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* Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Munich, Germany;
Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Munich, Germany; and
Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B signaling pathway in host cells by inhibiting inhibitory B kinase-
. This leads to macrophage apoptosis under infection conditions. Experiments with mouse macrophages deficient for TLR2, TLR4, or both receptors showed that, although yersiniae could activate signaling through both TLR2 and TLR4, loss of TLR4 solely diminished Yersinia-induced apoptosis. This suggests implication of TLR4, but not of TLR2, as a proapoptotic signal transducer in Yersinia-conferred cell death. In the same manner, agonist-specific activation of TLR4 efficiently mediated macrophage apoptosis in the presence of the proteasome inhibitor MG-132, an effect that was less pronounced for activation through TLR2. Furthermore, the extended stimulation of overexpressed TLR4 elicited cellular death in epithelial cells. A dominant-negative mutant of Fas-associated death domain protein could suppress TLR4-mediated cell death, which indicates that TLR4 may signal apoptosis through a Fas-associated death domain protein-dependent pathway. Together, these data show that TLR4 could act as a potent inducer of apoptosis in macrophages that encounter a bacterial pathogen. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, which regulates the production of various cytokines, adhesion molecules, and antiapoptotic factors (6, 7, 8, 9, 10). The up-regulation of synthesis of antiapoptotic proteins through NF-B activation prevents cellular apoptosis under multiple stress-induced conditions and is also essential for survival of bacteria-faced macrophages. This suggests that bacterial infection could trigger the activation of proapoptotic signals that are hindered in their cytotoxic effects by the antiapoptotic activity of NF-B (11, 12, 13).
In this study, we investigated the roles of TLRs as proapoptotic signal mediators in Yersinia-infected macrophages. Pathogenic Gram-negative Yersinia spp. disrupt the balances of pro- and antiapoptotic signaling in macrophages by down-regulating the activity of NF-B, which leads to macrophage apoptosis (11). These abilities depend on a specific virulence protein, which is Yersinia outer protein (Yop) P in Yersinia enterocolitica, or its homologue YopJ in Yersinia pseudotuberculosis and Yersinia pestis (14, 15). Y. pestis is the etiological agent of plague, whereas Y. pseudotuberculosis and Y. enterocolitica are enteric pathogens causing gastrointestinal syndromes and lymphadenitis (15). YopP/YopJ is injected by the virulence plasmid-encoded Yersinia type III protein secretion system into the host cell cytoplasm where it binds and inhibits the NF-B-activating inhibitory B kinase (IKK)-, leading to down-regulation of NF-B activation (14, 15). Our previous studies have shown that proapoptotic signals of innate immunity could synergize with the NF-B-inhibitory action of YopP/YopJ to mediate macrophage apoptosis (16). This suggests that yersiniae activate a conserved apoptotic pathway that induces cell death when NF-B activation is suppressed. The sources of the cytotoxic signals potentially generated by Yersinia are hitherto unclear. In this study, we show that signaling through TLR4, but not through TLR2, stimulates apoptosis in Y. enterocolitica-infected macrophages. Apparently, TLR2 and TLR4 differentially induce cytotoxic signals in stimulated cells. Our data indicate that activated TLR4 can potently signal apoptosis which could lead to the demise of the infected cell.
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Materials and Methods |
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The Y. enterocolitica strains used in this study were the serotype O8 wild-type strain WA-314, its isogenic yopP-knockout mutant WA-
yopP (16), the virulence plasmid-cured strain WA-C, and invasin-negative, virulence plasmid-cured WAinv-C (17). Overnight cultures grown at 27°C were diluted 1/20 in fresh Luria-Bertani broth and grown for another 2 h at 37°C (17). Shift of the growth temperature to 37°C initializes 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 x g for 5 min at a ratio of 20 bacteria per cell. For incubation times longer than 90 min, bacteria were killed by addition of gentamicin (100 µg/ml) after 90 min. The human embryonic kidney (HEK)293 cell line was cultured in DMEM cell growth medium supplemented with 10% heat-inactivated FCS. Murine J774A.1 macrophages were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FCS and 5 mM L-glutamine (11). In some experiments, the cells were pretreated with the proteasome inhibitory peptide Z-Leu-Leu-Leu-CHO (Mg-132, 2.5 µM; Biomol, Plymouth Meeting, PA). Stimulations were performed with LPS from Escherichia coli O55:B5 (Sigma-Aldrich, Munich, Germany), ultra-pure LPS from Salmonella minnesota R595 (List Biological Laboratories, Campbell, CA), a chemically synthesized analog of lipid A (18), the synthetic bacterial lipoprotein analog N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine P3CSK4 (Ref.12 ; EMC Microcollections, Tübingen, Germany), highly purified lipoteichoic acid (LTA) from Staphylococcus aureus (Refs. 19 and 20 ; kindly provided by T. Hartung, Biochemical Pharmacology, University of Konstanz, Konstanz, Germany), mouse TNF-
, and staurosporine (both Sigma-Aldrich).
