Polymorphonuclear Neutrophils Improve Replication of Chlamydia pneumoniae In Vivo upon MyD88-Dependent Attraction
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免疫学杂志 2005年第8期
Abstract
Chlamydia pneumoniae, an obligate intracellular bacterium, causes pneumonia in humans and mice. In this study, we show that GR1+/CD45+ polymorphonuclear neutrophils (PMN) surprisingly increase the bacterial load of C. pneumoniae in vivo. Upon intranasal infection of wild-type mice, the lung weight is increased; the cytokines TNF, IL-12p40, and IFN-, as well as the chemokines keratinocyte-derived chemokine, MCP-1, and MIP-2 are secreted; and GR1+/CD45+ PMN are recruited into lungs 3 days postinfection. In contrast, in infected MyD88-deficient mice, which lack a key adaptor molecule in the signaling cascade of TLRs and IL-1R family members, the increase of the lung weight is attenuated, and from the analyzed cyto- and chemokines, only IL-12p40 is detectable. Upon infection, almost no influx of inflammatory cells into lungs of MyD88-deficient mice can be observed. Six days postinfection, however, MyD88-deficient mice were able to produce TNF, IFN-, keratinocyte-derived chemokine, and MCP-1 in amounts similar to wild-type mice, but failed to secrete IL-12p40 and MIP-2. At this time point, the infection increased the lung weight to a level similar to wild-type mice. Curiously, the chlamydial burden in MyD88-deficient mice 3 days postinfection is lower than in wild-type mice, a finding that can be reproduced in wild-type mice by depletion of GR1+ cells. In analyzing how PMN influence the chlamydial burden in vivo, we find that PMN are infected and enhance the replication of C. pneumoniae in epithelial cells. Thus, the lower chlamydial burden in MyD88-deficient mice can be explained by the failure to recruit PMN.
Introduction
Polymorphonuclear neutrophils (PMN)3 are crucial for innate host defense against bacteria and fungi. Upon chemokine-mediated attraction, they rapidly ingest and kill extracellular microbial pathogens via release of reactive oxygen species, proteolytic enzymes, and antimicrobial peptides (1, 2). During pneumonia, the ELR+ CXC chemokines KC and MIP-2 were reported to control recruitment of PMN (1). Several obligate or facultative intracellular microbial pathogens such as Yersinia enterocolitica or Leishmania major developed mechanisms to evade destruction upon ingestion by PMN (3, 4). Among this group of pathogens, microorganisms such as Ehrlichia (3) or Chlamydia pneumoniae (5) even replicate in PMN, indicating that PMN might not participate in the control of these microorganisms in vivo.
C. pneumoniae is responsible for up to 10% of all cases of community-acquired pneumonia in humans (6), and also causes pneumonia in mice (7). The primary cell type of the lung infected by the microorganism are bronchial epithelial cells (8). Within these cells, C. pneumoniae enters a Chlamydia-specific cycle of replication. The cycle is initiated by metabolically inert, but infectious elementary bodies (EB) that subsequently develop into reticulate bodies (RB) and divide by binary fission. After 48–72 h, RB revert back to EB that leave the cell and infect neighboring cells. Within the first 2–3 days postinfection, infected pulmonary areas are characterized by a cellular infiltrate consisting among other cell types mainly of PMN (8).
The Gram-negative, obligate intracellular bacterium C. pneumoniae is recognized by the innate immune system via TLRs that detect microbes via recognition of pathogen-associated microbial patterns, such as endotoxin, peptidoglycan, bacterial DNA, and others (9). We have shown in a previous study that murine bone marrow-derived dendritic cells sense C. pneumoniae via TLR2 and TLR4 (10). The relative importance of the two TLRs to trigger cellular reactions varies with the cellular response analyzed. For instance, TNF release by bone marrow-derived dendritic cells stimulated with C. pneumoniae is clearly dependent on TLR2, but independent from TLR4, whereas IL-12p40 secretion depends on TLR2 and TLR4 (10). Because TLR2- and TLR4-induced signaling cascades result in different cellular reactions, more than one adaptor is probably recruited to TLRs upon recognition of this bacterium.
Upon interaction with their ligand, TLRs recruit up to three different adaptor molecules. Among these, MyD88 appears to interact with all TLRs (11, 12) and also with IL-1R family members, whereas Toll-IL-1R domain-containing adaptor protein (TIRAP, also known as Mal) associates with TLR1, 2, 4, and 6 (13, 14), and Toll-IL-1R domain-containing adaptor molecule-1 (TICAM-1, also known as Toll-IL-7R domain-containing adaptor inducing IFN-) with TLR3 and TLR4 (15). The majority of inflammatory responses, however, depend on MyD88. Yet, macrophages from MyD88-deficient mice are still able to activate the MAPKs JNK1, ERK1/2, and p38, as well as NF-B, albeit with a delayed kinetic (16). It was initially assumed that TIRAP represents the MyD88-independent adaptor (17), but later studies using gene-deficient mice indicated that MyD88 and TIRAP cooperate (13, 14). However, a recently defined third adaptor, TICAM-1, was shown to activate the IFN- promoter in a MyD88-independent manner (15, 18). Upon interaction of TLRs with MyD88, IL-1R-associated kinase 1 and 4 are recruited, which in turn interact with TNFR-associated factor 6 (12). This signaling cascade leads to the activation of NF-B, and MAPKs such as ERK1/2, p38, and JNK1/2 (16). As a final consequence, TLR signaling contributes to the control of microbial infections by the host in vivo. Thus, TLR4-mutant mice were shown to be highly susceptible to infection with Salmonella typhimurium (19). Furthermore, TLR2-deficient mice displayed a higher sensitivity to infection with Staphylococcus aureus (20) and were more susceptible to meningitis caused by Streptococcus pneumoniae (21). MyD88-deficient mice were also unable to control infections by Staphylococcus aureus (20). In contrast, in a model of polymicrobial peritonitis, MyD88 deficiency improved resistance against sepsis presumably via a reduction of hyperinflammation (22).
In this study, we infected mice intranasally with C. pneumoniae and analyzed the relevance of MyD88 for the initiation of inflammatory responses by the host as well as its influence on the replication of this obligate intracellular microorganism. The comparison of wild-type and MyD88-deficient mice revealed a new role of PMN in the replication of C. pneumoniae.
Materials and Methods
Strains of mice
C57BL/6 mice were purchased from Harlan Winkelmann. Breeding pairs of MyD88-deficient mice were kindly provided by Dr. S. Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) and bred in our own animal facility. Mice were 8x backcrossed to C57BL/6 mice.
Reagents and mAbs
The allophycocyanin-labeled mAb specific for CD11b (553312), PE-labeled anti-CD45 mAb (553081), FITC-labeled anti-GR1 mAb (553126), and CD16/32-specific mAb (553142) to block Fc receptors were purchased from BD Pharmingen. For depletion of GR1+ cells in vivo, purified GR1 mAb (RB6-8C5, 150 μg/mouse), kindly provided by Dr. J. Zerrahn (Max Planck Institute for Infection Biology, Berlin, Germany), was used.
Infection protocol
Mice were anesthesized with an i.p. injection of Ketamin (150 μl/mouse). Subsequently, mice were infected intranasally with C. pneumoniae by applying 30 μl of PBS containing 2.5 x 106 inclusion-forming units (IFU) of the microorganism or PBS alone.
Isolation of pulmonary cells
To isolate pulmonary cells, mice were sacrificed by CO2 inhalation 3 days postinfection with C. pneumoniae. The lungs were flushed with 10 ml of PBS applied through the right atrium of the heart to remove blood. Thereafter, the organ was cut into small pieces in a 60-mm plate and digested for 10 min with collagenase VIII (400 U/100 μl, room temperature (RT), C-2139; Sigma-Aldrich) and subsequently for another 30 min (400 U of collagenase/100 μl in 2 ml of RPMI 1640, 0% FCS, 37°C). The digested material was filtered through a cell strainer of 100 μm pore size (BD Discovery Labware Europe) to remove debris. The cells were incubated with ammonium chloride (0.15 M, 3 min, RT) to lyse erythrocytes. The remaining cells were washed in PBS containing 3% FCS and analyzed by FACS.
Detection of chemokines and cytokines
The chemokines keratinocyte-derived chemokine (KC), MCP-1, and MIP-2 as well as the cytokines TNF, IFN-, and IL-12p40 were determined in lungs of infected mice by commercially available ELISAs (duo set for KC, MIP-2, TNF, IFN-, and IL-12p40, R&D Systems; MCP-1 from BD Biosciences). The assays were performed, as described by the manufacturer. Lungs were isolated from mice and minced to homogeneity in 500 μl of PBS. After centrifugation (2000 x g, 5 min), the supernatant was analyzed for its cyto- or chemokine content in duplicates.
