MyD88-Dependent Responses Involving Toll-Like Receptor 2 Are Important for Protection and Clearance of Legionella pneumophila in a Mouse Mod
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感染与免疫杂志 2006年第6期
Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, Connecticut 06536
ABSTRACT
Legionella pneumophila is a gram-negative facultative intracellular parasite of macrophages. Although L. pneumophila is the causative agent of a severe pneumonia known as Legionnaires' disease, it is likely that most infections caused by this organism are cleared by the host innate immune system. It is predicted that host pattern recognition proteins belonging to the Toll-like receptor (TLR) family are involved in the protective innate immune responses. We examined the role of TLR-mediated responses in L. pneumophila detection and clearance using genetically altered mouse hosts in which the macrophages are permissive for L. pneumophila intracellular replication. Our data demonstrate that cytokine production by bone marrow-derived macrophages (BMMs) in response to L. pneumophila infection requires the TLR adapter protein MyD88 and is reduced in the absence of TLR2 but not in the absence of TLR4. Bacterial growth ex vivo in BMMs from MyD88-deficient mice was not enhanced compared to bacterial growth ex vivo in BMMs from heterozygous littermate controls. Wild-type mice were able to clear L. pneumophila from the lung, whereas respiratory infection of MyD88-deficient mice caused death that resulted from robust bacterial replication and dissemination. In contrast to an infection with virulent L. pneumophila, MyD88-deficient mice were able to clear infections with L. pneumophila dotA mutants, indicating that MyD88-independent responses in the lung are sufficient to clear bacteria that are unable to replicate intracellularly. In vivo growth of L. pneumophila was enhanced in the lungs of TLR2-deficient mice, which resulted in a delay in bacterial clearance. No significant differences were observed in the growth and clearance of L. pneumophila in the lungs of TLR4-deficient mice and heterozygous littermate control mice. Our data indicate that MyD88 is crucial for eliciting a protective innate immune response against virulent L. pneumophila and that TLR2 is one of the pattern recognition receptors involved in initiating this MyD88-dependent response.
INTRODUCTION
Legionella pneumophila is a ubiquitous gram-negative bacterium that parasitizes phagocytic protozoans in freshwater environments (14). When L. pneumophila gains access to the human respiratory system via inhalation of contaminated water sources, the bacteria have the capacity to replicate within alveolar macrophages (15, 21, 30). The Dot/Icm apparatus is a type IV secretion system that is required for L. pneumophila intracellular replication (29, 39, 45). Bacterial proteins translocated into host cells by the Dot/Icm system modulate trafficking of the vacuole containing L. pneumophila and promote biogenesis of a unique organelle that evades fusion with lysosomes and supports intracellular replication (7, 19, 20, 28, 33, 37). Although a severe type of pneumonia known as Legionnaires' disease can result from L. pneumophila replication in host macrophages (15, 21, 30), most infections are probably contained and cleared by the immune system without disease.
How the host detects L. pneumophila and initiates a protective response has not been clearly established. Previous studies have suggested that detection of L. pneumophila initiates a host response that leads to the production of gamma interferon (IFN-) and macrophage activation (4, 34). Treatment of macrophages ex vivo with IFN- is sufficient to upregulate effectors that restrict L. pneumophila intracellular growth (4, 34, 35). Using a mouse model of Legionnaires' disease, it has been shown that the cytokine interleukin-12 (IL-12) is important for host protection against L. pneumophila infection (6). IL-12 is able to stimulate production of IFN- by NK and NK T cells (44), suggesting that host susceptibility in IL-12-depleted mice is due primarily to a defect in macrophage activation by IFN- (6). Consistent with this hypothesis, mice lacking IFN- have lower survival rates upon infection with L. pneumophila than wild-type mice have (41).
Toll-like receptors (TLRs) are a well-characterized family of transmembrane pattern recognition receptors that detect conserved molecular patterns on microbes (23). TLRs can generate a rapid and potent response against a pathogen through induction of the transcription factor NF-B, leading to events that include production of inflammatory cytokines, activation of macrophages, and maturation of dendritic cells (31). Following TLR stimulation, the cytoplasmic adapter protein MyD88 is required for transduction of a signal that activates NF-B (32). MyD88-deficient mice have defects in most TLR signaling pathways and enhanced susceptibility to many different microbial pathogens (13, 24, 25, 40, 42).
L. pneumophila produces several factors that are potential TLR agonists, including lipopolysaccharide, lipopeptides, flagellin, unmethylated CpG DNA, and peptidoglycan (23). It was shown recently that macrophages derived from wild-type mice produce the IL-12 p40 subunit when they are infected with L. pneumophila and that IL-12 p40 production is attenuated in macrophages derived from TLR2-deficient mice, suggesting that TLR2 plays a role in detection of L. pneumophila (2). In contrast, no defect in cytokine production has been observed with macrophages derived from TLR4-deficient mice, suggesting that TLR4 does not respond to L. pneumophila (2). Interestingly, it has been shown that the L. pneumophila lipid A molecule, a canonical TLR4 agonist, is able to induce responses in bone marrow-derived cells from TLR4-deficient mice, but these responses are attenuated in bone marrow-derived cells from TLR2-deficient mice (16). Additionally, TLR4-deficient mice were shown to be as susceptible to L. pneumophila infection as wild-type mice (26). These findings suggest that the L. pneumophila lipid A molecule is a weak TLR4 agonist and potentially stimulates TLR2. This unusual pattern of detection might be explained by the presence of a fatty acid chain in the L. pneumophila lipid A molecule that is much longer than the fatty acid chain found at the same position in lipid A molecules that are known to function as TLR4 agonists (16).
The potential importance of TLR signaling in resistance to Legionnaires' disease was discovered during a genetic analysis of individuals who were infected with L. pneumophila at a flower show in The Netherlands (27). The researchers identified a polymorphism that introduces a stop codon in the human TLR5 gene (TLR5392STOP), which results in a dominant effect that abolishes signaling in response to the L. pneumophila flagellin protein (17). The frequency of the TLR5392STOP mutation was found to be higher in the attendees of the flower show who had radiologically confirmed pneumonia than in a control group of asymptomatic attendees who were likely exposed to L. pneumophila during the same epidemic (17). From these data it was concluded that defects in TLR5 signaling result in enhanced human susceptibility to Legionnaires' disease (17).
Genetically altered mice that have specific deficiencies in TLR proteins and downstream signaling molecules have been constructed; however, macrophages from these mice do not support growth of L. pneumophila because the mice harbor a dominant resistance allele on chromosome 13 known as Lgn1 (3, 9, 46). The presence of this resistance allele complicated previous studies in which the workers used a mouse model to study the role of TLRs in L. pneumophila infection, as the macrophages from these mice are normally nonpermissive for L. pneumophila intracellular replication (49). Although most mouse macrophages have this nonpermissive phenotype, it has been shown that macrophages from A/J mice support intracellular growth of L. pneumophila (49), which makes A/J mice an acceptable model for studying Legionnaires' disease (5). A/J mice are homozygous for an autosomal recessive mutation in the Lgn1 region that renders the macrophages permissive for L. pneumophila replication (3, 9, 10, 47, 48). The Lgn1 region of nonpermissive mouse macrophages encodes a functional Birc1e (Naip5) protein that restricts L. pneumophila growth by a process that includes a rapid cell death pathway regulated by dot/icm-dependent activation of caspase-1 (8, 50). Because the MyD88 gene and the TLR genes are not located on chromosome 13, TLR and MyD88 knockout mice homozygous for the permissive lgn1 allele can be generated easily by crossing F1 heterozygous mice obtained after a knockout mouse is mated with an A/J mouse. We utilized this approach to investigate the role of MyD88, TLR2, and TLR4 in the recognition and clearance of L. pneumophila in a mouse model of Legionnaires' disease.
