Differential Roles of Toll-Like Receptors 2 and 4 in In Vitro Responses of Macrophages to Legionella pneumophila
http://www.100md.com
感染与免疫杂志 2005年第1期
First Department of Internal Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa
Research Institute for Microbial Infectious Disease, Osaka University, Osaka, Japan
ABSTRACT
The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2–/–), TLR4–/–, and wild-type (WT) littermate (C57BL/6 x 129Sv) mice. Intracellular growth of L. pneumophila was enhanced within TLR2–/– macrophages compared to WT and TLR4–/– macrophages. There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice. Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L. pneumophila was attenuated in TLR2–/– macrophages compared to WT and TLR4–/– macrophages. Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L. pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain. There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria. An L. pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2–/– macrophages was significantly lower than those of WT and TLR4–/– macrophages. Treatment of L. pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production. Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L. pneumophila, although other TLRs may also contribute to innate immunity against this organism.
INTRODUCTION
Legionella pneumophila is the major etiologic agent of Legionnaires' disease, a potentially fatal type of pneumonia affecting immunocompromised and immunocompetent subjects (28, 30). This gram-negative bacterium can multiply within the mononuclear cells in vivo and in vitro (6, 14) and evades phagosome-lysosome fusion within these cells (3). Several L. pneumophila virulence factors that facilitate intracellular growth have been identified in screenings with macrophages or in an in vivo screening system with signature-tagged mutagenesis (12). One important set of virulence factors is the dot/icm system, which is a type IV secretion system and is required for evasion of phagosome-lysosome fusion (5, 37, 38) and for the establishment of phagosomes permissive for the growth of L. pneumophila within them (8). However, the effector molecule(s) of this system remains undetermined. The innate immunity to L. pneumophila has been extensively studied. The A/J mouse strain is permissive for the intracellular growth of L. pneumophila (48), whereas other inbred strains of mice, such as BALB/c (49), 57BL/6, and 129X1 (10), are not. In terms of permissiveness of A/J macrophages, the genetic determinant of this permissiveness has been confirmed to be within the Lgn locus, and specifically the Bircle/Naip5 gene (4, 9, 10, 40, 52, 53). However, the role of this gene in regulation of intracellular growth of the bacterium is still unknown.
Toll-like receptors (TLRs) have been identified as receptors of pathogen-associated molecular patterns (PAMPs) (29, 47, 50), and several studies have investigated the relationship between TLR deficiency in hosts and susceptibility to various bacteria (1, 25, 34, 47). TLR4 is the receptor for lipopolysaccharide (LPS), the representative PAMP of most gram-negative bacteria, in coordination with CD14 and MD2 molecules (35, 36, 44, 47). Interaction of PAMP with TLRs results in signal transduction for innate immunity through the activation of nuclear factor-B and/or AP-1 (29, 45, 47). TLR2 is important for recognition of PAMPs such as lipopeptides, lipoproteins, and peptidoglycan in various bacteria (7, 20, 26). Ligands for other TLRs are also being defined (2, 17-19, 22). The effect of TLR4 mutation on innate immunity against L. pneumophila has been investigated, but conflicting results have been reported (24, 51). Recently, LPS purified from L. pneumophila was identified as a possible ligand for TLR2 but not for TLR4 (15). The present study was designed to determine the role of TLRs in innate immunity against L. pneumophila. Specifically, we determined whether TLR2 and TLR4 mutations affect the response of murine macrophages to L. pneumophila infection by using genetically engineered mice lacking TLR2 and TLR4.
MATERIALS AND METHODS
Mice. Mutant mouse (interbred from C57BL/6 x 129Sv) strains deficient in TLR2 and TLR4 were generated by gene targeting as described previously (21, 44). For control of each experiment, wild-type (WT) littermates were used. Male mice (the F3 to F5 generations interbred from C57BL/6 x 129Sv mice) were used at the age of 8 to 10 weeks. Of note, C57BL/6 mice and their macrophages are genetically resistant to L. pneumophila infection (48, 52). The animal experimental protocols were approved by the University of the Ryukyus animal experimentation ethics review committee. Mice were housed in a pathogen-free environment and fed sterilized food and water at the Biomedical Science Laboratory Center of the University of the Ryukyus.
Bacterial strains. L. pneumophila serogroup1 strain AA100jm (12) is a spontaneous streptomycin-resistant mutant of strain 130b (30), which is virulent in guinea pigs, macrophages, and amoebae (12, 31). The avirulent dotO mutant was constructed by random transposon mutagenesis, as described previously (12); this mutation results in severe defects in intracellular growth and evasion of the endocytic pathway (3). These strains were kindly supplied by Paul H. Edelstein. L. pneumophila strains were grown at 35°C in a humidified incubator on either buffered charcoal-yeast extract-agar medium supplemented with -ketoglutarate (BCYE-; Becton Dickinson and Co.) or in buffered yeast extract broth supplemented with -ketoglutarate (BYE-) (11). UV-killed bacteria were prepared by overnight UV light irradiation of a bacterial suspension (108 CFU/ml) in an open petri dish. Heat-killed bacteria were prepared by heating the bacterial suspension at 100°C for 1 h. Both treated suspensions were confirmed to contain no viable bacteria by plating them on BCYE- agar.
