The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Watanabe, I.
Right arrow Articles by Nakanishi, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Watanabe, I.
Right arrow Articles by Nakanishi, Y.
The Journal of Immunology, 2007, 178: 4917-4925.
Copyright © 2007 by The American Association of Immunologists, Inc.

TLR2-Mediated Survival of Staphylococcus aureus in Macrophages: A Novel Bacterial Strategy against Host Innate Immunity1

Ikuko Watanabe*, Manami Ichiki{dagger}, Akiko Shiratsuchi*,{dagger} and Yoshinobu Nakanishi2,*,{dagger}

* Graduate School of Medical Science and {dagger} Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TLR2 plays a role as a pattern-recognition receptor in the innate immune response involving secreted proteins against microbial pathogens. To examine its possible involvement in the cellular response, we determined the levels of the engulfment and subsequent killing of bacteria by macrophages prepared from TLR2-deficient and wild-type mice. The level of the engulfment of Staphylococcus aureus or Escherichia coli was almost the same between TLR2-lacking and wild-type macrophages. However, the colony-forming ability of engulfed S. aureus, but not of E. coli, decreased to a greater extent in TLR2-lacking macrophages than in the wild-type control. The incubation with S. aureus caused activation of JNK in wild-type macrophages but not in TLR2-lacking macrophages, and the pretreatment of wild-type macrophages with a JNK inhibitor increased the rate of killing of engulfed S. aureus, but again not of E. coli. In addition, the number of colonies formed by engulfed S. aureus increased in the JNK-dependent manner when TLR2-lacking macrophages were pretreated with LPS. Furthermore, JNK seemed to inhibit the generation of superoxide, not of NO, in macrophages. These results collectively suggested that the level of superoxide is reduced in macrophages that have engulfed S. aureus through the actions of TLR2-activated JNK, resulting in the prolonged survival of the bacterium in phagosomes. The same regulation did not influence the survival of E. coli, because this bacterium was more resistant to superoxide than S. aureus. We propose a novel bacterial strategy for survival in macrophages involving the hijacking of an innate immune receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The coordinated actions of innate and adaptive immunity protect mammals against infectious diseases, with innate immunity at the front line of host defense (1). Innate immune responses consist of humoral and cellular reactions both of which are initiated through the actions of innate immune receptors that specifically recognize invading microbes and alert the body to their presence (2). The nature of such immune receptors and corresponding microbial ligands has been characterized: they are often called pattern-recognition receptor(s) (PRR)3 and pathogen-associated molecular pattern(s) (PAMP), respectively (2). TLR are representative of PRR responsible for the induction of responses that direct the production of secreted factors or cytokines and consist of 12 structurally related proteins, each of which basically recognizes a distinct PAMP present at either the periphery or the inside of microbes (3). There is another group of PRR in charge of the surveillance of microbes that have entered immune cells: such PRR include nucleotide-binding oligomerization domain proteins, NACHT-LRR-PYD domain proteins, and neuronal apoptosis inhibitor proteins that share some structural features with each other (4, 5, 6). All these PRR, when they are bound by corresponding PAMP, activate signaling pathways in immune cells. Such pathways lead to the activation of transcription factors, being exemplified by NF-{kappa}B, AP-1, and IFN regulatory factor, that in turn induce transcription of genes coding for a variety of proteins including inflammatory cytokines and IFN (3). These newly synthesized proteins evoke various reactions against invading microbes.

In contrast, our understanding of the mechanisms of cellular innate immune responses has been relatively poor. Bacteria are phagocytosed by macrophages and neutrophils, the professional phagocytes, and this phagocytosis is often accomplished with the aid of proteins present in serum, such as C components, collectins, C-reactive protein, and thrombospondin (7, 8, 9). These serum proteins bind to the surface of bacteria and at the same time to phagocytes; this action to bridge phagocytes and targets is called opsonization. Characterization of receptors that recognize bacteria-bound opsonins has not progressed far enough, although members of the C receptor, collectin receptor, and integrin are presumably in charge (7, 8, 9). In contrast, there is another type of phagocytosis receptor, including the macrophage mannose receptor and class A scavenger receptor, that directly recognizes bacterial components (7, 8, 9). Entry into host cells is not obligatory for most bacteria, but some appear to actively enter host cells to evade host immunity though how they are incorporated into or actively enter host cells is mostly unclear (10).

It is still not clear whether PRR that play roles in the induction of innate immune responses involving secreted proteins also regulate other responses including cellular ones. This issue was first addressed several years ago; Underhill et al. (11) suggested that TLR2 recognizes microbes that are engulfed and exist in phagosomes to evoke humoral innate immune responses in macrophages. We (12) and other investigators (13) more recently indicated that TLR2 and TLR4 are involved in the phagocytosis of apoptotic cells and bacteria not at the engulfment but at the step of phagosome maturation, though the data provided by the two groups are not completely consistent. TLR regulation of phagocytosis appears to be important for Ag presentation by dendritic cells (14, 15, 16). In contrast, another research group reported that the maturation of phagosomes proceeds independently of either TLR2 or TLR4 (17). More recent studies showed that TLR2 plays roles in the phagocytosis of amyloid beta peptide by microglia (18), a phagocyte specifically located in the brain, and of fungi by macrophage (19), and that TLR4 is involved in the phagocytosis of bacteria by enterocytes (20), a nonprofessional phagocyte. These results have led us to anticipate that TLR participate in the regulation of not only humoral responses but also cellular responses, in particular phagocytosis. Another important issue to be resolved is whether all TLR act as innate immune receptors to stimulate immunity. Although it is generally accepted that TLR2 is an immunostimulatory receptor against invading pathogenic microbes such as Gram-positive bacteria (21), some types of microbes seem to activate TLR2 so that immunosuppressive reactions are induced (22, 23, 24). To clarify the role of TLR2 in the cellular innate immune response against pathogenic microbes, we here examined the involvement of TLR2 in the phagocytosis and subsequent killing of bacteria by macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Chemicals

Ab recognizing both phosphorylated and unphosphorylated forms of JNK, ERK1/ERK2, and p38, and those specifically recognizing the phosphorylated forms were purchased from Cell Signaling Technology. Anti-phosphorylated c-Jun Ab was obtained also from Cell Signaling Technology. FITC was from Molecular Probes. LPS (from Salmonella serotype enteritidis or Escherichia coli 0111:B4), SP600125, and N-acetyl-L-cysteine were obtained from Sigma-Aldrich. Diogenes was purchased from National Diagnostics.