MATERIALS AND METHODS
Bacterial strains. L. pneumophila serogroup 1 strain JR32 (38), serogroup 1 clinical isolate F2111 (11), and an isogenic dotA mutant of strain F2111 obtained by sodium selection were used in this study. L. pneumophila strains were cultured on charcoal-yeast extract (CYE) agar (12) for 2 days and then cultured overnight in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) broth (10 g/liter yeast extract, 10 g/liter ACES, 0.4 g/liter L-cysteine HCl-H2O, 0.135 g/liter ferric nitrate) prior to use in experiments. For enzyme-linked immunosorbent assay (ELISA) studies and in vivo growth assays, bacteria were grown to an optical density at 600 nm of 1 in AYE broth. For ex vivo growth assays, bacteria were grown to an optical density at 600 nm of 3.4 in AYE broth.
Mice. MyD88–/– (1), TLR2–/– (43), and TLR4–/– (22) mice in a partially backcrossed 129/SvJ x C57BL/6 background were the parental mice used for mating. These knockout mice were crossed with A/J mice (Harlan Sprague Dawley), and the resulting heterozygous F1 generation was bred to generate F2 mice. Tail DNA from F2 mice was screened using PCR to determine the genotype of mice at the Lgn1 locus (46) and the knockout allele of interest (1, 22, 43), as described previously. Only mice homozygous for the permissive lgn1 allele from A/J mice were selected for further breeding. For experiments in which we examined the requirements for an individual TLR protein or MyD88, F2 permissive mice that were homozygous for the knockout allele of interest were mated with siblings that were heterozygous for the knockout allele of interest, which resulted in littermate-matched mice that either were homozygous for the knockout allele or had a functional allele. Preliminary studies in which we compared heterozygous mice with homozygous sufficient mice did not reveal significant differences in L. pneumophila growth in the lung, suggesting that haploinsufficiency should not be a complicating factor in the analysis of homozygous knockout mice with heterozygous littermate controls (data not shown).
Bone marrow-derived macrophages (BMMs). Bone marrow was collected from the femurs and tibiae of mice. Cells were plated on petri dishes and incubated at 37°C in RPMI 1640 containing 20% fetal bovine serum, 30% macrophage colony-stimulating factor (M-CSF)- conditioned medium, and 1% penicillin-streptomycin. On day 6, cells were harvested and resuspended in RPMI 1640 containing 10% fetal bovine serum and 5% M-CSF. Cells were then plated in 24-well tissue culture-treated plates and incubated at 37°C. M-CSF was obtained from an L-929 fibroblast cell line (ATCC).
Cytokine production assays. Bone marrow-derived macrophages were added to 24-well plates at a concentration of 2.5 x 105 cells/well. The cells were infected with L. pneumophila strain JR32 at a multiplicity of infection of 5 and incubated at 37°C. Supernatants were collected 24 h postinfection. IL-12 p40 production and IL-6 production were measured by ELISA using the BD Pharmingen IL-12 (p40/p70) and IL-6 reagents, respectively. Tumor necrosis factor alpha (TNF-) production was measured by ELISA using a BD Biosciences OptEIA mouse TNF kit. Each data point in the figures below is the value for cells from one mouse for which the average cytokine content of three independent wells was determined. The data in each figure are the data from a single experiment in which macrophages from the mice used were derived and infected together. Each experiment was repeated at least once, and similar results were obtained.
Ex vivo macrophage infections and growth assays. BMMs were infected with L. pneumophila strain JR32 at a multiplicity of infection of 5 and incubated at 37°C. For growth assays, BMMs were washed 1 h postinfection with warm Dulbecco's phosphate-buffered saline (PBS) to remove extracellular bacteria. Then either the BMMs were lysed immediately with sterile H2O (day 0) or fresh medium was added until the cells were harvested at 72 h postinfection. Cell lysates were plated on CYE agar to determine the number of bacterial CFU. Each data point is the value for one mouse for which the average bacterial content of three independent wells was determined. The fold differences were determined by dividing the values obtained at 72 h by the values obtained on day 0. Ex vivo growth assays were repeated at least once, and the results obtained were similar.
In vivo mouse infections. Initial studies revealed that there was enhanced proliferation of laboratory strain JR32 and clinical isolate F2111 in the lungs of TLR2-deficient mice compared to the proliferation in the lungs of littermate control heterozygous mice; however, F2111 grew better than JR32 in the lungs of immune-sufficient animals (data not shown). Thus, F2111 and an isogenic F2111 dotA mutant were used for all of the subsequent mouse infection studies. Mice were anesthetized by subcutaneous injection of a ketamine (100 mg/kg)-xylazine (10 mg/kg) PBS solution and infected intranasally with 1 x 106 L. pneumophila F2111 cells in 40 μl (total volume) of PBS. Mice were euthanized with CO2 either 4 h postinfection (day 0) or at different times, and lungs were harvested. Lungs, spleens, or livers were placed in sterile double-distilled H2O and homogenized using a PowerGen 125 handheld homogenizer (Fisher) for 30 s, and lysates were plated on CYE agar to determine the numbers of bacterial CFU. In each in vivo experiment, three mice were used per group unless indicated otherwise. Each data point in the figures below is the CFU count for a single mouse. Although infrequent, contamination of the CYE agar plate used to determine the number of CFU prevented accurate measurement in some of the experiments; this resulted in plotting of the data for only two mice for a time when a contaminated plate was encountered. All experiments were repeated at least two times independently, and similar results were obtained. The lower limit of detection in this assay was 100 CFU of L. pneumophila. To determine IFN- levels in bronchoalveolar lavage (BAL) fluid, mice were intranasally infected with 1 x 106 L. pneumophila F2111 cells. The mice were euthanized 2 days postinfection by subcutaneous injection of a ketamine (250 mg/kg)-xylazine (25 mg/kg) PBS solution. The mouse lungs were lavaged once with 400 μl of PBS, and the BAL fluids were stored at –80°C. The IFN- levels in the BAL fluids were determined by ELISA using BD Pharmingen capture and detection antibodies. The lower limit of detection in this assay was 250 pg/ml of IFN-.
Statistical analysis. Statistical significance was calculated using the unpaired Student t test for cytokine assays and bacterial growth assays. Differences were considered statistically significant if the P value was <0.05.
RESULTS
MyD88 is required for cytokine production by macrophages infected with L. pneumophila. To determine the collective contribution of TLR proteins to cytokine production by macrophages infected with L. pneumophila, BMMs derived from mice deficient in the adapter protein MyD88, heterozygous littermate controls, and A/J mice were infected ex vivo. Production of the cytokines IL-12 p40, IL-6, and TNF- was measured by assaying supernatants 24 h after infection by wild-type L. pneumophila (Fig. 1). The cytokine production by BMMs derived from three heterozygous mice was compared to the cytokine production by BMMs derived from three MyD88-deficient littermates. The cytokine production by MyD88-deficient BMMs was below the limit of detection. Thus, MyD88 is essential for production of cytokines by BMMs in response to L. pneumophila, indicating that TLR signaling has an important role in early innate responses by infected macrophages.
MyD88-dependent signaling pathways do not affect intracellular growth of L. pneumophila in macrophages ex vivo. To determine whether MyD88 signaling may be important for an autocrine response that limits replication of L. pneumophila intracellularly, BMMs from MyD88-deficient mice and heterozygous littermates were infected with L. pneumophila, and intracellular growth was monitored. L. pneumophila CFU were measured for 3 days (Fig. 2), and the fold increases over this time were plotted for BMMs from three different mice. The replication of L. pneumophila in A/J and MyD88+/– macrophages was not impaired compared to the replication in MyD88–/– macrophages. These data indicate that TLR signaling through MyD88 and the subsequent cytokine production do not have a dramatic autocrine effect that restricts L. pneumophila intracellular growth in macrophages.