FACS analysis and cell sorting
The cellular composition in lungs postinfection was determined by FACS. The isolated cells were preincubated with ethydium monacide (2 μg/ml, 20 min, 4°C; Molecular Probes) to exclude dead cells and anti-CD16/32 (100 μg/ml, 10 min, 4°C) to block Fc receptors. Subsequently, cells were double stained (30 min, 4°C) with PE-labeled mAb directed against CD45 (1 μg/ml) and FITC-labeled GR1 mAb (1 μg/ml). After three wash steps, the cells were fixed with 2% paraformaldehyde and flow cytometric analysis was performed (BD Biosiences). All FACS data were analyzed using FlowJo (Tree Star).
Pulmonary cells were stained with APC-labeled anti-CD11b mAb, PE-labeled anti-CD45 mAb, and FITC-labeled anti-GR1 mAb. Cells were gated on CD45, and CD11b+/GR1+ and CD11b+/GR1– subsets were sorted with a MoFlow instrument (DakoCytomation).
Giemsa stain
For Giemsa staining, sorted GR1+/CD45+ cells isolated from lungs of control and infected mice were cytospinned (72.3 x g, 5 min). Thereafter, cells were fixed with methanol (100%, 5 min, RT) and stained with Giemsa (20 min, RT).
Disruption of PMN
Sorted GR1+/CD45+ cells were vortexed with glass beads in 2.5 ml of cell culture medium (RPMI 1640, 0% FCS, 10 min). Supernatants were transferred to another tube to discard glass beads and again centrifuged (400 x g, 5 min, RT) to remove cellular debris. The supernatant containing the equivalent of 8.25 x 105 cells/well was added to HEp2 cells (3 x 105 cells/well). After 48 h of culture, chlamydial inclusions were visualized by confocal microscopy.
Histology and immunohistochemistry
Infected wild-type (C57BL/6) and MyD88-deficient mice were sacrificed by CO2 inhalation, and lungs were perfusion fixed with buffered 10% Formalin and in total embedded in paraffin. Sections were stained with H&E.
Immunohistochemistry with a mAb specific for heat shock protein 60 of C. pneumoniae (Affinity BioReagents) in a dilution of 1/1000 was performed on an automated immunostainer (Benchmark; Ventana Medical Systems) using established protocols. In brief, slides were dewaxed, and the subsequent procedure including heat-induced Ag retrieval as well as blocking of nonspecific binding was performed in the immunostainer. Detection of bound Ab was performed with a biotinylated secondary Ab and alkaline phosphatase-conjugated streptavidin (Ventana Medical Systems), using Fast Red as substrate. The same procedure with omission of the primary Ab was performed as negative control.
For quantitative evaluation, immunoreactive inclusion bodies were counted over 50 randomly selected high power fields (x400). Inclusion bodies in the bronchial epithelium and in alveolar lining cells were evaluated separately.
Confocal microscopy
To quantify the numbers of C. pneumoniae in lungs of infected mice, the supernatant of homogenized lung specimens was used to infect HEp2 cells. After 48 h, HEp2 cells were fixed and permeabilized with methanol/acetone (1:1 v/v, 5 min), stained with FITC-labeled mAb specific for chlamydial endotoxin, and counterstained with Evans blue (ACI-FITC, Progen; Biotechnik). Cells were washed and embedded in glycerin/PBS (50%) and analyzed by confocal microscopy (LSM510; Carl Zeiss Jena).
To detect C. pneumoniae in GR1+ cells, CD45+ cells were purified by MACS (Miltenyi Biotec) from lungs taken from mice 3 days postinfection or from mock-infected mice. FcRs were blocked, as described above, and cells were stained with a FITC-labeled GR1 mAb (1 μg/ml, 30 min, 4°C). After washing (PBS containing 3% FCS), cells were fixed with paraformaldehyde (2%, 30 min), washed again, and permeabilized with saponin (1%, 30 min). Simultaneously, cells were stained with an Alexa546-labeled mAb specific for chlamydial endotoxin. Cells were washed three times (1% saponin/PBS containing 3% FCS), followed by three wash steps with PBS and 3% FCS.
Electron microscopy
GR1+/CD45+ cells were purified by cell sorting on day 3 postinfection with C. pneumoniae from collagenase-treated lungs. The cells were washed with PBS, fixed with paraformaldehyde (2%) plus glutaraldehyde (0.1%) for 20 min, 37°C, and postfixed in osmium tetroxide. Subsequently, samples were dehydrated with a graded ethanol series and embedded in Epon, and ultrathin sections were prepared. Sections were washed carefully with deionized H2O and stained with uranyl acetate (0.5%) plus lead citrate (3%). Sections were examined at 80 kV with a Zeiss EM 10 GR transmission electron microscope.
Statistics
Multiple comparisons were analyzed by ANOVA, and as post hoc test the Holm-Sidak method was used. The t test was used to compare two groups.
Results
Inflammatory responses of MyD88-deficient mice upon infection with C. pneumoniae are severely impaired on day 3 postinfection
To analyze the ability of MyD88-deficient mice to recognize the obligate intracellular bacterium C. pneumoniae, gene-deficient and wild-type mice were infected intranasally with 2.5 x 106 IFU of the microorganism. Three days postinfection, lungs of wild-type mice contained elevated levels of TNF, IL-12p40, and IFN-, as well as the chemokines KC, MCP-1, and MIP-2 (Fig. 1A). In contrast, the cyto- and chemokine response of MyD88-deficient mice was much weaker, with the notable exception of IL-12p40, indicating that this cytokine was secreted in vivo during C. pneumoniae infection in a MyD88-independent fashion (Fig. 1A). Total cellular numbers of the lungs of wild-type mice increased 2- to 3-fold 3 days after infection, but did not change in lungs of MyD88-deficient mice (data not shown). As a consequence of the inflammatory response, the lung weight of wild-type mice rose substantially (Fig. 1B). The increment of lung weight was attenuated in the case of MyD88-deficient mice and significantly lower than in wild-type mice (Fig. 1B). In line with these findings, a histologic analysis of lungs 3 days postinfection with C. pneumoniae revealed that lungs of wild-type mice showed an extensive infiltration of pulmonary tissue with leukocytes, which was almost not detectable in MyD88-deficient mice (Fig. 2). However, 6 days postinfection, MyD88-deficient mice were able to produce TNF, IFN-, KC, and MCP-1 in amounts similar to wild-type mice, but failed to secrete IL-12p40 and MIP-2 (Fig. 1A). In addition, the infection increased the lung weight in both strains of mice to the same level 6 days postinfection (Fig. 1B). Thus, it appears that MyD88 is of critical importance to trigger inflammatory responses induced by C. pneumoniae during the early period of infection.
FIGURE 1. Impaired inflammatory response in MyD88-deficient mice upon infection with C. pneumoniae. A, C57BL/6 (wild type (WT), n = 6, ) or MyD88-deficient mice (n = 6, ) were intranasally infected with 2.5 x 106 IFU/mouse and sacrificed 3 days later. Of each strain, two uninfected mice served as negative controls (). In addition, C57BL/6 (WT, n = 3, ) or MyD88-deficient mice (n = 3, ) were infected for 6 days with 2.5 x 106 IFU/mouse. One mouse of each strain served as negative control (). Cyto- and chemokines were determined in the supernatant of lung homogenates via ELISA, as described in Materials and Methods. Error bars represent SD. *, p < 0.001; #, p < 0.05; ANOVA post hoc Holm-Sidak. B, Lung weight of C57BL/6 (WT, n = 6, ) or MyD88-deficient mice (n = 6, ) 3 days upon infection with 2.5 x 106 IFU/mouse. Of each mouse strain, two uninfected mice were used as negative controls (). The lung weight was also determined 6 days after infection with 2.5 x 106 IFU/mouse (C57BL/6, n = 5, ; MyD88-deficient mice, n = 5, ). Two uninfected mice of each strain served as negative controls (). *, p < 0.001; ANOVA post hoc Holm-Sidak.
FIGURE 2. Histologic analysis of the cellular infiltrate in wild-type vs MyD88-deficient mice. Lung histology of wild-type (a–d) and MyD88-deficient mice (e–h) 3 days after infection with C. pneumoniae (2.5 x 106 IFU/mouse). H&E stain, x250 (a–c and e–g); x40 (d and h). Mock-infected animals (a and e) show normal bronchi and alveoli. Infected wild-type animals (b and c) show massive infiltrates of PMN in a peribronchiolar and perivascular distribution. The low power view (d) demonstrates the wide extent of inflammatory changes, accompanied by focal hemorrhage. MyD88-deficient mice show essentially no (f) or only mild and focal (g) inflammatory changes with occasional PMN (arrow). The low power view (h) demonstrates the preservation of the lung architecture and the absence of major inflammatory changes in MyD88-deficient mice.