Preparation of bone marrow-derived macrophages (BMMs) and bone marrow-derived dendritic cells (BMDCs). Mice were sacrificed, and their tibias and femurs were removed aseptically. The bone marrow was irrigated with RPMI 1640 medium (Nipro, Osaka, Japan) to harvest bone marrow cells. Bone marrow cells were cultured in glass dishes with 20% L929 cell culture supernatant and 10% fetal calf serum (Cansera, Rexdale, Ontario, Canada)-supplemented endotoxin-free RPMI 1640 for 6 days, and adherent cells were referred to as BMMs and suspended at 2.5 x 105 to 5.0 x 105 cells/well in 24-well plate (Falcon 3047; Becton Dickinson), followed by incubation at 37°C in 5% CO2 for more than 12 h before infection under 5% CO2 (43). BMDCs were prepared as described previously (23). In brief, bone marrow cells were cultured for 6 days with interleukin-4 (IL-4; 2 ng/ml) and granulocyte/macrophage colony-stimulating factor (10 ng/ml). Most differentiated cells (>70%) were determined to be CD11c positive when the cells were analyzed by flow cytometry.
Infection of macrophages and dendritic cells with L. pneumophila. The cultured macrophages and dendritic cells in 24-well microplate were inoculated with L. pneumophila and cultured for 2 h. The extracellular fluid and bacteria were removed by washing with warm tissue culture medium, and the plate was further incubated for up to 72 h. Lack of growth of Legionella in the cell culture medium was confirmed in preliminary experiments (data not shown). The infected cells and supernatant in each well were harvested at the indicated time intervals by washing the wells several times with sterile distilled water. Detachment of macrophages was confirmed microscopically. The washings were combined into one tube at up to 10 ml in total volume, which was then vortexed for 20 s for complete lysis of the cells. These bacterial suspensions were diluted in sterile water and plated in known volume onto BCYE- agar. The number of viable legionellae in each well was determined by counting CFU after incubation of the plates at 35°C for 3 days and is expressed as the CFU count/well. Washing in distilled water only caused a <10% decrease in CFU within 2 h, and such a decrease did not affect the data. In some experiments, the dotO mutant or heat-killed bacteria were inoculated in the same manner.
Cytopathic effect of bacteria on macrophages. Cell viability was assessed by the Amido Black method described previously (41) with a minor modification. Briefly, BMMs were treated with bacteria at various concentrations. The stimulated macrophages were further cultured for another 24 h. After incubation of the BMMs with bacteria, the supernatant of culture wells was decanted, and 100 μl of 3.7% formaldehyde in 0.1 M sodium acetate-9% acetate was added to each well. After incubation for 20 min at room temperature, the supernatant of each well was decanted, and 100 μl of 0.05% Amido Black 10B (Wako Chemicals, Osaka, Japan) in 0.1 M sodium acetate and 9% acetate was added to each well of the plate. The well was later washed with distilled water, and 100 μl of 0.025 N NaOH was added. The optical density of each well was measured at 575 nm with an automatic plate reader.
Determination of IL-12p40, IL-10, and TNF- concentrations. The concentrations of IL-12p40, IL-10, and tumor necrosis factor alpha (TNF-) in culture supernatants were measured by using specific enzyme-linked immunosorbent assay (ELISA) kits (purchased from BioSource International [Camarillo, Calif.] for IL-12p40, and from R&D Systems [Minneapolis, Minn.] for IL-10 and TNF-, respectively).
Preparation of sonicated bacterial lysates. Bacteria (109 CFU/ml in 10 ml of distilled water) were sonicated on ice by using by an Astrason XL 2020 ultrasonic disrupter (Misonix, Farmingdale, N.Y.) at a setting of 7 and intermittent 1-min pulses (1 min on and 1 min off for 12 cycles). A portion (5.0 ml) of the sonic extract was passed through an ultrafiltration filter UFV2BCC (Millipore, Bedford, Mass.) that fractionates molecules into those more and less than 5 kDa. When needed, a fraction (1 mg of protein/ml) was treated with proteinase K (25 μg/ml; Sigma Chemical Co., St. Louis, Mo.), incubated for 15 min at 37°C, and then heated for 5 min at 60°C. This procedure was repeated twice and subsequently heated at 95°C for 10 min to stop the enzyme reaction. The protein concentration of each fraction was measured by the method of Lowry et al. (27). Bovine serum albumin was used as a standard.
Statistical analysis. Data were expressed as means ± the standard deviations (SD). Differences between group means were tested for statistical significance by using analysis of variance and Scheffe's post-hoc test. Analysis of data was conducted by using StatView software (Abacus Concepts, Inc., Berkeley, Calif.). A P value of <0.05 denotes the presence of a statistically significant difference.
RESULTS
Cell-associated growth of Legionella within macrophages and dendritic cells. BMMs from WT, TLR2–/–, and TLR4–/– mice (F3 generation) were infected with L. pneumophila at multiplicities of infection (MOIs) of 1 and 10. L. pneumophila multiplied more than one 1,000-fold in TLR2–/– macrophages but only 10-fold in WT and TLR4–/– macrophages at 3 days after infection (Fig. 1); the differences between growth in TLR2–/– macrophages and other macrophages were significantly different at both 2 and 3 days postinfection (P < 0.05). These findings indicate that TLR2 is an important factor for the innate resistance of macrophages against intracellular growth of L. pneumophila. Dendritic cells from WT, TLR2–/–, and TLR4–/– mice were infected with L. pneumophila at an MOI 10. L. pneumophila multiplied by 10-fold in the dendritic cells of each group at 72 h after infection, and no significant difference was observed between the groups (Fig. 1).