Mice and peritoneal macrophages
C3H/HeN and TLR4-defective C3H/HeJ TLR4d/d mice (1, 21, 22) were purchased from Charles River Breeding Laboratories (Sulzfeld, Germany). C57BL/6, CD95/Fas-defective B6.MRL-lpr, TNFR-1-defective B6.129-Tnf1a, and TNFR-1 and -2-defective B6.129-Tnf1a/1b mice were obtained from The Jackson Laboratory (Bar Harbor, ME). 129/Sv x B57BL/6 wild-type and 129/Sv x B57BL/6 TLR2-/- mice were kindly provided by Tularik (South San Francisco, CA) and Deltagen (Menlo Park, CA) (23). TLR2-deficient 129/Sv x C57BL/6 TLR2-/- mice were bred back against the genetic C3H/HeN background for five generations to obtain C3H/HeN TLR2-/- mice. These were crossed with C3H/HeJ TLR4d/d mice to generate C3H/HeJ TLR4d/d/TLR2-/- doubly deficient mice lacking TLR2 expression as well as expression of functional TLR4 (24). 129/Sv x C57BL/6 TLR2-/-/TLR4-/- mice lacking expression of TLR2 and TLR4 were generated on the 129/Sv x C57BL/6 genetic background by crossing 129/Sv x C57BL/6 TLR2-/- mice with gene-targeted TLR4-/- mice kindly provided by S. Akira (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) (25). Homozygous (TLR2- and/or TLR4-deficient) breeding pairs were established with corresponding genetic backgrounds. Matched groups of mice descending from these were applied synchronically regarding wild-type controls for experiments. Elicited peritoneal macrophages were obtained from mice 3 days after i.p. inoculation of 10% proteose peptone broth as described (11).
Fluorescent labeling of apoptotic cells
For quantification of cell death, apoptotic cells were labeled with fluorescein-conjugated annexin V (Boehringer Mannheim, Mannheim, Germany), which binds to phosphatidylserine exposed on the outer leaflet of cells undergoing apoptosis (26). The simultaneous application of the DNA stain propidium iodide (Sigma-Aldrich) allowed discrimination of apoptotic from necrotic cells. The rate of cell death was determined by visual scoring of a minimum of 200 cells per sample in a fluorescence microscope. Results are expressed as mean percentages ± SD of apoptotic fluorescent cells vs the total number of cells from several independent experiments.
Measurement of macrophage TNF- production
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 (Ref.27 ; R&D Systems, McKinley Place, MN).
Cell transfection and measurement of NF-B activation
HEK293 cells were seeded in 24-well cell culture plates and transfected with human DNA constructs for CD14, TLR2, TLR4 (each 10 ng; Tularik), endothelial leukocyte adhesion molecule (ELAM)-1-NF-B luciferase (100 ng), and the phRL-null vector (100 ng; Promega, Madison, WI) as described (28). MD-2 plasmids (1 ng) and murine TLR2 were kindly provided by K. Miyake (Division of Infectious Genetics, University of Tokyo, Tokyo, Japan) (29) and H. Heine (Department of Immunology and Cell Biology, Research Center Borstel, Borstel, Germany), respectively. The total amount of DNA was kept constant with empty vector for each transfection (250 ng). Twenty-four hours after transfection, HEK293 cells were serum-starved for 18–20 h, then stimulated for 18 h as indicated. J774A.1 cells were transfected with ELAM-1-NF-B luciferase plasmid (500 ng), phRL-null vector (500 ng), and the plasmid of interest (500 ng) using the ExGen 500 transfection reagent according to the manufacturer’s instructions (Fermentas, Hanover, MD). Dominant-negative expression constructs used for the inhibition studies were myeloid differentiation factor 88 (MyD88) (30), Toll-IL-1R adapter protein (TIRAP) (31), IL-1R-associated kinase (IRAK)4 (32), Fas-associated death domain protein (FADD) (33), and furthermore, expression plasmids for Bfl-1/A1 (34) and catalytic inactive dsRNA-dependent protein kinase (PKR) (35, 36). The plasmids were kindly provided by M. Muzio (Mario Negri Institute, Milano, Italy), R. Medzhitov (Yale University, New Haven, CT), Tularik, C. Vincenz (Department for Pathology, University of Michigan Medical School, Ann Arbor, MI), A. Werner and J. Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands), and A. E. Koromilas (Lady Davis Institute, Montreal, Quebec, Canada), respectively. An empty expression vector containing no insert was used as negative control. Three hours after transfection, cells were stimulated as indicated. E. coli LPS and P3CSK4 were used at concentrations of 2 µg/ml. Cells were then lysed and luciferase activities were measured with a microtiter plate chemiluminometer (Berthold, Bad Wildbad, Germany) according to the manufacturer’s instructions (Ref.27 ; Dual-Luciferase Reporter System; Promega). The NF-B-directed firefly luciferase activities were normalized to Renilla luciferase activities to compensate for differences in transfection efficiencies. Data on NF-B activities are the means ± SD of at least three independent experiments.
