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


     
 

The Journal of Immunology, 2007, 179, 3937-3946
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bhan, U.
Right arrow Articles by Standiford, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bhan, U.
Right arrow Articles by Standiford, T. J.

TLR9 Is Required for Protective Innate Immunity in Gram-Negative Bacterial Pneumonia: Role of Dendritic Cells

Urvashi Bhan*, Nicholas W. Lukacs{dagger}, John J. Osterholzer*, Michael W. Newstead*, Xianying Zeng*, Thomas A. Moore*, Tracy R. McMillan*, Arthur M. Krieg{ddagger}, Shizuo Akira§ and Theodore J. Standiford1,*

* Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine and {dagger} Department of Pathology, University of Michigan Medical Center, Ann Arbor, MI 48109; {ddagger} Coley Pharmaceutical Group, Wellesley, MA 02481; and § Department of Host Defense, Research Institute for Microbial Defenses, Osaka University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In this study, experiments were performed to determine the contribution of TLR9 to the generation of protective innate immunity against virulent bacterial pathogens of the lung. In initial studies, we found that the intratracheal administration of Klebsiella pneumoniae in wild-type (WT) BALB/c mice resulted in the rapid accumulation of dendritic cells (DC) expressing TLR9. As compared with WT mice, animals deficient in TLR9 (TLR9–/–) displayed significantly increased mortality that was associated with a >50-fold increase in lung CFU and a >400-fold increase in K. pneumoniae CFU in blood and spleen, respectively. Intrapulmonary bacterial challenge in TLR9–/– mice resulted in reduced lung DC accumulation and maturation as well as impaired activation of lung macrophages, NK cells, and {alpha}beta and {gamma}{delta} T cells. Mice deficient in TLR9 failed to generate an effective Th1 cytokine response following bacterial administration. The adoptive transfer of bone marrow-derived DC from syngeneic WT but not TLR9–/– mice administered intratracheally reconstituted antibacterial immunity in TLR9–/– mice. Collectively, our findings indicate that TLR9 is required for effective innate immune responses against Gram-negative bacterial pathogens and that approaches to maximize TLR9-mediated DC responses may serve as a means to augment antibacterial immunity in pneumonia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Innate immunity is the primary pathway for elimination of most bacterial organisms from the respiratory tract. The primary phagocytic cells that constitute innate immunity in the lung are the resident alveolar macrophages (AM)2 and recruited neutrophils (1, 2, 3, 4, 5). In addition, local lung dendritic cells (DC) and rapidly mobilized DC internalize and process bacteria, which promotes the elaboration of type 1 cytokines and chemokines, the expression of costimulatory molecules, and promotion of Ag-specific cellular and humoral immunity (6, 7, 8). Local and recruited DC are also active participants in pulmonary innate immune responses via the production of cytokines that induce the early expression of IFN-{gamma} in a non-Ag specific fashion by lung macrophages, NK cells, NK T cells, and {gamma}{delta} T cells. Moreover, DC produce chemokines that facilitate the recruitment and/or activation of specific leukocyte populations that contribute to the innate response (9, 10, 11, 12, 13, 14, 15).

TLRs are a family of type I transmembrane receptor proteins that are required for the recognition of various pathogen-associated molecular patterns expressed by a diverse group of infectious microorganisms, resulting in the activation of host immune responses (16, 17). Binding of these ligands to most TLRs initiates a signaling cascade involving MyD88, IL-1R-associated kinase (IRAK), and TNFR-associated factor 7 (TRAF6), resulting in NF-{kappa}B and MAPK activation and culminating in the expression of genes involved in antimicrobial defense (18). Certain TLRs can also initiate protective innate responses in a MyD88-independent fashion, which requires the adaptor molecule Toll/IL-1R domain-containing adaptor protein (TIRAP) (19). TLR4 has previously been shown to be required for effective innate immunity against selected extracellular Gram-negative pathogens, including Haemophilus influenza and Klebsiella pneumoniae (20, 21). However, although innate signals produced early (at 4 h) in response to challenge with K. pneumoniae are markedly diminished in mice with defective TLR4 signaling, later responses (at 16 h) remain intact (21). Moreover, host innate responses against both extracellular and intracellular bacterial pathogens are more dramatically impaired in mice that lack the common adaptor molecule MyD88 than in mice that are deficient in a single TLR (e.g., TLR4 or TLR2; Refs. 22, 23, 24, 25, 26). Collectively, these data indicate that multiple MyD88-dependent TLRs are required for the maintenance and/or full expression of protective innate antibacterial responses.

A TLR that is well positioned to respond to microbial invasion is TLR9. TLR9 is a Toll receptor that is localized intracellularly within endocytic vesicles and is activated by unmethylated CpG motifs that are present in high frequency in DNA from various microbes, including bacteria, viruses, and certain fungi (27, 28, 29, 30, 31). The activation of TLR9 requires the uptake of microbes (or synthetic CpG oligodeoxynucleotides) within endosomes, the formation of DNA:TLR9 complexes within the endocytic vesicles, and the subsequent acidification and maturation of the endosomes (32, 33, 34). Stimulation of immune cells with synthetic CpG motifs or microbial DNA results in a variety of effects primarily characterized by the stimulation of type 1-associated cytokines and chemokines as well as the up-regulation of costimulatory and MHC molecules on the cell surface of professional APC (35, 36). A variety of cells express TLR9, most notably cells of the myeloid lineage. In mice, TLR9 is primarily expressed on DC (plasmacytoid and myeloid), B cells, and, to a lesser extent, on macrophages (37, 38, 39, 40, 41, 42, 43, 44). Structural cells, including alveolar epithelial cells and endothelial cells, express TLR9, although the function of TLR9 in these cells is not known (45, 46).

Studies performed in diverse animal model systems indicate that stimulation with exogenous synthetic CpG oligodeoxynucleotides (ODN) can promote, in vivo, type 1 immune responses characterized by enhanced IL-12 and IFN-{gamma} production while inhibiting type 2 immune responses (47, 48, 49, 50, 51, 52, 53, 54, 55, 56). However, little is known about the contribution of TLR9 and naturally occurring TLR9 ligands to the development of a protective immune response in infection. It has recently been shown that whole live bacteria, including both Gram-positive and Gram-negative organisms, can directly activate TLR9 in vitro (57). Furthermore, TLR9-deficient mice display diminished antiviral activity against the DNA virus murine CMV in vivo (40). Moreover, TLR9 knockout mice have reduced type 1 immunity against the intracellular parasite Toxoplasma gondii (58). Finally, TLR9-deficient mice have recently been shown to have reduced clearance of the Gram-positive organism Streptococcus pneumoniae from the lung, which was associated with impaired bacterial uptake and killing by lung macrophages (59).

In this study, we investigated the contribution of TLR9 to the generation of innate immune responses against extracellular Gram-negative bacterial pathogens. Our findings show for the first time that TLR9-mediated DC responses are required for effective antibacterial immunity in a murine model of invasive bacterial pneumonia and that the adoptive transfer of DC isolated from wild-type (WT) BALB/c, but not from TLR9–/– mice, can restore innate antibacterial immunity in mice deficient in TLR9.


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

Murine recombinant cytokines for ELISA were purchased from R&D Systems. The TLR9 Ab used was a rat IgG2a anti-mouse Ab purchased from eBioscience.

Mice

Female, specific pathogen-free, 6- to 8-wk-old BALB/c mice were purchased from The Jackson Laboratory. Breeding pairs of TLR9–/– mice generated by S. Akira (Osaka University, Osaka, Japan) were obtained from Coley Pharmaceutical Group and a colony was established at the University of Michigan (Ann Arbor, MI). These mice were generated on a BALB/c background (more than five backcrosses), are phenotypically normal in the uninfected state, and reproduce without difficulty. The studies were approved by the animal use committee at the University of Michigan (Ann Arbor, MI).