Maintenance of animals and cells

All experiments involving animals were conducted according to the protocols that had been approved by the Committee on Animal Experimentation (Kanazawa University, Kanazawa, Japan). Mice carrying a disrupted tlr2, a gene coding for TLR2, in a C57BL/6 background (21) (provided by Dr. S. Akira, Research Institute for Microbial Diseases, Osaka University, Suita, Japan) and those with a normal tlr2 (wild-type control) were used throughout the study. Macrophages were prepared from peritoneal fluids of thioglycolate-administered mice as described previously (25) and maintained on coverslips with RPMI 1640 medium containing 10% (v/v) heat-inactivated FBS at 37°C with 5% (v/v) CO2 in air until used. The Staphylococcus aureus strain Smith (provided by Dr. K. Sekimizu, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan) or NCTC8325 that carries the plasmid pND50 containing a gene for chloramphenicol resistance (provided by Dr. K. Kurokawa, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan) and the E. coli strain W3110 (provided by Dr. K. Sekimizu) that had been manipulated to become resistant to kanamycin were grown with Luria broth, and those at the logarithmic phase were used in all experiments. We conducted most experiments with the two S. aureus strains, except for a colony-forming assay where only drug-resistant NCTC8325 was used, and essentially obtained similar results. Surface labeling of bacteria with FITC was done according to a standard procedure (26). The bacteria were harvested and suspended with 0.1 M carbonate buffer (pH 9.5) containing FITC (1 mg/ml); the mixture was incubated for 30 min at room temperature. The bacteria were collected by centrifugation, washed three times with PBS, suspended with PBS, and used as targets in assays of phagocytosis.

Assays for macrophage phagocytosis of bacteria and colony-forming ability of engulfed bacteria

Thioglycolate-elicited mouse peritoneal macrophages maintained on coverslips with serum-free RPMI 1640 medium were mixed with FITC-labeled bacteria at the ratios indicated in figure legends and the mixture was incubated at 37°C with 5% CO2 in air for the periods indicated in figure legends. Reactions for the determination of the extent of phagocytosis were conducted in the presence of chloroquine (100 µM), which prevents degradation of engulfed bacteria in lysosomes of macrophages (12). After incubation, the medium containing unincorporated bacteria was discarded and the samples were supplemented with PBS and agitated using a pipette to detach bacteria from the surface of macrophages. They were finally examined by fluorescence microscopy for the level of phagocytosis. To determine the colony-forming ability of engulfed bacteria, macrophages were incubated with unlabeled bacteria and washed as described above: note that we were unable to use antibiotics to kill bacteria present outside of macrophages because engulfed bacteria died quickly during the period necessary for the action of antibiotics. We determined beforehand the ratios of macrophages to S. aureus (NCTC8325) and E. coli so that almost the same number of the two bacteria strains was incorporated into macrophages. Macrophages, after being incubated with bacteria and washed, were lysed by treatment with water. The lysates containing bacteria were plated at serial dilutions on agar-solidified Luria broth containing chloramphenicol (12.5 µg/ml; for S. aureus) or 2x YT medium containing kanamycin (10 µg/ml; for E. coli). The plates were incubated at 37°C for a day or two and the number of colonies was determined.

Western blotting

The levels of either phosphorylated or unphosphorylated MAPK and c-Jun were determined by Western blotting, as described previously (27). In brief, macrophages were lysed by incubation with buffer containing SDS and inhibitors of phosphatases and proteases; proteins were subjected to SDS-PAGE. The separated proteins were transferred to polyvinylidene difluoride membranes and reacted with Ab. Signals were visualized by chemiluminescence reactions and data were processed using Fluor-S MultiImager (Bio-Rad).

Assay for determination of NO and superoxide

NO was determined by the Griess assay. Thioglycolate-elicited mouse peritoneal macrophages maintained on coverslips with serum-free RPMI 1640 medium were mixed with unlabeled bacteria and the mixture was incubated for 1 h at 37°C with 5% CO2 in air and centrifuged. The supernatants were collected and supplemented with the Griess reagent consisting of sulfanilamide (0.25% (w/v)) and N-1-naphthylethylenediamine (0.025% (w/v)), and the mixture was incubated for 10 min at 30°C. Absorbance was measured at 540 nm and compared with a standard curve prepared with sodium nitrite. To determine the level of superoxide, culture supernatants (20 µl) of macrophages that had been incubated with bacteria were prepared as described above and mixed with Diogenes (100 µl), a superoxide chemiluminescent enhancer, and chemiluminescence in the samples was subsequently measured in a luminometer for the relative amounts of superoxide. In some experiments, the amount of superoxide, as equivalent to hydrogen peroxide, was determined using a standard curve prepared with hydrogen peroxide.

Data processing and statistical analysis

Data are representative of at least two independent experiments that yielded similar results. Data from quantitative analyses are expressed as the mean ± SD (n > 3). Statistical analyses were performed using the Student t test and p values of <0.05 were considered significant. The data significantly different from controls are marked with asterisks.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Accelerated killing of S. aureus, but not of E. coli, after phagocytosis by TLR2-lacking macrophages

