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Previous Article | Next Article 
Infection and Immunity, March 2003, p. 1513-1519, Vol. 71, No. 3
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.3.1513-1519.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Center for Infectious Diseases, Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794-5120
Received 23 September 2002/ Returned for modification 12 November 2002/ Accepted 29 November 2002
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ABSTRACT |
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B by TLR2 or TLR4 signaling promotes proinflammatory and cell survival responses. Alternatively, TLR2 or TLR4 signaling can promote apoptosis if the activation of NF-B is blocked. The gram-negative bacterial pathogen Yersinia pseudotuberculosis secretes into macrophages a protease (YopJ) that inhibits the activation of NF-B and promotes apoptosis. We show that primary macrophages expressing constitutively active inhibitor B kinase ß (IKKß) are completely resistant to YopJ-dependent apoptosis, indicating that YopJ inhibits signaling upstream of IKKß. Apoptosis is reduced two- to threefold in TLR4-/- macrophages infected with Y. pseudotuberculosis, while the apoptotic response of TLR2-/- macrophages to Y. pseudotuberculosis infection is equivalent to that of wild-type macrophages. Therefore, TLR4 is the primary source of apoptotic signaling in Yersinia-infected macrophages. Our results also show that a small percentage of macrophages can die as a result of an apoptotic process that is YopJ dependent but does not require TLR2 or TLR4 signaling. |
INTRODUCTION |
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The signaling pathways that are activated downstream of TLRs are currently being elucidated. TLR family members contain a cytoplasmic Toll-interleukin 1 receptor homology region. After the activation of TLR4 by LPS, a series of events lead to the activation of ubiquitin ligase TRAF6 by a unique self-polyubiquitination reaction (reviewed in reference 1). TRAF6 then activates the TAK1 complex (31). This step leads to the phosphorylation and activation of mitogen-activated protein kinase kinases and the inhibitor B (IB) kinase (IKK) complex (18, 31). The IKK complex is comprised of two kinases, IKK
and IKKß, and a third protein, NEMO (also called IKK
). Upon activation, IKKß phosphorylates IB, triggering its polyubiquitination and degradation (6, 11). In nonstimulated cells, IB interacts with and traps NF-B in the cytosol. Degradation of IB releases NF-B to translocate into the nucleus and to activate proinflammatory and prosurvival gene expression. Thus, multiple signaling pathways can be activated through TLR4, leading to the production of cytokines and other factors that protect the host against infection.
In addition to activating gene expression, signaling through TLRs may activate a programmed cell death (apoptosis) response in eukaryotic cells. For example, the activation of TLR2 by bacterial lipopeptides can promote apoptosis (3). In this situation, the activation of caspase 8 and, subsequently, the effector caspases leads to the execution of the apoptotic program. The inhibition of NF-B activation has been shown to potentiate apoptosis (30, 32), indicating that the production of survival factors under the control of NF-B can counteract apoptotic signaling by TLR2 (3).
In this study, we investigated the role of TLR signaling in the apoptotic response of macrophages to the gram-negative bacterial pathogen Yersinia pseudotuberculosis. Y. pseudotuberculosis is closely related to Yersinia pestis, the agent of plague, and causes a systemic infection in rodents that resembles plague in humans. Previous studies established that Y. pseudotuberculosis stimulates apoptosis in infected macrophages (17). In addition, the ability to trigger macrophage apoptosis is important for systemic infection of mice by this pathogen (16). The toxin YopJ (also known as YopP in Yersinia enterocolitica) is required for apoptosis in macrophages (17). YopJ is one of six toxins secreted by Yersinia into the cytosol of macrophages by a plasmid-encoded bacterial secretion apparatus known as a type III secretion system (5). YopJ is a cysteine protease of the family of ubiquitin-like proteins that inhibits multiple signaling pathways (20). YopJ binds to IKKß (20), and the translocation of YopJ into macrophages is associated with decreased IKKß and NF-B activities (25). It has been suggested, but not demonstrated, that YopJ acts upstream of IKKß to block its activation (20).