On day 3 postinfection, 30% of the CD45+ pulmonary infiltrate was also GR1 positive in wild-type mice (Fig. 3, A and B). This cellular subset did not increase in MyD88-deficient mice (Fig. 3, A and B). Purification of GR1+/CD45+ cells by cell sorting and subsequent Giemsa staining revealed that the vast majority of cells displayed the morphology of PMN (Fig. 3B).
FIGURE 3. Recruitment of PMN into infected lungs of wild-type, but not MyD88-deficient mice. A, Lungs of C. pneumoniae-infected (2.5 x 106 IFU/mouse, day 3) C57BL/6 mice (n = 2) and MyD88-deficient mice (n = 2) as well as mock-infected controls (n = 1) were digested with collagenase. Single cell suspensions were stained with a FITC-labeled mAb specific for GR1 and a PE-labeled mAb specific for CD45. Top graphs, Show the FACS analysis from mock infected; middle and bottom graphs, from C. pneumoniae-infected animals. B, Shows the percentage of GR1+/CD45+ cells from infected C57BL/6 (wild type (WT), n = 4, ) and MyD88-deficient mice (n = 4, ). Two uninfected mice of each strain served as controls. *, p < 0.001; ANOVA post hoc Holm-Sidak. C, The experiment was performed, as described in A, with the exception that GR1+/CD45+ cells of C57BL/6 mice were sorted and their morphology was analyzed by Giemsa staining.
Chlamydial burden in MyD88-deficient mice is lower than in wild-type mice
Because the majority of inflammatory responses analyzed in this study were not induced in MyD88-deficient mice, we expected the chlamydial burden in these mice to be higher than in wild-type mice. In contrast to our expectation, we found that the microorganism was detected in even lower amounts in MyD88-deficient than wild-type mice (Fig. 4). A semiquantitative real-time PCR approach confirmed these results (data not shown). When chlamydial burden was analyzed 6 days postinfection, the number of microorganisms found in lungs of MyD88-deficient mice was about the same as on day 3 postinfection (Fig. 4). However, wild-type mice decreased chlamydial burden to the level of MyD88-deficient mice 6 days postinfection (Fig. 4). Thus, although MyD88-deficient mice are severely impaired to mount an inflammatory response against C. pneumoniae, the replication of the microorganism during the early phase of infection appears to be less effective in these mice.
FIGURE 4. Lower chlamydial burden in MyD88-deficient mice. Three and 6 days postinfection (2.5 x 106 IFU/mouse), chlamydial burden was determined in the supernatant of lung homogenates of C57BL/6 (wild type (WT), day 3, n = 6; day 6, n = 5, ) and MyD88–/– mice (day 3, n = 6; day 6, n = 5, ). Chlamydial burden was quantified by transfer of lung supernatants to HEp2 cells. Forty-eight hours later, IFU in HEp2 cells were counted. The graph represents the pooled data from two independent experiments. *, p < 0.001; ANOVA post hoc Holm-Sidak.
Depletion of GR1+ cells in vivo reduces chlamydial burden
Because GR1+/CD45+ cells were not attracted into infected lungs of MyD88-deficient mice, we wondered whether these cells may influence the chlamydial burden in wild-type mice. Therefore, we depleted wild-type mice from GR1+ cells and infected them 1 day later with C. pneumoniae. Fig. 5A shows that GR1+/CD45+ cells were completely absent in depleted mice 3 days postinfection. Furthermore, the chlamydial burden was lower in GR1-depleted mice compared with untreated wild-type mice at this time point (Fig. 5B). Thus, GR1+ cells appear to support the replication of C. pneumoniae in vivo.
FIGURE 5. Depletion of GR1+ cells in vivo decreases chlamydial burden. A, C57BL/6 mice (n = 2) were depleted from GR1+ cells by injecting i.p. 150 μg/mouse anti-GR1 mAb 1 day before infection with C. pneumoniae (2.5 x 106 IFU/mouse, right graphs). Control C57BL/6 mice (n = 2) were pretreated i.p. with PBS and subsequently infected (2.5 x 106 IFU/mouse, left graphs). One mouse was neither depleted nor infected (top graph). Three days postinfection, lungs were removed and digested with collagenase. Single cell suspensions were stained with a FITC-labeled mAb specific for GR1 and a PE-labeled mAb specific for CD45. B, C57BL/6 mice (n = 4) were depleted from GR1+ cells and infected, as described in A (). PBS-pretreated and infected C57BL/6 mice (n = 5) served as controls (). Chlamydial burden was determined 3 days after infection. The graph represents the pooled data from two independent experiments. *, t test, p = 0.012.
PMN are infected with and support the replication of C. pneumoniae in epithelial cells
In an attempt to identify the mechanism in which PMN increase the chlamydial burden in wild-type mice in vivo, we examined whether PMN are infected with C. pneumoniae in vivo. Confocal microscopy of CD45+ cells purified from infected lungs demonstrated that a substantial fraction of the GR1+ subset contained C. pneumoniae (Fig. 6a). Next, we analyzed the possibility that the microorganism replicates in GR1+ PMN by electron microscopy, as claimed recently for human PMN (5). Electron microscopy of ex vivo purified GR1+/CD45+ cells confirmed the results obtained with confocal microscopy, and EB could clearly be visualized in PMN (Fig. 6, b and c, arrow). Also, a few RB were found, indicating that C. pneumoniae replicated to some extent in these cells (Fig. 6c, arrowhead). Some of the EB isolated from PMN at day 3 postinfection were indeed living, because coincubation of HEp2 cells with a lysate of purified PMN gave rise to chlamydial inclusions (Fig. 6d).
FIGURE 6. C. pneumoniae infects and generates small inclusions in PMN in vivo. a, CD45+ cells were purified by magnetic separation from collagenase-treated lungs 3 days after infection of C57BL/6 mice with C. pneumoniae (2.5 x 106 IFU/mouse). Cells were stained with FITC-labeled anti-GR1 mAb. C. pneumoniae inclusions were visualized by an Alexa546-labeled mAb specific for chlamydial endotoxin. Cells were analyzed by confocal microscopy. b, GR1+/CD45+ cells were selected by cell sorting from collagenase-treated lungs 3 days after infection of C57BL/6 mice with C. pneumoniae (2.5 x 106 IFU/mouse) and analyzed by electron microscopy (magnification x22,000). Arrow points to a chlamydial inclusion. c, Further electronic close-up. Arrow points to an EB, and arrowhead to an RB. d, FACS-purified GR1+/CD45+ cells were minced by vortexing with glass beads. After centrifugation of the lysate, the supernatant (equivalent of 8.25 x 105 cells/well) was transferred to HEp2 cells (3 x 105 cells/well). After 48 h of culture, chlamydial inclusions were visualized by confocal microscopy. (arrows).
The total number of chlamydial inclusions found in PMN per infected lung is much smaller than the difference in chlamydial burden between untreated and GR1-depleted mice. Therefore, we speculated that PMN in addition stimulate the replication of C. pneumoniae in bronchial epithelial cells. We addressed this assumption by sorting PMN from lungs of untreated or infected wild-type mice and cocultured the cells with HEp2 cells infected with C. pneumoniae just before the addition of PMN. Confocal microscopy revealed that PMN isolated from mock or infected mice increased the number of chlamydial inclusions more than 2.5-fold, while GR1–/CD11b+ cells were less effective (Fig. 7).
FIGURE 7. PMN support the replication of C. pneumoniae in epithelial HEp2 cells. GR1+/CD11b+ or GR1–/CD11b+ cells were sorted from mock-infected () or day 3 infected C57BL/6 mice (). Both cell types were added at a density of 2 x 105 cells/well to HEp2 cell cultures (2 x 105 cells/well), which were infected with C. pneumoniae at an multiplicity of infection of 1 just before the addition of lung cells. , Represents C. pneumoniae-infected HEp2 cell cultures without addition of inflammatory cells. After 48 h of culture, cells were lysed, and the supernatant of the lysate was transferred to a new well seeded with uninfected HEp2 cells (2.5 x 105 cells/well). After another culture period of 30 h, chlamydial inclusions were counted using confocal microscopy. Error bars represent SD of results obtained with five to eight individual cultures. The graph represents data from three pooled experiments. *, p < 0.001; ANOVA post hoc Holm-Sidak.