We compared the level of phagocytosis of bacteria by macrophages prepared from TLR2-deficient mice and wild-type controls. To assure the accuracy of this assay, we first conducted a control experiment. For this purpose, S. aureus (Smith) surface labeled with FITC were mixed with thioglycolate-elicited peritoneal macrophages of wild-type mice and the mixtures were incubated for 30 min in the presence of an inhibitor of the lysosomal action. The samples were then agitated with a pipette and the supernatants were discarded. A biotinylating reagent was added to the remaining samples for the surface labeling of unincorporated bacteria and macrophages. They were then supplemented with Alexa 546-conjugated streptavidin and examined by fluorescence microscopy. This procedure enabled us to discriminate engulfed bacteria from those residing outside macrophages, which were respectively fluoresceinated with FITC alone and both FITC and Alexa 546, and we found that most bacteria existed within macrophages after washing (Fig. 1A). Adequateness of this procedure was further confirmed by extinguishing fluorescence derived from unengulfed bacteria using crystal violet, a quenching dye (data not shown). These results indicated that the assay used in this study allows us to analyze only bacteria that have been phagocytosed by macrophages. Assays of phagocytosis were then performed with peritoneal macrophages of TLR2-deficient and wild-type mice using FITC-labeled S. aureus (Smith and NCTC8325) or E. coli as targets. Lack of the expression of tlr2 did not influence either the ratio of macrophages that had phagocytosed bacteria or the number of bacteria that had been incorporated into each macrophage (Fig. 1B). We then compared the rate of killing of bacteria after phagocytosis by macrophages of the mutant and wild-type mice. To do so, macrophages after the phagocytosis reactions were lysed and engulfed bacteria released from macrophages were recovered and examined for the ability to form colonies on agar plates. We found that the level of the colony-forming activity of S. aureus (NCTC8325) recovered from TLR2-lacking macrophages was always lower than that of the bacteria obtained from wild-type macrophages, while no such difference was observed when E. coli were used as targets of phagocytosis (Fig. 2). These results suggested that TLR2 participates in the regulation of phagocytosis of bacteria by macrophages, not at the recognition or engulfment step but at the step of processing of engulfed bacteria. This regulation by TLR2 seemed to be beneficial to bacteria and dependent on the strain of bacteria; TLR2 helped engulfed S. aureus but not E. coli to remain alive longer in macrophages.


Figure 1
View larger version (65K):
[in this window]
[in a new window]

  		FIGURE 1. No effect of TLR2 deficiency on macrophage phagocytosis of S. aureus or E. coli. A, Wild-type macrophages were incubated with FITC-labeled S. aureus (Smith) (macrophages:bacteria = 1:30) for 30 min at 37°C. The mixtures were treated with a biotinylating reagent before (before) and after (after) a wash to remove unincorporated bacteria and supplemented with Alexa 546-conjugated streptavidin followed by examination by fluorescence microscopy. Phase contrast and fluorescent views of the same microscopic fields are shown in each row and the insets are magnified views of portions of the cells. The arrowheads point to unincorporated bacteria residing outside of macrophages. Scale bars, 3 µm. B, Wild-type (WT) ({circ}) or TLR2-lacking (tlr2–/–) (•) macrophages were incubated with bacteria (macrophages:bacteria = 1:30 for Smith, 1:100 for NCTC8325, and 1:1000 for E. coli) for the indicated period of time at 37°C and washed. The extent of phagocytosis was determined under a fluorescence microscope by measuring the ratio of macrophages that had phagocytosed bacteria (in percentage terms: the phagocytic index) or the number of bacteria engulfed by 100 macrophages. Fluorescence microscopic views of wild-type macrophages after the phagocytosis reactions are shown at the top. Scale bars, 10 µm. Data are representative of three (with S. aureus) and two (with E. coli) independent experiments that yielded similar results.

 

Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 2. Accelerated killing of engulfed S. aureus in TLR2-lacking macrophages. Wild-type (WT) ({circ}) or TLR2-lacking (tlr2–/–) (•) macrophages were incubated with unlabeled bacteria (macrophages:bacteria = 1:740 for S. aureus (NCTC8325) and 1:100 for E. coli) for 30 min at 37°C and washed. The macrophages were further incubated for the indicated period of time and lysed; the recovered bacteria were subjected to a colony-forming assay. The number of colonies formed on agar plates was determined and is shown relative to that at time 0. Data are representative of three (with S. aureus) and four (with E. coli) independent experiments that yielded similar results.

 
Involvement of JNK in TLR2-mediated mitigation of killing of engulfed S. aureus

To know how TLR2 regulates the killing of engulfed S. aureus in macrophages, we examined the involvement of MAPK. We first determined the level of phosphorylated MAPK in macrophages before and after the phagocytosis of S. aureus or E. coli (Fig. 3). We found that the level of the phosphorylated forms of all three MAPK, JNK, ERK1/ERK2, and p38, increased in wild-type macrophages upon incubation with S. aureus. When macrophages of TLR2-deficient mice were similarly analyzed, the level of the phosphorylated JNK remained low even after incubation with S. aureus while the activation of ERK1/ERK2 and p38 occurred normally. In contrast, JNK as well as ERK and p38 seemed to be effectively phosphorylated in TLR2-lacking macrophages when they were incubated with E. coli. These results suggested that of the three MAPK only JNK is phosphorylated, thus activated, in S. aureus-stimulated macrophages in a manner dependent on TLR2.


Figure 3
View larger version (49K):
[in this window]
[in a new window]

  		FIGURE 3. TLR2-dependent phosphorylation of JNK in macrophages upon incubation with S. aureus. Wild-type (WT) or TLR2-lacking (tlr2–/–) macrophages were incubated with unlabeled S. aureus (Smith, NCTC8325) or E. coli (macrophages:bacteria = 1:10) for the indicated period of time at 37°C and washed. Whole cell lysates of the macrophages were then prepared and analyzed by Western blotting for the presence of the phosphorylated (P) and total (T) forms of the three MAPK, JNK, ERK1/ERK2, and p38. Data are representative of two independent experiments that yielded similar results.

 
We then examined whether JNK is involved in TLR2-mediated survival of engulfed S. aureus in macrophages. For this purpose, we first tested the effect of the chemical JNK inhibitor SP600125. The presence of this compound almost completely inhibited the phosphorylation of c-Jun, a target of JNK, in wild-type macrophages that had been incubated with either S. aureus or E. coli (Fig. 4A). We then compared the extent of phagocytosis of bacteria and their killing by wild-type macrophages in the absence and presence of this inhibitor. The data showed that the level of the phagocytosis of either S. aureus or E. coli did not change irrespective of the addition of the inhibitor (Fig. 4B), whereas the colony-forming activity of engulfed S. aureus (NCTC8325) but not E. coli was significantly reduced in its presence (Fig. 4C). These results suggested that the action of JNK is necessary not for the engulfment of S. aureus or E. coli but for the regulation of killing of engulfed S. aureus in macrophages. We further confirmed this by testing the possibility that forced activation of JNK in TLR2-lacking macrophages leads to an increase in the colony-forming ability of engulfed S. aureus. Macrophages lacking the expression of tlr2 were incubated with LPS, a ligand for TLR4, and examined for the level of phosphorylated JNK. We found that the phosphorylation of JNK in TLR2-deficient macrophages was stimulated after the incubation with LPS (Fig. 5A). We then determined the level of killing of engulfed S. aureus (NCTC8325) in the mutant macrophages that had been pretreated with LPS. The data clearly showed that CFU of the engulfed bacteria was raised in LPS-treated macrophages and this stimulation was almost abolished in the presence of the JNK inhibitor (Fig. 5B). These results collectively indicated that the activation of JNK is sufficient for an increase in the rate of survival of engulfed S. aureus in macrophages.