Two models have been proposed to explain how YopJ causes apoptosis in macrophages. In one model, YopJ directly activates a programmed cell death pathway through a process that involves the proteolytic cleavage of the caspase 8 substrate Bid (7). In a second model, YopJ acts to potentiate the apoptotic signaling of LPS by inhibiting the activation of NF-B (24, 25). Transient overexpression of IKKß in macrophages reduced apoptosis in response to Yersinia infection by twofold (25). In addition, when a vector producing YopJ was transfected into macrophages, the percentage of cells that underwent apoptosis increased significantly in the presence of LPS (25). However, up to 50% of transfected macrophages not treated with LPS died, suggesting that other mechanisms of YopJ-dependent apoptosis may exist (25). Taken together, these results support the concept that YopJ potentiates the apoptotic signaling of LPS by inhibiting the activation of NF-B. However, an essential role for TLR4 or IKKß in YopJ-dependent apoptosis of macrophages has not been established.
We used retroviral transfection of primary macrophages and macrophages from mice deficient in TLR2 or TLR4 (TLR2-/- and TLR4-/-, respectively) to further characterize the mechanism of YopJ-dependent apoptosis. We found that macrophages expressing a constitutively activated derivative of IKKß are completely protected from YopJ-dependent apoptosis. This result establishes IKKß as a key target of Yersinia pathogenesis. Although signaling through TLR2 is dispensable for YopJ-dependent apoptosis, signaling through TLR4 contributes significantly to apoptosis but is not absolutely required for this activity.
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MATERIALS AND METHODS |
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/ß (H-470; sc-7607) and IB (C-21; sc-371) were purchased from Santa Cruz Biotechnology. Polyclonal antibodies to c-Jun N-terminal kinase (JNK) (9252), phospho-JNK (9251), and phospho-IB (9241) were obtained from Cell Signaling Technology. Anti-FLAG M2 monoclonal antibody and affinity gel and LPS from Escherichia coli O26:B6 were obtained from Sigma. Secondary antibodies conjugated to horseradish peroxidase were obtained from Jackson ImmunoResearch Laboratories. Lipopeptide Pam3CSK4 was obtained from Guenther Jung (University of Tübingen). Protein A- Sepharose CL-4B and glutathione-Sepharose 4B were obtained from Pharmacia Biotech. Tissue culture reagents were obtained from Invitrogen Life Technologies. Tumor necrosis factor alpha (TNF-) was obtained from the National Institute for Biological Standards and Control. The RetroMax retroviral system was obtained from IMGENEX. A plasmid containing the encephalomyocarditis virus internal ribosome entry site (IRES) was provided by Eckard Wimmer (Stony Brook, N.Y.). Polybrene and plasmids encoding wild-type IKKß, IKKß with S177E and S181E mutations, and glutathione S-transferase (GST)-IB(1-62) were provided by Kenneth Marcu (Stony Brook, N.Y.). GST- IB(1- 62) (where 1-62 is amino acids 1 to 62 of IB) was purified from E. coli by using glutathione-sepharose 4B and a procedure recommended by the supplier (Pharmacia Biotech). A protease inhibitor cocktail was obtained from Roche. Plasmid pEGFP-C1, encoding green fluorescent protein (GFP), was obtained from Clontech. Mice and bone marrow-derived macrophages. C57BL/6, C3H/HeJ, and C3HeB/FeJ mice were obtained from Jackson ImmunoResearch Laboratories. TLR2-/- mice (derived from 129/SvJ crossed with C57BL/6) (28), TLR4-/- mice (derived from 129/Ola crossed with C57BL/6) (10, 28), and age-matched wild-type control mice were provided by Shizuo Akira (Osaka University) and Ruslan Medzhitov (Yale University). Bone marrow-derived macrophages were obtained as previously described (4).