Bacterial preparation and intratracheal (i.t.) or i.v. administration

K. pneumoniae strain 43816 serotype 2 (American Type Culture Collection) was used in our studies (13, 56). K. pneumoniae was grown overnight in tryptic soy broth (Difco) at 37°C and quantitated using spectrophotometry. For i.t. administration, mice were anesthetized with an i.p. ketamine and xylazine mixture. Next, the trachea was exposed and 30 µl of inoculum was administered via a sterile 26-gauge needle. The skin incision was closed using surgical staples. For i.v. administration, bacteria were diluted in 500 µl of PBS and then administered by tail vein injection using a sterile 26-gauge needle.

Lung, spleen, and blood harvesting for bacterial CFU determination and cytokine analysis

At designated time points, the mice were killed by CO2 asphyxia. Before lung removal, the pulmonary vasculature was perfused with 1 ml of PBS containing 5 mM EDTA via the right ventricle. Whole lungs and spleen were then harvested for the assessment of bacterial numbers and cytokine protein expression. After removal, whole organs were homogenized in 1.0 ml of PBS with protease inhibitor (Boehringer Mannheim Biochemicals) using a tissue homogenizer (Biospec Products) under a vented hood. Portions of homogenates or heparized blood collected from the right ventricle (10 µl) were inoculated on blood agar after serial 1/10 dilutions with PBS. Homogenates were incubated on ice for 30 min and then centrifuged at 1,100 x g for 10 min. Supernatants were collected, passed through a 0.45-µm pore size filter (Gelman Sciences), and stored at –20°C for the assessment of cytokine levels.

Total lung leukocyte preparation

Lungs were removed from euthanized animals and leukocytes were prepared as previously described (56). Briefly, lungs were minced with scissors to a fine slurry in 15 ml of digestion buffer (RPMI 1640 medium, 10% FCS,1 mg/ml collagenase (Boehringer Mannheim Biochemical), and 30 µg/ml DNase (Sigma-Aldrich)) per lung. Lung slurries were enzymatically digested for 30 min at 37°C. Any undigested fragments were further dispersed by drawing the solution up and down through the bore of a 10-ml syringe. The total lung cell suspension was pelleted, resuspended, and spun through a 20% Percoll gradient to enrich for leukocytes. Cell counts and viability were determined using trypan blue exclusion counting on a hemacytometer. Cytospin slides were prepared and stained with a modified Wright-Giemsa stain. To assess spontaneous inducible NO synthase (iNOS) expression in lung macrophages, cells were isolated from lung digest cells by Percoll gradient enrichment and adherence purification at a concentration of 1–2 x 106 cells/well. Cells were washed three times and then RNA was immediately isolated.

Multiparameter flow cytometric analyses

Cells were isolated from lung digests as described above (56). For analyses of T cell subsets, isolated leukocytes were stained with the following FITC- or PE-labeled Abs: anti-{alpha}beta TCR, anti-{gamma}{delta} TCR, anti-DX5, anti-CD11c, anti-MHC class II, anti-Gr1, anti-CD40, anti-CD80, anti-CD86, and anti-CD69 (all reagents were from BD Pharmingen unless otherwise noted). In addition, cells were stained with anti-CD45-tricolor (Caltag Laboratories), allowing for the discrimination of leukocytes from nonleukocytes and thus eliminating any nonspecific binding of T cell surface markers on nonleukocytes. T and NK cell subsets were analyzed by first gating on CD45-positive "lymphocyte-sized" leukocytes and then examined for FL1 and FL2 fluorescence expression using four color flow cytometry. Cells were collected on a FACScan or FACScalibur cytometer (BD Biosciences) by using CellQuest software (Becton Dickinson). Analyses of data were performed using the CellQuest software package.

Intracellular TLR9 staining

Leukocytes from the lungs of K. pneumoniae-infected and uninfected control mice were enriched by Percoll gradient centrifugation. Cells were incubated with anti-CD11c Abs coupled to magnetic beads (Miltenyi Biotec) and then positively selected by running the cell suspension through a magnetic column. Intracytoplasmic TLR9 staining was performed using the Cytofix/Cytoperm Plus kit and manufacturers’ protocol (BD Pharmingen). Adherent CD11c+ cells were removed and then nonadherent CD11c+ cells were stained for surface expression of MHC class II. Cells were then fixed and permeabilized for 20 min on ice. After washing, cells were stained for intracytoplasmic TLR9 expression with biotinylated rat anti-mouse TLR9 Abs (eBioscience) diluted in wash solution for 30 min. Cells were then analyzed by using a FACSCalibur cytometer (BD Biosciences) with CellQuest software (BD Biosciences).

Isolation and culture of bone marrow-derived DC

Bone marrow was harvested from the long bones of mice using a previously described technique (60). Recovered marrow cells were seeded in tissue culture flasks in RPMI 1640-based complete medium with murine GM-CSF (10 ng/ml). Media and cytokines were replaced after 3 days, loosely adherent cells were collected after 6–7 days, and cells were positively selected for CD11c+ by magnetic bead separation. CD11c+ DC were plated overnight and resuspended in fresh medium the following day. Flow cytometry of cells verified >90% purity for DC.

Murine cytokine ELISA

Murine TNF-{alpha}, CXCL10/IFN-inducible protein 10 (IP-10), IFN-{gamma}, IL-12, and CCL2/MCP-1 were quantitated using a modification of a double-ligand method as previously described (13, 56). The ELISA method used consistently detected murine cytokine concentrations of >20 pg/ml. The ELISAs did not cross-react with the other cytokines tested.

Real-time quantitative RT-PCR

Measurement of gene expression was performed using the ABI Prism 7000 sequence detection system (Applied Biosystems) as previously described (56). Primer and probe nucleotide sequences were as follows: murine TNF-{alpha}, 5'-CAGCCGATGGGTTGTACCTT-3' (forward), 5'-TGTGGGTGAGGAGCACGTAGT-3' (reverse), and 5'-TCCCAGGTTCTCTTCAAGGGACAAGGC-3' (probe); CXCL10/murine IP-10, 5'-CCAGTGAGAATGAGGGCCATA-3' (forward), 5'-CTCAACACGTGGGCAGGAT-3' (reverse), and 5'-FAM-TTTGGGCATCATCTTCCTGGA-TAMRA-3' (TaqMan probe); murine IL-12 p40, 5'-AGACCCTGCCCATTGAACTG-3' (forward), 5'-GAAGCTGGTGCTGTAGTTCTCATATT-3' (reverse), and 5'-CGTTGGAAGCACGGCAGCAGAA-3' (probe); MCP-1/CCL2, 5'-GGCTCAGCCAGATGCAGTTAAC-3' (forward), 5'-CCTACTCATTGGGATCATCTTGCT-3' (reverse), and 5'-CCCCACTCACCTGCTGCTACTCATTCAC-3' (reverse); IFN-{gamma}, '-CTGCGGCCTAGCTCTGAGA-3' (forward), 5'-CAGCCAGAAACAGCCATGAG-3' (reverse), and 5'-CACACTGCATCTTGGCTTTGCAGCTTCTA-3' (probe); murine iNOS, 5'-CCCTCCTGATCTTGTGTTGGA-3' (forward), 5'-CAACCCGAGCTCCTGGAA-3' (reverse), and 5'-TGACCATGGAGCATCCCAAGTACGAGT-3' (probe); and murine beta-actin, 5'-CCGTGAAAAGATGACCCAGATC-3' (forward), 5'-CACAGCCTGGATGGCTACGT-3' (reverse), and 5'-TTTGAGACCTTCAACACCCCAGCCA-3' (probe). Specific thermal cycling parameters used with the TaqMan One-Step RT-PCR Master Mix Reagents kit included 30 min at 48°C, 10 min at 95°C, and 40 cycles involving denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Relative quantitation of cytokine mRNA levels was plotted as fold change compared with an untreated control lung. All experiments were performed in duplicate.