IL-12p40, TNF-, and IL-10 production by macrophages infected with L. pneumophila. We next examined IL-12p40, TNF-, and IL-10 production in BMMs of each type of TLR-deficient mouse when the cells were infected with L. pneumophila. IL-12p40 levels in BMM culture supernatants were determined at days 0, 1, 2, and 3 postinfection. At day 1 postinfection, IL-12p40 was secreted by WT and TLR4–/– macrophages but not by TLR2–/– macrophages (P < 0.05, Fig. 2). At day 2, IL-12p40 secretion from TLR2–/– macrophages was still significantly attenuated compared to those from WT and TLR4–/– macrophages (P < 0.05). At day 3 postinfection, TLR2–/– macrophages secreted IL-12p40 at concentrations that were not significantly different from those produced by WT and TLR4–/– macrophages (P > 0.05). IL-10 production by TLR2–/– macrophages was attenuated at days 2 and 3 postinfection compared to those from WT and TLR4–/– macrophages (Fig. 3).
To investigate the relationship between the level of macrophage-secreted IL-12p40 during the early stage of infection and the virulence and viability of L. pneumophila, we compared the IL-12p40 infection with a live virulent strain versus that by an avirulent mutant and a heat-killed virulent strain (Fig. 4). For live virulent bacteria, IL-12p40 secretion was significantly lower in macrophages of TLR2–/– mice at day 1 after infection than for macrophages from TLR4–/– and WT mice. Macrophage incubation with the dotO mutant, or dead bacteria, induced extremely low amounts of IL-12p40 from all three types of macrophages. The pattern of TNF- production by macrophages from WT, TLR2–/–, and TLR4–/– mice was almost identical to that of IL-12p40 production when the macrophages were infected with the same bacteria (data now shown).
Because virulent L. pneumophila grew within macrophages but the dotO mutant did not (data not shown), it was not clear if the differences in the response of macrophages represented quantitative or qualitative differences of stimuli by the different bacterial types. Accordingly, we evaluated the response of macrophages to various concentrations of live and dead L. pneumophila. The IL-12p40 production level from macrophages infected by the virulent strain, avirulent dotO mutant, or heat-killed bacteria was dose dependent. This IL-12p40 response was roughly equivalent to the corresponding inocula of live bacteria, regardless of bacterial virulence properties and viability (Fig. 5). At a relatively low level of bacterial load in macrophages, IL-12p40 secretion from TLR2–/– macrophages was much lower than in WT or TLR4–/– macrophages. However, with an extremely high bacterial load (109/ml), IL-12p40 secretion by WT and TLR4–/– macrophages was significantly attenuated, whereas IL-12p40 secretion from TLR2–/– macrophages was much less attenuated and IL-12p40 secretion by TLR2–/– macrophages seemed to be higher than that by WT or TLR4–/– macrophages. This paradoxical dose-response pattern in IL-12p40 secretion by WT, TLR4–/–, and TLR2–/– macrophages was almost the same when live dotO mutant and heat-killed bacteria were used as stimuli. It was also noted that heat-killed bacteria induced relatively higher IL-12 p40 production than live bacteria.
Evaluation of IL-10 secretion from macrophages infected by the virulent strain, avirulent dotO mutant, or heat-killed bacteria, also showed a dose-dependent secretion of IL-10 (Fig. 6). At a relatively low heat-killed bacterial load and various doses of virulent strain and avirulent dotO mutant, IL-10 secretion from TLR2–/– macrophages was much lower than in WT or TLR4–/– macrophages. A paradoxical dose-response pattern in IL-10 secretion by WT, TLR4–/–, and TLR2–/– macrophages was demonstrated only when heat-killed bacteria were used as the stimulus. Neither live virulent strain nor live avirulent dotO mutant showed paradoxical dose-response of IL-10 secretion, suggesting that the modes of production of IL-12 and IL-10 were distinct, and the production of these two cytokines may be controlled in different manners. We examined the cytopathic effect of bacteria against each macrophage type, and no difference was noted in the WT and its mutants with respect to. the susceptibility to the cytopathic effects of bacteria (Fig. 7).
IL-12p40 production by bacterial sonicate-stimulated macrophages. When sonicated bacterial extracts (50 μg/ml) were used as stimuli, IL-12p40 secretion by BMMs was attenuated in TLR2–/– macrophages compared to WT and TLR4–/– macrophages. When the sonicates were fractionated by ultrafiltration, the fraction that contained >5-kDa molecules stimulated macrophages to secrete IL-12p40. Proteinase K treatment and heat inactivation of the sonicate resulted in reduction of IL-12p40 production by macrophages in each group, but did not abolish the difference in IL-12p40 production between TLR2–/– macrophages and macrophages from WT and TLR4–/– mice (Fig. 8).