Retroviral expression of IKKß in cultured cells. DNA restriction fragments encoding wild-type or constitutively active IKKß, the IRES, and GFP were ligated into the EcoRI site in retroviral vector pCLXSN (IMGENEX). The resulting vectors, pCLXSN-IKKß(wt)-IRES-GFP, pCLXSN-IKKß(S178E/S181E)-IRES-GFP, and pCLXSN-IRES-GFP, were verified by sequencing. Retroviral particles were collected in the culture supernatants of transfected 293T cells according to the manufacturer's instructions (IMGENEX). To transfect NIH 3T3 cells, the cells were cultured in 1 ml of a 9:1 mixture of Dulbecco modified Eagle medium (DMEM) containing 10% calf serum- 8 µg of Polybrene/ml and 293T-cell supernatant in six-well plates (105 cells/well) for 8 h. The medium was removed, and the cells were cultured in DMEM containing 10% calf serum for 24 h. Macrophages grown for 5 days were used for retroviral transfections. The macrophages were cultured in 0.4 ml of a 3:1 mixture of bone marrow medium (BMM [DMEM containing 20% fetal bovine serum, 30% L-cell-conditioned medium, 2 mM glutamine, and 1 mM pyruvate]) and 293T-cell supernatant on glass coverslips in 24-well dishes (5 x 104 cells/well) for 40 h. The transfected macrophages were washed and incubated in modified BMM (BMM containing 10% fetal bovine serum and 15% L-cell-conditioned medium) 30 min prior to bacterial infection.
Immunoprecipitation and kinase activity assay.
The procedures for immunoprecipitation and the kinase activity assay were those of McKenzie et al. (14). Briefly, transfected NIH 3T3 cells were cultured in DMEM lacking serum overnight. The cells were left untreated or were stimulated with TNF- for 15 min. The cells were washed twice with ice-cold phosphate-buffered saline containing 1 mM sodium orthovanadate and 10 mM NaF and then incubated in lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM ß-glycerophosphate, 200 µM sodium orthovanadate, 1% Triton X-100, protease inhibitor cocktail) on ice for 15 min. Lysates clarified by centrifugation were incubated with anti-FLAG M2 affinity gel for 2 h at 4°C. The immune complexes were washed three times with lysis buffer and then twice with kinase assay buffer (20 mM HEPES [pH 7.4], 20 mM MgCl2, 1 mM dithiothreitol, 10 mM p-nitrophenyl phosphate). IKKß activity was assayed by incubating the immune complexes in 20 µl of kinase assay buffer containing 50 µM ATP and 2.5 µg of GST- IB(1- 62). After 10 min at 30°C, an equal volume of 2x Laemmli sample buffer (9a) was added, and the sample was boiled and then analyzed by Western blotting.
Bacterial strains and infection conditions. Y. pseudotuberculosis strains YP126 (wild type) and YP26 (YopJ-) have been described elsewhere (21). To prepare bacteria for infection, overnight cultures grown at 26°C in Luria broth were diluted to an optical density at 600 nm of 0.1 in Luria broth supplemented with 20 mM MgCl2 and 20 mM sodium oxalate. The cultures were shaken at 26°C for 1 h and at 37°C for 2 h. The bacteria were washed once and resuspended in Hanks balanced salt solution and then used to infect macrophages at a multiplicity of infection of 50. To initiate the infection, the plates were centrifuged for 5 min at 200 x g to bring the bacteria in contact with the macrophages.
Western blot analysis. Macrophages (5 x 105 cells/well) cultured in 2 ml of fresh modified BMM in six-well plates were stimulated or infected as described in the figure legends. The cells were lysed in boiling 1x Laemmli sample buffer. Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide). Western blotting with commercial primary antibodies was carried out as suggested by the suppliers. Immunoblots were developed with enhanced chemiluminescence reagents as recommended by the supplier (New England Nuclear).
