome components ASC
and caspase-1. Interestingly, silencing of cathepsin D with siRNAs
reduced dsRNA-induced IL-18 secretion (Fig. 4G), demonstrating
a role for cathepsin D in inflammasome activation. It is likely that
cathepsins have redundant functions, and knockdown of a single
cathepsin does not result in complete inhibition of inflammasome
activation, as was also shown in our study. In conclusion, our re-
sults suggest an important role for cathepsins in the innate-immune
response to virus infection, but further studies are required to
show the precise role of different cathepsins in inflammasome
activation and apoptosis.
We thank Dr. Jesper Melchjorsen for kindly providing EMCV and VSV.
The authors have no financial conflicts of interest.
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