MyD88 is necessary for host protection against L. pneumophila. Although MyD88-dependent responses did not adversely affect L. pneumophila replication in macrophages ex vivo, the cytokines produced by MyD88-dependent signaling pathways could be essential for clearance of L. pneumophila from the lungs of infected animals. To examine the role of MyD88 in vivo, the susceptibility of mice deficient for MyD88 was compared to the susceptibility of MyD88+/– littermates in a Legionnaires' disease model. Mice were infected intranasally with a clinical isolate of L. pneumophila, and the numbers of bacterial CFU in the lungs were determined over a 9-day period (Fig. 3A). The kinetics of L. pneumophila replication and clearance from the lungs of heterozygous MyD88+/– control mice were similar to the kinetics observed in A/J mice (5). The number of L. pneumophila CFU in the MyD88+/– mice peaked within the first 2 days after infection and then decreased over the next 7 days. By contrast, L. pneumophila replication in the lungs of the MyD88-deficient mice was more pronounced during the first 2 days after infection, and then the number of bacterial CFU remained high for the next 7 days (Fig. 3A). The MyD88-deficient mice were moribund on day 9 after infection, whereas the MyD88+/– mice appeared to be healthy. To evaluate systemic dissemination of L. pneumophila in these animals, the numbers of bacterial CFU in the livers and spleens of mice sacrificed on day 9 were determined (Fig. 3B). High numbers of bacteria were detected in the livers and spleens of MyD88-deficient animals, while bacteria were not detected in the livers and spleens of the heterozygous littermate mice. The IFN- levels in the lungs of mice infected with L. pneumophila were found to peak on day 2 postinfection (data not shown). BAL fluid from MyD88-deficient mice infected with L. pneumophila was compared to BAL fluid from infected heterozygous littermates on day 2 postinfection, and the IFN- levels were low in the lungs of all the MyD88-deficient mice (Fig. 3C). These data are consistent with the hypothesis that IFN- is a critical determinant in clearance of L. pneumophila in the lung and demonstrate that MyD88 is necessary for host protection in a mouse model of Legionnaires' disease.
MyD88-independent responses are sufficient for clearance of L. pneumophila intracellular growth mutants. Although intracellular growth mutants of L. pneumophila are avirulent for animals with a wild-type immune system (29), it is conceivable that extracellular growth of L. pneumophila can occur in the lungs of infected animals. Since MyD88-deficient mice were unable to control proliferation of L. pneumophila in the lungs, these animals were infected with an isogenic dotA mutant of L. pneumophila that is defective for intracellular replication in order to investigate whether MyD88-dependent responses are required for pulmonary clearance of L. pneumophila that may become established in an extracellular niche. MyD88-deficient mice and heterozygous littermates were infected intranasally with an L. pneumophila dotA mutant, and the bacterial CFU in the lungs of infected animals were monitored over a 7-day period (Fig. 4). The data show that the dotA mutant was unable to proliferate following infection of MyD88-deficient mice and was cleared from the lungs with kinetics that closely resembled the kinetics of clearance in heterozygous littermates (Fig. 4). Thus, the data established that MyD88-independent responses are sufficient for clearance of L. pneumophila that is unable to replicate in macrophages and demonstrated that intracellular replication of L. pneumophila in the lungs of infected animals is required for virulence.
TLR4 is not required for detection or clearance of L. pneumophila in a mouse model of Legionnaires' disease. Although TLR4-dependent responses to L. pneumophila were not observed in previous studies (2, 26), these studies were performed using macrophages that were nonpermissive for L. pneumophila replication. For this reason, cytokine production was evaluated using BMMs that were deficient for TLR4 and homozygous for the permissive lgn1 allele from the A/J mouse. The differences between cytokine production by TLR4-deficient BMMs in response to L. pneumophila infection and cytokine production by BMMs derived from either A/J mice or TLR4+/– mice were not significant (Fig. 5). Thus, no effect on macrophage recognition of L. pneumophila was detected in the absence of TLR4. To further examine whether TLR4 plays an important role in the innate immune response to L. pneumophila, TLR4-deficient mice were infected intranasally with L. pneumophila, and bacterial growth in the lungs was monitored over a 7-day period (Fig. 6). Compared to heterozygous littermate control mice, there were no significant differences in the proliferation and clearance of L. pneumophila in the lungs of the TLR4-deficient mice. Thus, TLR4 is not required for macrophage detection of L. pneumophila or bacterial clearance in a mouse model of Legionnaires' disease.
L. pneumophila stimulation of TLR2 enhances macrophage cytokine production and bacterial clearance in a mouse model of Legionnaires' disease. Previous studies have implicated TLR2 as an important receptor on macrophages used for L. pneumophila detection (2, 16). To further examine the importance of TLR2 in the innate immune response to L. pneumophila, mice deficient for TLR2 and homozygous for the permissive lgn1 allele from the A/J mouse were generated. BMMs from TLR2-deficient mice were infected with L. pneumophila, and cytokine production was assayed after 24 h (Fig. 7). The data show that there was a significant decrease in cytokine production by the TLR2-deficient BMMs compared to the cytokine production by BMMs from littermate-matched heterozygous TLR2+/– mice and A/J controls. The data demonstrate that TLR2 contributes to L. pneumophila detection and cytokine production by permissive mouse macrophages.
Because BMMs derived from TLR2–/– mice exhibit reduced cytokine production in response to L. pneumophila infection ex vivo, TLR2-deficient mice were infected intranasally to determine whether this reduced response enhances host susceptibility to infection. TLR2-deficient mice and heterozygous littermates were infected with L. pneumophila, and bacterial growth was monitored over a 7-day period (Fig. 8A). The data showed that there was a slight increase in the replication of L. pneumophila in the lungs of TLR2-deficient mice over the first several days of infection. Unlike MyD88-deficient mice, mice deficient in TLR2 were able to clear L. pneumophila from the lungs, resulting in host survival. To more accurately determine whether replication of L. pneumophila in the lungs of the TLR2-deficient mice was enhanced significantly compared to the replication in TLR2+/– mice, infections were repeated using a larger population of mice. By sacrificing groups of six mice on day 2 and day 3 postinfection, the replication of L. pneumophila in the TLR2-deficient mice was determined to be statistically significant on day 2 (P = 0.013) and day 3 (P = 0.004) compared to the replication in the TLR2+/– littermate control group (Fig. 8B). The IFN- levels in BAL fluids on day 2 were not significantly different for infected TLR2-deficient mice and heterozygous control mice, which is probably the reason that host protection was maintained (Fig. 8C). These results indicate that TLR2-dependent responses contribute to the containment and clearance of L. pneumophila in a mouse model of Legionnaires' disease.
DISCUSSION
TLR signaling is a critical component of the innate immune system required for detection of many types of bacteria (23). Data presented here show that MyD88, the major adaptor protein of the TLR signaling cascade, is essential for cytokine production ex vivo in response to L. pneumophila infection. MyD88–/– BMMs were unable to produce IL-12 p40, IL-6, and TNF- upon L. pneumophila infection (Fig. 1A), suggesting that signaling through MyD88 is important for alerting the host that there is an infection. In vivo, we observed that MyD88-deficient mice were unable to control L. pneumophila infection in the lungs. The L. pneumophila counts in the lungs remained high, and bacterial dissemination to the liver and spleen was observed. This was in contrast to the results obtained for the heterozygous MyD88+/– littermates, which were able to restrict L. pneumophila replication in the lungs as early as day 3 postinfection (Fig. 3). These results demonstrate that MyD88 has a critical role in stimulating a host response that protects animals from Legionnaires' disease.