Figure 4
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 4. Requirement for JNK in TLR2-mediated inhibition of the killing of engulfed S. aureus in macrophages. A, Wild-type macrophages were incubated with unlabeled S. aureus (Smith) or E. coli (macrophages:bacteria = 1:10) for 1 h (with S. aureus) or 15 min (with E. coli) at 37°C in the presence of the JNK inhibitor SP600125 (10 µM) or DMSO (solvent alone) and washed. Whole cell lysates of the macrophages were analyzed for the level of phosphorylated c-Jun (P-c-Jun) by Western blotting. Data are representative of two independent experiments that yielded similar results. B, Wild-type macrophages were incubated with FITC-labeled bacteria (macrophages:bacteria = 1:30 for Smith, 1:100 for NCTC8325, and 1:1000 for E. coli) for 1 h at 37°C in the presence of the JNK inhibitor SP600125 or DMSO and washed. The macrophages were examined by fluorescence microscopy for the extent of phagocytosis. Data are representative of three (with S. aureus) and five (with E. coli) independent experiments that yielded similar results. C, Wild-type macrophages were incubated with unlabeled bacteria (macrophages:bacteria = 1:740 for S. aureus (NCTC8325) and 1:100 for E. coli) for 30 min at 37°C in the presence of the JNK inhibitor SP600125 or DMSO and washed. The macrophages were further incubated for 1 h at 37°C in the absence and presence of SP600125 and lysed; the recovered bacteria were subjected to a colony-forming assay. The number of colonies is shown relative to that at time 0. Data are representative of eight (with S. aureus) and three (with E. coli) independent experiments that yielded similar results.

 

Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 5. Prolonged survival of engulfed S. aureus in TLR2-lacking macrophages by treatment with LPS. A, TLR2-lacking macrophages were preincubated with LPS (0.1 mg/ml) or left untreated (none) for 10 min at 37°C and whole cell lysates of the macrophages were analyzed for the levels of the phosphorylated (P) and all (T) forms of JNK by Western blotting. Data are representative of two independent experiments that yielded similar results. B, TLR2-lacking macrophages that had been pretreated with LPS (0.1 mg/ml) in the presence of the JNK inhibitor SP600125 (10 µM) or solvent alone (DMSO), or left untreated (none) for 10 min at 37°C were incubated with unlabeled S. aureus (NCTC8325) (macrophages:bacteria = 1:740) for 30 min at 37°C. The mixture was washed to remove unincorporated bacteria and the macrophages were further incubated for 1 h at 37°C in the absence of LPS and lysed; the recovered bacteria were subjected to a colony-forming assay. The number of colonies is shown relative to that at time 0. Data are representative of seven independent experiments that yielded similar results.

 
Role of superoxide in TLR2/JNK regulation of the survival of engulfed S. aureus

We next tried to identify a direct effector(s) that lowers the colony-forming ability of engulfed S. aureus in wild-type macrophages. To examine whether newly synthesized proteins are needed for this regulation, a colony-forming assay was conducted in the absence and presence of the protein synthesis inhibitor cycloheximide with wild-type macrophages that had phagocytosed S. aureus. CFU of S. aureus recovered from macrophages was almost the same irrespective of the presence of the inhibitor (data not shown). The involvement of lysosomal enzymes was next examined by incubating the macrophages with ammonium chloride, which inhibits the acidification of lysosomes. However, this treatment had no effect on the colony formation by engulfed bacteria (data not shown). We next examined the involvement of NO, one of key molecules responsible for the killing of bacteria by macrophages (28), in the JNK-mediated inhibition of killing of engulfed S. aureus. The production of NO in macrophages was induced by the incubation with either S. aureus or E. coli (Fig. 6A), but the levels of NO released from wild-type and TLR2-deficient macrophages were almost the same (Fig. 6B). We then determined the level of NO produced by wild-type macrophages in the presence of the JNK inhibitor SP600125. The presence of this compound caused a significant reduction in the level of NO in macrophages that had been incubated with E. coli or LPS while NO production in S. aureus-challenged macrophages was minimally affected (Fig. 6C). These results suggested that JNK positively regulates the synthesis of NO in macrophages that have engulfed S. aureus. However, NO is unlikely to be involved in the TLR2/JNK-mediated inhibition of killing of engulfed S. aureus, because an increase of NO, if it occurs at all, is opposite to prolonging the survival time of bacteria in macrophages. The above data collectively indicated that JNK regulation of the survival of engulfed S. aureus in macrophages does not require newly synthesized proteins, lysosomal function, or NO.


Figure 6
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 6. JNK-mediated production of NO in E. coli- but not S. aureus-phagocytosing macrophages. A, Wild-type macrophages (105 cells) were incubated with the indicated numbers of unlabeled S. aureus (NCTC8325) or E. coli for 1 h at 37°C and the culture supernatants were analyzed for the level of NO. Data are representative of six (with S. aureus) and two (with E. coli) independent experiments that yielded similar results. B, Wild-type (WT) or TLR2-lacking (tlr2–/–) macrophages were incubated with unlabeled S. aureus (Smith, NCTC8325) or E. coli (macrophages:bacteria = 1:1–2 x 105) for 1 h at 37°C; the supernatants were analyzed for the level of NO. The level of NO is shown relative to that with wild-type macrophages. Data are representative of two independent experiments that yielded similar results. C, Wild-type macrophages were incubated with unlabeled S. aureus (Smith, NCTC8325) or E. coli (macrophages:bacteria = 1: 1–2 x 105) for 1 h or with LPS (0.1 mg/ml) for 48 h, as described previously (46 ), at 37°C in the presence of SP600125 (50 µM) or DMSO (solvent alone); the supernatants were analyzed for the level of NO. The level of NO is shown relative to that in the reaction with no additives. Data are representative of three (with S. aureus), four (with E. coli), or four (with LPS) independent experiments that yielded similar results.