Analysis of morphology of transfected cells
To determine the influence of TLR2 and TLR4 on viability of HEK293 cells, the cells were seeded in 24-well cell culture plates and transfected with expression plasmids for CD14 and TLR2 or TLR4 (each 190 ng), MD-2 (19 ng), and pSV--galactosidase expression vector (400 ng; Promega). The total amount of DNA was kept constant with empty vector for each transfection (1 µg). Twenty-four hours after transfection, HEK293 cells were serum-starved for 18–20 h, then stimulated as indicated. J774A.1 cells were transfected with 0.33 µg of pSV--galactosidase expression vector and 0.66 µg of the plasmid of interest (16). Three hours after transfection, J774A.1 cells were treated with MG-132 and stimulated 30 min later. To identify the transfected cells, the cells were fixed and stained with 5-bromo-4-chloro-3-indolyl -D-galactoside (X-gal) at the time points indicated. For assessment of cell death, the morphology of blue transfected cells was determined using light microscopy (7, 8, 9, 10, 16). Every single 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 mean percentages ± SD from three independent experiments.
Immunoprecipitations and Western immunoblotting
For the detection of transiently overexpressed TLRs, TLR2 or TLR4 expression plasmids or empty control plasmid (1 µg) were transfected into HEK293 cells seeded in six-well cell culture plates. Total cell lysates were prepared and incubated with anti-Flag mAbs (Sigma-Aldrich) and protein A/G-Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) as described (28). The immune complexes were washed, fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Immunoblot analysis was performed with anti-Flag epitope Abs. To determine cleavage of Bid in LPS- and MG-132-treated J774A.1 macrophages, a total of 105 cells per sample was lysed in 4x Laemmli sample buffer after stimulation as indicated. The lysates were fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membrane and probed with polyclonal anti-Bid Abs (R&D Systems). Immunoreactive bands were visualized using appropriate secondary Abs and ECL detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
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Results |
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To find out whether TLR signaling could play a role in the mechanism of apoptosis induction by Y. enterocolitica, we investigated apoptosis induced by Yersinia in primary mouse macrophages that are deficient for functional TLR2, TLR4, or both receptors (Fig. 1). Peritoneal macrophages were elicited from mice with different genetic backgrounds. C3H/HeN TLR2-/- and 129/Sv x B57BL/6 TLR2-/- mice harbor a TLR2 null mutation, whereas C3H/HeJ TLR4d/d mice bear a point mutation within the cytoplasmic portion of TLR4 conferring a TLR4-/- phenotype (1, 2, 21, 22). Apoptosis in the single TLR mutant macrophages was analyzed in comparison to wild-type macrophages (C3H/HeN; 129/Sv x B57BL/6) and to macrophages that are doubly deficient for TLR2 and TLR4 (C3H/HeJ TLR4d/d/TLR2-/-; 129/Sv x B57BL/6 TLR2-/-/TLR4-/-).
