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Signaling of Apoptosis through TLRs
Critically Involves Toll/IL-1 Receptor
Domain-Containing Adapter Inducing IFN-β,
but Not MyD88, in Bacteria-Infected Murine
Macrophages
Klaus Ruckdeschel, Gudrun Pfaffinger, Rudolf Haase,
Andreas Sing, Heike Weighardt, Georg Häcker, Bernhard
Holzmann and Jürgen Heesemann
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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 © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 173:3320-3328; ;
doi: 10.4049/jimmunol.173.5.3320
http://www.jimmunol.org/content/173/5/3320
The Journal of Immunology
Signaling of Apoptosis through TLRs Critically Involves Toll/
IL-1 Receptor Domain-Containing Adapter Inducing IFN-␤,
But Not MyD88, in Bacteria-Infected Murine Macrophages1
Klaus Ruckdeschel,2* Gudrun Pfaffinger,* Rudolf Haase,* Andreas Sing,* Heike Weighardt,†
Georg Häcker,‡ Bernhard Holzmann,† and Jürgen Heesemann*
T
he TLRs are a class of evolutionarily conserved transmembrane receptors that are critically involved in
mammalian host defense. They serve to identify common patterns of microbial pathogens, which enables the innate
immune system to recognize invading microorganisms and to
induce a protective immune response (1– 4). Ten TLRs have
been described in mammals, which can discriminate various
microbial components. These include LPS, which activates
TLR4; bacterial lipoproteins, which signal through TLR2; and
viral dsRNA, which stimulates TLR3. The TLR-mediated intracellular signaling cascades converge to activate the NF-␬B
and MAPK pathways, which induce the transcription of a series
of cytokine and chemokine genes that are involved in the initiation of the inflammatory response (1– 4). The activation of
NF-␬B furthermore promotes cell survival through the induction of antiapoptotic genes (5–9). This ability appears important
for the signaling of apoptosis through TLRs, a TLR function
that is less well understood. It has been reported that TLR2 and
TLR4 can transmit proapoptotic signals that may induce the
*Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Munich,
Germany; †Department of Surgery, Technical University of Munich, Munich, Germany; and ‡Institute for Medical Microbiology, Immunology and Hygiene, Technical
University of Munich, Munich, Germany
Received for publication December 2, 2003. Accepted for publication June 29, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Deutsche Forschungsgemeinschaft (Grants DFG
Ru788/1 and -2 as well as SFB 576 Projects A7, B8, and B11).
2
Address correspondence and reprint requests to Dr. Klaus Ruckdeschel, Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Pettenkoferstrasse 9a, 80336
Munich, Germany. E-mail address: [email protected]
Copyright © 2004 by The American Association of Immunologists, Inc.
demise of the stimulated cell (10 –15). The induction of apoptosis through TLR stimulation is potentiated by the inhibition
of NF-␬B activation, which indicates that TLR-mediated cytotoxicity is normally balanced by the induction of the NF-␬B
pathway (11, 12, 16 –18).
Numerous bacterial pathogens apparently have developed strategies to exploit cytotoxic TLR signaling to interfere with the immune response of the host. One example is given by the Gramnegative bacterium Yersinia, which triggers apoptosis in infected
macrophages (19). Yersiniae down-regulate the activation of NF␬B, which disrupts the fine-tuned balances of pro- and antiapoptotic signaling in host cells. This sensitizes macrophages to undergo TLR-dependent apoptosis upon Yersinia infection (18). The
inhibition of NF-␬B activation depends on the presence of a specific virulence protein, which is Yersinia outer protein P (YopP)3
in Y. enterocolitica, or its homologue YopJ in Y. pseudotuberculosis and Y. pestis (18, 20, 21). Y. pestis is the etiological agent of
plague, whereas Y. pseudotuberculosis and Y. enterocolitica are
enteric pathogens causing gastrointestinal syndromes and lymphadenitis. YopP/YopJ is injected by the Yersinia type III protein
secretion system into the host cell cytoplasm, where it binds and
inhibits the NF-␬B-activating I␬B kinase-␤ (IKK␤), leading to impairment of NF-␬B activation and onset of macrophage apoptosis
(18, 20, 21). In a previous study we specified the involvement of
single TLRs in the Yersinia-conferred proapoptotic response. We
showed that, although Yersinia can activate both TLR2 and TLR4,
3
Abbreviations used in this paper: Yop, Yersinia outer protein; CrmA, cytokine response modifier A; FADD, Fas-associated death domain protein; IKK, I␬B kinase;
IRF-3, IFN regulatory factor 3; PKR, dsRNA-dependent protein kinase; poly(I:C),
polyinosinic-polycytidylic acid; RIP1, receptor-interacting protein-1; TIR, Toll/IL1R; TRIF, TIR domain-containing adapter inducing IFN-␤.
0022-1767/04/$02.00
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TLRs are important sensors of the innate immune system that serve to identify conserved microbial components to mount a
protective immune response. They furthermore control the survival of the challenged cell by governing the induction of pro- and
antiapoptotic signaling pathways. Pathogenic Yersinia spp. uncouple the balance of life and death signals in infected macrophages,
which compels the macrophage to undergo apoptosis. The initiation of apoptosis by Yersinia infection specifically involves TLR4
signaling, although Yersinia can activate TLR2 and TLR4. In this study we characterized the roles of downstream TLR adapter
proteins in the induction of TLR-responsive apoptosis. Experiments using murine macrophages defective for MyD88 or Toll/IL-1R
domain-containing adapter inducing IFN-␤ (TRIF) revealed that deficiency of TRIF, but not of MyD88, provides protection
against Yersinia-mediated cell death. Similarly, apoptosis provoked by treatment of macrophages with the TLR4 agonist LPS in
the presence of a proteasome inhibitor was inhibited in TRIF-defective, but not in MyD88-negative, cells. The transfection of
macrophages with TRIF furthermore potently promoted macrophage apoptosis, a process that involved activation of a Fasassociated death domain- and caspase-8-dependent apoptotic pathway. These data indicate a crucial function of TRIF as proapoptotic signal transducer in bacteria-infected murine macrophages, an activity that is not prominent for MyD88. The ability to
elicit TRIF-dependent apoptosis was not restricted to TLR4 activation, but was also demonstrated for TLR3 agonists. Together,
these results argue for a specific proapoptotic activity of TRIF as part of the host innate immune response to bacterial or viral
infection. The Journal of Immunology, 2004, 173: 3320 –3328.