Statistical analysis

Survival curves were compared using the log-rank test. For other data, statistical significance was determined using the unpaired t test or one-way ANOVA corrected for multiple comparisons as appropriate. All calculations were performed using the Prism 3.0 software program for Windows (GraphPad Software). All mean data shown are expressed as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Accumulation of DC expressing TLR9 in the lungs of mice challenged i.t. with K. pneumoniae

Previous studies have indicated that DC are one of the predominant cell types expressing TLR9 (38, 39, 40). To determine the number and percentage of DC in lung expressing TLR9 at baseline and during bacterial pneumonia, whole lungs were harvested from uninfected WT mice and from mice 48 h after i.t. K. pneumoniae administration. Leukocytes were then purified from lung digest preparations using a Percoll gradient and CD11c+ cells positively selected by magnetic sorting. CD11c+ cells were then permeabilized and costained for the expression of TLR9 and MHC class II. The lung DC population was gated based on forward and side scatter characteristics. As shown in Fig. 1, there was a low but detectable number of myeloid DC expressing TLR9 present in the lungs of uninfected mice at baseline (0.65 x 105 per lung). However, the i.t. administration of K. pneumoniae resulted in a nearly 2-fold increase in the number of CD11c+ cells that coexpressed both MHC class II (Ia-d) and TLR9 as compared with that observed in the lungs of uninfected mice. This increase in TLR9-expressing DC was consistent with a maximal 2.2-fold increase in the expression of TLR9 mRNA in the lungs of mice infected with K. pneumoniae at 48 h (data not shown). The population of CD11c+ cells that were MHC class IIlow also expressed F4/80, indicative of macrophages. These MHC class IIlow cells were autofluorescent in the uninfected state, and no specific staining for TLR9 was detected during infection.


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 1. FACS dot plot showing total number and percentage of CD11c+MHC class II+ cells expressing TLR9 in lungs of uninfected and K. pneumoniae (Kp)-infected BALB/c mice. BALB/c mice were i.t. administered 5 x 102 CFU of K. pneumoniae and then lungs were harvested 48 h later and CD11c+ cells were selected by MACS, gated for DC based on forward and side scatter characteristics, and analyzed by three-color flow cytometry. The percentage value represents the percentage of total CD11c+ cells expressing TLR9. Control IgG:FITC and IgG:PE are shown in left panel; n = 3 for uninfected mice and n = 4 animals combined for infected mice. Autofluorescent CD11c+MHC class IIlow cells coexpressed F4/80, indicative of macrophages.

 
Survival in WT and TLR9–/– mice following i.t. K. pneumoniae administration

To determine whether mice deficient in TLR9 displayed altered susceptibility to intrapulmonary challenge with K. pneumoniae, age- and sex-matched WT and TLR9–/– mice were administered either an LD30 (5 x 102 CFU) or an LD80 (5 x 103 CFU) dose of K. pneumoniae i.t. and then survival was assessed out to 10 days postadministration. As shown in Fig. 2, TLR9–/– mice died earlier and had a significantly higher overall mortality as compared with the WT mice when challenged with either a high dose (5 x 103 CFU; upper panel) or a lower dose (5 x 102 CFU; lower panel) of K. pneumoniae.


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 2. Survival in WT and TLR9–/– mice after i.t. administration of K. pneumoniae (Kp) at a dose of 5 x 103 (upper panel) or 5 x 102 (lower panel); n = 10 animals per group. *, p < 0.05 as compared with K. pneumoniae-infected WT mice. Balb, BALB/c.

 
Bacterial clearance in WT and TLR9–/– mice following i.t. K. pneumoniae administration

Having observed decreased survival in TLR9-deficient mice challenged with K. pneumoniae, we next evaluated the mechanism of increased mortality in TLR9–/– mice. K. pneumoniae (5 x 102 CFU) was administered to WT and TLR9–/– mice i.t. and then we assessed local bacterial clearance (lung CFU) and systemic dissemination (CFU in blood and spleen) at 24 and 72 h postchallenge. We observed a >15-fold and a >50-fold increase in K. pneumoniae CFU in the lungs of TLR9–/– mice at 24 and 72 h, respectively, as compared with controls (Fig. 3A; p < 0.05). Histologically, inflammatory cells were present in similar quantities within the alveoli and interstitium of both infected WT and mutant mice. However, a substantial increase in the numbers of free bacteria localized within the airspace and the numbers of K. pneumoniae found intracellularly within AM of TLR9–/– mice were noted, raising the possibility of suboptimal activation of AM in mutant mice (Fig. 3B). To quantitate the difference in the accumulation of intracellular bacteria within AM, we found 5.5 ± 0.3 and 7.5 ± 0.4 intracellular bacteria per AM at 24 and 72 h, respectively, after K. pneumoniae administration in TLR9–/– mice as compared with 2.2 ± 0.1 and 2.7 ± 0.3 intracellular bacteria per AM in infected WT mice (p < 0.001; Fig. 3C).


Figure 3
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 3. Lung K. pneumoniae CFU (A) and bronchoalveolar lavage cytospin analysis (B) from WT and TLR9–/– mice after i.t. bacterial challenge. WT and TLR9–/– mice were i.t. administered 5 x 102 CFU of K. pneumoniae and then lungs were harvested 72 h later. A, Lung CFU determination; n = 8–10 mice per group and expressed in log10 scale. *, p < 0.05 as compared with infected WT mice. B, Bronchoalveolar lavage cytospin analysis shown for WT (right panel) and TLR9–/– (left panel); representative of two separate experiments. The presence of intracellular bacteria in AM from TLR9–/– mice is indicated by the arrow. C, Mean number of intracellular bacteria present within AM obtained by bronchoalveolar lavage from WT and TLR9–/– mice 24 and 72 h after bacterial challenge. Data shown represent mean ± SEM from eight mice per group with 20 random AM counted per animal. *, p < 0.001 as compared with AM from infected WT mice. D, Expression of iNOS mRNA by lung macrophages isolated from WT and TLR9–/– mice isolated 24 and 48 h after K. pneumoniae (Kp) administration. Data represent mean expression of cells from four animals combined at each time point. *, p < 0.01 as compared with macrophages isolated from the lungs of infected WT mice. Balb, BALB/c.

 
NO has previously been shown to be a required component of effective lung innate immunity in Gram-negative bacterial pneumonia (61). To determine whether the intracellular accumulation of bacteria in the AM of TLR9–/– mice was attributable to impaired NO synthesis and the resultant reduced intracellular bacterial killing, we assessed the time-dependent expression of iNOS mRNA from ex vivo cultured lung macrophages isolated from WT or TLR9–/– mice at 24 and 48 h after i.t. K. pneumoniae administration. In these experiments, macrophages were isolated from lung digest cells after Percoll gradient centrifugation and adherence purification. As shown in Fig. 3D, spontaneous iNOS mRNA expression by lung macrophages peaked at 48 h after bacterial administration in WT macrophages. As compared with WT lung macrophages, the expression of iNOS mRNA was substantially blunted in macrophages isolated from infected TLR9–/– mice (p < 0.05).

Interestingly, we observed an even greater disparity in the dissemination of bacteria from the lung, as K. pneumoniae CFU in blood and spleen at 24 h postinoculation were >400 and >1,000-fold higher, respectively, in TLR9–/– mice as compared with infected WT animals (Fig. 4A). Bacterial CFU in blood and spleen remained persistently higher in TLR9–/– mice at later time points (72 h).


Figure 4
View larger version (12K):
[in this window]
[in a new window]

  		FIGURE 4. Systemic bacterial clearance in WT and TLR9–/– mice after i.t. or i.v. K. pneumoniae administration. A, Blood and spleen K. pneumoniae CFU from WT and TLR9–/– mice after i.t. bacterial challenge. WT and TLR9–/– mice were i.t. administered 5 x 102 CFU of K. pneumoniae and then blood was harvested 24 and 72 h later. Values represent mean ± SEM CFU values expressed on a log10 scale; n = 6–8 per group. *, p < 0.05, #, p < 0.01 as compared with K. pneumoniae-infected WT mice. B, Blood K. pneumoniae CFU from WT and TLR9–/– mice after i.v. bacterial administration. WT and TLR9–/– mice were administered 1 x 104 CFU of K. pneumoniae by tail vein injection and then blood was harvested 48 h later. Values represent mean CFU ± SEM values expressed on a log10 scale; n = 8 per group. *, p < 0.01 as compared with K. pneumoniae-infected WT mice. Balb, BALB/c.