TUNEL assay. Macrophages cultured on coverslips (5 x 105 cells/well) were infected for 4 h. Gentamicin was added to a final concentration of 100 µg/ml at the 2-h time point to inhibit further bacterial growth. The cells were then fixed and processed to detect apoptosis by using an in situ cell death detection kit with TMR red or fluorescein (Roche) according to the manufacturer's instructions. The coverslips were mounted in SlowFadeLight antifading medium with glycerol (Molecular Probes) and analyzed by fluorescence microscopy with a Zeiss Axioplan2 microscope. Digital images were captured by using a SPOT camera (Diagnostic Instruments). For transfected macrophages, fluorescence microscopy was used to determine the percentage of transfected macrophages that were terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) positive in at least 10 randomly chosen nonoverlapping fields. For nontransfected macrophages, phase microscopy and fluorescence microscopy were used to determine the percentage of TUNEL-positive cells in at least four randomly chosen nonoverlapping fields.
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RESULTS |
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for 10 min to activate IKKß. The FLAG-tagged IKKß proteins were immunoprecipitated from detergent extracts of the cells by using anti-FLAG antibody. Purified GST- IB(1- 62) was used as a substrate in an in vitro kinase reaction to measure the activities of the immunoprecipitated IKKß proteins. No IKKß activity was immunoprecipitated from cells transfected with virus expressing GFP alone (Fig. 1B, lanes 3 and 6). On the other hand, cells transfected with virus expressing IKKß-EE contained active kinase in the presence or absence of TNF- stimulation (Fig. 1B, lanes 2 and 5). Cells transfected with virus encoding wild-type IKKß contained a low but detectable level of kinase activity in the absence of TNF- (Fig. 1B, lane 1) and a greatly increased level of kinase activity in the presence of TNF- (lane 4). Thus, the kinases encoded by the retroviruses functioned as expected. The retroviruses were used to transfect bone marrow-derived macrophages obtained from C57BL/6 mice. At 2 days after transfection, between 15 and 30% of the cells expressed GFP, depending on the individual experiment and the stock of retrovirus used (data not shown). Detergent extracts of the macrophages were prepared and analyzed by immunoblotting to confirm that IKKß and IKKß-EE were expressed at similar levels (Fig. 2A).
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B through a TLR4-dependent pathway (23). The activation of NF-B prior to Yersinia infection is likely to promote some degree of protection from YopJ-dependent apoptosis. Nevertheless, the impact of IKKß and IKKß-EE expression on YopJ-dependent apoptosis was clearly evident: while 17.5% of macrophages expressing GFP alone underwent apoptosis, apoptosis was decreased to 8% in macrophages expressing IKKß and to a negligible 0.8% in macrophages expressing IKKß-EE (Fig. 2C). Thus, the expression of IKKß-EE decreased apoptosis to background levels. The partial protection from apoptosis provided by wild-type IKKß was consistent with its low level of constitutive activity (Fig. 1B), a result which confirms the findings of Ruckdeschel et al. (25). However, by using a catalytically active form of IKKß, we further demonstrated that inhibition of the catalytic activity of IKKß is essential for Yersinia-induced apoptosis. This result suggests that YopJ acts upstream of IKKß to inhibit its activation.
Role of TLR4 in YopJ-dependent apoptosis.