The Journal of Immunology
Materials and Methods
Yersinia strains, cell lines, and stimulation conditions
The Y. enterocolitica strains used in this study were the serotype O8 wildtype strain WA-314 and its isogenic yopP-knockout mutant WA314⌬yopP (18). For infection, overnight cultures grown at 27°C were diluted 1/20 in fresh Luria-Bertoni broth and grown for another 2 h at 37°C
(19). Shifting the growth temperature to 37°C initialized activation of the
Yersinia type III secretion machinery for efficient translocation of Yops
into the host cell upon cellular contact. To equalize and synchronize infection, bacteria were seeded on the cells by centrifugation at 400 ⫻ g for
5 min at a ratio of 20 bacteria/cell. For incubation times ⬎90 min, bacteria
were killed by addition of gentamicin (100 ␮g/ml) after 90 min. Murine
J774A.1 macrophages were grown in RPMI 1640 medium supplemented
with 10% heat-inactivated FCS and 5 mM L-glutamine (19). In some experiments cells were pretreated with the proteasome inhibitory peptide ZLeu-Leu-Leu-CHO (MG-132; 5 ␮M; Biomol Research Laboratories, Plymouth Meeting, PA), or with inhibitors for caspase-1 and -5 (z-WEHDfmk), caspase-2 (z-VDVAD-fmk), caspase-8 (z-IETD-fmk), or caspase-9
(z-LEHD-fmk; all from Calbiochem, La Jolla, CA) at 40 ␮M for 30 min,
then stimulated as indicated. Stimulations were performed with LPS from
Escherichia coli O55:B5 (1 ␮g/ml; Sigma-Aldrich, Munich, Germany),
polyinosinic-polycytidylic acid (poly(I:C); 40 ␮g/ml; Sigma-Aldrich), or
the synthetic bacterial lipoprotein analog N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine P3CSK4 (4 ␮g/ml;
EMC Microcollections, Tubingen, Germany) (10). The human embryonic
kidney HEK293 cell line was grown in DMEM cell growth medium
supplemented with 10% heat-inactivated FCS (12). Jurkat T cells were
cultured in RPMI 1640 medium containing 5% heat-inactivated FCS in the
absence or the presence of anti-Fas mAb CH11 (Upstate, Charlottesville,
VA) for 4 h (38).
Mice and peritoneal macrophages
MyD88-deficient mice were provided by Shizuo Akira (Research Institute
for Microbial Diseases, Osaka University, Japan) (39) and were used after
backcrossing for 10 generations to C57BL/6. TRIF-defective TRIFLps2/Lps2
mice on the C57BL/6 background were gifts from B. Beutler (The Scripps
Research Institute, La Jolla, CA) (26). C57BL/6 and C3H/HeN wild-type
mice and TLR4-defective C3H/HeJ TLR4d/d mice (40, 41) were purchased
from Charles River (Sulzfeld, Germany). Elicited peritoneal macrophages
were obtained from mice 3 days after i.p. inoculation of 10% proteose
peptone broth as previously described (12).
Expression vectors, transfection, and analysis of quantity and
morphology of transfected cells
J774A.1 cells were transfected with the ExGen 500 transfection reagent
according to the manufacturer’s instructions (Fermentas, Hanover, MD)
(18). HEK293 cells were transfected by the calcium phosphate transfection
method (12). Unless indicated otherwise, transfections were conducted as
cotransfection experiments using 0.2 ␮g of pSV-␤-galactosidase expression vector (Promega, Madison, WI) and 0.8 ␮g of the plasmids of interest.
␤-Galactosidase expression correlates with expression of proteins encoded
by cotransfected plasmids in the transfected cells (18). The influence of
TRIF on cellular viability was determined by transfecting cells with
epitope-tagged, full-length, wild-type TRIF and a mutant version encoding
only the TRIF TIR domain (⌬TRIF) (42). For apoptosis inhibition studies,
0.15 ␮g of the full-length TRIF expression plasmid was combined with
0.65 ␮g of expression plasmids for dominant negative FADD protein
(⌬FADD) (43), cytokine response modifier A (CrmA) (44), Bcl-2 (38), or
a CD4/TLR4 chimeric construct (45). The TRIF, ⌬FADD, CrmA, and
CD4/TLR4 plasmids were provided by S. Akira and S. Sato (Research
Institute for Microbial Diseases, Osaka University, Japan), C. Vincenz
(University of Michigan Medical School, Ann Arbor, MI), D. Vaux
(Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia), and C. J. Kirschning (Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Munich, Germany),
respectively. Empty expression vector containing no insert was used as a
negative control. To identify the transfected cells, the cells were fixed and
stained with X-galactosidase 18 h later. For assessment of cell death, the
morphology of blue transfected cells was determined using light microscopy (6 –9, 18). Every transfected cell was analyzed for an apoptotic appearance. A minimum of eight microscopic fields were investigated for
each sample. For quantification, the number of apoptotic blue cells was
assayed in relation to the total number of transfected cells. Results are
expressed as the mean percentage ⫾ SD from three independent experiments. For specific fluorescent labeling of apoptotic TRIF-transfected cells,
0.2 ␮g of the TRIF expression constructs were cotransfected with 0.05 ␮g
of enhanced GFP reporter plasmid (pEGFP-C1; BD Clontech, San Diego,
CA) and stained as indicated above.