 
Additional experiments were performed to more precisely discern the rate of clearance of bacteria from the bloodstream using a well-established K. pneumoniae bacteremia model (62). WT and TLR9–/– mice were administered 5 x 103 CFU of K. pneumoniae by tail vein injection and then bacterial CFU were quantitated in blood 48 h later. As compared with WT animals, TLR9–/– mice had a > 400-fold increase in CFU in blood at 48 h postinoculation (0.94 ± 0.4 CFU (log10) for wild type vs 3.58 ± 0.15 CFU (log10) for TLR9–/–, p < 0.01; Fig. 4B). These studies suggest that TLR9-deficient mice have innate immune defects in multiple compartments, including the lung and the systemic circulation.

Cellular recruitment/activation in WT and TLR9–/– mice following i.t. K. pneumoniae administration

To determine the possible mechanism of impaired lung innate immunity in TLR9–/– mice, we examined the influx and activation of selected leukocyte populations at 24 and 72 h after K. pneumoniae administration in WT and TLR9 knockout mice. We observed no differences in the total number of leukocytes or the numbers of neutrophils and macrophages in lung digests from infected TLR9–/– mice as compared with WT controls (Table I). The recruitment and activation of specific lung DC, NK, and T cell populations were evaluated by flow cytometry. To assess for DC accumulation, T cells, B cells, and autofluorescent macrophages were eliminated by forward and side scatter characteristics and the remaining cells were assessed for expression of MHC class II, CD11c, and GR-1 (63). We found no difference in the number of myeloid DC (MHC class II+CD11c+) in the lungs of WT and TLR9–/– mice in the uninfected state (Fig. 5A). However, the administration of K. pneumoniae resulted in a 3-fold increase in the number of myeloid DC in lungs of WT mice 48 h postchallenge, whereas there was a more modest increase in the total number and percentage of these cells in infected TLR9–/– mice (p < 0.05), indicating impaired accumulation of myeloid DC after i.t. bacterial challenge. Furthermore, using four-color flow cytometry we observed an impairment in the activation/maturation of myeloid DC as indicated by a reduced expression of the costimulatory molecules of CD40 and especially of CD80 in the lungs of infected TLR9–/– mice 48 h after K. pneumoniae administration as compared with infected WT animals (Fig. 5B). We did not observe appreciable differences in the number of plasmacytoid DC (CD11c+GR-1moderate) in the lungs of uninfected or Klebsiella-infected TLR9–/– animals as compared with WT mice (data not shown).


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

  		Table I. Leukocyte population in lung digest from BALB/c and TLR9-deficient mice 48 h after i.t. K. pneumoniae administrationa

 

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

  		FIGURE 5. Lung DC recruitment and activation in WT and TLR9–/– mice during Klebsiella pneumoniae infection. A, FACS plot showing absolute number and percentage of myeloid DC in lungs of uninfected and K. pneumoniae-infected WT and TLR9–/– mice. WT and TLR9–/– mice were i.t. administered 5 x 102 CFU of K. pneumoniae and then lungs were harvested 48 h later and analyzed by four-color flow cytometry. The percentage value represents the percentage of total leukocytes and represents a composite from three separate experiments; n = 6–7 per condition; p < 0.05 compared with infected WT mice. B, Flow histogram showing cell surface expression of CD40 and CD80 by myeloid DC (CD11c+MHC class II+) from the lungs of uninfected and K. pneumoniae-infected WT and TLR9–/– mice. WT and TLR9–/– mice were i.t. administered 5 x 102 CFU of K. pneumoniae (Kp) and then lungs were harvested 48 h later and analyzed by four-color flow cytometry. Representative of n = 3 for uninfected mice and n = 6 animals for infected mice. Balb, BALB/c.

 
Having found impaired accumulation and maturation of conventional DC in infected TLR9–/– mice, we next assessed the accumulation of other cell types involved in type 1 immune response generation. As compared with the K. pneumoniae-challenged WT mice, we observed a trend toward a reduced total number of DX5+ cells (both NK and NKT cells combined) and {alpha}beta T cells as well as a significant and substantial decrease in the number of DX5+, {alpha}beta, and {gamma}{delta} T cells expressing the activation marker CD69 (Table I), indicating impaired activation of these leukocyte subsets in mutant mice during bacterial pneumonia.

Lung cytokine and chemokine production in WT and TLR9–/– mice following i.t. K. pneumoniae administration

To explore the mechanism for defects in accumulation of DC, we assessed the mRNA expression of chemokines involved in the trafficking of immature DC or DC precursors to peripheral sites (64, 65). We observed a marked reduction in the mRNA expression of the CC chemokine CCL2/MCP-1 in TLR9–/– mice at both the 24 and 48 h after i.t. K. pneumoniae challenge (Fig. 6). In contrast, there was no appreciable change in CCL3/MIP-1{alpha} expression and even an increase in CCL20/MIP 3{alpha} expression in infected TLR9–/– mice as compared with the WT mice (data not shown).


Figure 6
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 6. Levels of chemokines, TNF-{alpha}, and type 1 cytokine mRNA in lungs of WT and TLR9 –/– mice after i.t. K. pneumoniae (Kp) challenge. WT and TLR9–/– mice were i.t. administered 5 x 102 CFU of K. pneumoniae and then lungs were harvested 1, 2, or 3 days later, homogenates were prepared, and cytokine mRNA levels were quantitated by real-time PCR. Each value represents the mean of 4–5 mice per value. "Untreated" means uninfected control. *, p < 0.05 as compared with K. pneumoniae-infected WT mice. Balb, BALB/c.

 
Expression of the mRNA of TNF-{alpha} and the type 1 cytokines and chemokines IL-12, IFN-{gamma}, and CXCL10/IP-10 in the lungs of infected WT and TLR9–/– mice was also assessed. In WT mice, bacteria challenge induced a robust expression of TNF-{alpha}, IL-12 p40, IFN-{gamma}, and CXCL10/IP-10 mRNA at 24 h as determined by real-time quantitative PCR, with continued expression 48 h after bacterial challenge (Fig. 6). In contrast, mRNA expression of TNF-{alpha} and type 1 cytokines was substantially blunted in the lungs of TLR9–/– mice challenged i.t. with K. pneumoniae, especially at the 24-h time point postchallenge. We also measured the levels of MCP-1/CCL2, TNF-{alpha}, IL-12, IFN-{gamma}, and CXCL10/IP-10 in lung homogenates of WT and TLR9–/– mice 24 and 48 h after i.t. K. pneumoniae challenge. Similar to what was observed at the message level, we found decreased production of these cytokines in TLR9-deficient mice at the 24 and/or 48 h time points compared with the infected WT controls (Table II). In contrast, we observed no differences in the protein levels of CXCL1/MIP-2 or IL-17 (data not shown).


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

  		Table II. Protein levels of cytokines and chemokines in lung homogenates from Balb and TLR9–/– mice at 24 and 48 hrs after i.t. K pneumoniae challengea

 
Reconstitution of immunity in TLR9–/– mice after adoptive transfer of syngeneic bone marrow-derived DC

Previous studies demonstrated impaired DC accumulation and effector cell function in TLR9–/– mice after i.t Klebsiella challenge. To determine whether these DC defects contributed meaningfully to the impaired phenotype observed in TLR9-deficient mice, we adoptively transferred bone marrow-derived DC into WT or mutant mice and then assessed the effects on bacterial clearance and cytokine/chemokine production. For these experiments, we administered DC intratracheally, an approach that has previously been shown to stimulate intrapulmonary immunity in other model systems in which the disease was localized to the lung (66). In preliminary experiments using bone marrow-derived DC labeled with the vital fluorochrome CFSE, we found that the labeled DC that were coadministered i.t. with K. pneumoniae (2 x 103 CFU) migrated to regional lymph nodes as early as 4 h postadministration, whereas no migration of labeled DC was noted in the absence of exposure to live bacteria (data not shown). Bone marrow-derived DC (1 x 106 cells) obtained from WT mice or TLR9–/– mice were administered i.t. concomitantly with the administration of high-dose K. pneumoniae (5 x 103 CFU). Lungs and spleen were harvested 48 h later and bacterial CFU were determined. As expected, lung and spleen CFU were substantially higher in infected TLR9–/– mice as compared with WT mice (Fig. 7A). Importantly, the intratracheal delivery of WT DC into infected TLR9–/– mice resulted in a marked reduction in bacterial CFU in both lung (Fig. 7A) and spleen (Fig. 7B), whereas WT DC transfer into K. pneumoniae-infected WT mice had minimal effect on bacterial clearance in these animals. In contrast, the i.t. transfer of DC isolated from TLR9–/– mice had no effect on bacterial clearance in either WT or TLR9–/– mice.