DISCUSSION
Several studies have investigated the relationship between TLR deficiency in hosts and susceptibility to various bacteria (1, 25, 34). Our study showed that TLR2, but not TLR4, was an important molecule for host resistance against the intracellular growth of L. pneumophila, as demonstrated by the ability of the bacterium to grow within macrophages from TLR2–/– mice. To our knowledge, there are no published data that elucidate the role of TLR2 in L. pneumophila-infected macrophages and our study provides the first description of the role of TLR2 in the regulation of intracellular growth of the bacterium. The role of TLR4 in Legionella infection was previously evaluated with C3H/HeJ mice, which have a mutated tlr4 gene (24, 51). Our findings with genetically engineered TLR4–/– BMMs are in agreement with the studies of Lettinga et al. (24), who showed that C3H/HeJ mice are not susceptible to pulmonary Legionella infection but are in conflict with the finding that C3H/HeJ peritoneal macrophages are permissive for the intracellular growth of L. pneumophila (51). Another study (48) showed that a small subset of lymphocytes contaminating the peritoneal lavage enhances the resistance of peritoneal macrophages to Legionella, which may explain the discrepancies between the above studies. Furthermore, the permissiveness varies when different cell populations are used. A/J dendritic cells are not permissive for the intracellular growth of L. pneumophila in contrast to A/J macrophages (33). In the present study, no enhancement of bacterial growth within TLR2–/– dendritic cells was observed. Thus, we speculate that the resistance of host cells to intracellular growth of bacteria occurs through a multifactorial system and elucidation of the mechanism requires further investigation.
Macrophages recognize various pathogens, and the signal is transduced for gene expression of appropriate proinflammatory cytokines such as IL-12, IL-10, and TNF-. In our study, macrophages produced significant amounts of these cytokines when infected with L. pneumophila, but the production of these cytokines was attenuated in TLR2–/– macrophages and TLR2–/– dendritic cells in the early stages of infection. IL-12 secreted by the cells subsequently stimulates natural killer cells to produce gamma interferon, with a resultant Th1 polarization (42). Several studies have shown that IL-12 is critical for immunoregulation against L. pneumophila (6, 46). Salins et al. (39) demonstrated that exogenous IL-12 inhibits the intracellular growth of L. pneumophila in A/J peritoneal macrophages. As stated above, the precise mechanism of enhanced growth of the bacteria within TLR2-deficient macrophages is complex and unknown, but the decrease of autocrine IL-12 during the early stage of infection may play a role in this phenomenon.
Our findings suggest that TLR4 plays little, if any, role in the recognition of L. pneumophila because the response of TLR4-deficient macrophages was similar to that of the WT macrophages. This may be because TLR4 does not recognize the bacteria at all or other receptors compensate for TLR4 deficiency. It is possible that the unique LPS of L. pneumophila results in poor interaction with TLR4, similar to the situation with Rhizobium species (15). In experiments with fractionated lysates, TLR2-dependent IL-12p40 secretion from macrophages was not abolished by treatment of the lysates with proteinase K and heating. Our findings seem to corroborate those of Girard et al. (15), who showed that TNF- production by bone marrow-derived granulocytes was induced by purified L. pneumophila LPS via TLR2. However, Zahringer et al. (54) recently showed that LPS of Bartonella henselae, whose conformation is very close to L. pneumophila LPS, does not interact with TLR2 at all and rather interacts with TLR4 very weakly but definitively when the LPS product is deliberately free of protein contamination. We should therefore be very careful to determine the ligand(s) of L. pneumophila for TLR2. Avirulent dotO mutant induced IL-12 secretion similar to the virulent strain of bacteria when the bacterial load was adjusted, indicating that the icm/dot system, the major virulence factor of L. pneumophila, is not involved in IL-12p40 secretion via TLR2 and TLR4 by macrophages. TLR5 has recently been reported to be responsible for the immune response to L. pneumophila through the recognition of bacterial flagellin (16). Our data show that IL-12p40 production by macrophages is in part independent of both TLR2 and TLR4. We speculate that other receptors, such as TLR5, may be involved in recognition of the bacterium.
A paradoxical response to an extremely high concentration of bacteria was observed in the present study. IL-12p40 production from TLR2–/– macrophages was higher than that from WT and TLR4–/– macrophages when the macrophages were stimulated with extremely high concentration of bacteria (corresponding to 109 CFU/ml). This phenomenon was reproducible, and no difference in the susceptibility of macrophages from WT, TLR2–/–, TLR4–/– mice to bacterial cytotoxicity was observed. Therefore, the paradoxical response might be explained by the following mechanisms: (i) loss of negative feedback via TLR2-mediated signal transduction and (ii) enhanced positive response via receptors other than TLR2 in TLR2-deficient macrophages. Interestingly, the production of IL-10, a Th2 cytokine, was also TLR2 dependent, but the paradoxical response of IL-10 production was not observed other than when heat-killed bacteria was used as the stimulus. Recent studies have shown that phosphoinositide 3-kinase, an endogenous suppressor of IL-12 production, is triggered by TLR signaling and limits excessive Th1 polarization (13). It has been shown that induction of IRAK-M and inhibition of kinase activity of IRAK-1 are crucial to peptidoglycan-induced tolerance in macrophages (32). Negative regulator(s), such as phosphoinositide 3-kinase and IRAK-M, may be involved in this paradoxical response of macrophages against Legionella but this hypothesis needs to be investigated in more detail in the future.
In summary, our study examined the role of TLRs in innate immune responses against L. pneumophila infection in vitro. The results suggest that TLR2 is an important molecule responsible for resistance against intracellular growth of the bacterium and IL-12p40 production by macrophages and during the early stage of infection. However, the response of macrophages against the bacterium through TLRs seems to be rather complex and further investigation is needed to dissect this response.