We also determined the anatomical localization of chlamydial inclusions in infected lungs of wild-type and MyD88-deficient mice and their spatial relationship to PMN using immunohistochemistry. Although similar numbers of large chlamydial inclusions were found within bronchial epithelial cells in both strains of mice, which indicates that MyD88-deficient epithelial cells are in principle able to replicate the microorganism as efficiently as epithelial cells from wild-type mice, alveolar epithelial cells were rarely infected in MyD88-deficient mice compared with wild-type mice (Fig. 8 and Table I). Of note, infected alveolar epithelial cells were regularly surrounded by PMN (Figs. 8 and 9), suggesting that PMN might support chlamydial replication in alveolar epithelial cells in vivo. In agreement with data detailed above, PMN of wild-type mice contained small chlamydial inclusions (Fig. 9).
FIGURE 8. C. pneumoniae-infected pulmonary cell types of C57BL/6 and MyD88-deficient mice. Sections of paraffin-embedded lungs from C57BL/6 (animal 1, a and c; animal 2, b and d) or MyD88-deficient mice (animal 1, e and g; animal 2, f and h) 3 days postinfection (2.5 x 106 IFU/mouse) were stained with a mAb specific for chlamydial heat shock protein 60 to visualize chlamydial inclusions. Large chlamydial inclusions were detected in bronchial (a, b, e, and f, magnification x400) and alveolar epithelial cells (c, d, g, and h, magnification x200) of C57BL/6 and MyD88-deficient mice. Infected alveolar cells were less frequent in MyD88-deficient mice (see also Table I).
Table I. Quantification of chlamydial inclusions in lung epithelial cells of C57BL/6 and MyD88-deficient mice
FIGURE 9. PMN are close to alveolar epithelial cells containing chlamydial inclusions. Lung sections of two C57BL/6 mice (animal 1, a; animal 2, b) of the experiment described in Fig. 8 were analyzed with higher magnification (x1000) to visualize the proximity of PMN (arrows) and alveolar epithelial cells containing large chlamydial inclusions. Small chlamydial inclusions were found within PMN.
We conclude from these findings that PMN increased chlamydial burden in two ways: they act as cellular site of infection by C. pneumoniae and, in addition, amplify the replication of the microorganism in epithelial cells.
Discussion
MyD88 is the central adaptor of the TLR-signaling cascade interacting with all known TLRs, but also with IL-1R family members. In this study, we analyzed the importance of this adaptor molecule for host defense against the obligate intracellular bacterium C. pneumoniae. In the early phase of infection, i.e., 3 days postinfection, most of the inflammatory responses analyzed were switched off, as expected. In addition, gene microarray analysis of lungs revealed that 312 genes were induced >2- and up to 27-fold by C. pneumoniae infection in wild-type mice. Of these genes, 197 were below a 2-fold induction in MyD88-deficient mice, indicating that MyD88 is crucial for the initiation of host responses against this intracellular pathogen (data not shown). Surprisingly, chlamydial replication in MyD88-deficient was less efficient during the early phase of infection, a conclusion based on the finding that bacterial burden in the lung was lower in comparison with wild-type mice. This unexpected result is explained by the observation that PMN support the replication of C. pneumoniae in wild-type mice. MyD88-deficient mice, however, are unable to recruit this population of cells to sites of inflammation.
At present, it is unclear whether the inflammatory defects observed in MyD88-deficient mice upon infection with C. pneumoniae are due to impaired TLR signaling and/or failure of signaling through IL-1R family members. Our preliminary results, however, suggest that TLR signaling is important. Thus, mice double deficient for TLR2 and TLR4 display a similar phenotype upon infection with C. pneumoniae, i.e., the numbers of microorganisms in the lung are lower and pulmonary recruitment of PMN is highly attenuated. In addition, murine dendritic cells sense C. pneumoniae in a TLR2- and TLR4-dependent fashion (10). Furthermore, gene microarray results revealed that transcription of IL-1, IL-1, and IL-18 genes is not induced in MyD88-deficient mice, suggesting that IL-1R family members are not triggered in vivo (our unpublished results).
How C. pneumoniae persists in PMN is unknown to date. In epithelial cells, in which C. pneumoniae forms large inclusions within 48–72 h, inclusions avoid fusion with late endosomes and lysosomes, but accumulate early transferrin receptor-positive endosomes (23). In human monocytes, however, C. pneumoniae-containing inclusions are small and characterized by large atypical RBs (24), which may be explained by the finding that in the case of infection with Chlamydia psittaci, early vacuoles fused with lysosomes in the monocytic cell line THP1 (25). Nevertheless, the microorganism persists in human monocytes (24), indicating that lysosomal effector molecules are either unable to completely kill the bacterium or lysosomal fusion with chlamydial inclusion is incomplete. In PMN, we also found only small inclusions containing few EBs and RBs. This indicates that C. pneumoniae replicates to some extent in PMN, and that lysosomal fusion, if it occurs in these cells, is also only partially effective. To maximize the weak replication inside PMN, the microorganism appears to elongate the life span of PMN by interference with the apoptotic signal cascade (5).
Infection of PMN with C. pneumoniae and enhancement of chlamydial replication in epithelial cells by PMN are functionally not related, because PMN from uninfected wild-type mice increase chlamydial replication in epithelial cells. However, the molecular mechanism(s) involved in the latter process is unclear. A similar function has been reported for the monocyte-like cell U937. Thus, coculture of U937 cells with human endothelial cells enhanced the infection of the latter cells by C. pneumoniae (26). Subsequently, it was suggested that the infectivity-enhancing factor is identical with human insulin-like growth factor 2 (27). Using a different cellular system, a recent study demonstrated that human monocytes also enhance the growth of C. pneumoniae in arterial smooth muscle cells (28). In this case, cell to cell contact was required, and it was suggested that mannose 6-phosphate receptor expressed on monocytes is implicated in this process. Other possible mechanisms include the regulation of intracellular tryptophan levels in epithelial cells by PMN because C. pneumoniae lacks tryptophan biosynthesis genes (29) and thus is completely dependent on host-derived tryptophan (30). Increasing the supply of this amino acid via TGF--mediated down-regulation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase or the tryptophanyl-tRNA synthetase could represent potential mechanisms (31). TGF-, in turn, might be produced by PMN because it was reported that TGF- mRNA was induced after stimulation of human PMN with Mycobacterium bovis bacillus Calmette-Guerin (32).
In comparison with wild-type mice, the chlamydial load appears to be more reduced in MyD88-deficient mice than in GR1-depleted mice, indicating that in addition to PMN, another cell type might support the replication of C. pneumoniae. As discussed above, the human monocyte-like cell U937 was able to amplify chlamydial replication in smooth muscle and epithelial cells (26, 28). In our experiments, the GR1–/CD11b+ cell population, which may include monocytes/macrophages, also weakly amplified chlamydial replication, although the differences were not statistically significant. Thus, further experimentation is needed to clarify this issue.
It remains to be seen whether other adaptors aside from MyD88 are relevant for host defense against C. pneumoniae. However, release of IL-12p40 at day 3 postinfection appears to be MyD88 independent. Also, our experiments show that on day 6 postinfection, TNF, IFN-, KC, and MCP-1 are secreted MyD88 independently. IL-12 participates in the control of C. pneumoniae replication in vivo, because anti IL-12p40 treatment results in higher chlamydial titers, but less severe pathological changes within the lung of infected mice (33). From the adaptors identified to date, only TICAM-1 (Toll-IL-7R domain-containing adaptor-inducing IFN-) appears to operate MyD88 independently (15, 18). TICAM-1 is crucially involved in the induction of IFN- (34), but also in endotoxin-mediated secretion of IL-12p40 (15). In vitro, IL-12p40 secretion induced by C. pneumoniae depends partially on TLR4 (10), and TICAM-1 not only interacts with TLR3 (34), but also with TLR4 (18). On the one hand, these findings argue for a role of TICAM-1 during recognition of C. pneumoniae. In contrast, our preliminary data show that TLR4 appears not to participate in release of IL-12p40 in vivo.
In conclusion, the results of this study describe a new role for PMN in the replication of the intracellular pathogen C. pneumoniae in vivo: attracted MyD88 dependently to the site of inflammation, they increase chlamydial burden during the first days of infection.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. S. Akira for providing MyD88-deficient mice. We are also grateful to Dr. J. Zerrahn for the generous supply with anti-GR1 mAb. We thank Carmen Hartmann for expert technical assistance with immunohistochemistry. Many thanks to Dr. B. Holzmann for critical reading of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Deutsche Forschungsgemeinschaft MI 471/1-1.
2 Address correspondence and reprint requests to Dr. Thomas Miethke, Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Trogerstr. 9, 81675 Munich, Germany. E-mail address: Thomas.Miethke{at}lrz.tu-muenchen.de
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; EB, elementary body; IFU, inclusion-forming unit; RB, reticulate body; RT, room temperature; TICAM-1, Toll-IL-1R domain-containing adaptor molecule-1; TIRAP, Toll-IL-1R domain-containing adaptor protein; KC, keratinocyte-derived chemokine.