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yopP. This confirms that the NF-B inhibitory activity of YopP is required for efficient execution of apoptosis in Yersinia-infected macrophages (16). Interestingly, macrophages of C3H/HeJ TLR4d/d mice with a nonfunctional TLR4 were considerably protected against Yersinia-induced apoptosis. Accordingly, 60–70% of TLR4-mutagenized macrophages survived, when already >90% of the control cells were apoptotic. This points out a role of TLR4 as inducer of apoptosis. Notably, apoptosis in C3H/HeN TLR2-/- and 129/Sv x B57BL/6 TLR2-/- knockout macrophages was comparable to cell death induced in wild-type cells, which indicates that absence of TLR2 does not influence survival of Yersinia-infected macrophages. Macrophages with a double deficiency for both TLR2 and TLR4 exhibited the same degree of apoptosis as macrophages with a single TLR4 mutation. Together, these findings suggest that signaling through TLR4, but not through TLR2, plays an important role in Yersinia-conferred apoptosis. Y. enterocolitica induces cellular signaling via TLR2 and TLR4
Because TLR4 solely appears to be of critical importance in signaling macrophage apoptosis upon Y. enterocolitica infection, we wondered whether yersiniae are impaired in their ability to activate TLR2. The activation of TLRs largely contributes to determine the macrophage TNF- production in response to bacterial stimuli. Thus, we investigated the roles of TLR2 and TLR4 in Yersinia-induced TNF- release by peritoneal macrophages isolated from wild-type (C3H/HeN), TLR2-deficient (C3H/HeN TLR2-/-), and TLR4-mutagenized (C3H/HeJ TLR4d/d) mice. Yersiniae were grown under the same conditions by which wild-type bacteria efficiently mediate macrophage apoptosis. Wild-type yersiniae (WA-314), which down-regulate NF-B activities through YopP at the level of IKK (14, 16, 37), efficiently suppressed TNF- production of macrophages from all three mice strains (Fig. 2A), which correlates with previously described effects of YopP (11, 14, 15). In contrast, the YopP-negative mutant WA-yopP induced a TNF- release in wild-type macrophages that was remarkably reduced in macrophages deficient for either functional TLR2 or TLR4 (Fig. 2A). This implies that both TLR2 and TLR4 participate in the induction of the macrophage TNF- response elicited by YopP-negative yersiniae.
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B-dependent ELAM-luciferase reporter indicates activation of the NF-B pathway in response to TLR stimulation. TLR2 and TLR4 were transfected in combinations with the coreceptors CD14 and MD-2 and expression of TLRs was checked by immunoprecipitation and Western blotting with Abs directed against the Flag-epitope tags of TLR2 and TLR4 (Fig. 2B). As expected, the Y. enterocolitica wild-type strain WA-314 did not trigger substantial activation of the NF-B-dependent reporter gene in any of the transfected cells (Fig. 2C). This potentially results from YopP-mediated IKK inhibition (16, 37). In contrast, the YopP-negative mutant WA-yopP mediated prominent NF-B responses in TLR2- and TLR4-expressing cells (Fig. 2C). Comparable NF-B signals were generated by the TLR2-stimulating synthetic lipopeptide P3CSK4 and by TLR4-activating E. coli LPS, respectively. A modest but significant NF-B signal generated by WA-yopP in control vector-transfected cells was also induced by a virulence plasmid-cured Yersinia strain (WA-C), but not by a mutant additionally negative for the chromosomally encoded invasin gene (WAinv-C). Invasin is an outer surface protein that binds to eukaryotic 1 integrins and mediates NF-B activation (38). Thus, invasin apparently also triggers modest NF-B activation in HEK293 cells probably through 1 integrins. The TLR2- and TLR4-dependent NF-B signals induced by the diverse YopP-negative Y. enterocolitica strains indicate that both TLR2 and TLR4 can confer cellular reactivity to Yersinia infection. Use of murine TLR2, TLR4, and MD-2 expression vectors instead of the human constructs produced similar results (data not shown). TLR4 potently signals agonist-dependent apoptosis
To find out whether the ability of TLR4 to activate apoptosis is restricted to infection of macrophages by Yersinia or a more general phenomenon, we analyzed the apoptosis-conferring abilities of specific TLR2 and TLR4 agonists. LPS is a potent activator of TLR4, whereas the synthetic lipopeptide P3CSK4 and highly purified LTA from S. aureus mediate activation of TLR2 (1, 2, 3, 4, 5, 12, 20, 39). 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 with LPS or YopP-negative yersiniae (11). MG-132 suppresses degradation of the NF-B inhibitory IB proteins through the proteasome pathway (40), which substantially inhibits NF-B activation in macrophages (11). In these conditions, LPS can trigger an apoptotic response. We compared the induction of apoptosis by TLR2 and TLR4 agonists in J774.A1 macrophages that were pretreated with MG-132 (Fig. 3A). In correlation with our previous studies, the stimulation with E. coli LPS elicited robust apoptosis (11), whereas P3CSK4 and LTA could not provoke substantial cell death (Fig. 3A). To ensure cellular activation in these experiments, the TLR agonists were used at relatively high concentrations (1 µg/ml LPS, 4 µg/ml P3CSK4, 25 µg/ml LTA). In these concentrations, LPS as well as P3CSK4 and LTA elicited a strong TNF- response in J774A.1 macrophages (Fig. 3B), which indicates successful stimulation of the cells by these reagents. Similar to E. coli LPS, ultra-pure LPS from S. minnesota and a chemically synthesized analog of lipid A (18), the component of LPS that harbors the biological activity, efficiently induced macrophage apoptosis in the presence of MG-132 (Fig. 3A). At lower concentrations of 0.1 and 0.01 µg/ml, E. coli LPS, S. minnesota LPS, and synthetic lipid A still mediated macrophage cell death (80–90% and 60–80% apoptotic cells within 4.5 h, respectively), an effect not observed for P3CSK4 (<10% apoptotic cells). These experiments suggest that activation of TLR4 is superior to activation of TLR2 in eliciting an apoptotic response in macrophages upon inhibition of the proteasome pathway.