ACKNOWLEDGMENTS
We are grateful to Paul H. Edelstein for reviewing the manuscript and for providing L. pneumophila strain and its mutant.
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Sports of Japan.
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Research Institute for Microbial Infectious Disease, Osaka University, Osaka, Japan
ABSTRACT
The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2–/–), TLR4–/–, and wild-type (WT) littermate (C57BL/6 x 129Sv) mice. Intracellular growth of L. pneumophila was enhanced within TLR2–/– macrophages compared to WT and TLR4–/– macrophages. There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice. Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L. pneumophila was attenuated in TLR2–/– macrophages compared to WT and TLR4–/– macrophages. Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L. pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain. There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria. An L. pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2–/– macrophages was significantly lower than those of WT and TLR4–/– macrophages. Treatment of L. pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production. Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L. pneumophila, although other TLRs may also contribute to innate immunity against this organism.
INTRODUCTION
Legionella pneumophila is the major etiologic agent of Legionnaires' disease, a potentially fatal type of pneumonia affecting immunocompromised and immunocompetent subjects (28, 30). This gram-negative bacterium can multiply within the mononuclear cells in vivo and in vitro (6, 14) and evades phagosome-lysosome fusion within these cells (3). Several L. pneumophila virulence factors that facilitate intracellular growth have been identified in screenings with macrophages or in an in vivo screening system with signature-tagged mutagenesis (12). One important set of virulence factors is the dot/icm system, which is a type IV secretion system and is required for evasion of phagosome-lysosome fusion (5, 37, 38) and for the establishment of phagosomes permissive for the growth of L. pneumophila within them (8). However, the effector molecule(s) of this system remains undetermined. The innate immunity to L. pneumophila has been extensively studied. The A/J mouse strain is permissive for the intracellular growth of L. pneumophila (48), whereas other inbred strains of mice, such as BALB/c (49), 57BL/6, and 129X1 (10), are not. In terms of permissiveness of A/J macrophages, the genetic determinant of this permissiveness has been confirmed to be within the Lgn locus, and specifically the Bircle/Naip5 gene (4, 9, 10, 40, 52, 53). However, the role of this gene in regulation of intracellular growth of the bacterium is still unknown.
Toll-like receptors (TLRs) have been identified as receptors of pathogen-associated molecular patterns (PAMPs) (29, 47, 50), and several studies have investigated the relationship between TLR deficiency in hosts and susceptibility to various bacteria (1, 25, 34, 47). TLR4 is the receptor for lipopolysaccharide (LPS), the representative PAMP of most gram-negative bacteria, in coordination with CD14 and MD2 molecules (35, 36, 44, 47). Interaction of PAMP with TLRs results in signal transduction for innate immunity through the activation of nuclear factor-B and/or AP-1 (29, 45, 47). TLR2 is important for recognition of PAMPs such as lipopeptides, lipoproteins, and peptidoglycan in various bacteria (7, 20, 26). Ligands for other TLRs are also being defined (2, 17-19, 22). The effect of TLR4 mutation on innate immunity against L. pneumophila has been investigated, but conflicting results have been reported (24, 51). Recently, LPS purified from L. pneumophila was identified as a possible ligand for TLR2 but not for TLR4 (15). The present study was designed to determine the role of TLRs in innate immunity against L. pneumophila. Specifically, we determined whether TLR2 and TLR4 mutations affect the response of murine macrophages to L. pneumophila infection by using genetically engineered mice lacking TLR2 and TLR4.
MATERIALS AND METHODS
Mice. Mutant mouse (interbred from C57BL/6 x 129Sv) strains deficient in TLR2 and TLR4 were generated by gene targeting as described previously (21, 44). For control of each experiment, wild-type (WT) littermates were used. Male mice (the F3 to F5 generations interbred from C57BL/6 x 129Sv mice) were used at the age of 8 to 10 weeks. Of note, C57BL/6 mice and their macrophages are genetically resistant to L. pneumophila infection (48, 52). The animal experimental protocols were approved by the University of the Ryukyus animal experimentation ethics review committee. Mice were housed in a pathogen-free environment and fed sterilized food and water at the Biomedical Science Laboratory Center of the University of the Ryukyus.
Bacterial strains. L. pneumophila serogroup1 strain AA100jm (12) is a spontaneous streptomycin-resistant mutant of strain 130b (30), which is virulent in guinea pigs, macrophages, and amoebae (12, 31). The avirulent dotO mutant was constructed by random transposon mutagenesis, as described previously (12); this mutation results in severe defects in intracellular growth and evasion of the endocytic pathway (3). These strains were kindly supplied by Paul H. Edelstein. L. pneumophila strains were grown at 35°C in a humidified incubator on either buffered charcoal-yeast extract-agar medium supplemented with -ketoglutarate (BCYE-; Becton Dickinson and Co.) or in buffered yeast extract broth supplemented with -ketoglutarate (BYE-) (11). UV-killed bacteria were prepared by overnight UV light irradiation of a bacterial suspension (108 CFU/ml) in an open petri dish. Heat-killed bacteria were prepared by heating the bacterial suspension at 100°C for 1 h. Both treated suspensions were confirmed to contain no viable bacteria by plating them on BCYE- agar.