Received for publication July 21, 2004. Accepted for publication January 3, 2005.
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Chlamydia pneumoniae, an obligate intracellular bacterium, causes pneumonia in humans and mice. In this study, we show that GR1+/CD45+ polymorphonuclear neutrophils (PMN) surprisingly increase the bacterial load of C. pneumoniae in vivo. Upon intranasal infection of wild-type mice, the lung weight is increased; the cytokines TNF, IL-12p40, and IFN-, as well as the chemokines keratinocyte-derived chemokine, MCP-1, and MIP-2 are secreted; and GR1+/CD45+ PMN are recruited into lungs 3 days postinfection. In contrast, in infected MyD88-deficient mice, which lack a key adaptor molecule in the signaling cascade of TLRs and IL-1R family members, the increase of the lung weight is attenuated, and from the analyzed cyto- and chemokines, only IL-12p40 is detectable. Upon infection, almost no influx of inflammatory cells into lungs of MyD88-deficient mice can be observed. Six days postinfection, however, MyD88-deficient mice were able to produce TNF, IFN-, keratinocyte-derived chemokine, and MCP-1 in amounts similar to wild-type mice, but failed to secrete IL-12p40 and MIP-2. At this time point, the infection increased the lung weight to a level similar to wild-type mice. Curiously, the chlamydial burden in MyD88-deficient mice 3 days postinfection is lower than in wild-type mice, a finding that can be reproduced in wild-type mice by depletion of GR1+ cells. In analyzing how PMN influence the chlamydial burden in vivo, we find that PMN are infected and enhance the replication of C. pneumoniae in epithelial cells. Thus, the lower chlamydial burden in MyD88-deficient mice can be explained by the failure to recruit PMN.
Introduction
Polymorphonuclear neutrophils (PMN)3 are crucial for innate host defense against bacteria and fungi. Upon chemokine-mediated attraction, they rapidly ingest and kill extracellular microbial pathogens via release of reactive oxygen species, proteolytic enzymes, and antimicrobial peptides (1, 2). During pneumonia, the ELR+ CXC chemokines KC and MIP-2 were reported to control recruitment of PMN (1). Several obligate or facultative intracellular microbial pathogens such as Yersinia enterocolitica or Leishmania major developed mechanisms to evade destruction upon ingestion by PMN (3, 4). Among this group of pathogens, microorganisms such as Ehrlichia (3) or Chlamydia pneumoniae (5) even replicate in PMN, indicating that PMN might not participate in the control of these microorganisms in vivo.
C. pneumoniae is responsible for up to 10% of all cases of community-acquired pneumonia in humans (6), and also causes pneumonia in mice (7). The primary cell type of the lung infected by the microorganism are bronchial epithelial cells (8). Within these cells, C. pneumoniae enters a Chlamydia-specific cycle of replication. The cycle is initiated by metabolically inert, but infectious elementary bodies (EB) that subsequently develop into reticulate bodies (RB) and divide by binary fission. After 48–72 h, RB revert back to EB that leave the cell and infect neighboring cells. Within the first 2–3 days postinfection, infected pulmonary areas are characterized by a cellular infiltrate consisting among other cell types mainly of PMN (8).
The Gram-negative, obligate intracellular bacterium C. pneumoniae is recognized by the innate immune system via TLRs that detect microbes via recognition of pathogen-associated microbial patterns, such as endotoxin, peptidoglycan, bacterial DNA, and others (9). We have shown in a previous study that murine bone marrow-derived dendritic cells sense C. pneumoniae via TLR2 and TLR4 (10). The relative importance of the two TLRs to trigger cellular reactions varies with the cellular response analyzed. For instance, TNF release by bone marrow-derived dendritic cells stimulated with C. pneumoniae is clearly dependent on TLR2, but independent from TLR4, whereas IL-12p40 secretion depends on TLR2 and TLR4 (10). Because TLR2- and TLR4-induced signaling cascades result in different cellular reactions, more than one adaptor is probably recruited to TLRs upon recognition of this bacterium.
Upon interaction with their ligand, TLRs recruit up to three different adaptor molecules. Among these, MyD88 appears to interact with all TLRs (11, 12) and also with IL-1R family members, whereas Toll-IL-1R domain-containing adaptor protein (TIRAP, also known as Mal) associates with TLR1, 2, 4, and 6 (13, 14), and Toll-IL-1R domain-containing adaptor molecule-1 (TICAM-1, also known as Toll-IL-7R domain-containing adaptor inducing IFN-) with TLR3 and TLR4 (15). The majority of inflammatory responses, however, depend on MyD88. Yet, macrophages from MyD88-deficient mice are still able to activate the MAPKs JNK1, ERK1/2, and p38, as well as NF-B, albeit with a delayed kinetic (16). It was initially assumed that TIRAP represents the MyD88-independent adaptor (17), but later studies using gene-deficient mice indicated that MyD88 and TIRAP cooperate (13, 14). However, a recently defined third adaptor, TICAM-1, was shown to activate the IFN- promoter in a MyD88-independent manner (15, 18). Upon interaction of TLRs with MyD88, IL-1R-associated kinase 1 and 4 are recruited, which in turn interact with TNFR-associated factor 6 (12). This signaling cascade leads to the activation of NF-B, and MAPKs such as ERK1/2, p38, and JNK1/2 (16). As a final consequence, TLR signaling contributes to the control of microbial infections by the host in vivo. Thus, TLR4-mutant mice were shown to be highly susceptible to infection with Salmonella typhimurium (19). Furthermore, TLR2-deficient mice displayed a higher sensitivity to infection with Staphylococcus aureus (20) and were more susceptible to meningitis caused by Streptococcus pneumoniae (21). MyD88-deficient mice were also unable to control infections by Staphylococcus aureus (20). In contrast, in a model of polymicrobial peritonitis, MyD88 deficiency improved resistance against sepsis presumably via a reduction of hyperinflammation (22).
In this study, we infected mice intranasally with C. pneumoniae and analyzed the relevance of MyD88 for the initiation of inflammatory responses by the host as well as its influence on the replication of this obligate intracellular microorganism. The comparison of wild-type and MyD88-deficient mice revealed a new role of PMN in the replication of C. pneumoniae.
Materials and Methods
Strains of mice
C57BL/6 mice were purchased from Harlan Winkelmann. Breeding pairs of MyD88-deficient mice were kindly provided by Dr. S. Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) and bred in our own animal facility. Mice were 8x backcrossed to C57BL/6 mice.
Reagents and mAbs
The allophycocyanin-labeled mAb specific for CD11b (553312), PE-labeled anti-CD45 mAb (553081), FITC-labeled anti-GR1 mAb (553126), and CD16/32-specific mAb (553142) to block Fc receptors were purchased from BD Pharmingen. For depletion of GR1+ cells in vivo, purified GR1 mAb (RB6-8C5, 150 μg/mouse), kindly provided by Dr. J. Zerrahn (Max Planck Institute for Infection Biology, Berlin, Germany), was used.
Infection protocol
Mice were anesthesized with an i.p. injection of Ketamin (150 μl/mouse). Subsequently, mice were infected intranasally with C. pneumoniae by applying 30 μl of PBS containing 2.5 x 106 inclusion-forming units (IFU) of the microorganism or PBS alone.
Isolation of pulmonary cells
To isolate pulmonary cells, mice were sacrificed by CO2 inhalation 3 days postinfection with C. pneumoniae. The lungs were flushed with 10 ml of PBS applied through the right atrium of the heart to remove blood. Thereafter, the organ was cut into small pieces in a 60-mm plate and digested for 10 min with collagenase VIII (400 U/100 μl, room temperature (RT), C-2139; Sigma-Aldrich) and subsequently for another 30 min (400 U of collagenase/100 μl in 2 ml of RPMI 1640, 0% FCS, 37°C). The digested material was filtered through a cell strainer of 100 μm pore size (BD Discovery Labware Europe) to remove debris. The cells were incubated with ammonium chloride (0.15 M, 3 min, RT) to lyse erythrocytes. The remaining cells were washed in PBS containing 3% FCS and analyzed by FACS.
Detection of chemokines and cytokines
The chemokines keratinocyte-derived chemokine (KC), MCP-1, and MIP-2 as well as the cytokines TNF, IFN-, and IL-12p40 were determined in lungs of infected mice by commercially available ELISAs (duo set for KC, MIP-2, TNF, IFN-, and IL-12p40, R&D Systems; MCP-1 from BD Biosciences). The assays were performed, as described by the manufacturer. Lungs were isolated from mice and minced to homogeneity in 500 μl of PBS. After centrifugation (2000 x g, 5 min), the supernatant was analyzed for its cyto- or chemokine content in duplicates.