The above data suggest that the inhibition of IKKß activation by YopJ is required for apoptosis. We next investigated the source of the apoptotic signaling that is normally counteracted by IKKß. Specifically, we examined whether TLR signaling pathways are required to trigger the apoptotic response. Macrophages deficient in TLR4 function were isolated from C3H/HeJ mice and used in infection assays to test directly the role of this receptor in YopJ-dependent apoptosis. C3H/HeJ mice harbor a naturally occurring codon substitution in Tlr4; this mutation results in a dominant-negative receptor and a TLR4-/- phenotype (22). Initially, we examined the activation of signaling pathways in C3H/HeJ macrophages to confirm that they were hyporesponsive to LPS. Macrophages from C3H/HeJ mice or the LPS-sensitive mouse strain C3HeB/FeJ were stimulated by exposure to osmotic shock or to increasing concentrations of LPS or by infection with a Y. pseudotuberculosis YopJ- mutant for 15 min. The activation of IKKß or the mitogen-activated protein kinase JNK was examined by immunoblotting. The degradation of IB indicates IKKß activation, while the phosphorylation of JNK is indicative of JNK activation. Both isoforms of JNK (p46 and p54) were activated to equivalent levels in C3H/HeJ and C3HeB/FeJ macrophages following osmotic shock with 0.2 M NaCl (Fig. 3A, middle panel, lanes 7 and 15), confirming that signaling pathways are intact in C3H/HeJ macrophages. Higher levels of LPS were needed to achieve full activation of IKKß or JNK in C3H/HeJ macrophages than in C3HeB/FeJ macrophages (Fig. 3A, upper and middle panels, compare lanes 1 to 6 with lanes 9 to 14). The fact that C3H/HeJ macrophages responded at all to this preparation of LPS likely is a result of its contamination with lipoproteins, as a previous study showed (8). The activation of IKKß or JNK in response to infection with the YopJ- mutant was also significantly decreased in C3H/HeJ macrophages compared to C3HeB/FeJ macrophages (Fig. 3A, upper and middle panels, lanes 8 and 16). These results confirm that the C3H/HeJ macrophages that we isolated were hyporesponsive to LPS and that infection of wild-type macrophages with Y. pseudotuberculosis stimulates signaling through TLR4.
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50%. After infection with the YopJ- mutant, 17.6% of C3HeB/FeJ macrophages underwent apoptosis, while only 6.7% of C3H/HeJ macrophages died, again, a reduction of 50%. Extremely low levels of apoptosis occurred in uninfected macrophages of both types. To confirm that TLR4-/- signaling is important for YopJ-dependent apoptosis, we next examined the infection-induced responses of macrophages obtained from mice with null mutations in the Tlr4 gene. Macrophages obtained from mice congenic with the TLR4-/- animals were used as controls. Like CH3/HeJ macrophages, TLR4-/- macrophages showed decreased signaling responses to LPS or YopJ- Y. pseudotuberculosis (Fig. 4A, top and middle panels, compare lanes 2, 3, and 4 with lanes 14, 15, and 16). In addition, YopJ-dependent apoptosis was significantly reduced but not eliminated in TLR4-/- macrophages infected with Y. pseudotuberculosis (71.2% for wild type versus 21.9% for TLR4-/-) (Fig. 4B). These results further demonstrate that TLR4 is the primary source of signaling that triggers YopJ-dependent apoptosis.
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DISCUSSION |
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Our results extend previous conclusions about the relationship between YopJ and IKKß. It has been shown that the inhibition of NF-B activation is required for YopJ-dependent apoptosis (25). In addition, the translocation of YopJ into macrophages is associated with decreased IKK activity (25). We show here that macrophages overexpressing a constitutively active mutant of IKKß are completely protected from YopJ-dependent apoptosis (Fig. 2). These results demonstrate that YopJ acts upstream of IKKß to block its activation and rule out the possibility that YopJ interferes with the access of activated IKKß to IB. Thus, inhibiting the activation of this single kinase is a key event in Yersinia pathogenesis.
How YopJ inhibits the activation of IKKß remains unanswered. YopJ is a cysteine protease that appears to cleave ubiquitin or ubiquitin-like modifications from proteins (20). Although YopJ binds to IKKß and inhibits its activation, there is currently no evidence that IKKß is modified by ubiquitin or is itself a substrate of YopJ. Since ubiquitination is required for TRAF6 activation (31), it is possible that YopJ acts on TRAF6 to block the downstream activation of IKKß (20). Recently, Ruckdeschel et al. showed that the transfection of macrophages with a dominant-negative form of TRAF6 increases apoptosis in response to LPS stimulation (26). These findings support the idea that TRAF6, like IKKß, is important for the expression of survival factors in macrophages exposed to apoptotic stimuli.