A defect in the early innate immune response triggered by MyD88 likely explains the dramatic susceptibility phenotype observed for MyD88-deficient mice infected with L. pneumophila. The production and secretion of proinflammatory cytokines by macrophages are predicted to be important for the host defense against L. pneumophila infection. In particular, IL-12 is known to be a potent activator of innate IFN--producing cells, such as NK and NK T cells (44), and it has been shown that IFN- is a critical cytokine for restricting L. pneumophila growth in animals (41). Consistent with this hypothesis, the IFN- levels in the lungs of L. pneumophila-infected MyD88–/– mice were significantly reduced (Fig. 3C). Thus, the inability of MyD88-deficient macrophages to produce measurable IL-12 and other proinflammatory cytokines upon L. pneumophila infection is likely to affect the production of IFN-, which would interfere with activation of macrophages capable of restricting the intracellular growth of L. pneumophila.
To test whether intracellular replication of L. pneumophila is essential for bacterial proliferation in the lungs of MyD88-deficient animals, mice were infected with a dotA mutant that is unable to establish an intracellular niche that permits replication. The data revealed that intracellular growth of L. pneumophila is essential for bacterial proliferation and dissemination in animals that are unable to mount a MyD88-dependent innate immune response (Fig. 4). Clearance of an L. pneumophila dotA mutant in a MyD88-deficient mouse probably occurs by removal of extracellular bacteria through the actions of the mucocilliary escalator of the lung epithelium, in combination with the inability of internalized dotA mutant bacteria to replicate within alveolar macrophages following phagocytosis. Thus, the data establish that the MyD88-dependent cytokine response is important primarily for clearance of L. pneumophila replicating intracellularly.
It was determined that L. pneumophila intracellular replication in MyD88-deficient BMMs homozygous for the permissive lgn1 gene from the A/J mouse was comparable to replication in control BMMs either from heterozygous MyD88 littermates or from A/J mice (Fig. 2). The data indicate that autocrine MyD88-dependent signaling upon TLR stimulation does not restrict intracellular replication of L. pneumophila. Similarly, we did not detect enhanced replication in TLR2-deficient or TLR4-deficient BMMs homozygous for the permissive lgn1 locus (data not shown). Thus, our findings indicate that MyD88 and TLR signaling does not greatly affect cell autonomous pathways that restrict L. pneumophila replication in permissive mouse macrophages.
Our conclusions differ from those of an independent study which showed that TLR2-deficient BMMs are less effective in restricting L. pneumophila growth ex vivo (2). Interpretation of the results of this previous study is complicated by the fact that the TLR2-deficient mice used were derived from founders with a mixed 129/SvJ-C57BL/6 genetic background. Because macrophages from mice having the 129/SvJ lgn1 region are more permissive for L. pneumophila replication than macrophages from mice having the C57BL/6 Lgn1 region are and the Lgn1 genotype of the mice used in this study was not determined, it is possible that the difference in L. pneumophila replication measured in the previous study was related to differences in the Lgn1 region and was not a result of a TLR2 deficiency. Recent studies have indicated that MyD88 signaling is not involved in Lgn1-mediated restriction of L. pneumophila growth in C57BL/6 macrophages (36). Thus, the question of whether L. pneumophila growth is enhanced in TLR2-deficient macrophages that are homozygous for the nonpermissive C57BL/6 Lgn1 region is important because enhanced replication would suggest a novel MyD88-independent role for TLR2 in Lgn1-mediated restriction of L. pneumophila intracellular replication.
L. pneumophila is a gram-negative bacterium that possesses lipopolysaccharide, the canonical TLR4 agonist (22, 23). Similar to data obtained in previous studies, data obtained in this study show that TLR4-deficient BMMs that permit L. pneumophila replication respond to L. pneumophila like littermate control macrophages respond, as indicated by similar levels of cytokine production (Fig. 5). Having TLR4-deficient mice homozygous for the permissive lgn1 allele allowed us to examine the role of TLR4 in L. pneumophila clearance in a mouse model of Legionnaires' disease. The data show that L. pneumophila proliferation and clearance in the lungs of TLR4-deficient animals were similar to L. pneumophila proliferation and clearance in the lungs of heterozygous littermate control mice (Fig. 6). Thus, TLR4 signaling is not critical for L. pneumophila recognition or clearance. It is important to note that the data do not rule out the possibility that TLR4 can respond to L. pneumophila. TLR4 signaling in response to L. pneumophila infection may occur; however, in the absence of TLR4, stimulation of the other TLRs is sufficient to obtain a response that is similar to the response in wild-type mice.
There are several lines of evidence that indicate that TLR2 is involved in the macrophage response to L. pneumophila. Using cells from a nonpermissive mouse host, TLR2-dependent responses to purified L. pneumophila lipid A and intact L. pneumophila bacteria have been demonstrated (16). Additionally, it has been shown that TLR2 stimulation of activated mouse macrophages by L. pneumophila is important for triggering the production of prostaglandins that inhibit T-cell secretion of IFN- (35). These ex vivo studies suggested that stimulation of TLR2 by L. pneumophila during macrophage infection facilitates early innate immune responses that lead to protection of the host and also later responses that dampen the immune response once bacterial containment has been achieved.
Here, it was shown using BMMs from mice homozygous for the permissive lgn1 allele that TLR2 is important for cytokine production by macrophages infected with L. pneumophila (Fig. 7), which is consistent with observations made by using macrophages from a nonpermissive mouse (2). More importantly, infection of these TLR2-deficient mice with L. pneumophila revealed a significant increase in L. pneumophila replication in the lungs over the first 3 days of infection (Fig. 8B). Unlike the MyD88-deficient mice, the TLR2-deficient mice retained the ability to clear L. pneumophila from the lungs (Fig. 8A), indicating that other MyD88-dependent signaling pathways are operational in the TLR2-deficient mice. Importantly, cytokine production by the TLR2-deficient macrophages was reduced, but not eliminated, in response to L. pneumophila (Fig. 7), and the IFN- levels in the lungs of infected TLR2-deficient mice were similar to the levels in heterozygous control mice (Fig. 8). Thus, a possible explanation for the in vivo TLR2-deficient phenotype is that the induction of a protective innate immune in response to L. pneumophila in these animals was delayed due to the reduced levels of cytokine production. It is also possible that once a protective response to L. pneumophila has been mounted, it is sustained longer or is exaggerated slightly in the TLR2-deficient animals due to the defect in prostaglandin-mediated suppression of IFN- production by T cells reported previously (35), which might enhance the ability of the TLR2-deficient animals to efficiently clear the infection.
In addition to TLR2, there are several other TLRs that require the MyD88 protein for signaling that are probably stimulated during infection by L. pneumophila. It has been shown that the L. pneumophila flagellin protein stimulates TLR5 (17), and epidemiological evidence supports the hypothesis that TLR5 has a role in protection of humans against Legionnaires' disease (17). Additionally, it is known that roughly 20% of the L. pneumophila cells internalized by macrophages are unsuccessful in their attempts to evade fusion with lysosomes (37). Degradation of bacteria that traffic incorrectly would result in the release of unmethylated DNA with CpG motifs, an agonist that stimulates TLR9 signaling pathways (18). TLR11 has also been implicated in detection of bacterial pathogens, although the bacterial ligand that stimulates TLR11 is not well defined (51), so it is difficult to predict whether TLR11 could be involved in L. pneumophila detection. The presence of multiple TLR proteins capable of detecting molecular determinants presented by L. pneumophila is likely the reason that MyD88-deficient mice are exquisitely sensitive to L. pneumophila infection and mice deficient in a single TLR retain the ability to effectively combat and clear L. pneumophila infections. Future studies performed with mice that are homozygous for the permissive lgn1 allele and deficient in multiple TLRs might eventually reveal the principal receptors used for the MyD88-dependent signaling pathway that is essential for host protection against Legionnaires' disease.
ACKNOWLEDGMENTS
We thank S. Akira (Osaka, Japan) for permission to use knockout mice in this study, R. Medzhitov for providing these mice, H. Shuman (Columbia University) for providing the JR32 strain, and P. Edelstein (University of Pennsylvania) for providing the F2111 isolate. We also thank A. Neild, D. Zamboni, and S. Shin for their suggestions and technical assistance.