Preparation of bone marrow-derived macrophages (BMMs) and bone marrow-derived dendritic cells (BMDCs). Mice were sacrificed, and their tibias and femurs were removed aseptically. The bone marrow was irrigated with RPMI 1640 medium (Nipro, Osaka, Japan) to harvest bone marrow cells. Bone marrow cells were cultured in glass dishes with 20% L929 cell culture supernatant and 10% fetal calf serum (Cansera, Rexdale, Ontario, Canada)-supplemented endotoxin-free RPMI 1640 for 6 days, and adherent cells were referred to as BMMs and suspended at 2.5 x 105 to 5.0 x 105 cells/well in 24-well plate (Falcon 3047; Becton Dickinson), followed by incubation at 37°C in 5% CO2 for more than 12 h before infection under 5% CO2 (43). BMDCs were prepared as described previously (23). In brief, bone marrow cells were cultured for 6 days with interleukin-4 (IL-4; 2 ng/ml) and granulocyte/macrophage colony-stimulating factor (10 ng/ml). Most differentiated cells (>70%) were determined to be CD11c positive when the cells were analyzed by flow cytometry.
Infection of macrophages and dendritic cells with L. pneumophila. The cultured macrophages and dendritic cells in 24-well microplate were inoculated with L. pneumophila and cultured for 2 h. The extracellular fluid and bacteria were removed by washing with warm tissue culture medium, and the plate was further incubated for up to 72 h. Lack of growth of Legionella in the cell culture medium was confirmed in preliminary experiments (data not shown). The infected cells and supernatant in each well were harvested at the indicated time intervals by washing the wells several times with sterile distilled water. Detachment of macrophages was confirmed microscopically. The washings were combined into one tube at up to 10 ml in total volume, which was then vortexed for 20 s for complete lysis of the cells. These bacterial suspensions were diluted in sterile water and plated in known volume onto BCYE- agar. The number of viable legionellae in each well was determined by counting CFU after incubation of the plates at 35°C for 3 days and is expressed as the CFU count/well. Washing in distilled water only caused a <10% decrease in CFU within 2 h, and such a decrease did not affect the data. In some experiments, the dotO mutant or heat-killed bacteria were inoculated in the same manner.
Cytopathic effect of bacteria on macrophages. Cell viability was assessed by the Amido Black method described previously (41) with a minor modification. Briefly, BMMs were treated with bacteria at various concentrations. The stimulated macrophages were further cultured for another 24 h. After incubation of the BMMs with bacteria, the supernatant of culture wells was decanted, and 100 μl of 3.7% formaldehyde in 0.1 M sodium acetate-9% acetate was added to each well. After incubation for 20 min at room temperature, the supernatant of each well was decanted, and 100 μl of 0.05% Amido Black 10B (Wako Chemicals, Osaka, Japan) in 0.1 M sodium acetate and 9% acetate was added to each well of the plate. The well was later washed with distilled water, and 100 μl of 0.025 N NaOH was added. The optical density of each well was measured at 575 nm with an automatic plate reader.
Determination of IL-12p40, IL-10, and TNF- concentrations. The concentrations of IL-12p40, IL-10, and tumor necrosis factor alpha (TNF-) in culture supernatants were measured by using specific enzyme-linked immunosorbent assay (ELISA) kits (purchased from BioSource International [Camarillo, Calif.] for IL-12p40, and from R&D Systems [Minneapolis, Minn.] for IL-10 and TNF-, respectively).
Preparation of sonicated bacterial lysates. Bacteria (109 CFU/ml in 10 ml of distilled water) were sonicated on ice by using by an Astrason XL 2020 ultrasonic disrupter (Misonix, Farmingdale, N.Y.) at a setting of 7 and intermittent 1-min pulses (1 min on and 1 min off for 12 cycles). A portion (5.0 ml) of the sonic extract was passed through an ultrafiltration filter UFV2BCC (Millipore, Bedford, Mass.) that fractionates molecules into those more and less than 5 kDa. When needed, a fraction (1 mg of protein/ml) was treated with proteinase K (25 μg/ml; Sigma Chemical Co., St. Louis, Mo.), incubated for 15 min at 37°C, and then heated for 5 min at 60°C. This procedure was repeated twice and subsequently heated at 95°C for 10 min to stop the enzyme reaction. The protein concentration of each fraction was measured by the method of Lowry et al. (27). Bovine serum albumin was used as a standard.
Statistical analysis. Data were expressed as means ± the standard deviations (SD). Differences between group means were tested for statistical significance by using analysis of variance and Scheffe's post-hoc test. Analysis of data was conducted by using StatView software (Abacus Concepts, Inc., Berkeley, Calif.). A P value of <0.05 denotes the presence of a statistically significant difference.
RESULTS
Cell-associated growth of Legionella within macrophages and dendritic cells. BMMs from WT, TLR2–/–, and TLR4–/– mice (F3 generation) were infected with L. pneumophila at multiplicities of infection (MOIs) of 1 and 10. L. pneumophila multiplied more than one 1,000-fold in TLR2–/– macrophages but only 10-fold in WT and TLR4–/– macrophages at 3 days after infection (Fig. 1); the differences between growth in TLR2–/– macrophages and other macrophages were significantly different at both 2 and 3 days postinfection (P < 0.05). These findings indicate that TLR2 is an important factor for the innate resistance of macrophages against intracellular growth of L. pneumophila. Dendritic cells from WT, TLR2–/–, and TLR4–/– mice were infected with L. pneumophila at an MOI 10. L. pneumophila multiplied by 10-fold in the dendritic cells of each group at 72 h after infection, and no significant difference was observed between the groups (Fig. 1).