Using macrophages deficient in TLR function, we demonstrate that signaling through TLR4 is important for YopJ-dependent apoptosis. This finding confirms the previous finding that LPS stimulation of macrophages can trigger apoptotic signaling (25). How TLR4 signaling triggers apoptosis is not fully understood. YopJ-dependent apoptosis is reduced when dominant-negative forms of MyD88 and FADD are expressed in macrophages (26). Inhibitors of caspase 8 and caspase 9 also significantly reduce YopJ-dependent apoptosis in macrophages (26). It has not been shown that caspase 8 is activated in response to Yersinia infection, although its substrate, Bid, is cleaved (7). These results suggest that apoptotic signaling through TLR4 likely is mechanistically similar to apoptotic signaling mediated by TLR2 (3).
A significant finding of this study is that TLR4-/- macrophages were not completely resistant to YopJ-dependent apoptosis (Fig. 3 and 4). In addition, infection-induced signaling was delayed but not eliminated in TLR4-/- macrophages (Fig. 5). TLR2-/- macrophages displayed wild-type levels of apoptosis and wild-type signaling responses to infection (Fig. 4 and 5). We tentatively conclude that TLR2 signaling is not responsible for the YopJ-dependent apoptosis that we observed in TLR4-/- macrophages. Other studies have failed to demonstrate a role for TLR2 in recognizing gram-negative bacteria as well. For example, Underhill et al. found that macrophages expressing dominant-negative TLR2 produced wild-type levels of TNF- when challenged with Salmonella (29). Other PAMPs associated with Yersinia could contribute to the signaling responses of TLR4-/- macrophages to infection. For example, Y. pseudotuberculosis produces flagella that would be recognized by TLR5. Another interesting possibility is that a structural component of the type III secretion system could act as a PAMP. Sing et al. recently showed that LcrV stimulates macrophages to produce interleukin 10 independently of TLR4 function (27). They speculated that LcrV is recognized by a TLR on the surface of macrophages (27). Yet another possibility is that intracellular LPS receptors are involved in sensing Yersinia infection. For example, the NOD1 and NOD2 proteins contain a leucine-rich region, a structure found in the TLR4 extracellular domain, which is required for LPS signaling (12). It has been reported that the NOD proteins are able to activate the NF-B pathway following LPS injection into cells (9, 19). However, it remains unknown how LPS could gain access to the NOD proteins under our infection conditions.
The involvement of receptors other than TLR4 in stimulating infection responses (Fig. 5) is suggestive of a hierarchy in the PRRs of the innate immune system for recognizing gram-negative pathogens. The use of pure PAMPs, such as LPS, has facilitated the identification of TLRs as PRRs and the characterization of TLR signaling pathways. However, new approaches will be needed to discover additional PRRs and to study complex functional relationships between subfamilies of PRRs. The use of live, intact pathogens is one approach that may prove useful in this context. Here, using whole live Yersinia to infect macrophages, we found that the activation of proinflammatory responses can occur in two waves in wild-type macrophages. The fast response is clearly TLR4 dependent, since it was abolished in TLR4-/- cells (Fig. 5). Studies to identify the receptors responsible for the slower response are ongoing.
In summary, our data support the concept that YopJ promotes the apoptotic signaling activity of TLR4 and potentially other PRRs by inhibiting the NF-B-dependent survival response of macrophages. Even in the absence of YopJ, stimulation by LPS or by infection with Y. pseudotuberculosis can lead to low levels of apoptosis in macrophages. The level of apoptosis that we observed in macrophages infected with YopJ- bacteria varied depending on the genetic background of the macrophages used (Fig. 3 and 4). These results reinforce the idea that the relative strength of the prosurvival response versus the proapoptotic response critically determines whether macrophages live or die when infected by pathogenic bacteria.
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ACKNOWLEDGMENTS |
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We thank members of the laboratory of James B. Bliska for assistance, helpful discussions, and comments on the manuscript. In addition, we thank C. Roy for helpful comments on the retrovirus expression system; S. Akira, R. Medzhitov, K. Marcu, E. Wimmer, and K. Orth for reagents and suggestions; and J. E. Galán and S. Miller for stimulating discussions.
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FOOTNOTES |
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