FACS analysis and cell sorting
The cellular composition in lungs postinfection was determined by FACS. The isolated cells were preincubated with ethydium monacide (2 μg/ml, 20 min, 4°C; Molecular Probes) to exclude dead cells and anti-CD16/32 (100 μg/ml, 10 min, 4°C) to block Fc receptors. Subsequently, cells were double stained (30 min, 4°C) with PE-labeled mAb directed against CD45 (1 μg/ml) and FITC-labeled GR1 mAb (1 μg/ml). After three wash steps, the cells were fixed with 2% paraformaldehyde and flow cytometric analysis was performed (BD Biosiences). All FACS data were analyzed using FlowJo (Tree Star).
Pulmonary cells were stained with APC-labeled anti-CD11b mAb, PE-labeled anti-CD45 mAb, and FITC-labeled anti-GR1 mAb. Cells were gated on CD45, and CD11b+/GR1+ and CD11b+/GR1– subsets were sorted with a MoFlow instrument (DakoCytomation).
Giemsa stain
For Giemsa staining, sorted GR1+/CD45+ cells isolated from lungs of control and infected mice were cytospinned (72.3 x g, 5 min). Thereafter, cells were fixed with methanol (100%, 5 min, RT) and stained with Giemsa (20 min, RT).
Disruption of PMN
Sorted GR1+/CD45+ cells were vortexed with glass beads in 2.5 ml of cell culture medium (RPMI 1640, 0% FCS, 10 min). Supernatants were transferred to another tube to discard glass beads and again centrifuged (400 x g, 5 min, RT) to remove cellular debris. The supernatant containing the equivalent of 8.25 x 105 cells/well was added to HEp2 cells (3 x 105 cells/well). After 48 h of culture, chlamydial inclusions were visualized by confocal microscopy.
Histology and immunohistochemistry
Infected wild-type (C57BL/6) and MyD88-deficient mice were sacrificed by CO2 inhalation, and lungs were perfusion fixed with buffered 10% Formalin and in total embedded in paraffin. Sections were stained with H&E.
Immunohistochemistry with a mAb specific for heat shock protein 60 of C. pneumoniae (Affinity BioReagents) in a dilution of 1/1000 was performed on an automated immunostainer (Benchmark; Ventana Medical Systems) using established protocols. In brief, slides were dewaxed, and the subsequent procedure including heat-induced Ag retrieval as well as blocking of nonspecific binding was performed in the immunostainer. Detection of bound Ab was performed with a biotinylated secondary Ab and alkaline phosphatase-conjugated streptavidin (Ventana Medical Systems), using Fast Red as substrate. The same procedure with omission of the primary Ab was performed as negative control.
For quantitative evaluation, immunoreactive inclusion bodies were counted over 50 randomly selected high power fields (x400). Inclusion bodies in the bronchial epithelium and in alveolar lining cells were evaluated separately.
Confocal microscopy
To quantify the numbers of C. pneumoniae in lungs of infected mice, the supernatant of homogenized lung specimens was used to infect HEp2 cells. After 48 h, HEp2 cells were fixed and permeabilized with methanol/acetone (1:1 v/v, 5 min), stained with FITC-labeled mAb specific for chlamydial endotoxin, and counterstained with Evans blue (ACI-FITC, Progen; Biotechnik). Cells were washed and embedded in glycerin/PBS (50%) and analyzed by confocal microscopy (LSM510; Carl Zeiss Jena).
To detect C. pneumoniae in GR1+ cells, CD45+ cells were purified by MACS (Miltenyi Biotec) from lungs taken from mice 3 days postinfection or from mock-infected mice. FcRs were blocked, as described above, and cells were stained with a FITC-labeled GR1 mAb (1 μg/ml, 30 min, 4°C). After washing (PBS containing 3% FCS), cells were fixed with paraformaldehyde (2%, 30 min), washed again, and permeabilized with saponin (1%, 30 min). Simultaneously, cells were stained with an Alexa546-labeled mAb specific for chlamydial endotoxin. Cells were washed three times (1% saponin/PBS containing 3% FCS), followed by three wash steps with PBS and 3% FCS.
Electron microscopy
GR1+/CD45+ cells were purified by cell sorting on day 3 postinfection with C. pneumoniae from collagenase-treated lungs. The cells were washed with PBS, fixed with paraformaldehyde (2%) plus glutaraldehyde (0.1%) for 20 min, 37°C, and postfixed in osmium tetroxide. Subsequently, samples were dehydrated with a graded ethanol series and embedded in Epon, and ultrathin sections were prepared. Sections were washed carefully with deionized H2O and stained with uranyl acetate (0.5%) plus lead citrate (3%). Sections were examined at 80 kV with a Zeiss EM 10 GR transmission electron microscope.
Statistics
Multiple comparisons were analyzed by ANOVA, and as post hoc test the Holm-Sidak method was used. The t test was used to compare two groups.
Results
Inflammatory responses of MyD88-deficient mice upon infection with C. pneumoniae are severely impaired on day 3 postinfection
To analyze the ability of MyD88-deficient mice to recognize the obligate intracellular bacterium C. pneumoniae, gene-deficient and wild-type mice were infected intranasally with 2.5 x 106 IFU of the microorganism. Three days postinfection, lungs of wild-type mice contained elevated levels of TNF, IL-12p40, and IFN-, as well as the chemokines KC, MCP-1, and MIP-2 (Fig. 1A). In contrast, the cyto- and chemokine response of MyD88-deficient mice was much weaker, with the notable exception of IL-12p40, indicating that this cytokine was secreted in vivo during C. pneumoniae infection in a MyD88-independent fashion (Fig. 1A). Total cellular numbers of the lungs of wild-type mice increased 2- to 3-fold 3 days after infection, but did not change in lungs of MyD88-deficient mice (data not shown). As a consequence of the inflammatory response, the lung weight of wild-type mice rose substantially (Fig. 1B). The increment of lung weight was attenuated in the case of MyD88-deficient mice and significantly lower than in wild-type mice (Fig. 1B). In line with these findings, a histologic analysis of lungs 3 days postinfection with C. pneumoniae revealed that lungs of wild-type mice showed an extensive infiltration of pulmonary tissue with leukocytes, which was almost not detectable in MyD88-deficient mice (Fig. 2). However, 6 days postinfection, MyD88-deficient mice were able to produce TNF, IFN-, KC, and MCP-1 in amounts similar to wild-type mice, but failed to secrete IL-12p40 and MIP-2 (Fig. 1A). In addition, the infection increased the lung weight in both strains of mice to the same level 6 days postinfection (Fig. 1B). Thus, it appears that MyD88 is of critical importance to trigger inflammatory responses induced by C. pneumoniae during the early period of infection.
FIGURE 1. Impaired inflammatory response in MyD88-deficient mice upon infection with C. pneumoniae. A, C57BL/6 (wild type (WT), n = 6, ) or MyD88-deficient mice (n = 6, ) were intranasally infected with 2.5 x 106 IFU/mouse and sacrificed 3 days later. Of each strain, two uninfected mice served as negative controls (). In addition, C57BL/6 (WT, n = 3, ) or MyD88-deficient mice (n = 3, ) were infected for 6 days with 2.5 x 106 IFU/mouse. One mouse of each strain served as negative control (). Cyto- and chemokines were determined in the supernatant of lung homogenates via ELISA, as described in Materials and Methods. Error bars represent SD. *, p < 0.001; #, p < 0.05; ANOVA post hoc Holm-Sidak. B, Lung weight of C57BL/6 (WT, n = 6, ) or MyD88-deficient mice (n = 6, ) 3 days upon infection with 2.5 x 106 IFU/mouse. Of each mouse strain, two uninfected mice were used as negative controls (). The lung weight was also determined 6 days after infection with 2.5 x 106 IFU/mouse (C57BL/6, n = 5, ; MyD88-deficient mice, n = 5, ). Two uninfected mice of each strain served as negative controls (). *, p < 0.001; ANOVA post hoc Holm-Sidak.
FIGURE 2. Histologic analysis of the cellular infiltrate in wild-type vs MyD88-deficient mice. Lung histology of wild-type (a–d) and MyD88-deficient mice (e–h) 3 days after infection with C. pneumoniae (2.5 x 106 IFU/mouse). H&E stain, x250 (a–c and e–g); x40 (d and h). Mock-infected animals (a and e) show normal bronchi and alveoli. Infected wild-type animals (b and c) show massive infiltrates of PMN in a peribronchiolar and perivascular distribution. The low power view (d) demonstrates the wide extent of inflammatory changes, accompanied by focal hemorrhage. MyD88-deficient mice show essentially no (f) or only mild and focal (g) inflammatory changes with occasional PMN (arrow). The low power view (h) demonstrates the preservation of the lung architecture and the absence of major inflammatory changes in MyD88-deficient mice.