Fluorescent labeling of apoptotic cells
For quantification of apoptosis in mouse peritoneal macrophages, the cells
were labeled with fluorescein-conjugated annexin V (Roche, Mannheim,
Germany), which binds to phosphatidylserine exposed on the outer leaflet
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only TLR4 signaling contributes to induce macrophage apoptosis
(12). Also, agonist-specific activation of TLR4, but not TLR2, efficiently elicits cell death under NF-␬B inhibitory conditions.
These results identify TLR4 as a major inducer of apoptosis in
bacteria-infected macrophages (12). This observation is opposed to
previous studies that reported an apoptosis-inducing capacity of
TLR2 upon treatment with conserved bacterial components (10,
13–15, 17). To elucidate these apparently discrepant findings, we
investigated the roles of TLR adapter proteins as proapoptotic signal transmitters downstream from TLR activation.
Several Toll/IL-1R (TIR) domain-containing adapter molecules
have been shown to be recruited to the cytoplasmic TIR domains
of activated TLRs and to mediate downstream TLR signaling
(1– 4, 22). These adapters determine the signaling pathways that
confer NF-␬B activation. There are two major NF-␬B-inducing
pathways that diverge at the level of the TLR adapter proteins
(1– 4, 22). One of them depends on MyD88, which is required for
NF-␬B activation by all known TLRs, with the possible exception
of TLR3 (22–26). MyD88-driven NF-␬B signaling upon TLR2
and TLR4 stimulation furthermore involves TIR adapter protein,
also termed MyD88-adapter-like protein (2, 3, 22). The MyD88transmitted signal is relayed via IL-1R-associated kinase family
members and TNF receptor-associated factor 6 to the IKK complex, which ultimately mediates NF-␬B activation (1– 4, 22). The
second major pathway emanating from TLRs depends on TIR domain-containing adapter inducing IFN-␤ (TRIF), also known as
TIR-containing adapter molecule-1. TRIF plays the predominant
role in TLR3-mediated NF-␬B activation and promotes MyD88independent NF-␬B activation in TLR4 signaling (22–26). TLR4induced TRIF activation furthermore requires the TRIF-related
adapter molecule (or TIR-containing adapter molecule-2) (27–29).
The TRIF pathway appears to be specifically induced by activation
of TLR3 and TLR4. In addition to NF-␬B induction, TRIF signaling facilitates activation of the IFN regulatory factor 3 (IRF-3)
transcription factor via IKK⑀ and TRAF family member-associated NF-␬B activator-binding kinase 1, followed by the expression
of IFN-␤ genes (22–31).
Numerous reports have shown the involvement of TLR adapters
in proapoptotic signaling in stimulated cells (17, 26, 32–37). These
studies suggested that both the MyD88- as well as the TRIF-dependent signaling pathways can play roles in monocyte or macrophage signaling of apoptosis upon stimulation with conserved bacterial components, such as bacterial lipoproteins or LPS. In this
study we characterized Yersinia-mediated cell death in macrophages deficient for functional MyD88 or TRIF. Our data show
that TRIF, but not MyD88, plays a critical role in the induction of
apoptosis in Y. enterocolitica-infected macrophages. The ability of
TRIF to promote apoptosis is common to treatment of macrophages with TLR4 as well as with TLR3 agonists. This suggests a
major role for the TLR4- and TLR3-responsive TRIF pathway in
the induction of apoptosis in innate immunity signaling. The initiation of apoptosis through TRIF, which occurs through activation
of the death receptor-related Fas-associated death domain (FADD)
and caspase-8 apoptotic pathway, may be part of an immune effector mechanism that enables the host to remove infected cells
and control infection.
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of cells undergoing apoptosis (19). The simultaneous application of the
DNA stain propidium iodide (Sigma-Aldrich) allowed discrimination of
apoptotic from necrotic cells. The rates of cell death were determined by
visual scoring of a minimum of 200 cells/sample in a fluorescence microscope. Results are expressed as the mean percentage ⫾ SD of apoptotic
fluorescent cells vs the total number of cells from several independent
experiments. To evaluate caspase-8 activity at the single-cell level, unfixed
cells were stained with carboxyfluorescein-labeled FAM-LETD-fmk according to the manufacturer’s instructions (ApoFluor caspase activity assay; ICN, Irvine, CA). FAM-LETD-fmk covalently binds to active
caspase-8, and the rate of fluorescent cells was analyzed by fluorescence
microscopy. The viability and nuclear morphology of cells treated with
specific caspase inhibitory peptides were assessed by immunofluorescent
labeling with propidium iodide (34). The initiation of apoptosis in J774A.1
macrophages that were cotransfected with a GFP reporter plasmid was
analyzed by staining with Cy3-labeled annexin V (Sigma-Aldrich), which
confers Cy3-dependent red fluorescence to cells undergoing apoptosis (34).
Measurement of macrophage TNF-␣ production
Immunoblotting
For the detection of transiently overexpressed TRIF and ⌬TRIF in
HEK293 cells, lysates were prepared from 3 ⫻ 106 cells/sample 20 h after
transfection. The lysis buffer was composed of 50 mM HEPES (pH 7.6),
150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, and protease and phosphatase inhibitors (Roche). Cellular lysates
were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Immunoblot analysis was performed with anti-FLAG
(Sigma-Aldrich) or anti-c-Myc (BD Clontech) epitope mouse mAbs to detect the expression of TRIF or ⌬TRIF, respectively. To determine
caspase-8 processing in TRIF-transfected HEK293 and Fas-activated Jurkat cells, the membranes were probed with mouse monoclonal anti-human
caspase-8 Abs (Cell Signaling Technology, Beverly, MA). The Jurkat cell
lysates were prepared using a buffer that contained 150 mM NaCl, 50 mM
Tris-HCl (pH 8.0), 1% Igepal CA-630, and a standard mix of protease
inhibitors (Roche). For analysis of cleavage of caspase-8 and Bid in peritoneal macrophages, a total of 2 ⫻ 106 cells/sample was lysed in 4⫻
Laemmli sample buffer after stimulation as indicated. The lysates were
fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with rat monoclonal anti-mouse caspase-8 Abs (Alexis
Biochemicals, Carlsbad, CA). Immunoreactive bands were visualized using appropriate secondary Abs and ECL detection reagents (Amersham
Pharmacia Biotech, Piscataway, NJ). Thereafter, the membrane was
stripped in 62.5 mM Tris (pH 6.7), 0.1 mM 2-ME, and 2% SDS for 30 min
at 50°C. The membrane was then reprobed with polyclonal anti-Bid (R&D
Systems) and secondary Abs, and developed by chemiluminescence.