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production in wild-type macrophages (data not shown). Interestingly, the apoptosis-inducing capability of LPS was completely abolished in TLR4-defective macrophages from C3H/HeJ TLR4d/d mice, which points out that LPS specifically signals apoptosis through TLR4. Furthermore, LPS in combination with MG-132 was fully active on peritoneal macrophages obtained from TNFR-1 and TNFR2-deficient mice, which suggests that apoptosis does not result from TNF- that is released from the cells upon LPS treatment (data not shown).
Because it has been shown that expression of the tlr2 gene is up-regulated after NF-B activation (41, 42), we wondered whether the low rate of apoptosis in TLR2-stimulated and MG-132-pretreated cells could necessarily result from the prevention of TLR2 up-regulation by MG-132-mediated NF-B inhibition. Thus, we stimulated peritoneal mouse macrophages prepared from C3H/HeN mice with mouse TNF- (40 ng/ml) for 3 or 16 h, which significantly increases TLR2 expression (43, 44, 45), before treatment with MG-132 and P3CSK4. Although the preactivation with TNF- nearly doubled macrophage apoptosis in response to P3CSK4 (from 6 ± 2% without TNF- to 11 ± 3% with TNF- pretreatment after 8 h P3CSK4 stimulation), the overall degree of apoptosis remained moderate as compared with TLR4 responsive cell death. This implies that the reduced ability of TLR2 in inducing apoptosis does not merely result from impaired TLR2 expression.
To substantiate our observations on the relations between TLR activation and apoptosis, we overexpressed TLR2 and TLR4 in HEK293 cells and analyzed their influences on cellular viability. TLR2 and TLR4 were transfected together with CD14 and MD-2 expression plasmids to obtain functional TLR complexes. For identification of the transfected cells, a -galactosidase-encoding reporter vector was included (7, 8, 9, 10, 16). Staining with X-gal allows the detection of -galactosidase expression by conferring a blue color to the transfected cells. Apoptosis in these cells is characterized by typical cellular shrinkage and condensation (7, 8, 9, 10, 16), which was microscopically evaluated. Fig. 4 shows that the extended stimulation of TLR4 with E. coli LPS for 20–48 h initiated death in transfected HEK293 cells. The cytotoxic effect was less pronounced in cells transfected with TLR2 and stimulated with P3CSK4 (Fig. 4). The dying cells also displayed enhanced membrane binding of the apoptosis marker annexin V, which was determined by cotransfection of the cells with green fluorescent protein-reporter plasmid and labeling with red fluorescent Cy3-annexin V (data not shown). From these data it can be concluded that the overexpression and activation of TLR4 can conspicuously confer death to epithelial cells.
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Our data indicate that LPS-stimulated TLR4 can efficiently signal apoptosis when activation of NF-B is impaired. To identify the signal transducers that are potentially involved in the TLR4-dependent proapoptotic response, we transfected J774A.1 macrophages with different dominant-negative versions of components of the proximal LPS signaling cascade and analyzed the impact of these constructs on LPS-induced NF-B activation and initiation of apoptosis after MG-132 pretreatment (Fig. 5). Four of the investigated molecules, MyD88, TIRAP/MyD88-adapter-like protein (Mal), PKR, and FADD have been previously shown to play roles in signaling apoptosis upon stimulation with bacteria or bacterial components (46, 47, 48, 49, 50). All the constructs analyzed in Fig. 5 were expressed at the expected m.w. sizes in HEK293 cells, which was determined by Western immunoblotting using Abs directed against the epitope tags of the overexpressed proteins (MyD88, TIRAP, IRAK4, FADD, Bfl-1/A1) or against the protein itself (kinase inactive PKR; data not shown). Because of a lower transfection efficiency in J774A.1 macrophages, expression of the proteins in J774A.1 cells was checked by immunofluorescence microscopy at the single cell level using the Abs that recognized the respective overexpressed constructs in HEK293 cells (Ref.48 ; data not shown). For analysis of NF-B activation, J774A.1 cells were cotransfected with the NF-B-dependent ELAM-luciferase reporter plasmid and luciferase activities were monitored 5 h after LPS stimulation (Fig. 5B). The LPS-dependent induction of apoptosis was investigated at the same time point in MG-132-pretreated cells (Fig. 5A). For identification of apoptotic transfected cells, the expression plasmids were cotransfected with a -galactosidase-encoding reporter vector and apoptosis in single transfected cells was microscopically evaluated after staining with X-gal (7, 8, 9, 10, 16). None of the overexpressed constructs induced substantial cell death in the absence of MG-132 (Ref.48 ; data not shown).