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

  		FIGURE 7. Effect of i.t. administration of DC on bacterial clearance and cytokine production in WT and TLR9–/– mice with K. pneumoniae. A and B, Bone marrow cells were isolated from WT and TLR9–/– mice, incubated with GM-CSF (10 ng/ml) for 6 days, positively selected for CD11c, and then DC were administered i.t. (106) in a volume of 30 µl with K. pneumoniae (5 x 103 CFU). K. pneumoniae CFU in lung (A) and spleen (B) homogenates were measured 48 h after i.t. K. pneumoniae administration. Bacterial CFU are expressed in log10 scale ± SEM; n = 5–7 per group; composite of two separate experiments. *, p < 0.05 as compared with K. pneumoniae-infected WT mice. C, Cytokine levels in lung homogenates from infected TLR9–/– mice 48 h after the i.t. administration of WT DC or DC from TLR9–/– mice, *, p < 0.05 as compared with K. pneumoniae-infected TLR9–/– mice without DC transfer. Balb, BALB/c.

 
In additional studies, we found that the i.t. transfer of WT DC, but not DC from TLR9–/– mice, substantially increased the protein levels of IL-12 and TNF-{alpha} in the lung homogenates of K. pneumoniae-infected TLR9–/– mice (Fig. 7C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we observed impaired bacterial clearance and reduced survival in TLR9–/– mice as compared with the WT mice after i.t. challenge with the extracellular bacterial pathogen K. pneumoniae. Impaired bacterial clearance was associated with reduced DC accumulation and activation and the defective generation of type 1 cytokine responses. This is the first time that TLR9 has been shown to be required for the effective clearance of virulent Gram-negative bacterial pathogens. Other TLRs, including TLR2, TLR4, and TLR5, are known to be involved in the generation of an innate immune response to Klebsiella and other extracellular bacterial pathogens (19, 20, 21, 22, 23). In contrast to TLRs2, TLR4, and TLR5, which are expressed at the cell surface, TLR9 is primarily localized and activated intracellularly within endocytic vesicles just below the plasma membrane (32, 33). The presence of a system to sense the accumulation of internalized pathogens may be a means to trigger the generation of activating signals that results in optimal intracellular killing by innate phagocytic cells. Moreover, there is strong evidence that TLRs can interact synergistically or be activated in a sequential fashion to maximally amplify the innate response (20, 22). Evidence for cooperative interactions include: 1) the observation that host responses to bacterial pathogens are more substantially impaired in MyD88-deficient mice as compared with that observed in animals in which individual TLRs are absent or dysfunctional; and 2) the additive or even synergistic effects of TLR4 and TLR9 agonists on leukocyte activation in vitro (22, 23, 24, 25, 26). Collectively, these data indicate that TLRs present in several different cellular compartments may act in concert to appropriately detect and respond to invasive microbial pathogens.

TLR9 is expressed on both myeloid and nonmyeloid cells. Our studies, combined with the observations of others, suggest that DC represent one of the major cell populations responding to microbial challenge in a TLR9-dependent fashion and that DC function is altered in mice deficient in TLR9 (40). First, we found that there is an increase in CD11c+MHC class IIhigh cells expressing TLR9 in the lung during bacterial pneumonia. Moreover, we found that DC isolated from the lungs of Klebsiella-infected TLR9–/– mice produced less IL-12 and CCL2/MCP-1 when cultured ex vivo as compared with DC recovered from infected WT mice (data not shown). In vivo, a significant reduction in the accumulation and maturation of myeloid DC was noted in K. pneumoniae-infected TLR9–/– mice. Decreased DC numbers could be attributable to reduced recruitment of immature DC to the lung or, alternatively, to impaired maturation of DC precursors into functional DC, which has previously been shown to be mediated by TLR9 in response to certain intracellular microbes (67). Our data supports both an alteration of DC influx and defects in DC maturation. Specifically, we found markedly reduced expression of CCR2/MCP-1 in lung tissue and DC isolated from TLR9–/– mice. CCR2/MCP-1 has been shown to mediate the recruitment of immature DC to peripheral sites during inflammation induced by toxic and/or infectious insults (Refs. 56 and 57 and J. Osterholzer, unpublished observations). We also observed reductions in the expression of DC maturation markers (CD40 and CD80) by myeloid DC in TLR9–/– mice during pneumonia as compared with similarly treated WT mice. Our findings are consistent with the recent observation that TLR9–/– mice have diminished antiviral activity against the murine CMV in vivo that was associated with impairment in DC function (40).

Intrapulmonary bacterial challenge in TLR9–/– mice resulted in delayed and/or reduced type 1 cytokine and chemokine expression, which occurred in conjunction with diminished activation of the major IFN-{gamma}-producing cells, including NK cells and {alpha}beta and {gamma}{delta} T cells. Attenuated type 1 responses in TLR9–/– mice likely occur as a result of altered DC function, including but not limited to defects in the elaboration of IL-12. Diminished type 1 responses are in line with the observation of reduced granulomatous inflammation in TLR9–/– mice in response to heat-killed Propionibacterium acnes and impaired type 1 immunity against the parasite Toxoplasma gondii in these animals (58, 59). Conversely, the exogenous administration of the synthetic TLR9 agonist CpG ODN to mice can bolster type 1 responses against live intracellular and extracellular microbial pathogens or microbial Ags (49, 50, 51, 52, 53, 54, 55, 56).

Although DC represent a rich cellular source of TLR9 and respond robustly to CpG ODN, macrophages and neutrophils have also been reported to produce inflammatory cytokines and/or reactive oxygen species in response to CpG, albeit weakly relative to other pathogen-associated leukocyte activators (21, 44, 68, 69). For that reason, we cannot exclude a direct effect of TLR9 on the antimicrobial function of macrophages or neutrophils. It is noteworthy that our studies and the studies of other indicate that resting rodent AM express minimal quantities of TLR9 mRNA or protein (70). Expression of TLR9 by these cells in the setting of infection or other inflammatory states has not been defined. It has recently been reported that AM and bone marrow-derived macrophages isolated from TLR9–/– mice display impaired ingestion and killing of S. pneumoniae in vitro (59). In our study we did not observe impaired internalization of K. pneumoniae by AM in vivo. In fact, we found rather striking intracellular accumulation of bacteria within AM in TLR9–/– mice during pneumonia, which could occur simply due to an increase in the number of free bacterial within the airspace or as a consequence of a diminished ability of AM to kill internalized microbes. Classical activation of macrophages is driven by selected host-derived cytokines, including IFN-{gamma} and TNF-{alpha}, and the elaboration of activating cytokines is mitigated in TLR9-deficient mice. Consistent with this, we found that lung macrophages recovered from K. pneumoniae-infected TLR9–/– mice displayed reduced expression of iNOS relative to that observed in cells recovered from infected WT mice. NO is an important component of antimicrobial host defense in Gram-negative pneumonia (61), and impaired NO production would account for reduced intracellular killing without compromising phagocytic function.

Mice deficient in TLR9 demonstrate defects in innate response in both the lung and systemically as clearly indicated by the impaired clearance of K. pneumoniae from the lung after i.t. administration and the reduced clearance of bacteria from the blood after i.v. administration. Bacteria that invade the bloodstream are generally removed in the liver and spleen, implicating impaired innate antibacterial responses in these organs in TLR9–/– mice. The nature of this defective response in liver and spleen of mutant mice has not yet been defined, but because these organs have a rich supply of DC it is likely that impaired DC responses contribute to the defects observed.