Results
TRIF signals apoptosis in Yersinia-infected macrophages
To identify TLR adapter proteins mediating apoptosis induction by
Y. enterocolitica downstream from the TLR complexes, we investigated Yersinia-conferred cell death in primary murine macrophages deficient for functional MyD88 or TRIF (Fig. 1).
MyD88⫺/⫺ mice harbor a MyD88-null mutation (39), whereas
TRIFLps2/Lps2 mice bear a frameshift mutation within the C terminus of TRIF that generates a nonfunctional TRIF (26). Apoptosis
in peritoneal macrophages obtained from these mice was analyzed
in comparison with that in wild-type macrophages. Apoptotic cells
were identified by labeling with fluorescein-conjugated annexin V
and propidium iodide. Annexin V binds with high affinity to phosphatidylserine exposed on the outer leaflet of cells undergoing apoptosis (19). In these experiments the WA-314 Y. enterocolitica
wild-type strain induced robust apoptosis in wild-type control
cells, which was not observed after infection with the YopP-negative mutant WA-⌬yopP. This confirms that the NF-␬B inhibitory
activity of YopP is required for efficient execution of apoptosis in
FIGURE 1. The TRIFLps2/Lps2 mutation provides protection against Y.
enterocolitica-induced apoptosis. Peritoneal macrophages elicited from
C57BL/6 wild-type (WT), MyD88⫺/⫺, or TRIFLps2/Lps2 mice remained untreated or were infected with wild-type yersiniae (WA-314) or the YopPnegative mutant (WA-⌬yopP). Apoptosis was analyzed 7 h after the onset
of stimulation by labeling apoptotic cells with fluorescein-conjugated annexin V and propidium iodide. The rate of cell death was determined by
visual scoring of a minimum of 200 cells/sample in a fluorescence microscope. Results are expressed as the mean percentage ⫾ SD of apoptotic
cells vs the total number of cells from three independent experiments.
Yersinia-infected macrophages (18). Notably, apoptosis in
MyD88⫺/⫺ macrophages was not reduced compared with that in
wild-type cells. This shows that the absence of MyD88 does not
improve the survival of Yersinia-infected macrophages. In contrast, macrophages elicited from TRIFLps2/Lps2 mice were considerably protected against Yersinia-induced apoptosis. Accordingly,
60 –70% of TRIF-mutagenized macrophages survived when
⬎80% of control or MyD88⫺/⫺ cells were already apoptotic. This
indicates that signaling through TRIF, but not through MyD88,
plays a role in Yersinia-conferred cell death, suggesting a critical
function of TRIF as a signal transmitter of apoptosis.
TLR4 and TLR3 agonists signal apoptosis via TRIF
To examine whether the ability of TRIF to activate apoptosis is
restricted to infection of macrophages by Yersinia or may represent a more general phenomenon, we analyzed the apoptosis-conferring abilities of specific TLR agonists. LPS is an activator of
TLR4, poly(I:C) signals via TLR3, and the synthetic lipopeptide
P3CSK4 mediates activation of TLR2 (1– 4, 10, 12, 23, 46). In
previous studies we showed that pretreatment of macrophages with
the proteasome inhibitory peptide Z-Leu-Leu-Leu-CHO (MG-132)
sensitizes the cells to undergo apoptosis upon stimulation of TLR4
with LPS (12). MG-132 suppresses degradation of the NF-␬B inhibitory I␬B proteins through the proteasome pathway (47), which
inhibits NF-␬B activation in macrophages (48). Treatment with
MG-132 could thus mimic the NF-␬B inhibitory activity of YopP.
In fact, activation of TLR4, but not of TLR2, triggers an efficient
apoptotic response in MG-132-treated macrophages (12), which is
probably an indication of the roles of these receptors in Yersiniamediated cell death. We compared the induction of apoptosis by
the diverse TLR agonists in macrophages prepared from
MyD88⫺/⫺ and TRIFLps2/Lps2 mice (Fig. 2A). In correlation with
our previous studies, LPS stimulation elicited robust apoptosis of
wild-type macrophages in presence of MG-132, whereas P3CSK4
did not (12). Notably, the TLR3 stimulus poly(I:C) could also
provoke substantial cell death in MG-132-pretreated cells. This
effect was not due to a potential LPS contamination of the
poly(I:C) preparation, because poly(I:C), unlike LPS, could also
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For quantitation of TNF-␣ production, macrophages were treated as
indicated, and cell culture supernatants were removed after a final 20-h
incubation. The TNF-␣ levels in the supernatants were evaluated by a
commercially available capture ELISA using goat anti-mouse TNF-␣
mAbs as recommended by the manufacturer (R&D Systems, Minneapolis, MN) (12).
PROAPOPTOTIC ROLE OF TRIF
The Journal of Immunology
3323
mediate apoptosis in TLR4-defective C3H/HeJ TLR4d/d macrophages (Fig. 2B). C3H/HeJ TLR4d/d mice harbor a loss of function
mutation within the cytoplasmic portion of TLR4 (40, 41). In C3H/
HeN wild-type cells, LPS and poly(I:C) induced a similar degree
of apoptosis as in C57BL/6 control macrophages (Fig. 2, B and A,
first graphs each). Despite exhibiting distinct effects on cellular
viability, all three TLR agonists elicited a significant TNF-␣ response in wild-type cells (Fig. 2C), which was highest for LPS.