On day 3 postinfection, 30% of the CD45+ pulmonary infiltrate was also GR1 positive in wild-type mice (Fig. 3, A and B). This cellular subset did not increase in MyD88-deficient mice (Fig. 3, A and B). Purification of GR1+/CD45+ cells by cell sorting and subsequent Giemsa staining revealed that the vast majority of cells displayed the morphology of PMN (Fig. 3B).
FIGURE 3. Recruitment of PMN into infected lungs of wild-type, but not MyD88-deficient mice. A, Lungs of C. pneumoniae-infected (2.5 x 106 IFU/mouse, day 3) C57BL/6 mice (n = 2) and MyD88-deficient mice (n = 2) as well as mock-infected controls (n = 1) were digested with collagenase. Single cell suspensions were stained with a FITC-labeled mAb specific for GR1 and a PE-labeled mAb specific for CD45. Top graphs, Show the FACS analysis from mock infected; middle and bottom graphs, from C. pneumoniae-infected animals. B, Shows the percentage of GR1+/CD45+ cells from infected C57BL/6 (wild type (WT), n = 4, ) and MyD88-deficient mice (n = 4, ). Two uninfected mice of each strain served as controls. *, p < 0.001; ANOVA post hoc Holm-Sidak. C, The experiment was performed, as described in A, with the exception that GR1+/CD45+ cells of C57BL/6 mice were sorted and their morphology was analyzed by Giemsa staining.
Chlamydial burden in MyD88-deficient mice is lower than in wild-type mice
Because the majority of inflammatory responses analyzed in this study were not induced in MyD88-deficient mice, we expected the chlamydial burden in these mice to be higher than in wild-type mice. In contrast to our expectation, we found that the microorganism was detected in even lower amounts in MyD88-deficient than wild-type mice (Fig. 4). A semiquantitative real-time PCR approach confirmed these results (data not shown). When chlamydial burden was analyzed 6 days postinfection, the number of microorganisms found in lungs of MyD88-deficient mice was about the same as on day 3 postinfection (Fig. 4). However, wild-type mice decreased chlamydial burden to the level of MyD88-deficient mice 6 days postinfection (Fig. 4). Thus, although MyD88-deficient mice are severely impaired to mount an inflammatory response against C. pneumoniae, the replication of the microorganism during the early phase of infection appears to be less effective in these mice.
FIGURE 4. Lower chlamydial burden in MyD88-deficient mice. Three and 6 days postinfection (2.5 x 106 IFU/mouse), chlamydial burden was determined in the supernatant of lung homogenates of C57BL/6 (wild type (WT), day 3, n = 6; day 6, n = 5, ) and MyD88–/– mice (day 3, n = 6; day 6, n = 5, ). Chlamydial burden was quantified by transfer of lung supernatants to HEp2 cells. Forty-eight hours later, IFU in HEp2 cells were counted. The graph represents the pooled data from two independent experiments. *, p < 0.001; ANOVA post hoc Holm-Sidak.
Depletion of GR1+ cells in vivo reduces chlamydial burden
Because GR1+/CD45+ cells were not attracted into infected lungs of MyD88-deficient mice, we wondered whether these cells may influence the chlamydial burden in wild-type mice. Therefore, we depleted wild-type mice from GR1+ cells and infected them 1 day later with C. pneumoniae. Fig. 5A shows that GR1+/CD45+ cells were completely absent in depleted mice 3 days postinfection. Furthermore, the chlamydial burden was lower in GR1-depleted mice compared with untreated wild-type mice at this time point (Fig. 5B). Thus, GR1+ cells appear to support the replication of C. pneumoniae in vivo.
FIGURE 5. Depletion of GR1+ cells in vivo decreases chlamydial burden. A, C57BL/6 mice (n = 2) were depleted from GR1+ cells by injecting i.p. 150 μg/mouse anti-GR1 mAb 1 day before infection with C. pneumoniae (2.5 x 106 IFU/mouse, right graphs). Control C57BL/6 mice (n = 2) were pretreated i.p. with PBS and subsequently infected (2.5 x 106 IFU/mouse, left graphs). One mouse was neither depleted nor infected (top graph). Three days postinfection, lungs were removed and digested with collagenase. Single cell suspensions were stained with a FITC-labeled mAb specific for GR1 and a PE-labeled mAb specific for CD45. B, C57BL/6 mice (n = 4) were depleted from GR1+ cells and infected, as described in A (). PBS-pretreated and infected C57BL/6 mice (n = 5) served as controls (). Chlamydial burden was determined 3 days after infection. The graph represents the pooled data from two independent experiments. *, t test, p = 0.012.
PMN are infected with and support the replication of C. pneumoniae in epithelial cells
In an attempt to identify the mechanism in which PMN increase the chlamydial burden in wild-type mice in vivo, we examined whether PMN are infected with C. pneumoniae in vivo. Confocal microscopy of CD45+ cells purified from infected lungs demonstrated that a substantial fraction of the GR1+ subset contained C. pneumoniae (Fig. 6a). Next, we analyzed the possibility that the microorganism replicates in GR1+ PMN by electron microscopy, as claimed recently for human PMN (5). Electron microscopy of ex vivo purified GR1+/CD45+ cells confirmed the results obtained with confocal microscopy, and EB could clearly be visualized in PMN (Fig. 6, b and c, arrow). Also, a few RB were found, indicating that C. pneumoniae replicated to some extent in these cells (Fig. 6c, arrowhead). Some of the EB isolated from PMN at day 3 postinfection were indeed living, because coincubation of HEp2 cells with a lysate of purified PMN gave rise to chlamydial inclusions (Fig. 6d).
FIGURE 6. C. pneumoniae infects and generates small inclusions in PMN in vivo. a, CD45+ cells were purified by magnetic separation from collagenase-treated lungs 3 days after infection of C57BL/6 mice with C. pneumoniae (2.5 x 106 IFU/mouse). Cells were stained with FITC-labeled anti-GR1 mAb. C. pneumoniae inclusions were visualized by an Alexa546-labeled mAb specific for chlamydial endotoxin. Cells were analyzed by confocal microscopy. b, GR1+/CD45+ cells were selected by cell sorting from collagenase-treated lungs 3 days after infection of C57BL/6 mice with C. pneumoniae (2.5 x 106 IFU/mouse) and analyzed by electron microscopy (magnification x22,000). Arrow points to a chlamydial inclusion. c, Further electronic close-up. Arrow points to an EB, and arrowhead to an RB. d, FACS-purified GR1+/CD45+ cells were minced by vortexing with glass beads. After centrifugation of the lysate, the supernatant (equivalent of 8.25 x 105 cells/well) was transferred to HEp2 cells (3 x 105 cells/well). After 48 h of culture, chlamydial inclusions were visualized by confocal microscopy. (arrows).
The total number of chlamydial inclusions found in PMN per infected lung is much smaller than the difference in chlamydial burden between untreated and GR1-depleted mice. Therefore, we speculated that PMN in addition stimulate the replication of C. pneumoniae in bronchial epithelial cells. We addressed this assumption by sorting PMN from lungs of untreated or infected wild-type mice and cocultured the cells with HEp2 cells infected with C. pneumoniae just before the addition of PMN. Confocal microscopy revealed that PMN isolated from mock or infected mice increased the number of chlamydial inclusions more than 2.5-fold, while GR1–/CD11b+ cells were less effective (Fig. 7).
FIGURE 7. PMN support the replication of C. pneumoniae in epithelial HEp2 cells. GR1+/CD11b+ or GR1–/CD11b+ cells were sorted from mock-infected () or day 3 infected C57BL/6 mice (). Both cell types were added at a density of 2 x 105 cells/well to HEp2 cell cultures (2 x 105 cells/well), which were infected with C. pneumoniae at an multiplicity of infection of 1 just before the addition of lung cells. , Represents C. pneumoniae-infected HEp2 cell cultures without addition of inflammatory cells. After 48 h of culture, cells were lysed, and the supernatant of the lysate was transferred to a new well seeded with uninfected HEp2 cells (2.5 x 105 cells/well). After another culture period of 30 h, chlamydial inclusions were counted using confocal microscopy. Error bars represent SD of results obtained with five to eight individual cultures. The graph represents data from three pooled experiments. *, p < 0.001; ANOVA post hoc Holm-Sidak.
We also determined the anatomical localization of chlamydial inclusions in infected lungs of wild-type and MyD88-deficient mice and their spatial relationship to PMN using immunohistochemistry. Although similar numbers of large chlamydial inclusions were found within bronchial epithelial cells in both strains of mice, which indicates that MyD88-deficient epithelial cells are in principle able to replicate the microorganism as efficiently as epithelial cells from wild-type mice, alveolar epithelial cells were rarely infected in MyD88-deficient mice compared with wild-type mice (Fig. 8 and Table I). Of note, infected alveolar epithelial cells were regularly surrounded by PMN (Figs. 8 and 9), suggesting that PMN might support chlamydial replication in alveolar epithelial cells in vivo. In agreement with data detailed above, PMN of wild-type mice contained small chlamydial inclusions (Fig. 9).