In this study, we made the particularly novel observation that the adoptive transfer of bone marrow-derived DC administered directly into the lung markedly improved bacterial clearance in TLR9–/– mice. Intratracheal delivery of DC has been used previously in a murine bronchoalveolar cell cancer model in which the administration of CCL21 gene-modified DC intratracheally resulted in decreased lung tumor burden and improved type 1 cytokine responses (66). The fact that the beneficial effects of DC transfer occurred rapidly (within a 48-h time period) strongly argue against the development of Ag-specific acquired immunity. Also, the observation that DC obtained from WT but not TLR9-deficient mice can reconstitute antibacterial immunity in TLR9–/– mice provides further evidence of defective DC responses in the mutant mice. The immune mechanisms by which DC transfer augments the clearance of K. pneumoniae have not been completely defined, but we did observe a substantial increase in the intrapulmonary expression of activating/chemotactic cytokines, including IL-12 and TNF-{alpha}. Intravenous adoptive transfer of genetically modified DC has been used previously to stimulate adaptive immunity against bacterial (Pseudomonas aeruginosa) and fungal (Pneumocystis carinii) pathogens (71, 72), but this is the first study to demonstrate beneficial effects of DC transfer on innate immune responses in bacterial pneumonia.

We have focused our studies on an investigation of innate immune response and have not yet fully explored the contribution of TLR9 to the development of acquired immunity. Given that TLR9 appears to play a critical role in DC and T cell recruitment/activation and that B cells in mice highly express TLR9, it is tempting to speculate that humoral responses will be substantially impaired in TLR9-deficient mutant mice. However, a previous study has shown that while innate responses to murine CMV are impaired in TLR9–/– mice, there were no changes in anti-CMV Ig production (40). Experiments are ongoing in the bacterial pneumonia model to address this issue.

In conclusion, our studies indicate that TLR9 serves as an important signal in the generation of protective innate responses to bacterial pathogens of the lung and that approaches to maximize TLR9-mediated DC responses may serve as an important means to augment antibacterial immunity in pneumonia.


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


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Theodore J. Standiford, University of Michigan Medical Center, Division of Pulmonary and Critical Care Medicine, 109 Zina Pitcher Place, 4062 Biomedical Science Research Building, Ann Arbor, MI 48109-2200. E-mail address: tstandif{at}umich.edu Back

2 Abbreviations used in this paper: AM, alveolar macrophage; DC, dendritic cell; iNOS, inducible NO synthase; IP-10, IFN-inducible protein 10; i.t., intratracheal; ODN, oligodeoxynucleotide; WT, wild type. Back