This indicates that all agonists can successfully stimulate the cells
under the experimental conditions. Interestingly, the apoptosis-inducing capabilities of LPS and poly(I:C) were almost completely
abolished in TRIF-defective macrophages (Fig. 2A). In contrast,
MyD88 deficiency could not provide protection against agonistdependent apoptosis. This indicates a crucial involvement of the
TRIF signaling pathway in transducing the TLR4- and TLR3-responsive proapoptotic signals. MyD88 apparently is not mandatory
for this process. In the absence of MG-132, neither LPS nor
poly(I:C) could elicit macrophage apoptosis. Thus, inhibition of
the antiapoptotic NF-␬B signaling pathway by MG-132 appears to
be required for the initiation of apoptosis in response to these stimuli, which correlates well with cell death in Yersinia-infected macrophages that only undergo apoptosis when NF-␬B activation is
suppressed (18, 48). The data in Fig. 2A furthermore show that a
small fraction of TRIF-mutagenized cells could undergo LPS- or
poly(I:C)-dependent apoptosis upon inhibition of the proteasome
pathway (10 –25% apoptotic cells 7 h after onset of stimulation;
Fig. 2A, right graph). The rate of apoptosis under these conditions
increased to ⬎60% after 20 h of stimulation (data not shown). This
effect was not equally observed in LPS-stimulated, TLR4-defective C3H/HeJ TLR4d/d cells (5–10% after 7 h of stimulation (Fig.
2B, right graph) and 10 –30% after 20 h of stimulation). Thus,
TRIF-deficient macrophages were not completely protected
against LPS- and poly(I:C)-responsive cell death, which implies
that another TLR adapter could confer moderate and delayed apoptosis in the absence of functional TRIF.
TRIF signals apoptosis through a FADD-caspase-8-dependent
pathway
To strengthen the indications for a proapoptotic function of TRIF,
we tested whether TRIF could induce apoptosis when overexpressed in eukaryotic cells. We first transfected murine J774A.1
macrophages with eukaryotic expression vectors encoding either
Flag epitope-tagged full-length TRIF or a c-Myc epitope-tagged
⌬TRIF version that produces only the TRIF-TIR domain (42).
The ⌬TRIF construct has been reported to act as a dominant
negative inhibitor of TLR-mediated NF-␬B activation (42). TRIF
and ⌬TRIF were expressed in single transfected cells, and this was
controlled by immunofluorescence microscopy using anti-Flag or
anti-c-Myc epitope Abs, respectively (34) (data not shown). For
analysis of apoptosis specifically in transfected cells, a GFP
reporter plasmid was cotransfected. The expression of GFP enables the detection of single transfected cells by fluorescence
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FIGURE 2. TRIF transmits apoptotic signals generated by TLR4 and TLR3 agonists. A, Apoptosis mediated by LPS and poly(I:C) in the presence of
the proteasome inhibitor MG-132 depends on functional TRIF. Peritoneal macrophages elicited from C57BL/6 wild-type (WT), MyD88⫺/⫺, or TRIFLps2/Lps2
mice remained untreated or were stimulated with LPS, P3CSK4, or poly(I:C) under conditions with or without pretreatment with the proteasome inhibitor
MG-132. B, Poly(I:C) confers apoptosis independently from TLR4. Peritoneal macrophages elicited from C3H/HeN wild-type or TLR4-defective C3H/HeJ
TLR4d/d mice remained untreated or were stimulated with LPS, P3CSK4, or poly(I:C) under conditions with or without pretreatment with the proteasome
inhibitor MG-132. Apoptosis in A and B was analyzed 7 h after the onset of stimulation by labeling apoptotic cells with fluorescein-conjugated annexin
V and propidium iodide. Apoptotic cells were analyzed by fluorescence microscopy. Results are expressed as the mean percentage ⫾ SD of apoptotic cells
vs the total number of cells from three independent experiments. C, TNF-␣ production in response to LPS, P3CSK4, and poly(I:C) stimulation. Peritoneal
macrophages from C57BL/6 wild-type mice remained untreated or were stimulated with LPS, P3CSK4, or poly(I:C). The amount of TNF-␣ released was
analyzed 20 h later. Results are expressed as the mean percentage of picograms of TNF-␣ per milliliter ⫾ SD from three independent experiments.
3324
microscopy without preceding immunostaining. Simultaneous labeling with Cy3-conjugated annexin V confers a red fluorescence
to cells undergoing apoptosis (34). In these experiments, 50 –70%
of the TRIF-transfected cells displayed enhanced membrane binding of annexin V 8 h after transfection, indicating the onset of
apoptosis (Fig. 3A). Apoptosis induction was not detected in cells
transfected with the TRIF-TIR domain (⌬TRIF; Fig. 3A). Because
expression of the cotransfected GFP decreased during apoptosis, a
␤-galactosidase-encoding reporter vector was cotransfected for
quantitation of cell death at later time points. Apoptosis in ␤-galactosidase-expressing cells, which is characterized by typical
cellular shrinkage and condensation, was microscopically evaluated (6 –9, 18). Almost all cells transfected with full-length TRIF
were apoptotic 18 h after transfection (Fig. 3B, left panel). This
pronounced proapoptotic effect was not observed in cells transfected with ⌬TRIF or empty vector control. These data show that
TRIF overexpression is sufficient to elicit apoptosis in macrophages. This could indicate that the signaling of apoptosis is a
prominent function of TRIF. However, because apoptosis in TRIFtransfected cells did not require blockage of NF-␬B activation by
either Yersinia infection or proteasome inhibitor treatment, it
cannot be ruled out that TRIF overexpression triggers proapoptotic
events that are normally not operative under physiological conditions of TRIF expression.
Our previous studies suggested that Yersinia- and TLR4-dependent
apoptosis could both involve signaling via FADD and caspase-8.