 
We next examined the possible involvement of superoxide, another key molecule for macrophage killing of bacteria (28), in the prolonged survival of S. aureus in wild-type macrophages. Wild-type macrophages produced superoxide in response to incubation with S. aureus (Fig. 7A). The colony-forming ability of S. aureus recovered from wild-type macrophages that had been maintained in the presence of N-acetyl-L-cysteine, a superoxide scavenger, was determined. The rate of killing of engulfed S. aureus was reduced in the presence of this scavenger (Fig. 7B), indicating a role for superoxide in killing of the bacteria in macrophages. We then compared the level of superoxide in culture supernatants of wild-type and TLR2-lacking macrophages that had phagocytosed S. aureus. TLR2-lacking macrophages produced more superoxide than wild-type macrophages upon phagocytosis of S. aureus (Fig. 7C). We then examined the involvement of JNK in the production of superoxide by macrophages. For this purpose, wild-type or the mutant macrophages that had phagocytosed S. aureus were maintained in the absence and presence of SP600125 and the level of superoxide in culture supernatants was determined. We found that the addition of the JNK inhibitor brought about an increase of superoxide production in S. aureus-phagocytosing wild-type macrophages but not in TLR2-lacking macrophages (Fig. 7D). Furthermore, the treatment with LPS, which led to the phosphorylation of JNK independent of TLR2 (see Fig. 5A), caused a reduction in the generation of superoxide in TLR2-lacking macrophages, and this effect was completely abolished in the presence of the JNK inhibitor (Fig. 7E). All together, superoxide has become a likely candidate for the substance responsible for the TLR2/JNK regulation of killing of engulfed S. aureus in macrophages. Finally, a possible difference in the sensitivity to superoxide between S. aureus and E. coli was tested. To do so, the bacteria were treated with hydrogen peroxide and the viability was examined by a colony-forming assay. We found that E. coli were much more resistant to this treatment than S. aureus (Table I), explaining why TLR2/JNK-mediated inhibition of superoxide production did not influence the colony-forming activity of engulfed E. coli.


Figure 7
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 7. JNK-mediated inhibition of superoxide generation in macrophages. A, Wild-type macrophages (105 cells) were incubated with the indicated numbers of unlabeled S. aureus (Smith, NCTC8325) for 30 min at 37°C and the culture supernatants were analyzed for the level of superoxide. Data are representative of two independent experiments that yielded similar results. B, Wild-type macrophages were pretreated with N-acetyl-L-cysteine (LNAC) (2 mM) or left untreated for 1 h at 37°C and incubated with unlabeled S. aureus (NCTC8325) (macrophages:bacteria = 1:740) for 30 min at 37°C in the absence and presence of N-acetyl-L-cysteine. The samples were washed to remove unincorporated bacteria; the macrophages were further incubated for 1 h at 37°C in the absence and presence of N-acetyl-L-cysteine followed by a colony-forming assay. The number of colonies is shown relative to that at the time of a wash. Data are representative of five independent experiments that yielded similar results. C, Wild-type (WT) or TLR2-lacking (tlr2–/–) macrophages were incubated with unlabeled S. aureus (macrophages:bacteria = 1:10 for Smith and 1:1000 for NCTC8325) for 2 (with Smith) or 1 (with NCTC8325) h at 37°C; the culture supernatants were subjected to an assay of superoxide determination. The level of superoxide is shown relative to that with wild-type macrophages. Data are representative of three (with Smith) and five (with NCTC8325) independent experiments that yielded similar results. D, Wild-type (WT) or TLR2-lacking (tlr2–/–) macrophages were pretreated with SP600125 (10 µM) or DMSO (solvent alone) for 1 h at 37°C and incubated with unlabeled S. aureus (macrophages:bacteria = 1:1000 for Smith and 1:740 for NCTC8325) for 30 min at 37°C in the absence and presence of SP600125. The culture supernatants were then subjected to an assay of superoxide determination. The level of superoxide is shown relative to that in the samples prepared in the absence of SP600125. Data are representative of three (with wild-type macrophages and Smith), five (with wild-type macrophages and NCTC8325), and three (with mutant macrophages and NCTC8325) independent experiments that yielded similar results. E, TLR2-lacking macrophages that had been pretreated with LPS (0.1 mg/ml) in the presence of the JNK inhibitor SP600125 (10 µM) or solvent alone (DMSO) or left untreated (none) for 10 min at 37°C and washed. The macrophages were further incubated with unlabeled S. aureus (NCTC8325) (macrophages:bacteria = 1:740) for 30 min at 37°C; the culture supernatants were subjected to an assay of superoxide determination. The level of superoxide is shown relative to that in the samples prepared without treatment with LPS. Data are representative of three independent experiments that yielded similar results.

 

View this table:
[in this window]
[in a new window]

  		Table I. Resistance of E. coli to hydrogen peroxide

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have confirmed the previous suggestion that TLR2 is involved in the phagocytosis of invading microbes (11, 13, 14, 19). However, our data indicated that TLR2 acts not to accelerate the elimination of bacteria but actually to prolong the survival of engulfed bacteria in macrophages. This is in accord with the previous observation that TLR2 responds to pathogenic microbes and activates signaling pathways that lead to the production of the immunosuppressive cytokine IL-10 (22, 23). Moreover, it was recently reported the Porphyromonas gingivalis, a Gram-negative bacterium, is cleared in TLR2-deficient mice more rapidly than in wild-type mice (24). It is therefore possible that TLR2 is an immunosuppressive PRR, in both humoral and cellular innate immune responses, at least for certain types of microbes. However, it remains to be clarified whether TLR2-mediated survival of engulfed bacteria is accomplished as a part of the host immune response or as a bacterial strategy to effectively propagate in the host organism. To gain some insight into this issue, it is necessary to determine whether bacteria that have been afforded a longer survival time actively propagate within macrophages. In addition, the microbial ligands, which bind to TLR2 and activate it for the induction of immunosuppressive responses, need to be identified and characterized. Peptidoglycan (18) and lipoproteins (29) of the Gram-positive bacterium S. aureus, and {alpha}-glucan of the fungus Pseudallescheria boydii (30), have been proposed to be such ligands for TLR2 as a cellular innate immune receptor, although S. aureus could be sensed by a receptor other than TLR2 (31). In contrast, a protein named LcrV that is released from the Gram-negative bacterium Yersinia could serve as an immunosuppressive ligand for TLR2 (22). It is necessary to identify a S. aureus ligand that binds to and activates TLR2 for the inhibition of superoxide generation in macrophage phagosomes.