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MyD88), TIRAP (TIRAP), and IRAK4 (IRAK4), a signaling molecule that acts downstream of MyD88 and TIRAP/Mal in the TLR signaling cascade (32, 51), considerably impeded NF-B activation (Fig. 5B). This correlates with the described inhibitory effects of these constructs on NF-B activation (30, 31, 32, 52, 53), indicating that the molecules are apparently functionally expressed in J774A.1 macrophages. MG-132 pretreatment and LPS challenge induced apoptosis in empty control vector-transfected cells to the same extent as in nontransfected cells (Fig. 5A). The overexpression of MyD88 and TIRAP, but not of IRAK4, could provide partial protection against LPS-promoted cell death, which suggests that the TLR4-induced apoptotic signal relay could involve MyD88 and TIRAP and diverge from the TLR-NF-B cascade at the level of the TLR adapter proteins. A catalytic inactive mutant version of PKR also impaired LPS-induced NF-B activation (Fig. 5B). This observation is in line with a number of reports that suggest involvement of the PKR kinase activity in TLR-driven NF-B activation (50, 54, 55, 56). However, kinase inactive PKR did not exert a protective effect against LPS-mediated apoptosis (Fig. 5A), which implies redundancy of the proposed apoptotic function of PKR in our experimental conditions (50). Notably, expression of the death domain of FADD (FADD), which acts as a dominant-negative molecule on FADD-dependent apoptosis (33, 57, 58), potently suppressed apoptosis in LPS-challenged cells, similar to the antiapoptotic Bcl-2 family member Bfl-1/A1 (Fig. 5A). Both FADD and Bfl-1/A1 play regulatory roles in the caspase-8/Bid apoptotic signaling cascade (33, 34, 57, 58), which suggests that this pathway could be critically involved in the proapoptotic response triggered by TLR4. However, the influences of FADD and Bfl-1/A1 on LPS-conferred NF-B activation were considerably different (Fig. 5B). Bfl-1/A1 expression remarkably increased NF-B activities in nonstimulated and LPS-treated cells, which suggests that the Bfl-1/A1 antiapoptotic activities may at least partially result from enhancement of NF-B activation. In contrast, FADD suppressed the LPS-induced NF-B response. The FADD death domain has already previously been shown to counteract NF-B activation in LPS-stimulated endothelial cells, which could be a consequence of binding and inhibition of MyD88 by FADD (59). The blockage of NF-B activation and apoptosis by FADD suggests a regulatory function of FADD in the induction of both pathways in LPS-treated macrophages. Interestingly, while MyD88, TIRAP, and IRAK4 almost equally impaired LPS- and P3CSK4-mediated NF-B activation, FADD was 3.5 times more effective in suppressing macrophage NF-B activation in response to LPS stimulation, as compared with treatment with P3CSK4 (Fig. 5C). This could argue for a certain degree of specificity by which FADD is engaged by TLR4 signaling, that is less obvious for signaling through TLR2. To achieve more indication of whether the involvement of FADD is direct or rather indirect via activation of the CD95/Fas death receptor, we investigated LPS/MG-132-induced cell death in peritoneal macrophages that were derived from mice lacking a functional CD95/Fas receptor (lpr mice). Onset of apoptosis was similar in C57/BL6 wild-type and CD95/Fas-mutagenized macrophages (50–60% apoptosis after 6 h stimulation with LPS in presence of MG-132), which rules out crucial involvement of the CD95/Fas receptor for the engagement of FADD in TLR4-dependent apoptosis.
To elucidate a nonspecific function of FADD in apoptosis prevention in LPS/MG-132-treated J774A.1 cells, we explored the protective effect of FADD on staurosporine-induced cell death. In these experiments, the overexpression of FADD did not inhibit apoptosis triggered by 5 µM staurosporine within 4 h (78 ± 4% apoptosis), as compared with control vector-transfected cells (80 ± 5% apoptosis), whereas Bfl-1/A1 could significantly preserve cellular viability (49 ± 5% apoptosis). This result indicates that FADD does not globally desensitize J774A.1 cells to apoptotic stimuli, whereas Bfl-1/A1 appears to be a more general inhibitor of apoptosis. Because FADD controls activation of initiator caspase-8, we furthermore examined cleavage of the proapoptotic Bcl-2 family member Bid. Bid is a specific proximal substrate of caspase-8 in the FADD signaling pathway to apoptosis (34, 57, 58). We found that the proapoptotic truncated 14–15 kDa Bid fragment (tBid) was produced in LPS-stimulated J774A.1 macrophages that were pretreated with MG-132, but not in control cells (Fig. 6). Cleaved Bid can activate the mitochondrial apoptosis pathway by mediating release of cytochrome c (34, 57, 58). Together, these data indicate a role of the FADD/caspase-8/Bid pathway in the signaling of apoptosis through TLR4.