Accordingly, the overexpression of a dominant negative version of
FADD (⌬FADD) could provide protection against TLR4- and
Yersinia-conferred cell death (12, 34). Because TRIF apparently
relays the apoptotic signals emanating from TLR4, we examined
whether FADD and caspase-8 may also play role in TRIFmediated apoptosis. In an attempt to obtain indications on involvement of FADD or caspase-8 in TRIF-dependent cell death, we
cotransfected the TRIF expression plasmid with vectors encoding
⌬FADD (43) or the viral serpin CrmA (44). CrmA acts as specific
inhibitor of caspase-1 and caspase-8 (49, 50). Both ⌬FADD and
CrmA constructs can prevent Fas- and TNF receptor-induced
apoptosis through inhibition of caspase-8 (43, 51). The expression
of TRIF in the cotransfection experiments displayed in Fig. 3B was
verified by immunofluorescence staining with anti-Flag Abs (data
not shown). In line with the data previously reported for TLR4induced apoptosis, transfection of dominant negative ⌬FADD
remarkably rescued macrophages from cell death resulting from
TRIF overexpression (Fig. 3B, right panel). Similarly, CrmA
transfection inhibited TRIF-dependent apoptosis of macrophages.
These data imply that TRIF engages the FADD-caspase-8 cytotoxic pathway to mediate apoptosis downstream of TLR4. Fig. 3B
(right panel) furthermore shows that overexpression of the antiapoptotic molecule Bcl-2 could not prevent apoptosis of TRIFtransfected cells. Under the same conditions, Bcl-2 transfection
reduced J774A.1 cell apoptosis in response to staurosporine treatment; 20 – 40% of Bcl-2-transfected cells underwent apoptosis 8 h
after onset of treatment with 5 ␮M staurosporine, whereas ⬎90%
of control vector-transfected cells were already apoptotic at that
time point. This demonstrates that overexpressed Bcl-2 in principle
can protect against apoptosis. However, because Bcl-2 predominantly counteracts cell death resulting from activation of intrinsic
mitochondrial, but not from extrinsic, death receptor-induced pathways (52, 53), the absence of an inhibitory effect of Bcl-2 reinforces the hypothesis that the death receptor-responsive FADDcaspase-8 pathway plays a critical role in TRIF-conferred
apoptosis.
Apoptosis as a consequence of TRIF overexpression was not
only confined to macrophages, but became evident also in transfected human embryonic kidney (HEK) 293 cells (Fig. 3C). Cotransfection of ⌬FADD and CrmA blocked TRIF-dependent apoptosis in the same manner as in J774A.1 cells, whereas Bcl-2 had
no protective effect. The expression of TRIF and ⌬TRIF in the
different experimental conditions was analyzed in cellular lysates
Downloaded from http://www.jimmunol.org/ by guest on June 12, 2017
FIGURE 3. ⌬FADD and CrmA inhibit TRIF-promoted apoptosis. A,
TRIF-transfected macrophages stain with the apoptotic marker annexin V.
J774A.1 macrophages were cotransfected with GFP reporter plasmid and
expression vectors for either full-length TRIF (TRIF) or the TRIF TIR
domain (⌬TRIF), and were labeled with Cy3-annexin V (red fluorescence)
8 h after transfection. The micrographs show GFP-producing transfected
cells (green fluorescence). B, TRIF transfection mediates J774A.1 macrophage cell death that is inhibited by ⌬FADD and CrmA. J774A.1 cells
were transfected in the left panel with empty expression vector control
(vector) or full-length TRIF or ⌬TRIF expression plasmids together with a
␤-galactosidase reporter vector. In the right panel, J774A.1 macrophages
were cotransfected with ␤-galactosidase reporter vector and either the
empty control vector (vector) or the full-length TRIF expression vector
(TRIF) together with ⌬FADD, CrmA, Bcl-2, or control expression plasmid. C, TRIF-transfected HEK293 undergo apoptosis similar to J774A.1
macrophages. HEK293 cells were cotransfected with ␤-galactosidase reporter vector and either the empty control vector (vector), or the full-length
TRIF expression vector (TRIF) together with the indicated expression plasmids. Transfected cells in B and C were stained with X-galactosidase 18 h
after transfection, and single transfected blue cells were analyzed for an
apoptotic morphology. Results are expressed as the mean percentage ⫾ SD
of apoptotic vs the total number of transfected cells from three independent
experiments. The expression levels of cotransfected TRIF and ⌬TRIF in
lysates of HEK293 cells were analyzed by Western immunoblotting using
anti-Flag or anti-c-Myc epitope Abs, respectively (C).
PROAPOPTOTIC ROLE OF TRIF
The Journal of Immunology
by Western immunoblotting (Fig. 3C), which became feasible because of the higher transfection efficiencies in HEK293 cells compared with macrophages. The differing rates of cellular survival
were not consequences of modified TRIF expression, because
TRIF was similarly expressed under the different cotransfection
conditions. These data indicate that TRIF is not acting as a specific
inducer of apoptosis in macrophages, but can activate similar apoptotic pathways in other cell types. Notably, the ability of TRIF
to elicit apoptosis in HEK293 was enhanced by concomitant TLR4
signaling. The coexpression of a constitutively active CD4/TLR4
receptor construct enhanced TRIF-mediated apoptosis (Fig. 3C).
In the CD4/TLR4 chimeric construct, the CD4 ectodomain is fused
to the transmembrane and cytoplasmic domains of TLR4, which
renders the TLR4 signaling domains constitutively active (45)
(data not shown). The promotion of apoptosis by this construct
supports the idea that the activation status of TLR4 may control the
ability of TRIF to deliver a cell death signal.