IL-12p40, TNF-, and IL-10 production by macrophages infected with L. pneumophila. We next examined IL-12p40, TNF-, and IL-10 production in BMMs of each type of TLR-deficient mouse when the cells were infected with L. pneumophila. IL-12p40 levels in BMM culture supernatants were determined at days 0, 1, 2, and 3 postinfection. At day 1 postinfection, IL-12p40 was secreted by WT and TLR4–/– macrophages but not by TLR2–/– macrophages (P < 0.05, Fig. 2). At day 2, IL-12p40 secretion from TLR2–/– macrophages was still significantly attenuated compared to those from WT and TLR4–/– macrophages (P < 0.05). At day 3 postinfection, TLR2–/– macrophages secreted IL-12p40 at concentrations that were not significantly different from those produced by WT and TLR4–/– macrophages (P > 0.05). IL-10 production by TLR2–/– macrophages was attenuated at days 2 and 3 postinfection compared to those from WT and TLR4–/– macrophages (Fig. 3).
To investigate the relationship between the level of macrophage-secreted IL-12p40 during the early stage of infection and the virulence and viability of L. pneumophila, we compared the IL-12p40 infection with a live virulent strain versus that by an avirulent mutant and a heat-killed virulent strain (Fig. 4). For live virulent bacteria, IL-12p40 secretion was significantly lower in macrophages of TLR2–/– mice at day 1 after infection than for macrophages from TLR4–/– and WT mice. Macrophage incubation with the dotO mutant, or dead bacteria, induced extremely low amounts of IL-12p40 from all three types of macrophages. The pattern of TNF- production by macrophages from WT, TLR2–/–, and TLR4–/– mice was almost identical to that of IL-12p40 production when the macrophages were infected with the same bacteria (data now shown).
Because virulent L. pneumophila grew within macrophages but the dotO mutant did not (data not shown), it was not clear if the differences in the response of macrophages represented quantitative or qualitative differences of stimuli by the different bacterial types. Accordingly, we evaluated the response of macrophages to various concentrations of live and dead L. pneumophila. The IL-12p40 production level from macrophages infected by the virulent strain, avirulent dotO mutant, or heat-killed bacteria was dose dependent. This IL-12p40 response was roughly equivalent to the corresponding inocula of live bacteria, regardless of bacterial virulence properties and viability (Fig. 5). At a relatively low level of bacterial load in macrophages, IL-12p40 secretion from TLR2–/– macrophages was much lower than in WT or TLR4–/– macrophages. However, with an extremely high bacterial load (109/ml), IL-12p40 secretion by WT and TLR4–/– macrophages was significantly attenuated, whereas IL-12p40 secretion from TLR2–/– macrophages was much less attenuated and IL-12p40 secretion by TLR2–/– macrophages seemed to be higher than that by WT or TLR4–/– macrophages. This paradoxical dose-response pattern in IL-12p40 secretion by WT, TLR4–/–, and TLR2–/– macrophages was almost the same when live dotO mutant and heat-killed bacteria were used as stimuli. It was also noted that heat-killed bacteria induced relatively higher IL-12 p40 production than live bacteria.
Evaluation of IL-10 secretion from macrophages infected by the virulent strain, avirulent dotO mutant, or heat-killed bacteria, also showed a dose-dependent secretion of IL-10 (Fig. 6). At a relatively low heat-killed bacterial load and various doses of virulent strain and avirulent dotO mutant, IL-10 secretion from TLR2–/– macrophages was much lower than in WT or TLR4–/– macrophages. A paradoxical dose-response pattern in IL-10 secretion by WT, TLR4–/–, and TLR2–/– macrophages was demonstrated only when heat-killed bacteria were used as the stimulus. Neither live virulent strain nor live avirulent dotO mutant showed paradoxical dose-response of IL-10 secretion, suggesting that the modes of production of IL-12 and IL-10 were distinct, and the production of these two cytokines may be controlled in different manners. We examined the cytopathic effect of bacteria against each macrophage type, and no difference was noted in the WT and its mutants with respect to. the susceptibility to the cytopathic effects of bacteria (Fig. 7).
IL-12p40 production by bacterial sonicate-stimulated macrophages. When sonicated bacterial extracts (50 μg/ml) were used as stimuli, IL-12p40 secretion by BMMs was attenuated in TLR2–/– macrophages compared to WT and TLR4–/– macrophages. When the sonicates were fractionated by ultrafiltration, the fraction that contained >5-kDa molecules stimulated macrophages to secrete IL-12p40. Proteinase K treatment and heat inactivation of the sonicate resulted in reduction of IL-12p40 production by macrophages in each group, but did not abolish the difference in IL-12p40 production between TLR2–/– macrophages and macrophages from WT and TLR4–/– mice (Fig. 8).