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Discussion |
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B activation (40). In these experiments, solely stimulation of TLR4 could trigger a pronounced apoptotic response, which indicates that signaling through TLR4 is superior to TLR2 signaling in eliciting macrophage apoptosis in the investigated conditions. However, our data show that TLR2 also could have the capability to evoke apoptosis. The transient overexpression and activation of TLR2 mediated apoptosis in HEK293 cells, although to a lesser extent as compared with TLR4. Previous studies, that were reporting apoptosis through TLR2 stimulation, were conducted predominantly on human monocytic THP-1 cells, THP-1-derived macrophages, and human Schwann cells (12, 47, 60, 61), while our experiments were done on mouse J774A.1 and primary peritoneal macrophages. This suggests that there may exist cell type-specific differences in TLR-responsive apoptosis signaling, which could be related to the distinct engagement of intracellular adapter proteins by TLR2 and TLR4.
TLR2-dependent apoptotic signaling is coupled to the cell death machinery through activation of MyD88 and FADD (47), a cytotoxic pathway that is potentially also involved in Yersinia-mediated apoptosis (48, 62). MyD88 binds to the TLR2 intracellular signaling domain and recruits FADD through death domain-death domain interaction (47). Mobilized FADD then initiates the apoptotic cascade by caspase-8 activation. Our experiments using dominant-negative expression constructs suggest that apoptotic signaling via TLR4 could similarly target the MyD88-FADD pathway. A dominant-negative version of FADD (FADD) was highly protective against TLR4-dependent apoptosis, which indicates that apoptotic signaling by TLR4 necessarily goes through FADD. LPS-mediated cell death was furthermore characterized by processing of the caspase-8 substrate Bid, which transmits the apoptotic signal from death receptors to mitochondria (56, 57). The overexpression of Bfl-1/A1, an antiapoptotic Bcl-2 family molecule that inhibits the collaboration of Bid with proapoptotic Bak or Bax (34), suppressed TLR4-mediated cell death. These data additionally argue for involvement of a FADD/caspase-8/Bid-dependent pathway in TLR4 proapoptotic signaling. Analyzing the implication of TLR adapter proteins, we found that a dominant-negative MyD88 mutant (MyD88) could diminish TLR4-mediated apoptosis. This suggests that MyD88 may interrupt recruitment and activation of FADD by TLR4 analogically as by TLR2. Interestingly, a dominant-negative version of TIRAP (TIRAP), another adapter of TLR2 and TLR4 (63, 64), was as efficient as MyD88 in reducing TLR4-conferred cell death, which indicates that also TIRAP/Mal could play a role in transducing an apoptotic signal. However, TIRAP/Mal does not possess a death domain (31, 65) and interaction between TIRAP/Mal and FADD has not yet been described, which suggests that the mechanism by which TIRAP reduces apoptosis potentially differs from the inhibitory mechanism of MyD88. Furthermore, it cannot be excluded that overexpression of any dominant-negative TLR adapter blocks activation of downstream pathways by competing with the functional endogenous adapter proteins, which could argue against a specific inhibitory effect of TIRAP or MyD88 in TLR4-dependent apoptosis. In this regard, it would be interesting to investigate the role of a third TLR adapter protein which has been recently identified (66, 67). The Toll-IL-1R-domain-containing adapter inducing IFN- transduces TLR4- and TLR3-, but not TLR2-, generated signals, which could have implication in the regulation of cellular viability.
Our data show that the death domain of FADD (FADD) is much more effective in counteracting TLR4- than TLR-2-dependent NF-B activation in J774A.1 macrophages. This gives indirect indication that the composition of intracellular protein signaling complexes differs for distinct TLRs. The conspicuous inhibition of TLR4-responsive signaling by FADD suggests reinforced mobilization of FADD by TLR4, which appears to be important for the activation of apoptosis in our experimental conditions. It has been shown that TLR4 but not TLR2 activation results in degradation of the IRAK1 molecule (68, 69). Binding of IRAK1 to the MyD88 death domain is an important step of the TLR-responsive signal relay that leads to NF-B activation (1, 2, 3, 4, 5, 6). The selective degradation of IRAK1 after LPS stimulation, which we also observed in J774A.1 macrophages (data not shown), could give a possible explanation for enhanced recruitment of FADD after TLR4 activation. The death domain of MyD88 may be more accessible to FADD after IRAK1 disappearance, concomitantly leading to increased induction of the apoptotic pathway. IRAK1 degradation upon LPS treatment has been suggested to occur via the proteasome pathway (70), which indicates that the proteasome function could play a role in the regulation of TLR-dependent apoptosis by controlling the turnover of TLR-responsive signal transmitters. Thus, although both TLR2 and TLR4 could potentially engage the FADD pathway to provoke apoptosis, the composition and stability of intracellular adapter protein complexes recruited by the respective TLR may determine the activation of apoptotic signaling and the extent of apoptosis.