Because TRIF- and CD4/TLR4-transfected cells efficiently underwent apoptosis, we tried to analyze caspase-8 activation in the
transfected cells by Western immunoblotting. Upon death receptor
ligation, FADD and caspase-8 are recruited into a multicomponent
death-inducing signaling complex, which triggers autoproteolytic
cleavage and activation of caspase-8. Mature caspase-8 then processes and activates downstream proapoptotic effector molecules,
which leads to execution of the apoptotic program (52–54). An
anti-caspase-8 Ab detected an immunoreactive band in lysates of
TRIF- and CD4/TLR4-transfected HEK293 cells that displayed
the same electrophoretic mobility as the active caspase-8 p18/20
subunit generated by Fas ligation in Jurkat cells (Fig. 4A). This
suggests that TRIF overexpression in CD4/TLR4-transfected cells
induced caspase-8 maturation. To strengthen the indications for a
role of TRIF in caspase-8 activation under physiological conditions of TRIF expression, we investigated caspase-8 processing in
mouse peritoneal macrophages that were treated with LPS and
MG-132 (Fig. 4B). In these experiments, caspase-8 was specifically cleaved and processed in MG-132-pretreated cells upon LPS
stimulation, followed by the onset of macrophage apoptosis (Fig.
2A). The processing of caspase-8 correlated with cleavage of the
proapoptotic Bcl-2 family member Bid, a proximal substrate of
caspase-8 (Fig. 4B). Cleaved Bid (tBid) activates a mitochondrial
apoptosis pathway downstream from FADD and caspase-8 (52,
54). The maturation of both caspase-8 and of Bid was not observed
in macrophages prepared from TRIFLps2/Lps2 mice, which demonstrates that TRIF controls the LPS-responsive induction of
caspase-8. The TRIF-dependent processing of caspase-8 correlated
with an increase in caspase-8 activity, which was determined by
labeling of apoptotic cells with a caspase-8-specific substrate (Fig.
4C). Furthermore, pretreatment of the macrophages with the
caspase-8 selective inhibitor z-IETD-fmk diminished LPS/MG132- and Yersinia-induced apoptosis by 40 –50% 10 h after the
onset of stimulation. Interestingly, inhibition of caspase-8 in these
conditions prevented chromatin condensation, but not membrane
damage, in the majority (70 –90%) of the dying cells, which was
assessed after staining with the membrane-impermeable DNA dye
propidium iodide (19, 34). Membrane damage and influx of propidium iodide without detectable nuclear chromatin condensation
untreated, were stimulated with LPS in the absence or the presence of the
proteasome inhibitor MG-132, or were infected with YopP-negative (WA⌬yopP) or wild-type (WA-314) yersiniae. The activation of caspase-8 in
single cells was assayed 8 h after the onset of stimulation by staining with
caspase-8-specific FAM-LETD-fmk. The percentage of fluorescent cells
exhibiting caspase-8 activity was determined by fluorescence microscopy.
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FIGURE 4. TRIF controls LPS-dependent caspase-8 activation. A,
TRIF overexpression can mediate processing of caspase-8. HEK293 cells
were cotransfected with CD4/TLR4 and either the empty control vector
(vector) or the full-length TRIF expression vector (TRIF). Twenty hours
after transfection, cellular lysates were prepared, separated by 15% SDSPAGE, and probed with anti-caspase-8 Abs. Lysates of untreated and antiFas Ab-treated Jurkat cells were included as negative and positive controls,
respectively. B, LPS-responsive processing of caspase-8 and Bid in MG132-pretreated macrophages depends on TRIF. Peritoneal macrophages
elicited from C57BL/6 wild-type (WT) or TRIFLps2/Lps2 mice remained
untreated or were stimulated with LPS in the absence or the presence of the
proteasome inhibitor MG-132. Cellular lysates were prepared 3 h after LPS
addition. Lysates were separated by 15% SDS-PAGE, probed with anticaspase-8 Abs, and visualized by ECL reaction (upper panel). The arrows
in A and B indicate full-length pro-caspase-8 and the processed active
p18/20 caspase-8 subunit. B, The intermediate p43 caspase-8 cleavage
product was additionally detected. A nonspecific band (n. s.) appearing
above caspase-8 p18/20 in B demonstrates equal loading of the gel with
cellular lysates. The same membrane was than stripped and reprobed with
anti-Bid Abs (B, lower panel). Arrows indicate cleaved, truncated (tBid),
and uncleaved forms of Bid (Bid). C, Macrophages treated with LPS- and
MG-132 or infected with wild-type Yersinia display caspase-8 activation.
Peritoneal macrophages elicited from C57BL/6 wild-type mice remained
3325
3326
are characteristics of necrotic cell death (55, 56), suggesting that
the dying macrophages undergo necrosis when caspase-8 activity
is blocked. This observation correlates with previous reports that
describe a FADD-dependent shift from apoptosis to necrosis in
cells that are impaired in their caspase activities (55, 56). Together,
these data indicate an important role for the TRIF-regulated
FADD-caspase-8 signaling pathway in determining the fate of
LPS/MG-132- and Yersinia-treated macrophages. No obvious implication of other caspases in the initiation of TRIF-related apoptosis could be uncovered by using peptide inhibitors for
caspase-1, -2, -5, and -9 (data not shown). These experiments furthermore did not reveal a salient role of one of these caspases in
the induction of the TRIF-independent, delayed apoptosis pathway
mentioned above (Fig. 2A; data not shown). However, because the
caspase-8 inhibitor exerted a 40 –50% inhibitory effect also in
these conditions, apoptosis that bypasses TRIF may also involve
caspase-8 activation.
Discussion
Thus, although TRIF clearly plays the major role in transmitting
TLR-dependent apoptosis in murine macrophages, alternative TLR
signaling pathways could be able to perform limited proapoptotic
activities in the absence of functional TRIF. Interestingly, it has
recently been shown that activation of dsRNA-dependent protein
kinase (PKR) by TLR4 can contribute to Yersinia-induced apoptosis in mouse bone marrow-derived macrophages (57). In these
cells, PKR mediates late onset of apoptosis that depends on PKR
kinase activity, mediating phosphorylation of elongation initiation
factor 2␣ and concomitant inhibition of protein synthesis. Although this mechanism appears to be of minor importance for
TRIF-dependent apoptosis in our studies, because a kinase-inactive PKR mutant did not interfere with wither TLR4-related (12) or
TRIF-related (data not shown) cell death, PKR could play a role as
signal transmitter that confers delayed cell death when expeditious
TRIF engagement is impaired.