This work was supported by an NSF predoctoral award (to K.A.A.) and by NIH grant R01-AI048770 (to C.R.R.).
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ABSTRACT
Legionella pneumophila is a gram-negative facultative intracellular parasite of macrophages. Although L. pneumophila is the causative agent of a severe pneumonia known as Legionnaires' disease, it is likely that most infections caused by this organism are cleared by the host innate immune system. It is predicted that host pattern recognition proteins belonging to the Toll-like receptor (TLR) family are involved in the protective innate immune responses. We examined the role of TLR-mediated responses in L. pneumophila detection and clearance using genetically altered mouse hosts in which the macrophages are permissive for L. pneumophila intracellular replication. Our data demonstrate that cytokine production by bone marrow-derived macrophages (BMMs) in response to L. pneumophila infection requires the TLR adapter protein MyD88 and is reduced in the absence of TLR2 but not in the absence of TLR4. Bacterial growth ex vivo in BMMs from MyD88-deficient mice was not enhanced compared to bacterial growth ex vivo in BMMs from heterozygous littermate controls. Wild-type mice were able to clear L. pneumophila from the lung, whereas respiratory infection of MyD88-deficient mice caused death that resulted from robust bacterial replication and dissemination. In contrast to an infection with virulent L. pneumophila, MyD88-deficient mice were able to clear infections with L. pneumophila dotA mutants, indicating that MyD88-independent responses in the lung are sufficient to clear bacteria that are unable to replicate intracellularly. In vivo growth of L. pneumophila was enhanced in the lungs of TLR2-deficient mice, which resulted in a delay in bacterial clearance. No significant differences were observed in the growth and clearance of L. pneumophila in the lungs of TLR4-deficient mice and heterozygous littermate control mice. Our data indicate that MyD88 is crucial for eliciting a protective innate immune response against virulent L. pneumophila and that TLR2 is one of the pattern recognition receptors involved in initiating this MyD88-dependent response.
INTRODUCTION
Legionella pneumophila is a ubiquitous gram-negative bacterium that parasitizes phagocytic protozoans in freshwater environments (14). When L. pneumophila gains access to the human respiratory system via inhalation of contaminated water sources, the bacteria have the capacity to replicate within alveolar macrophages (15, 21, 30). The Dot/Icm apparatus is a type IV secretion system that is required for L. pneumophila intracellular replication (29, 39, 45). Bacterial proteins translocated into host cells by the Dot/Icm system modulate trafficking of the vacuole containing L. pneumophila and promote biogenesis of a unique organelle that evades fusion with lysosomes and supports intracellular replication (7, 19, 20, 28, 33, 37). Although a severe type of pneumonia known as Legionnaires' disease can result from L. pneumophila replication in host macrophages (15, 21, 30), most infections are probably contained and cleared by the immune system without disease.
How the host detects L. pneumophila and initiates a protective response has not been clearly established. Previous studies have suggested that detection of L. pneumophila initiates a host response that leads to the production of gamma interferon (IFN-) and macrophage activation (4, 34). Treatment of macrophages ex vivo with IFN- is sufficient to upregulate effectors that restrict L. pneumophila intracellular growth (4, 34, 35). Using a mouse model of Legionnaires' disease, it has been shown that the cytokine interleukin-12 (IL-12) is important for host protection against L. pneumophila infection (6). IL-12 is able to stimulate production of IFN- by NK and NK T cells (44), suggesting that host susceptibility in IL-12-depleted mice is due primarily to a defect in macrophage activation by IFN- (6). Consistent with this hypothesis, mice lacking IFN- have lower survival rates upon infection with L. pneumophila than wild-type mice have (41).
Toll-like receptors (TLRs) are a well-characterized family of transmembrane pattern recognition receptors that detect conserved molecular patterns on microbes (23). TLRs can generate a rapid and potent response against a pathogen through induction of the transcription factor NF-B, leading to events that include production of inflammatory cytokines, activation of macrophages, and maturation of dendritic cells (31). Following TLR stimulation, the cytoplasmic adapter protein MyD88 is required for transduction of a signal that activates NF-B (32). MyD88-deficient mice have defects in most TLR signaling pathways and enhanced susceptibility to many different microbial pathogens (13, 24, 25, 40, 42).
L. pneumophila produces several factors that are potential TLR agonists, including lipopolysaccharide, lipopeptides, flagellin, unmethylated CpG DNA, and peptidoglycan (23). It was shown recently that macrophages derived from wild-type mice produce the IL-12 p40 subunit when they are infected with L. pneumophila and that IL-12 p40 production is attenuated in macrophages derived from TLR2-deficient mice, suggesting that TLR2 plays a role in detection of L. pneumophila (2). In contrast, no defect in cytokine production has been observed with macrophages derived from TLR4-deficient mice, suggesting that TLR4 does not respond to L. pneumophila (2). Interestingly, it has been shown that the L. pneumophila lipid A molecule, a canonical TLR4 agonist, is able to induce responses in bone marrow-derived cells from TLR4-deficient mice, but these responses are attenuated in bone marrow-derived cells from TLR2-deficient mice (16). Additionally, TLR4-deficient mice were shown to be as susceptible to L. pneumophila infection as wild-type mice (26). These findings suggest that the L. pneumophila lipid A molecule is a weak TLR4 agonist and potentially stimulates TLR2. This unusual pattern of detection might be explained by the presence of a fatty acid chain in the L. pneumophila lipid A molecule that is much longer than the fatty acid chain found at the same position in lipid A molecules that are known to function as TLR4 agonists (16).
The potential importance of TLR signaling in resistance to Legionnaires' disease was discovered during a genetic analysis of individuals who were infected with L. pneumophila at a flower show in The Netherlands (27). The researchers identified a polymorphism that introduces a stop codon in the human TLR5 gene (TLR5392STOP), which results in a dominant effect that abolishes signaling in response to the L. pneumophila flagellin protein (17). The frequency of the TLR5392STOP mutation was found to be higher in the attendees of the flower show who had radiologically confirmed pneumonia than in a control group of asymptomatic attendees who were likely exposed to L. pneumophila during the same epidemic (17). From these data it was concluded that defects in TLR5 signaling result in enhanced human susceptibility to Legionnaires' disease (17).
Genetically altered mice that have specific deficiencies in TLR proteins and downstream signaling molecules have been constructed; however, macrophages from these mice do not support growth of L. pneumophila because the mice harbor a dominant resistance allele on chromosome 13 known as Lgn1 (3, 9, 46). The presence of this resistance allele complicated previous studies in which the workers used a mouse model to study the role of TLRs in L. pneumophila infection, as the macrophages from these mice are normally nonpermissive for L. pneumophila intracellular replication (49). Although most mouse macrophages have this nonpermissive phenotype, it has been shown that macrophages from A/J mice support intracellular growth of L. pneumophila (49), which makes A/J mice an acceptable model for studying Legionnaires' disease (5). A/J mice are homozygous for an autosomal recessive mutation in the Lgn1 region that renders the macrophages permissive for L. pneumophila replication (3, 9, 10, 47, 48). The Lgn1 region of nonpermissive mouse macrophages encodes a functional Birc1e (Naip5) protein that restricts L. pneumophila growth by a process that includes a rapid cell death pathway regulated by dot/icm-dependent activation of caspase-1 (8, 50). Because the MyD88 gene and the TLR genes are not located on chromosome 13, TLR and MyD88 knockout mice homozygous for the permissive lgn1 allele can be generated easily by crossing F1 heterozygous mice obtained after a knockout mouse is mated with an A/J mouse. We utilized this approach to investigate the role of MyD88, TLR2, and TLR4 in the recognition and clearance of L. pneumophila in a mouse model of Legionnaires' disease.