Received for publication January 30, 2007. Accepted for publication July 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Nelson, S., C. M. Mason, J. Kolls, W. R. Summer. 1995. Pathophysiology of pneumonia. Clin. Chest Med. 16: 1-12. [Medline]
  2. Toews, G. B., G. N. Gross, A. K. Pierce. 1980. The relationship of inoculum size to lung bacterial clearance and phagocytic cell response in mice. Am. Rev. Respir. Dis. 120: 559-566.
  3. Lipscomb, M. F., J. M. Onofrio, E. J. Nash. 1983. A morphological study of the role of phagocytes in the clearance of Staphylococcus aureus from the lung. J. Reticuloendothel. Soc. 33: 429-442. [Medline]
  4. Broug-Holub, E., G. B. Toews, J. F. van Iwaarden, R. M. Strieter, S. L. Kunkel, R. Paine, T. J. Standiford. 1997. Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival. Infect. Immun. 65: 1139-1146. [Abstract]
  5. Tsai, W. C., R. M. Strieter, B. Mehrad, M. W. Newstead, X. Zeng, T. J. Standiford. 2000. CXC chemokine receptor CXCR2 is essential for protective innate host response in murine Pseudomonas aeruginosa pneumonia. Infect. Immun. 68: 289-296.
  6. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and control of immunity. Nature 392: 245-252. [Medline]
  7. Kradin, R. L., H. Sakamoto, F. I. Preffer, D. Dombkowski, K. M. Springer, C. P. Leary. 2000. Accumulation of macrophages with dendritic cell characteristics in the pulmonary response to Listeria. Am. J. Respir. Crit. Care Med. 161: 535-542. [Abstract/Free Full Text]
  8. McWilliam, A. S., D. Nelson, J. A. Thomas, P. G. Holt. 1994. Rapid dendritic cell recruitment is a hallmark of the acute inflammatory response at mucosal surfaces. J. Exp. Med. 179: 1331-1336. [Abstract/Free Full Text]
  9. Liu, C. H., Y. T. Fan, A. Dias, L. Esper, R. A. Corn, A. Bafica, F. S. Machado, J. Aliberti. 2006. Cutting edge: dendritic cells are essential for in vivo IL-12 production and development of resistance against Toxoplasma gondii infection in mice. J. Immunol. 177: 31-35. [Abstract/Free Full Text]
  10. Moore, T. A., B. B. Moore, T. J. Standiford. 2000. {gamma}{delta}-T cells are critical for survival and early proinflammatory cytokine gene expression during murine Klebsiella pneumonia. J. Immunol. 165: 2643-2650. [Abstract/Free Full Text]
  11. Ferlazzo, G., B. Morandi, A. D’Agostino, R. Meazza, G. Melioli, A. Moretta, L. Moretta. 2003. The interaction between NK cells and dendritic cells in bacterial infections results in rapid induction of NK cell activation and in the lysis of uninfected dendritic cells. Eur. J. Immunol. 33: 306-313. [Medline]
  12. Deng, J. C., K. Tateda, X. Zeng, T. J. Standiford. 2001. Transient transgenic expression of {gamma} interferon promotes Legionella pneumophila clearance in immunocompetent hosts. Infect. Immun. 69: 6382-6390. [Abstract/Free Full Text]
  13. Greenberger, M. J., S. L. Kunkel, R. M. Strieter, N. W. Lukacs, J. Bramson, J. Gauldie, F. L. Graham, M. Hitt, J. M. Danforth, T. J. Standiford. 1996. IL-12 gene therapy protects mice in lethal Klebsiella pneumonia. J. Immunol. 157: 3006-3012. [Abstract]
  14. Khan, I. A., J. A. Maclean, F. S. Lee, L. Casiotti, E. DeHaan, J. D. Schwartzman, A. D. Luster. 2000. IP-10 is critical to effector T cell trafficking and host survival in Toxoplasma gondii infection. Immunity 12: 483-494. [Medline]
  15. Zeng, X., T. A. Moore, M. W. Newstead, J. C. Deng, S. L. Kunkel, A. D. Luster, T. J. Standiford. 2005. IP-10, but not MIG, promotes protective type 1 immunity in murine Klebsiella pneumonia. Infect. Immun. 73: 8226-8236. [Abstract/Free Full Text]
  16. Aderem, A., R. J. Ulevitch. 2000. Toll-like receptors in the induction of the innate immune response. Nature 406: 782-787. [Medline]
  17. Akira, S., H. Hemmi. 2003. Recognition of pathogen-associated molecular patterns by TLR family. Immunol. Lett. 85: 85-95. [Medline]
  18. An, H., Y. Yu, M. Zhang, H. Xu, R. Qi, X. Yan, S. Liu, W. Wang, Z. Guo, J. Guo, et al 2002. Involvement of ERK, p38 and NF-{kappa} B signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells. Immunology 106: 38-45. [Medline]
  19. Jeyaseelan, S., S. K. Young, M. Yamamoto, P. G. Arndt, S. Akira, J. K. Kolls, S. K. Worthen. 2006. Toll/IL-1R domain-containing adaptor protein (TIRAP) is a critical mediator of antibacterial defense in the lung against Klebsiella pneumoniae but not Pseudomonas aeruginosa. J. Immunol. 177: 538-547. [Abstract/Free Full Text]
  20. Wang, X., C. Moser, J. Louboutin, E. S. Lysenko, D. J. Weiner, J. N. Weiser, J. M. Wilson. 2002. Toll-like receptor 4 mediates innate immune responses to Haemophilus influenza infection in mouse lung. J. Immunol. 168: 810-815. [Abstract/Free Full Text]
  21. Schuur, J. R., E. Young, P. Byrne, C. Steele, J. E. Shellito, J. K. Kolls. 2005. Central role of Toll-like receptor 4 signaling and host defense in experimental pneumonia caused by Gram-negative bacteria. Infect. Immun. 73: 532-545. [Abstract/Free Full Text]
  22. Weiss, D. S., B. Raupach, K. Takeda, S. Akira, A. Zychlinsky. 2004. Toll-like receptors are temporally involved in host defense. J. Immunol. 172: 4463-4469. [Abstract/Free Full Text]
  23. Skerrett, S. J., H. D. Liggitt, A. M. Hajjar, C. B. Wilson. 2004. Cutting edge: myeloid differentiation factor 88 is essential for pulmonary host defense against Pseudomonas aeruginosa but not Staphylococcus aureus. J. Immunol. 172: 3377-3381. [Abstract/Free Full Text]
  24. Feng, G. C., C. A. Scanga, C. M. Collazo-Custodio, A. W. Cheever, S. Hieny, P. Caspar, A. Sher. 2003. Mice lacking myeloid differentiation factor 88 display profound defects in host resistance and immune responses to Mycobacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-deficient animals. J. Immunol. 171: 4758-4764. [Abstract/Free Full Text]
  25. Archer, K. A., C. R. Roy. 2006. MyD88-dependent responses involving Toll-like receptor 2 are important for protection and clearance of Legionella pneumophila in a mouse model of Legionnaires’ disease. Infect. Immun. 74: 3325-3333. [Abstract/Free Full Text]
  26. Hawn, T. R., K. D. Smith, A. Aderem, S. J. Skerrett. 2006. Myeloid differentiation primary response gene (MyD88)- and Toll-like receptor 2-deficient mice are susceptible to infection with aerosolized Legionella pneumophila. J. Infect. Dis. 193: 1693-1702. [Medline]
  27. Krieg, A. M., A. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G. A. Koretzky, D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374: 546-549. [Medline]
  28. Klinman, D. M., A. Yi, S. L. Beaucage, J. Conover, A. M. Krieg. 1996. CpG motifs expressed by bacterial DNA rapidly induce lymphocytes to secrete IL-6, IL-12 and IFN{gamma}. Proc. Natl. Acad. Sci. USA 93: 2879-2883. [Abstract/Free Full Text]
  29. Hemmi, H., O. Takeuchi, T. Kawai, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408: 740-745. [Medline]
  30. Takeshita, F., C. A. Leifer, I. Gursel, K. J. Ishii, S. Takesita, M. Gursel, D. M. Klinman. 2001. Cutting edge: role of Toll-like receptor 9 in CpG DNA-induced activation of human cells. J. Immunol. 167: 3555-3558. [Abstract/Free Full Text]
  31. Malmgaard, L., J. Melchjorsen, A. G. Bowie, S. C. Mogensen, S. R. Paludan. 2004. Viral activation of macrophages through TLR-dependent and -independent pathways. J. Immunol. 173: 6890-6898. [Abstract/Free Full Text]
  32. Wagner, H.. 2004. The immunobiology of the TLR9 subfamily. Trends Immunol. 25: 381-386. [Medline]
  33. Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks, C. F. Knetter, E. Lien, N. J. Nilsen, T. Espevik, D. T. Golenbock. 2004. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5: 190-198. [Medline]
  34. Yasuda, K., Y. Ogawa, I. Yamane, M. Nishikawa, Y. Takakura. 2005. Macrophage activation by a DNA/cationic liposome complex requires endosomal acidification and TLR9-dependent and-independent pathways. J. Leukocyte Biol. 77: 71-80. [Abstract/Free Full Text]
  35. Huang, L. Y., K. J. Ishii, S. Akira, J. Aliberti, B. Golding. 2005. Th1-like cytokine induction by heat-killed Brucella abortus is dependent on triggering of TLR9. J. Immunol. 175: 3964-3970. [Abstract/Free Full Text]
  36. Matsumoto, S., M. Matsumoto, K. Umemori, Y. Ozeki, M. Furugen, T. Tatsuo, Y. Hirayama, S. Yamamoto, T. Yamada, K. Kobayashi. 2005. DNA augments antigenicity of mycobacterial DNA-binding protein 1 and confers protection against Mycobacterium tuberculosis infection in mice. J. Immunol. 175: 441-449. [Abstract/Free Full Text]
  37. Takeshita, F., I. Gursel, K. J. Ishii, K. Suzuki, M. Gursel, D. M. Klinman. 2004. Signal transduction pathways mediated by the interaction of CpG DNA with Toll-like receptor 9. Semin. Immunol. 16: 17-22. [Medline]
  38. Kadowaki, N., S. Ho, S. Antonenko, R. de Wall Malefyt, R. A. Kastelein, F. Bazan, Y. J. Liu. 2001. Subsets of human dendritic cell precursors express different Toll-like receptors and respond to different microbial antigens. J. Exp. Med. 194: 863-869. [Abstract/Free Full Text]