We showed in this study that having recognized S. aureus TLR2 activates the MAPK pathway to suppress the production of superoxide, resulting in the prolonged survival of engulfed bacteria. Similar findings have been reached by other investigators in a study on the killing of engulfed Candida albicans in macrophages, though the mechanism by which TLR2 inhibits the killing of this fungus is unknown (23). Reactive oxygen species including superoxide are produced by an active process involving the actions of the NADPH oxidase family of proteins (8, 28, 32). Upon the engulfment of microbes, a protein complex responsible for the production of superoxide is formed on the membrane of phagosomes. This complex consists of the phagocyte type NADPH oxidase Nox2, several phosphorylated proteins, and the small G protein Rac. We found that the activation of JNK resulted in a reduction in the level of superoxide production. This is most probably achieved through the inhibition of either the formation of the oxidase complex or the activity of particular components, with no need for newly synthesized proteins. There is an example of regulated formation of the oxidase complex in macrophages; effective association of p47phox, a cytosolic regulator of superoxide generation by the complex, with mycobacteria-containing phagosomes seems to require the actions of C5/C5a receptor and protein kinase C (33). Further investigation is necessary to clarify the mechanism of JNK-mediated negative regulation of superoxide production in microbe-containing phagosomes. Whatever the mechanism is, this regulation does not seem to be restricted to the function of TLR2 because we showed that the treatment of TLR2-lacking macrophages with LPS, which most probably caused TLR4-mediated activation of JNK, brought about the inhibition of superoxide production. Because many types of TLR activate signaling pathways leading to the phosphorylation of MAPK including JNK (3), any microbes recognized by TLR could cause an inhibition of superoxide production in phagosomes. Whether this regulation determines the fate of engulfed microbes should depend on how sensitive the microbes are to superoxide. In fact, the engulfment of E. coli also caused the JNK-mediated inhibition of superoxide production in macrophages, but the colony-forming ability of engulfed E. coli, which were more resistant to superoxide than S. aureus, remained the same before and after JNK activation.

Bacteria modulate both humoral and cellular responses by host immune cells (8, 34, 35, 36, 37) and host cells contrarily appear to possess a strategy to counteract these bacterial actions (38). Examples of the bacterial regulation of host humoral reactions are: inhibition of signaling pathways that lead to the production of proinflammatory cytokines, modification of signaling pathways so that anti-inflammatory cytokines are produced, changes in the structure of PAMP so that they are not bound by PRR, and prevention of bacterial component from being presented as Ag. There are also several examples of the inhibition of phagocytosis: bacteria inhibit the engulfment step, sneak out of phagosomes, and prevent the maturation of phagosomes. We have added another example to this list. Our findings are unique in that bacteria use the action of PRR to resist host immune responses. A recent report showed that Porphyromonas gingivalis activates integrin though the action of TLR2 so that the bacteria more efficiently internalize macrophages (39). However, it remains to be determined whether this action of the bacteria promotes their survival in macrophages. It is not known whether these modes of regulation to seemingly suppress immunity are a bacterial strategy to evade the host immune system or are still a part of the host defense mechanism. Engulfed cells, either invading microbes or altered self, i.e., apoptotic cells, undergo two degradation processes: killing by reactive nitrogen intermediates or reactive oxygen species and digestion by lysosomal enzymes. Both processes proceed only within phagosomes under strict regulation. One explanation for a delay in the killing and digestion of engulfed cells as a host defense mechanism is that prolonged existence of the engulfed materials allows phagocytes to use their components; some types of phagocytes process engulfed materials and use them for the defense mechanism, Ag presentation for the induction of adaptive immunity as an example (40). In fact, the rate of phagosome maturation, that is, the killing and digestion of engulfed cells, appears to be different between Ag-presenting phagocytes and those without such an activity (41). In contrast, it has been suggested that TLR2 plays a role in dendritic cells, not in the engulfment or phagosome maturation step, but in the inspection of engulfed materials, asking whether they are invading microbes (14, 15, 16).

It is not known what receptor defines the phagocytosis of S. aureus by macrophages in this study. Phagocytosis reactions were conducted in the absence of sera and therefore the involvement of serum opsonins is unlikely. It is not probable that TLR2 itself serves as a phagocytosis receptor, because the level of phagocytosis of S. aureus did not differ between wild-type and TLR2-lacking macrophages. Drosophila membrane proteins Croquemort (42) and Eater (43), and mammalian class B scavenger receptors (43, 44), have been suggested to be opsonin-independent phagocytosis receptors against S. aureus, although the corresponding bacterial ligand(s) remains to be identified. Therefore, a member of the class B scavenger receptor family of proteins is a likely candidate for the receptor responsible for the phagocytosis of S. aureus by macrophages. Opsonin-dependent phagocytosis receptors such as Fc{gamma}R activate signaling pathways involving a cascade of protein phosphorylation (7). Characterization of downstream effectors for opsonin-independent phagocytosis receptors has not been fully achieved, but the involvement of MAPK has been suggested for some receptors including a class B scavenger receptor (27, 45). It is therefore possible that signaling pathways located downstream of phagocytosis receptors and PRR have cross-talk to induce the engulfment and define the fate of engulfed target cells. Many issues remain to be resolved before we will completely understand the mechanism and role of PRR-mediated regulation of the phagocytosis of invading microbial pathogens and altered self as a cellular innate immune response.