MATERIALS AND METHODS
Bacterial strains. L. pneumophila serogroup 1 strain JR32 (38), serogroup 1 clinical isolate F2111 (11), and an isogenic dotA mutant of strain F2111 obtained by sodium selection were used in this study. L. pneumophila strains were cultured on charcoal-yeast extract (CYE) agar (12) for 2 days and then cultured overnight in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) broth (10 g/liter yeast extract, 10 g/liter ACES, 0.4 g/liter L-cysteine HCl-H2O, 0.135 g/liter ferric nitrate) prior to use in experiments. For enzyme-linked immunosorbent assay (ELISA) studies and in vivo growth assays, bacteria were grown to an optical density at 600 nm of 1 in AYE broth. For ex vivo growth assays, bacteria were grown to an optical density at 600 nm of 3.4 in AYE broth.
Mice. MyD88–/– (1), TLR2–/– (43), and TLR4–/– (22) mice in a partially backcrossed 129/SvJ x C57BL/6 background were the parental mice used for mating. These knockout mice were crossed with A/J mice (Harlan Sprague Dawley), and the resulting heterozygous F1 generation was bred to generate F2 mice. Tail DNA from F2 mice was screened using PCR to determine the genotype of mice at the Lgn1 locus (46) and the knockout allele of interest (1, 22, 43), as described previously. Only mice homozygous for the permissive lgn1 allele from A/J mice were selected for further breeding. For experiments in which we examined the requirements for an individual TLR protein or MyD88, F2 permissive mice that were homozygous for the knockout allele of interest were mated with siblings that were heterozygous for the knockout allele of interest, which resulted in littermate-matched mice that either were homozygous for the knockout allele or had a functional allele. Preliminary studies in which we compared heterozygous mice with homozygous sufficient mice did not reveal significant differences in L. pneumophila growth in the lung, suggesting that haploinsufficiency should not be a complicating factor in the analysis of homozygous knockout mice with heterozygous littermate controls (data not shown).
Bone marrow-derived macrophages (BMMs). Bone marrow was collected from the femurs and tibiae of mice. Cells were plated on petri dishes and incubated at 37°C in RPMI 1640 containing 20% fetal bovine serum, 30% macrophage colony-stimulating factor (M-CSF)- conditioned medium, and 1% penicillin-streptomycin. On day 6, cells were harvested and resuspended in RPMI 1640 containing 10% fetal bovine serum and 5% M-CSF. Cells were then plated in 24-well tissue culture-treated plates and incubated at 37°C. M-CSF was obtained from an L-929 fibroblast cell line (ATCC).
Cytokine production assays. Bone marrow-derived macrophages were added to 24-well plates at a concentration of 2.5 x 105 cells/well. The cells were infected with L. pneumophila strain JR32 at a multiplicity of infection of 5 and incubated at 37°C. Supernatants were collected 24 h postinfection. IL-12 p40 production and IL-6 production were measured by ELISA using the BD Pharmingen IL-12 (p40/p70) and IL-6 reagents, respectively. Tumor necrosis factor alpha (TNF-) production was measured by ELISA using a BD Biosciences OptEIA mouse TNF kit. Each data point in the figures below is the value for cells from one mouse for which the average cytokine content of three independent wells was determined. The data in each figure are the data from a single experiment in which macrophages from the mice used were derived and infected together. Each experiment was repeated at least once, and similar results were obtained.
Ex vivo macrophage infections and growth assays. BMMs were infected with L. pneumophila strain JR32 at a multiplicity of infection of 5 and incubated at 37°C. For growth assays, BMMs were washed 1 h postinfection with warm Dulbecco's phosphate-buffered saline (PBS) to remove extracellular bacteria. Then either the BMMs were lysed immediately with sterile H2O (day 0) or fresh medium was added until the cells were harvested at 72 h postinfection. Cell lysates were plated on CYE agar to determine the number of bacterial CFU. Each data point is the value for one mouse for which the average bacterial content of three independent wells was determined. The fold differences were determined by dividing the values obtained at 72 h by the values obtained on day 0. Ex vivo growth assays were repeated at least once, and the results obtained were similar.
In vivo mouse infections. Initial studies revealed that there was enhanced proliferation of laboratory strain JR32 and clinical isolate F2111 in the lungs of TLR2-deficient mice compared to the proliferation in the lungs of littermate control heterozygous mice; however, F2111 grew better than JR32 in the lungs of immune-sufficient animals (data not shown). Thus, F2111 and an isogenic F2111 dotA mutant were used for all of the subsequent mouse infection studies. Mice were anesthetized by subcutaneous injection of a ketamine (100 mg/kg)-xylazine (10 mg/kg) PBS solution and infected intranasally with 1 x 106 L. pneumophila F2111 cells in 40 μl (total volume) of PBS. Mice were euthanized with CO2 either 4 h postinfection (day 0) or at different times, and lungs were harvested. Lungs, spleens, or livers were placed in sterile double-distilled H2O and homogenized using a PowerGen 125 handheld homogenizer (Fisher) for 30 s, and lysates were plated on CYE agar to determine the numbers of bacterial CFU. In each in vivo experiment, three mice were used per group unless indicated otherwise. Each data point in the figures below is the CFU count for a single mouse. Although infrequent, contamination of the CYE agar plate used to determine the number of CFU prevented accurate measurement in some of the experiments; this resulted in plotting of the data for only two mice for a time when a contaminated plate was encountered. All experiments were repeated at least two times independently, and similar results were obtained. The lower limit of detection in this assay was 100 CFU of L. pneumophila. To determine IFN- levels in bronchoalveolar lavage (BAL) fluid, mice were intranasally infected with 1 x 106 L. pneumophila F2111 cells. The mice were euthanized 2 days postinfection by subcutaneous injection of a ketamine (250 mg/kg)-xylazine (25 mg/kg) PBS solution. The mouse lungs were lavaged once with 400 μl of PBS, and the BAL fluids were stored at –80°C. The IFN- levels in the BAL fluids were determined by ELISA using BD Pharmingen capture and detection antibodies. The lower limit of detection in this assay was 250 pg/ml of IFN-.
Statistical analysis. Statistical significance was calculated using the unpaired Student t test for cytokine assays and bacterial growth assays. Differences were considered statistically significant if the P value was <0.05.
RESULTS
MyD88 is required for cytokine production by macrophages infected with L. pneumophila. To determine the collective contribution of TLR proteins to cytokine production by macrophages infected with L. pneumophila, BMMs derived from mice deficient in the adapter protein MyD88, heterozygous littermate controls, and A/J mice were infected ex vivo. Production of the cytokines IL-12 p40, IL-6, and TNF- was measured by assaying supernatants 24 h after infection by wild-type L. pneumophila (Fig. 1). The cytokine production by BMMs derived from three heterozygous mice was compared to the cytokine production by BMMs derived from three MyD88-deficient littermates. The cytokine production by MyD88-deficient BMMs was below the limit of detection. Thus, MyD88 is essential for production of cytokines by BMMs in response to L. pneumophila, indicating that TLR signaling has an important role in early innate responses by infected macrophages.
MyD88-dependent signaling pathways do not affect intracellular growth of L. pneumophila in macrophages ex vivo. To determine whether MyD88 signaling may be important for an autocrine response that limits replication of L. pneumophila intracellularly, BMMs from MyD88-deficient mice and heterozygous littermates were infected with L. pneumophila, and intracellular growth was monitored. L. pneumophila CFU were measured for 3 days (Fig. 2), and the fold increases over this time were plotted for BMMs from three different mice. The replication of L. pneumophila in A/J and MyD88+/– macrophages was not impaired compared to the replication in MyD88–/– macrophages. These data indicate that TLR signaling through MyD88 and the subsequent cytokine production do not have a dramatic autocrine effect that restricts L. pneumophila intracellular growth in macrophages.