However, the downstream signal relay that couples apoptosis
signaling by TRIF to the cell death machinery is not yet completely understood. Our previous studies have indicated the engagement of FADD and caspase-8 in TLR4- and Yersinia-dependent apoptosis (12, 34), suggesting roles for these molecules in
TRIF-mediated cell death. FADD acts as a central adapter molecule in apoptosis signaling by the TNF receptor superfamily (43,
52–54). It activates the apoptotic machinery through induction of
caspase-8. In our hands, dominant negative FADD and the
caspase-8 inhibitor CrmA could counteract apoptosis elicited by
TRIF overexpression. In addition, TRIF controlled the maturation
and activation of caspase-8 and its substrate Bid in macrophages
that were forced to undergo LPS-dependent apoptosis. This demonstrates that TRIF critically participates in the induction of the
FADD-caspase-8 pathway downstream from TLR4. However, we
were unable to find a direct interaction of TRIF with FADD in
coprecipitation experiments, which suggests that TRIF cannot directly activate FADD (data not shown). This is not surprising,
because TRIF lacks a death domain that normally mediates interaction of FADD with its upstream activators (22, 25, 42). Interestingly, MyD88 bears such a death domain and can potentially
activate FADD in TLR2-dependent apoptosis (17). This appears to
be an evolutionary conserved signaling mechanism, because a
comparable interaction has been described for dMyD88 and
dFADD of Drosophila (58). Whether TRIF may engage MyD88 to
confer FADD activation is currently not clear. We did not observe
attenuation of TLR4- and TLR3-dependent apoptosis in MyD88negative macrophages. Furthermore, dominant negative MyD88
could not reduce TRIF-related apoptosis (data not shown), and we
were unable to detect an interaction of overexpressed TRIF and
MyD88 in coprecipitation experiments (data not shown). These
results could argue against a decisive role for MyD88 in TRIFrelated cell death. Other molecules that can associate with TRIF
include TRAF family member-associated NF-␬B activator-binding
kinase 1, TNF receptor-associated factor 6, and IRF-3 (30, 31, 42).
IRF-3 has been reported to induce apoptosisvia a caspase-8dependent pathway (59), which suggests that IRF-3 could be a
downstream signal transmitter of TRIF-directed cell death. However, apoptosis mediated by IRF-3 is supposed to occur through
up-regulation of the expression of proapoptotic genes (59). This
mechanism appears contrary to TLR4/TLR3- and TRIF-promoted
apoptosis, because treatment with the protein synthesis inhibitor
cycloheximide does not protect against, but, rather, renders macrophages susceptible to LPS- and poly(I:C)-elicited cell death (16,
60) (data not shown). This implies that the initiation of proapoptotic signaling through TLR4/TLR3 and TRIF does not depend on
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Our previous studies have shown that the bacterial pathogen Y.
enterocolitica inhibits antiapoptotic NF-␬B signaling and simultaneously engages receptors of the innate immune system to trigger
apoptosis in macrophages (18). The induction of apoptosis by Yersinia infection involves signaling through TLR4, but not through
TLR2, although both TLRs share the ability to recognize Yersinia
(12). To emphasize the implication of single TLR adapter proteins
in the transmission of proapoptotic signals downstream from the
TLR complexes, we investigated Yersinia-conferred cell death in
primary mouse macrophages that were defective for functional
MyD88 or TRIF. Both TLR adapter proteins can transduce TLR4responsive signaling and have been linked to the elicitation of
proapoptotic pathways in monocytic or macrophage cells upon
challenge with conserved bacterial components (17, 26, 34). Our
data show that selectively TRIF, but not MyD88, is involved in the
promotion of an apoptotic response in Yersinia-infected macrophages. Furthermore, TRIF plays the major role in conferring cell
death that is provoked by the activation of TLR4 through LPS
under conditions in which the proteasome pathway is inhibited.
Because TRIF is activated by signaling through TLR4, but not
through TLR2 (24 –26), these results explain the efficient and selective apoptosis-inducing capacity that we previously observed
for TLR4. Moreover, the ability of TRIF to confer apoptosis is not
restricted to TLR4 activation, but seems to also occur via TLR3. In
fact, the TLR3 ligand poly(I:C) could trigger apoptosis under
NF-␬B inhibitory conditions similar to LPS. These data demonstrate that the transmission of apoptosis signals is a major function
of TRIF signal transduction, an activity that is not prominent for
MyD88 signaling.
The unapparent role of MyD88 in promoting cell death in our
study questions the significance of previous reports that suggested
a proapoptotic function of MyD88 (17, 32–36). These conclusions
were mainly based on data resulting from the overexpression of
dominant negative MyD88, which implies that the observed effects
could merely reflect overexpression phenomena. However, most of
the studies that have shown apoptotic activities of the TLR2MyD88 pathway were conducted on human cell lines (10, 13–15,
17, 33), whereas our experiments and those described by Hoebe et
al. (26), which first have implicated TRIF as an apoptosis transmitter, were performed on primary mouse cells. This suggests that
there could be cell- or species-specific differences in the activation
of TLR-dependent proapoptotic pathways. In addition, our data
show that TRIF-mutagenized cells could undergo delayed, LPSdependent apoptosis upon inhibition of the proteasome pathway.
This effect may potentially be mediated by another TLR adapter.
PROAPOPTOTIC ROLE OF TRIF
The Journal of Immunology
Acknowledgments
We thank Martin Aepfelbacher and Carsten Kirschning for constructive
discussions and advice, and Susanne Bierschenk for TNF-␣ measurements.
We also thank Bruce Beutler and Shizuo Akira for providing us with mice,
and David Vaux, Carsten Kirschning, Claudius Vincenz, Shintaro Sato,
and Shizuo Akira for supplying us with expression vectors.
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PROAPOPTOTIC ROLE OF TRIF