It is shown here that TLR4 can efficiently signal apoptosis in bacteria-infected macrophages, which plays an important role in Yersinia-induced apoptosis. A study by Zhang and Bliska (71), which was published while our manuscript was in revision, supports our findings, elucidating TLR4 as the primary source of apoptotic signaling in Yersinia-infected macrophages. Although the authors consider the redundancy of TLR2 in Yersinia-induced apoptosis as a result of a putative inability of TLR2 to recognize yersiniae, our data clearly demonstrate that both TLR2 and TLR4 can confer responsiveness to Yersinia infection, but only TLR4 signals apoptosis. However, TLR4-deficient cells were not completely protected against Yersinia-induced cell death. This indicates that additional cytotoxic pathways are active in the absence of functional TLR4. It has been shown that constitutive NF-B activation is required for macrophage survival in nonstimulated conditions (72). This suggests the existence of a constitutively active cytotoxic pathway that is balanced by basic NF-B activation. In fact, the transfection of macrophages with YopP (16) or kinase-inactive IKK, as well as extended treatment with MG-132 (data not shown), significantly confers apoptosis, which could support this hypothesis. In this case, TLR4 signaling critically accelerates onset of apoptosis that is otherwise mediated by the NF-B inhibitory activity of YopP with delayed kinetics. Alternatively, pathways that signal apoptosis independently from TLR4 are activated in Yersinia-infected cells. Our preliminary studies do not indicate Yersinia-conferred activation of other known TLRs besides TLR2 and TLR4 when overexpressed in HEK293 cells. However, additional bacteria-responsive proapoptotic signal transmitters, such as the intracellular Nod proteins, which function as cytosolic peptidoglycan receptors (73), could be implicated in Yersinia-induced apoptosis. Whether Yersinia as extracellular pathogen induces the Nod pathways and whether these pathways cooperate with TLR4 to evoke Yersinia-mediated cell death has not yet been investigated.
The role of apoptotic signaling through TLR4 is still elusive. It could help to limit the lifetime of activated inflammatory cells to protect the organism from developing damage by inflammatory hyperactivation during infection. Furthermore, it could be part of an evolutionary conserved mechanism of innate immune defense. TLRs display some homology to plant disease resistance gene products, which mediate a so-called hypersensitive response when encountering a bacterial pathogen (47, 74, 75). The hypersensitive response resembles apoptosis in animal cells, leading to localized cell death at the site of pathogen invasion which limits dissemination of the microbes (75, 76). Analogously, apoptosis of macrophages could help to prevent spread of pathogenic intracellular bacteria, for instance Francisella tularensis which has been found to inhibit NF-B activation and to induce macrophage cell death (77, 78). However, under normal circumstances the activation of NF-B counteracts TLR-dependent apoptosis and macrophage viability is not necessarily restricted during infection. From this observation it appears that some extracellularly pathogenic bacteria, such as yersiniae, could take advantage of the conserved cytotoxic TLR4-signaling pathway to trigger macrophage cell death through the inhibition of NF-B. This suggests that apoptosis induced by TLRs could potentially fulfill diverse roles in the host immune response depending on the invading pathogen and on the challenged cell.
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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: ruckdeschel{at}m3401.mpk.med.uni-muenchen.de
3 Abbreviations used in this paper: TLR, Toll-like receptor; Yop, Yersinia outer protein; IKK, inhibitory B kinase; HEK, human embryonic kidney; LTA, lipoteichoic acid; ELAM, endothelial leukocyte adhesion molecule; MyD88, myeloid differentiation factor 88; TIRAP, Toll-IL-1R adapter protein; IRAK, IL-1R-associated kinase; FADD, Fas-associated death domain protein; PKR, dsRNA-dependent protein kinase; Mal, MyD88-adapter-like protein; tBid, truncated Bid; TRIF, TIR domain-containing adapter inducing IFN-.
Received for publication February 19, 2003. Accepted for publication August 13, 2003.
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