MyD88 is necessary for host protection against L. pneumophila. Although MyD88-dependent responses did not adversely affect L. pneumophila replication in macrophages ex vivo, the cytokines produced by MyD88-dependent signaling pathways could be essential for clearance of L. pneumophila from the lungs of infected animals. To examine the role of MyD88 in vivo, the susceptibility of mice deficient for MyD88 was compared to the susceptibility of MyD88+/– littermates in a Legionnaires' disease model. Mice were infected intranasally with a clinical isolate of L. pneumophila, and the numbers of bacterial CFU in the lungs were determined over a 9-day period (Fig. 3A). The kinetics of L. pneumophila replication and clearance from the lungs of heterozygous MyD88+/– control mice were similar to the kinetics observed in A/J mice (5). The number of L. pneumophila CFU in the MyD88+/– mice peaked within the first 2 days after infection and then decreased over the next 7 days. By contrast, L. pneumophila replication in the lungs of the MyD88-deficient mice was more pronounced during the first 2 days after infection, and then the number of bacterial CFU remained high for the next 7 days (Fig. 3A). The MyD88-deficient mice were moribund on day 9 after infection, whereas the MyD88+/– mice appeared to be healthy. To evaluate systemic dissemination of L. pneumophila in these animals, the numbers of bacterial CFU in the livers and spleens of mice sacrificed on day 9 were determined (Fig. 3B). High numbers of bacteria were detected in the livers and spleens of MyD88-deficient animals, while bacteria were not detected in the livers and spleens of the heterozygous littermate mice. The IFN- levels in the lungs of mice infected with L. pneumophila were found to peak on day 2 postinfection (data not shown). BAL fluid from MyD88-deficient mice infected with L. pneumophila was compared to BAL fluid from infected heterozygous littermates on day 2 postinfection, and the IFN- levels were low in the lungs of all the MyD88-deficient mice (Fig. 3C). These data are consistent with the hypothesis that IFN- is a critical determinant in clearance of L. pneumophila in the lung and demonstrate that MyD88 is necessary for host protection in a mouse model of Legionnaires' disease.
MyD88-independent responses are sufficient for clearance of L. pneumophila intracellular growth mutants. Although intracellular growth mutants of L. pneumophila are avirulent for animals with a wild-type immune system (29), it is conceivable that extracellular growth of L. pneumophila can occur in the lungs of infected animals. Since MyD88-deficient mice were unable to control proliferation of L. pneumophila in the lungs, these animals were infected with an isogenic dotA mutant of L. pneumophila that is defective for intracellular replication in order to investigate whether MyD88-dependent responses are required for pulmonary clearance of L. pneumophila that may become established in an extracellular niche. MyD88-deficient mice and heterozygous littermates were infected intranasally with an L. pneumophila dotA mutant, and the bacterial CFU in the lungs of infected animals were monitored over a 7-day period (Fig. 4). The data show that the dotA mutant was unable to proliferate following infection of MyD88-deficient mice and was cleared from the lungs with kinetics that closely resembled the kinetics of clearance in heterozygous littermates (Fig. 4). Thus, the data established that MyD88-independent responses are sufficient for clearance of L. pneumophila that is unable to replicate in macrophages and demonstrated that intracellular replication of L. pneumophila in the lungs of infected animals is required for virulence.
TLR4 is not required for detection or clearance of L. pneumophila in a mouse model of Legionnaires' disease. Although TLR4-dependent responses to L. pneumophila were not observed in previous studies (2, 26), these studies were performed using macrophages that were nonpermissive for L. pneumophila replication. For this reason, cytokine production was evaluated using BMMs that were deficient for TLR4 and homozygous for the permissive lgn1 allele from the A/J mouse. The differences between cytokine production by TLR4-deficient BMMs in response to L. pneumophila infection and cytokine production by BMMs derived from either A/J mice or TLR4+/– mice were not significant (Fig. 5). Thus, no effect on macrophage recognition of L. pneumophila was detected in the absence of TLR4. To further examine whether TLR4 plays an important role in the innate immune response to L. pneumophila, TLR4-deficient mice were infected intranasally with L. pneumophila, and bacterial growth in the lungs was monitored over a 7-day period (Fig. 6). Compared to heterozygous littermate control mice, there were no significant differences in the proliferation and clearance of L. pneumophila in the lungs of the TLR4-deficient mice. Thus, TLR4 is not required for macrophage detection of L. pneumophila or bacterial clearance in a mouse model of Legionnaires' disease.
L. pneumophila stimulation of TLR2 enhances macrophage cytokine production and bacterial clearance in a mouse model of Legionnaires' disease. Previous studies have implicated TLR2 as an important receptor on macrophages used for L. pneumophila detection (2, 16). To further examine the importance of TLR2 in the innate immune response to L. pneumophila, mice deficient for TLR2 and homozygous for the permissive lgn1 allele from the A/J mouse were generated. BMMs from TLR2-deficient mice were infected with L. pneumophila, and cytokine production was assayed after 24 h (Fig. 7). The data show that there was a significant decrease in cytokine production by the TLR2-deficient BMMs compared to the cytokine production by BMMs from littermate-matched heterozygous TLR2+/– mice and A/J controls. The data demonstrate that TLR2 contributes to L. pneumophila detection and cytokine production by permissive mouse macrophages.
Because BMMs derived from TLR2–/– mice exhibit reduced cytokine production in response to L. pneumophila infection ex vivo, TLR2-deficient mice were infected intranasally to determine whether this reduced response enhances host susceptibility to infection. TLR2-deficient mice and heterozygous littermates were infected with L. pneumophila, and bacterial growth was monitored over a 7-day period (Fig. 8A). The data showed that there was a slight increase in the replication of L. pneumophila in the lungs of TLR2-deficient mice over the first several days of infection. Unlike MyD88-deficient mice, mice deficient in TLR2 were able to clear L. pneumophila from the lungs, resulting in host survival. To more accurately determine whether replication of L. pneumophila in the lungs of the TLR2-deficient mice was enhanced significantly compared to the replication in TLR2+/– mice, infections were repeated using a larger population of mice. By sacrificing groups of six mice on day 2 and day 3 postinfection, the replication of L. pneumophila in the TLR2-deficient mice was determined to be statistically significant on day 2 (P = 0.013) and day 3 (P = 0.004) compared to the replication in the TLR2+/– littermate control group (Fig. 8B). The IFN- levels in BAL fluids on day 2 were not significantly different for infected TLR2-deficient mice and heterozygous control mice, which is probably the reason that host protection was maintained (Fig. 8C). These results indicate that TLR2-dependent responses contribute to the containment and clearance of L. pneumophila in a mouse model of Legionnaires' disease.
DISCUSSION
TLR signaling is a critical component of the innate immune system required for detection of many types of bacteria (23). Data presented here show that MyD88, the major adaptor protein of the TLR signaling cascade, is essential for cytokine production ex vivo in response to L. pneumophila infection. MyD88–/– BMMs were unable to produce IL-12 p40, IL-6, and TNF- upon L. pneumophila infection (Fig. 1A), suggesting that signaling through MyD88 is important for alerting the host that there is an infection. In vivo, we observed that MyD88-deficient mice were unable to control L. pneumophila infection in the lungs. The L. pneumophila counts in the lungs remained high, and bacterial dissemination to the liver and spleen was observed. This was in contrast to the results obtained for the heterozygous MyD88+/– littermates, which were able to restrict L. pneumophila replication in the lungs as early as day 3 postinfection (Fig. 3). These results demonstrate that MyD88 has a critical role in stimulating a host response that protects animals from Legionnaires' disease.
A defect in the early innate immune response triggered by MyD88 likely explains the dramatic susceptibility phenotype observed for MyD88-deficient mice infected with L. pneumophila. The production and secretion of proinflammatory cytokines by macrophages are predicted to be important for the host defense against L. pneumophila infection. In particular, IL-12 is known to be a potent activator of innate IFN--producing cells, such as NK and NK T cells (44), and it has been shown that IFN- is a critical cytokine for restricting L. pneumophila growth in animals (41). Consistent with this hypothesis, the IFN- levels in the lungs of L. pneumophila-infected MyD88–/– mice were significantly reduced (Fig. 3C). Thus, the inability of MyD88-deficient macrophages to produce measurable IL-12 and other proinflammatory cytokines upon L. pneumophila infection is likely to affect the production of IFN-, which would interfere with activation of macrophages capable of restricting the intracellular growth of L. pneumophila.