DISCUSSION
Several studies have investigated the relationship between TLR deficiency in hosts and susceptibility to various bacteria (1, 25, 34). Our study showed that TLR2, but not TLR4, was an important molecule for host resistance against the intracellular growth of L. pneumophila, as demonstrated by the ability of the bacterium to grow within macrophages from TLR2–/– mice. To our knowledge, there are no published data that elucidate the role of TLR2 in L. pneumophila-infected macrophages and our study provides the first description of the role of TLR2 in the regulation of intracellular growth of the bacterium. The role of TLR4 in Legionella infection was previously evaluated with C3H/HeJ mice, which have a mutated tlr4 gene (24, 51). Our findings with genetically engineered TLR4–/– BMMs are in agreement with the studies of Lettinga et al. (24), who showed that C3H/HeJ mice are not susceptible to pulmonary Legionella infection but are in conflict with the finding that C3H/HeJ peritoneal macrophages are permissive for the intracellular growth of L. pneumophila (51). Another study (48) showed that a small subset of lymphocytes contaminating the peritoneal lavage enhances the resistance of peritoneal macrophages to Legionella, which may explain the discrepancies between the above studies. Furthermore, the permissiveness varies when different cell populations are used. A/J dendritic cells are not permissive for the intracellular growth of L. pneumophila in contrast to A/J macrophages (33). In the present study, no enhancement of bacterial growth within TLR2–/– dendritic cells was observed. Thus, we speculate that the resistance of host cells to intracellular growth of bacteria occurs through a multifactorial system and elucidation of the mechanism requires further investigation.
Macrophages recognize various pathogens, and the signal is transduced for gene expression of appropriate proinflammatory cytokines such as IL-12, IL-10, and TNF-. In our study, macrophages produced significant amounts of these cytokines when infected with L. pneumophila, but the production of these cytokines was attenuated in TLR2–/– macrophages and TLR2–/– dendritic cells in the early stages of infection. IL-12 secreted by the cells subsequently stimulates natural killer cells to produce gamma interferon, with a resultant Th1 polarization (42). Several studies have shown that IL-12 is critical for immunoregulation against L. pneumophila (6, 46). Salins et al. (39) demonstrated that exogenous IL-12 inhibits the intracellular growth of L. pneumophila in A/J peritoneal macrophages. As stated above, the precise mechanism of enhanced growth of the bacteria within TLR2-deficient macrophages is complex and unknown, but the decrease of autocrine IL-12 during the early stage of infection may play a role in this phenomenon.
Our findings suggest that TLR4 plays little, if any, role in the recognition of L. pneumophila because the response of TLR4-deficient macrophages was similar to that of the WT macrophages. This may be because TLR4 does not recognize the bacteria at all or other receptors compensate for TLR4 deficiency. It is possible that the unique LPS of L. pneumophila results in poor interaction with TLR4, similar to the situation with Rhizobium species (15). In experiments with fractionated lysates, TLR2-dependent IL-12p40 secretion from macrophages was not abolished by treatment of the lysates with proteinase K and heating. Our findings seem to corroborate those of Girard et al. (15), who showed that TNF- production by bone marrow-derived granulocytes was induced by purified L. pneumophila LPS via TLR2. However, Zahringer et al. (54) recently showed that LPS of Bartonella henselae, whose conformation is very close to L. pneumophila LPS, does not interact with TLR2 at all and rather interacts with TLR4 very weakly but definitively when the LPS product is deliberately free of protein contamination. We should therefore be very careful to determine the ligand(s) of L. pneumophila for TLR2. Avirulent dotO mutant induced IL-12 secretion similar to the virulent strain of bacteria when the bacterial load was adjusted, indicating that the icm/dot system, the major virulence factor of L. pneumophila, is not involved in IL-12p40 secretion via TLR2 and TLR4 by macrophages. TLR5 has recently been reported to be responsible for the immune response to L. pneumophila through the recognition of bacterial flagellin (16). Our data show that IL-12p40 production by macrophages is in part independent of both TLR2 and TLR4. We speculate that other receptors, such as TLR5, may be involved in recognition of the bacterium.
A paradoxical response to an extremely high concentration of bacteria was observed in the present study. IL-12p40 production from TLR2–/– macrophages was higher than that from WT and TLR4–/– macrophages when the macrophages were stimulated with extremely high concentration of bacteria (corresponding to 109 CFU/ml). This phenomenon was reproducible, and no difference in the susceptibility of macrophages from WT, TLR2–/–, TLR4–/– mice to bacterial cytotoxicity was observed. Therefore, the paradoxical response might be explained by the following mechanisms: (i) loss of negative feedback via TLR2-mediated signal transduction and (ii) enhanced positive response via receptors other than TLR2 in TLR2-deficient macrophages. Interestingly, the production of IL-10, a Th2 cytokine, was also TLR2 dependent, but the paradoxical response of IL-10 production was not observed other than when heat-killed bacteria was used as the stimulus. Recent studies have shown that phosphoinositide 3-kinase, an endogenous suppressor of IL-12 production, is triggered by TLR signaling and limits excessive Th1 polarization (13). It has been shown that induction of IRAK-M and inhibition of kinase activity of IRAK-1 are crucial to peptidoglycan-induced tolerance in macrophages (32). Negative regulator(s), such as phosphoinositide 3-kinase and IRAK-M, may be involved in this paradoxical response of macrophages against Legionella but this hypothesis needs to be investigated in more detail in the future.
In summary, our study examined the role of TLRs in innate immune responses against L. pneumophila infection in vitro. The results suggest that TLR2 is an important molecule responsible for resistance against intracellular growth of the bacterium and IL-12p40 production by macrophages and during the early stage of infection. However, the response of macrophages against the bacterium through TLRs seems to be rather complex and further investigation is needed to dissect this response.
ACKNOWLEDGMENTS
We are grateful to Paul H. Edelstein for reviewing the manuscript and for providing L. pneumophila strain and its mutant.
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Sports of Japan.
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