   Acknowledgments
 
We thank Hideki Sumimoto (Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan) and Kohei Kakishima (Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan) for suggestions regarding the assays for the determination of superoxide and NO, respectively. We also thank Kenji Kurokawa and Kohei Kakishima for valuable discussions. We are grateful to Shizuo Akira Kazuhisa Sekimizu and Kenji Kurokawa for materials.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (No. 16570112), an institutional research grant from Kanazawa University (No. 1715102), and a grant from the Naito Memorial Foundation and in part by the Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (No. 18570123) and Ministry of Education, Culture, Sports, Science and Technology (No. 18057009). Back

2 Address correspondence and reprint requests to Dr. Yoshinobu Nakanishi, Graduate School of Medical Science, Kanazawa University, Shizenken, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan. E-mail address: nakanaka{at}kenroku.kanazawa-u.ac.jp Back

3 Abbreviations used in this paper: PRR, pattern-recognition receptor; PAMP, pathogen-associated molecular pattern. Back

Received for publication October 11, 2006. Accepted for publication February 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Janeway, C. A., Jr. 2001. How the immune system works to protect the host from infection: a personal view. Proc. Natl. Acad. Sci. USA 98: 7461-7468. [Free Full Text]
  2. Janeway, C. A., Jr, R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197-216. [Medline]
  3. Akira, S., S. Uematsu, O. Takeuchi. 2006. Pathogen recognition and innate immunity. Cell 124: 783-801. [Medline]
  4. Chamaillard, M., S. E. Girardin, J. Viala, D. J. Philpott. 2003. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell. Microbiol. 5: 581-592. [Medline]
  5. Martinon, F., J. Tschopp. 2005. NLRs join TLRs as innate sensors of pathogens. Trends Immunol. 26: 447-454. [Medline]
  6. Inohara, N., M. Chamaillard, C. McDonald, G. Nuñez. 2005. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu. Rev. Biochem. 74: 355-383. [Medline]
  7. Aderem, A., D. M. Underhill. 1999. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17: 593-623. [Medline]
  8. Underhill, D. M., A. Ozinsky. 2002. Phagocytosis of microbes: complexity in action. Annu. Rev. Immunol. 20: 825-852. [Medline]
  9. Stuart, L. M., R. A. B. Ezekowitz. 2005. Phagocytosis: elegant complexity. Immunity 22: 539-550. [Medline]
  10. Gruenberg, J., F. G. van der Goot. 2006. Mechanisms of pathogen entry through the endosomal compartments. Nat. Rev. Mol. Cell Biol. 7: 495-504. [Medline]
  11. Underhill, D. M., A. Ozinsky, A. M. Hajjar, A. Stevens, C. B. Wilson, M. Bassetti, A. Aderem. 1999. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature 401: 811-815. [Medline]
  12. Shiratsuchi, A., I. Watanabe, O. Takeuchi, S. Akira, Y. Nakanishi. 2004. Inhibitory effect of Toll-like receptor 4 on fusion between phagosomes and endosomes/lysosomes in macrophages. J. Immunol. 172: 2039-2047. [Abstract/Free Full Text]
  13. Blander, J. M., R. Medzhitov. 2004. Regulation of phagosome maturation by signals from Toll-like receptors. Science 304: 1014-1018. [Abstract/Free Full Text]
  14. Blander, J. M., R. Medzhitov. 2006. Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature 440: 808-812. [Medline]
  15. Blander, J. M.. 2006. Coupling Toll-like receptor signaling with phagocytosis: potentiation of antigen presentation. Trends Immunol. 28: 19-25. [Medline]
  16. Blander, J. M.. 2007. Signalling and phagocytosis in the orchestration of host defence. Cell. Microbiol. 9: 290-299. [Medline]
  17. Yates, R. M., D. G. Russell. 2005. Phagosome maturation proceeds independently of stimulation of Toll-like receptors 2 and 4. Immunity 23: 409-417. [Medline]
  18. Chen, K., P. Iribarren, J. Hu, J. Chen, W. Gong, E. H. Cho, S. Lockett, N. M. Dunlop, J. M. Wang. 2006. Activation of Toll-like receptor 2 on microglia promotes cell uptake of Alzheimer disease-associated amyloid beta peptide. J. Biol. Chem. 281: 3651-3659. [Abstract/Free Full Text]
  19. Luther, K., A. Torosantucci, A. A. Brakhage, J. Heesemann, F. Ebel. 2007. Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 beta-glucan receptor and Toll-like receptor 2. Cell. Microbiol. 9: 368-381. [Medline]
  20. Neal, M. D., C. Leaphart, R. Levy, J. Prince, T. R. Billiar, S. Watkins, J. Li, S. Cetin, H. Ford, A. Schreiber, D. J. Hackam. 2006. Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier. J. Immunol. 176: 3070-3079. [Abstract/Free Full Text]
  21. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11: 443-451. [Medline]
  22. Sing, A., D. Rost, N. Tvardovskaia, A. Roggenkamp, A. Wiedemann, C. J. Kirschning, M. Aepfelbacher, J. Heesemann. 2002. Yersinia V-antigen exploits Toll-like receptor 2 and CD14 for interleukin 10-mediated immunosuppression. J. Exp. Med. 196: 1017-1024. [Abstract/Free Full Text]
  23. Netea, M. G., R. Sutmuller, C. Hermann, C. A. A. Van der Graaf, J. W. M. Van der Meer, J. H. van Krieken, T. Hartung, G. Adema, B. J. Kullberg. 2004. Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells. J. Immunol. 172: 3712-3718. [Abstract/Free Full Text]
  24. Burns, E., G. Bachrach, L. Shapira, G. Nussbaum. 2006. Cutting edge: TLR2 is required for the innate response to Porphyromonas gingivalis: activation leads to bacterial persistence and TLR2 deficiency attenuates induced alveolar bone resorption. J. Immunol. 177: 8296-8300. [Abstract/Free Full Text]
  25. Shiratsuchi, A., S. Osada, S. Kanazawa, Y. Nakanishi. 1998. Essential role of phosphatidylserine externalization in apoptosing cell phagocytosis by macrophages. Biochem. Biophys. Res. Commun. 246: 549-555. [Medline]
  26. Hed, J.. 1986. Methods for distinguishing ingested from adhering particles. Methods Enzymol. 132: 198-204. [Medline]