FIGURE 8. C. pneumoniae-infected pulmonary cell types of C57BL/6 and MyD88-deficient mice. Sections of paraffin-embedded lungs from C57BL/6 (animal 1, a and c; animal 2, b and d) or MyD88-deficient mice (animal 1, e and g; animal 2, f and h) 3 days postinfection (2.5 x 106 IFU/mouse) were stained with a mAb specific for chlamydial heat shock protein 60 to visualize chlamydial inclusions. Large chlamydial inclusions were detected in bronchial (a, b, e, and f, magnification x400) and alveolar epithelial cells (c, d, g, and h, magnification x200) of C57BL/6 and MyD88-deficient mice. Infected alveolar cells were less frequent in MyD88-deficient mice (see also Table I).
Table I. Quantification of chlamydial inclusions in lung epithelial cells of C57BL/6 and MyD88-deficient mice
FIGURE 9. PMN are close to alveolar epithelial cells containing chlamydial inclusions. Lung sections of two C57BL/6 mice (animal 1, a; animal 2, b) of the experiment described in Fig. 8 were analyzed with higher magnification (x1000) to visualize the proximity of PMN (arrows) and alveolar epithelial cells containing large chlamydial inclusions. Small chlamydial inclusions were found within PMN.
We conclude from these findings that PMN increased chlamydial burden in two ways: they act as cellular site of infection by C. pneumoniae and, in addition, amplify the replication of the microorganism in epithelial cells.
Discussion
MyD88 is the central adaptor of the TLR-signaling cascade interacting with all known TLRs, but also with IL-1R family members. In this study, we analyzed the importance of this adaptor molecule for host defense against the obligate intracellular bacterium C. pneumoniae. In the early phase of infection, i.e., 3 days postinfection, most of the inflammatory responses analyzed were switched off, as expected. In addition, gene microarray analysis of lungs revealed that 312 genes were induced >2- and up to 27-fold by C. pneumoniae infection in wild-type mice. Of these genes, 197 were below a 2-fold induction in MyD88-deficient mice, indicating that MyD88 is crucial for the initiation of host responses against this intracellular pathogen (data not shown). Surprisingly, chlamydial replication in MyD88-deficient was less efficient during the early phase of infection, a conclusion based on the finding that bacterial burden in the lung was lower in comparison with wild-type mice. This unexpected result is explained by the observation that PMN support the replication of C. pneumoniae in wild-type mice. MyD88-deficient mice, however, are unable to recruit this population of cells to sites of inflammation.
At present, it is unclear whether the inflammatory defects observed in MyD88-deficient mice upon infection with C. pneumoniae are due to impaired TLR signaling and/or failure of signaling through IL-1R family members. Our preliminary results, however, suggest that TLR signaling is important. Thus, mice double deficient for TLR2 and TLR4 display a similar phenotype upon infection with C. pneumoniae, i.e., the numbers of microorganisms in the lung are lower and pulmonary recruitment of PMN is highly attenuated. In addition, murine dendritic cells sense C. pneumoniae in a TLR2- and TLR4-dependent fashion (10). Furthermore, gene microarray results revealed that transcription of IL-1, IL-1, and IL-18 genes is not induced in MyD88-deficient mice, suggesting that IL-1R family members are not triggered in vivo (our unpublished results).
How C. pneumoniae persists in PMN is unknown to date. In epithelial cells, in which C. pneumoniae forms large inclusions within 48–72 h, inclusions avoid fusion with late endosomes and lysosomes, but accumulate early transferrin receptor-positive endosomes (23). In human monocytes, however, C. pneumoniae-containing inclusions are small and characterized by large atypical RBs (24), which may be explained by the finding that in the case of infection with Chlamydia psittaci, early vacuoles fused with lysosomes in the monocytic cell line THP1 (25). Nevertheless, the microorganism persists in human monocytes (24), indicating that lysosomal effector molecules are either unable to completely kill the bacterium or lysosomal fusion with chlamydial inclusion is incomplete. In PMN, we also found only small inclusions containing few EBs and RBs. This indicates that C. pneumoniae replicates to some extent in PMN, and that lysosomal fusion, if it occurs in these cells, is also only partially effective. To maximize the weak replication inside PMN, the microorganism appears to elongate the life span of PMN by interference with the apoptotic signal cascade (5).
Infection of PMN with C. pneumoniae and enhancement of chlamydial replication in epithelial cells by PMN are functionally not related, because PMN from uninfected wild-type mice increase chlamydial replication in epithelial cells. However, the molecular mechanism(s) involved in the latter process is unclear. A similar function has been reported for the monocyte-like cell U937. Thus, coculture of U937 cells with human endothelial cells enhanced the infection of the latter cells by C. pneumoniae (26). Subsequently, it was suggested that the infectivity-enhancing factor is identical with human insulin-like growth factor 2 (27). Using a different cellular system, a recent study demonstrated that human monocytes also enhance the growth of C. pneumoniae in arterial smooth muscle cells (28). In this case, cell to cell contact was required, and it was suggested that mannose 6-phosphate receptor expressed on monocytes is implicated in this process. Other possible mechanisms include the regulation of intracellular tryptophan levels in epithelial cells by PMN because C. pneumoniae lacks tryptophan biosynthesis genes (29) and thus is completely dependent on host-derived tryptophan (30). Increasing the supply of this amino acid via TGF--mediated down-regulation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase or the tryptophanyl-tRNA synthetase could represent potential mechanisms (31). TGF-, in turn, might be produced by PMN because it was reported that TGF- mRNA was induced after stimulation of human PMN with Mycobacterium bovis bacillus Calmette-Guerin (32).
In comparison with wild-type mice, the chlamydial load appears to be more reduced in MyD88-deficient mice than in GR1-depleted mice, indicating that in addition to PMN, another cell type might support the replication of C. pneumoniae. As discussed above, the human monocyte-like cell U937 was able to amplify chlamydial replication in smooth muscle and epithelial cells (26, 28). In our experiments, the GR1–/CD11b+ cell population, which may include monocytes/macrophages, also weakly amplified chlamydial replication, although the differences were not statistically significant. Thus, further experimentation is needed to clarify this issue.
It remains to be seen whether other adaptors aside from MyD88 are relevant for host defense against C. pneumoniae. However, release of IL-12p40 at day 3 postinfection appears to be MyD88 independent. Also, our experiments show that on day 6 postinfection, TNF, IFN-, KC, and MCP-1 are secreted MyD88 independently. IL-12 participates in the control of C. pneumoniae replication in vivo, because anti IL-12p40 treatment results in higher chlamydial titers, but less severe pathological changes within the lung of infected mice (33). From the adaptors identified to date, only TICAM-1 (Toll-IL-7R domain-containing adaptor-inducing IFN-) appears to operate MyD88 independently (15, 18). TICAM-1 is crucially involved in the induction of IFN- (34), but also in endotoxin-mediated secretion of IL-12p40 (15). In vitro, IL-12p40 secretion induced by C. pneumoniae depends partially on TLR4 (10), and TICAM-1 not only interacts with TLR3 (34), but also with TLR4 (18). On the one hand, these findings argue for a role of TICAM-1 during recognition of C. pneumoniae. In contrast, our preliminary data show that TLR4 appears not to participate in release of IL-12p40 in vivo.
In conclusion, the results of this study describe a new role for PMN in the replication of the intracellular pathogen C. pneumoniae in vivo: attracted MyD88 dependently to the site of inflammation, they increase chlamydial burden during the first days of infection.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. S. Akira for providing MyD88-deficient mice. We are also grateful to Dr. J. Zerrahn for the generous supply with anti-GR1 mAb. We thank Carmen Hartmann for expert technical assistance with immunohistochemistry. Many thanks to Dr. B. Holzmann for critical reading of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Deutsche Forschungsgemeinschaft MI 471/1-1.
2 Address correspondence and reprint requests to Dr. Thomas Miethke, Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Trogerstr. 9, 81675 Munich, Germany. E-mail address: Thomas.Miethke{at}lrz.tu-muenchen.de
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; EB, elementary body; IFU, inclusion-forming unit; RB, reticulate body; RT, room temperature; TICAM-1, Toll-IL-1R domain-containing adaptor molecule-1; TIRAP, Toll-IL-1R domain-containing adaptor protein; KC, keratinocyte-derived chemokine.
Received for publication July 21, 2004. Accepted for publication January 3, 2005.
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