To test whether intracellular replication of L. pneumophila is essential for bacterial proliferation in the lungs of MyD88-deficient animals, mice were infected with a dotA mutant that is unable to establish an intracellular niche that permits replication. The data revealed that intracellular growth of L. pneumophila is essential for bacterial proliferation and dissemination in animals that are unable to mount a MyD88-dependent innate immune response (Fig. 4). Clearance of an L. pneumophila dotA mutant in a MyD88-deficient mouse probably occurs by removal of extracellular bacteria through the actions of the mucocilliary escalator of the lung epithelium, in combination with the inability of internalized dotA mutant bacteria to replicate within alveolar macrophages following phagocytosis. Thus, the data establish that the MyD88-dependent cytokine response is important primarily for clearance of L. pneumophila replicating intracellularly.
It was determined that L. pneumophila intracellular replication in MyD88-deficient BMMs homozygous for the permissive lgn1 gene from the A/J mouse was comparable to replication in control BMMs either from heterozygous MyD88 littermates or from A/J mice (Fig. 2). The data indicate that autocrine MyD88-dependent signaling upon TLR stimulation does not restrict intracellular replication of L. pneumophila. Similarly, we did not detect enhanced replication in TLR2-deficient or TLR4-deficient BMMs homozygous for the permissive lgn1 locus (data not shown). Thus, our findings indicate that MyD88 and TLR signaling does not greatly affect cell autonomous pathways that restrict L. pneumophila replication in permissive mouse macrophages.
Our conclusions differ from those of an independent study which showed that TLR2-deficient BMMs are less effective in restricting L. pneumophila growth ex vivo (2). Interpretation of the results of this previous study is complicated by the fact that the TLR2-deficient mice used were derived from founders with a mixed 129/SvJ-C57BL/6 genetic background. Because macrophages from mice having the 129/SvJ lgn1 region are more permissive for L. pneumophila replication than macrophages from mice having the C57BL/6 Lgn1 region are and the Lgn1 genotype of the mice used in this study was not determined, it is possible that the difference in L. pneumophila replication measured in the previous study was related to differences in the Lgn1 region and was not a result of a TLR2 deficiency. Recent studies have indicated that MyD88 signaling is not involved in Lgn1-mediated restriction of L. pneumophila growth in C57BL/6 macrophages (36). Thus, the question of whether L. pneumophila growth is enhanced in TLR2-deficient macrophages that are homozygous for the nonpermissive C57BL/6 Lgn1 region is important because enhanced replication would suggest a novel MyD88-independent role for TLR2 in Lgn1-mediated restriction of L. pneumophila intracellular replication.
L. pneumophila is a gram-negative bacterium that possesses lipopolysaccharide, the canonical TLR4 agonist (22, 23). Similar to data obtained in previous studies, data obtained in this study show that TLR4-deficient BMMs that permit L. pneumophila replication respond to L. pneumophila like littermate control macrophages respond, as indicated by similar levels of cytokine production (Fig. 5). Having TLR4-deficient mice homozygous for the permissive lgn1 allele allowed us to examine the role of TLR4 in L. pneumophila clearance in a mouse model of Legionnaires' disease. The data show that L. pneumophila proliferation and clearance in the lungs of TLR4-deficient animals were similar to L. pneumophila proliferation and clearance in the lungs of heterozygous littermate control mice (Fig. 6). Thus, TLR4 signaling is not critical for L. pneumophila recognition or clearance. It is important to note that the data do not rule out the possibility that TLR4 can respond to L. pneumophila. TLR4 signaling in response to L. pneumophila infection may occur; however, in the absence of TLR4, stimulation of the other TLRs is sufficient to obtain a response that is similar to the response in wild-type mice.
There are several lines of evidence that indicate that TLR2 is involved in the macrophage response to L. pneumophila. Using cells from a nonpermissive mouse host, TLR2-dependent responses to purified L. pneumophila lipid A and intact L. pneumophila bacteria have been demonstrated (16). Additionally, it has been shown that TLR2 stimulation of activated mouse macrophages by L. pneumophila is important for triggering the production of prostaglandins that inhibit T-cell secretion of IFN- (35). These ex vivo studies suggested that stimulation of TLR2 by L. pneumophila during macrophage infection facilitates early innate immune responses that lead to protection of the host and also later responses that dampen the immune response once bacterial containment has been achieved.
Here, it was shown using BMMs from mice homozygous for the permissive lgn1 allele that TLR2 is important for cytokine production by macrophages infected with L. pneumophila (Fig. 7), which is consistent with observations made by using macrophages from a nonpermissive mouse (2). More importantly, infection of these TLR2-deficient mice with L. pneumophila revealed a significant increase in L. pneumophila replication in the lungs over the first 3 days of infection (Fig. 8B). Unlike the MyD88-deficient mice, the TLR2-deficient mice retained the ability to clear L. pneumophila from the lungs (Fig. 8A), indicating that other MyD88-dependent signaling pathways are operational in the TLR2-deficient mice. Importantly, cytokine production by the TLR2-deficient macrophages was reduced, but not eliminated, in response to L. pneumophila (Fig. 7), and the IFN- levels in the lungs of infected TLR2-deficient mice were similar to the levels in heterozygous control mice (Fig. 8). Thus, a possible explanation for the in vivo TLR2-deficient phenotype is that the induction of a protective innate immune in response to L. pneumophila in these animals was delayed due to the reduced levels of cytokine production. It is also possible that once a protective response to L. pneumophila has been mounted, it is sustained longer or is exaggerated slightly in the TLR2-deficient animals due to the defect in prostaglandin-mediated suppression of IFN- production by T cells reported previously (35), which might enhance the ability of the TLR2-deficient animals to efficiently clear the infection.
In addition to TLR2, there are several other TLRs that require the MyD88 protein for signaling that are probably stimulated during infection by L. pneumophila. It has been shown that the L. pneumophila flagellin protein stimulates TLR5 (17), and epidemiological evidence supports the hypothesis that TLR5 has a role in protection of humans against Legionnaires' disease (17). Additionally, it is known that roughly 20% of the L. pneumophila cells internalized by macrophages are unsuccessful in their attempts to evade fusion with lysosomes (37). Degradation of bacteria that traffic incorrectly would result in the release of unmethylated DNA with CpG motifs, an agonist that stimulates TLR9 signaling pathways (18). TLR11 has also been implicated in detection of bacterial pathogens, although the bacterial ligand that stimulates TLR11 is not well defined (51), so it is difficult to predict whether TLR11 could be involved in L. pneumophila detection. The presence of multiple TLR proteins capable of detecting molecular determinants presented by L. pneumophila is likely the reason that MyD88-deficient mice are exquisitely sensitive to L. pneumophila infection and mice deficient in a single TLR retain the ability to effectively combat and clear L. pneumophila infections. Future studies performed with mice that are homozygous for the permissive lgn1 allele and deficient in multiple TLRs might eventually reveal the principal receptors used for the MyD88-dependent signaling pathway that is essential for host protection against Legionnaires' disease.
ACKNOWLEDGMENTS
We thank S. Akira (Osaka, Japan) for permission to use knockout mice in this study, R. Medzhitov for providing these mice, H. Shuman (Columbia University) for providing the JR32 strain, and P. Edelstein (University of Pennsylvania) for providing the F2111 isolate. We also thank A. Neild, D. Zamboni, and S. Shin for their suggestions and technical assistance.
This work was supported by an NSF predoctoral award (to K.A.A.) and by NIH grant R01-AI048770 (to C.R.R.).
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