  27. Osada, Y., A. Shiratsuchi, Y. Nakanishi. 2006. Involvement of mitogen-activated protein kinases in class B scavenger receptor type I-induced phagocytosis of apoptotic cells. Exp. Cell Res. 312: 1820-1830. [Medline]
  28. Halliwell, B.. 2006. Phagocyte-derived reactive species: salvation or suicide?. Trends Biochem. Sci. 31: 509-515. [Medline]
  29. Wardenburg, J. B., W. A. Williams, D. Missiakas. 2006. Host defenses against Staphylococcus aureus infection require recognition of bacterial lipoproteins. Proc. Natl. Acad. Sci. USA 103: 13831-13836. [Abstract/Free Full Text]
  30. Bittencourt, V. C. B., R. T. Figueiredo, R. B. da Silva, D. S. Mourão-Sá, P. L. Fernandez, G. L. Sassaki, B. Mulloy, M. T. Bozza, E. Barreto-Bergter. 2006. An {alpha}-glucan of Pseudallescheria boydii is involved in fungal phagocytosis and Toll-like receptor activation. J. Biol. Chem. 281: 22614-22623. [Abstract/Free Full Text]
  31. Mullaly, S. C., P. Kubes. 2006. The role of TLR2 in vivo following challenge with Staphylococcus aureus and prototypic ligands. J. Immunol. 177: 8154-8163. [Abstract/Free Full Text]
  32. Geiszt, M., T. L. Leto. 2004. The Nox family of NAD(P)H oxidases: host defense and beyond. J. Biol. Chem. 279: 51715-51718. [Free Full Text]
  33. Daniel, D. S., G. Dai, C. R. Singh, D. R. Lindsey, A. K. Smith, S. Dhandayuthapani, R. L. Hunter, Jr, C. Jagannath. 2006. The reduced bactericidal function of complement C5-deficient murine macrophages is associated with defects in the synthesis and delivery of reactive oxygen radicals to mycobacterial phagosomes. J. Immunol. 177: 4688-4698. [Abstract/Free Full Text]
  34. Ernst, J. D.. 2000. Bacterial inhibition of phagocytosis. Cell. Microbiol. 2: 379-386. [Medline]
  35. Pizarro-Cerdá, J., P. Cossart. 2004. Subversion of phosphoinositide metabolism by intracellular bacterial pathogens. Nat. Cell Biol. 6: 1026-1033. [Medline]
  36. Coombes, B. K., Y. Valdez, B. B. Finlay. 2004. Evasive maneuvers by secreted bacterial proteins to avoid innate immune responses. Curr. Biol. 14: R856-R867. [Medline]
  37. Rottner, K., T. E. B. Stradal, J. Wehland. 2005. Bacteria-host-cell interactions at the plasma membrane: stories on actin cytoskeleton subversion. Dev. Cell 9: 3-17. [Medline]
  38. Shintani, T., D. J. Klionsky. 2004. Autophagy in health and disease: a double-edged sword. Science 306: 990-995. [Abstract/Free Full Text]
  39. Hajishengallis, G., M. Wang, E. Harokopakis, M. Triantafilou, K. Triantafilou. 2006. Porphyromonas gingivalis fimbriae proactively modulate beta2 integrin adhesive activity and promote binding to and internalization by macrophages. Infect. Immun. 74: 5658-5666. [Abstract/Free Full Text]
  40. Ackerman, A. L., P. Cresswell. 2004. Cellular mechanisms governing cross-presentation of exogenous antigens. Nat. Immunol. 5: 678-684. [Medline]
  41. Erwig, L.-P., K. A. McPhilips, M. W. Wynes, A. Ivetic, A. J. Ridley, P. M. Henson. 2006. Differential regulation of phagosome maturation in macrophages and dendritic cells mediated by Rho GTPases and ezrin-radixin-moesin (ERM) proteins. Proc. Natl. Acad. Sci. USA 103: 12825-12830. [Abstract/Free Full Text]
  42. Stuart, L. M., J. Deng, J. M. Silver, K. Takahashi, A. A. Tseng, E. J. Hennessy, R. A. B. Ezekowitz, K. J. Moore. 2005. Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain. J. Cell Biol. 170: 477-485. [Abstract/Free Full Text]
  43. Kocks, C., J. H. Cho, N. Nehme, J. Ulvila, A. M. Pearson, M. Meister, C. Strom, S. L. Conto, C. Hetru, L. M. Stuart, et al 2005. Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila. Cell 123: 335-346. [Medline]
  44. Philips, J. A., E. J. Rubin, N. Perrimon. 2005. Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection. Science 309: 1251-1253. [Abstract/Free Full Text]
  45. Jehle, A. W., S. J. Gardai, S. Li, P. Linsel-Nitschke, K. Morimoto, W. J. Janssen, R. W. Vandivier, N. Wang, S. Greenberg, B. M. Dale, et al 2006. ATP-binding cassette transporter A7 enhances phagocytosis of apoptotic cells and associated ERK signaling in macrophages. J. Cell Biol. 174: 547-556. [Abstract/Free Full Text]
  46. Thoeni, G., P. Stoitzner, G. Brandacher, N. Romani, C. Heufler, G. Werner-Felmayer, E. R. Werner. 2005. Tetrahydro-4-aminobiopterin attenuates dendritic cell-induced T cell priming independently from inducible nitric oxide synthase. J. Immunol. 174: 7584-7591. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
R. M. McLoughlin, J. C. Lee, D. L. Kasper, and A. O. Tzianabos
IFN-{gamma} Regulated Chemokine Production Determines the Outcome of Staphylococcus aureus Infection
J. Immunol., July 15, 2008; 181(2): 1323 - 1332.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
O. S. Shin, R. R. Isberg, S. Akira, S. Uematsu, A. K. Behera, and L. T. Hu
Distinct Roles for MyD88 and Toll-Like Receptors 2, 5, and 9 in Phagocytosis of Borrelia burgdorferi and Cytokine Induction
Infect. Immun., June 1, 2008; 76(6): 2341 - 2351.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C.-L. Small, S. McCormick, N. Gill, K. Kugathasan, M. Santosuosso, N. Donaldson, D. E. Heinrichs, A. Ashkar, and Z. Xing
NK Cells Play a Critical Protective Role in Host Defense against Acute Extracellular Staphylococcus aureus Bacterial Infection in the Lung
J. Immunol., April 15, 2008; 180(8): 5558 - 5568.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Xu, H. Fang, and D. M. Frucht
Anthrax Lethal Toxin Increases Superoxide Production in Murine Neutrophils via Differential Effects on MAPK Signaling Pathways
J. Immunol., March 15, 2008; 180(6): 4139 - 4147.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
D. J. Weiss, C. D. Souza, O. A. Evanson, M. Sanders, and M. Rutherford
Bovine monocyte TLR2 receptors differentially regulate the intracellular fate of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium
J. Leukoc. Biol., January 1, 2008; 83(1): 48 - 55.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Watanabe, I.
Right arrow Articles by Nakanishi, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Watanabe, I.
Right arrow Articles by Nakanishi, Y.