  39. Jarrossay, D., G. Napolitani, M. Colonna, F. Sallustro, A. Lanzavecchia. 2001. Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. Eur. J. Immunol. 31: 3388-3393. [Medline]
  40. Krug, A., A. R. French, W. Barchet, J. A. A. Fischer, A. Dzionek, J. T. Pingel, M. M. Orihuela, S. Akira, W. M. Yokoyama, M. Colonna. 2004. TRL9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function. Immunity 21: 107-119. [Medline]
  41. Pichyangkul, S., K. Yongvanitchit, U. Kum-arb, H. Hemmi, S. Akira, A. M. Krieg, D. G. Heppner, V. A. Stewart, H. Hasegawa, S. Looareesuwan, et al 2004. Malaria blood stage parasites activate human plasmacytoid dendritic cells and murine dendritic cells through a toll-like receptor 9-dependent pathway. J. Immunol. 172: 4926-4933. [Abstract/Free Full Text]
  42. Liang, H., Y. Nishioka, C. F. Reich, D. S. Pisetsky, H. Wagner, K. Heeg. 1996. Activation of human B cells by phosphorothioate oligodeoxynucleotides. J. Clin. Invest. 98: 1119-1129. [Medline]
  43. Kikuchi, T., T. Kobayashi, K. Gomi, T. Suzuki, Y. Tokue, A. Watanabe, T. Nukiwa. 2004. Dendritic cells pulsed with live and dead Legionella pneumophila elicit distinct immune responses. J. Immunol. 172: 1727-1734. [Abstract/Free Full Text]
  44. Sester, D. P., K. J. Stacey, M. J. Sweet, S. J. Beasley, S. L. Cronau, D. A. Hume. 1999. The actions of bacterial DNA on murine macrophages. J. Leukocyte Biol. 66: 542-544. [Abstract]
  45. Li, J., Z. Ma, Z. L. Tang, T. Stevens, B. Pitt, S. Li. 2004. CpG DNA-mediated immune response in pulmonary endothelial cells. Am. J. Physiol. 287: L552-L558.
  46. Platz, J., C. Beisswenger, A. Dalpke, R. Koczulla, O. Pinkenburg, C. Vogelmeier, R. Bals. 2004. Microbial DNA induces a host defense reaction of human respiratory epithelial cells. J. Immunol. 173: 1219-1223. [Abstract/Free Full Text]
  47. Chu, R. S., O. S. Targoni, A. M. Krieg, P. V. Lehmann, C. V. Harding. 1997. CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. J. Exp. Med. 186: 1623-1631. [Abstract/Free Full Text]
  48. Kline, J. N., T. J. Waldschmidt, T. R. Businga, J. E. Lemish, J. V. Weinstock, P. S. Thorne, A. M. Krieg. 1998. Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J. Immunol. 160: 2555-2559. [Abstract/Free Full Text]
  49. Zimmermann, S., O. Egeter, S. Hausmann, G. B. Lipford, M. Rocken, H. Wagner, K. Heeg. 1998. CpG oligodeoxynucleotides trigger protective and curative Th1 responses in lethal murine leishmaniasis. J. Immunol. 160: 3627-3630. [Abstract/Free Full Text]
  50. Krieg, A. M., L. Love-Homan, A. K. Yi, J. T. Harty. 1998. CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge. J. Immunol. 161: 2428-2434. [Abstract/Free Full Text]
  51. Walker, P. S., T. Scharton-Kersten, A. M. Krieg, L. Love-Homan, E. D. Rowton, M. C. Udey, J. C. Vogel. 1999. Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFN-{gamma}-dependent mechanisms. Proc. Natl. Acad. Sci. USA 96: 6970-6975. [Abstract/Free Full Text]
  52. Klinman, D. M., J. Conover, C. Coban. 1999. Repeated administration of synthetic oligodeoxynucleotides expressing CpG motifs provides long-term protection against bacterial infection. Infect. Immun. 67: 5658-5663. [Abstract/Free Full Text]
  53. Elkins, K. L., T. R. Rhinehart-Jones, S. Stibitz, J. S. Conover, D. M. Klinman. 1999. Bacterial DNA containing CpG motifs stimulates lymphocyte-dependent protection of mice against lethal infection with intracellular bacteria. J. Immunol. 162: 2291-2298. [Abstract/Free Full Text]
  54. Freidag, B. L., G. B. Melton, F. Collins, D. M. Klinman, A. Cheever, L. Stobie, W. Suen, R. A. Seder. 2000. CpG oligodeoxynucleotides and interleukin-12 improve the efficacy of Mycobacterium bovis BCG vaccination in mice challenged with M. tuberculosis. Infect. Immun. 68: 2948-2953. [Abstract/Free Full Text]
  55. Juffermans, N. P., J. C. Leemans, S. Florquin, A. Verbon, A. H. Kolk, P. Speelman, S. J. van Deventer, T. van der Poll. 2002. CpG oligodeoxynucleotides enhance host defense during murine tuberculosis. Infect. Immun. 70: 147-152. [Abstract/Free Full Text]
  56. Deng, J. C., T. A. Moore, M. W. Newstead, X. Zeng, T. J. Standiford. 2004. CpG oligodeoxynucleotides stimulate protective innate immunity against pulmonary Klebsiella infection. J. Immunol. 173: 5148-5155. [Abstract/Free Full Text]
  57. Mogensen, T. H., S. R. Paludan, M. Kilian, L. Ostergaard. 2006. Live Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitides activate the inflammatory response through toll-like receptors 2, 4, and 9 in species-specific patterns. J. Leukocyte Biol. 80: 267-277. [Abstract/Free Full Text]
  58. Minns, L. A., L. C. Menard, D. M. Foureau, S. Darche, C. Ronet, D. W. Mielcarz, D. Buzoni-Gatel, L. H. Kasper. 2006. TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii. J. Immunol. 176: 7589-7597. [Abstract/Free Full Text]
  59. Albiger, B., S. Dahlberg, A. Sandgren, F. Wartha, K. Beiter, H. Katsuragi, S. Akira, S. Normark, B. Henriques-Normark. 2007. Toll-like receptor 9 acts as an early stage in host defence against pneumococcal infection. Cell. Microbiol. 9: 1162-1171. [Medline]
  60. Smit, J. J., B. C. Rudd, N. W. Lukacs. 2006. Plasmacytoid dendritic cells inhibit pulmonary immunopathology and promote clearance of respiratory syncytial virus. J. Exp. Med. 203: 1153-1159. [Abstract/Free Full Text]
  61. Tsai, W. C., R. M. Strieter, D. A. Zisman, J. M. Wilkowski, K. A. Bucknell, G.-H. Chen, T. J. Standiford. 1997. Nitric oxide is required for effective innate immunity against Klebsiella pneumoniae. Infect. Immun. 65: 1870-1875. [Abstract]
  62. Moore, T. A., H. Lau, A. L. Cogen, C. L. Monteleon, T. J. Standiford. 2003. Anti-TNF{alpha} therapy during murine Klebsiella pneumoniae bacteremia: increased mortality in the absence of liver injury. Shock 20: 309-315. [Medline]
  63. Van Rijt, L. S., J. B. Prins, P. J. M. Leenen, K. Thielemans, V. C. de Vries, H. C. Hoogsteden, B. N. Lambrecht. 2004. Allergen-induced accumulation of airway dendritic cells is supported by an increase in CD31hiLy-6Cneg bone marrow precursors in a mouse model of asthma. Blood 100: 3663-3671.
  64. Xu, L. L., M. K. Warren, W. L. Rose, W. Gong, J. M. Want. 1996. Human recombinant monocyte chemotactic protein and other C-C chemokines bind and induce directional migration of dendritic cells in vitro. J. Leukocyte Biol. 60: 365-371. [Abstract]
  65. Vanbervliet, B., B. Homey, I. Durand, C. Massacrier, S. Ait-Yahia, O. deBouteiller, A. Vicari, C. Caux. 2002. Sequential involvement of CCR2 and CCR6 ligands for immature dendritic cell recruitment: possible role at inflamed epithelial surfaces. Eur. J. Immunol. 32: 231-242. [Medline]
  66. Yang, S., R. K. Batra, S. Hillinger, K. L. Reckamp, R. M. Strieter, S. M. Dubinett, S. Sharma. 2006. Intrapulmonary administration of CCL21 gene-modified dendritic cells reduces tumor burden in spontaneous murine bronchoalveolar cell carcinoma. Cancer Res. 66: 3205-3213. [Abstract/Free Full Text]
  67. Saikh, K. U., T. L. Kissner, A. Sultana, G. Rythel, R. G. Ulrich. 2004. Human monocytes infected with Yersinia pestis express cell surface TLR9 and differentiate into dendritic cells. J. Immunol. 173: 7426-7434. [Abstract/Free Full Text]
  68. Hayashi, F., T. K. Means, A. D. Luster. 2003. Toll-like receptors stimulate human neutrophil function. Blood 102: 2660-2669. [Abstract/Free Full Text]
  69. Jozsef, L., T. Khreiss, D. El Kebir, J. G. Filep. 2006. Activation of TLR-9 induces IL-8 secretion through peroxynitrite signaling in human neutrophils. J. Immunol. 176: 1195-1202. [Abstract/Free Full Text]
  70. Suzuki, K., T. Suda, T. Naito, K. Ide, K. Chida, H. Nakamura. 2005. Impaired Toll-like receptor 9 expression in alveolar macrophages with no sensitivity to CpG DNA. Am. J. Respir. Crit. Care Med. 171: 707-713. [Abstract/Free Full Text]
  71. Kikuchi, T., S. Worgall, R. Singh, M. A. Moore, R. G. Crystal. 2000. Dendritic cells genetically modified to express CD40 ligand and pulsed with antigen can initiate antigen-specific humoral immunity independent of CD4+ T cells. Nat. Med. 6: 1154-1159. [Medline]
  72. Zheng, M., J. E. Shellito, L. Marrero, Q. Zhong, S. Julian, P. Ye, V. Wallace, P. Schwarzenberger, J. K. Kolls. 2001. CD4 T cell-independent vaccination against Pneumocystis carinii in mice. J. Clin. Invest. 108: 1469-1474. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
D. El Kebir, L. Jozsef, W. Pan, L. Wang, and J. G. Filep
Bacterial DNA Activates Endothelial Cells and Promotes Neutrophil Adherence through TLR9 Signaling
J. Immunol., April 1, 2009; 182(7): 4386 - 4394.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
A. Craig, J. Mai, S. Cai, and S. Jeyaseelan
Neutrophil Recruitment to the Lungs during Bacterial Pneumonia
Infect. Immun., February 1, 2009; 77(2): 568 - 575.
[Full Text] [PDF]

Home page
 J. Immunol.Home page
F. Hkima Abou Fakher, N. Rachinel, M. Klimczak, J. Louis, and N. Doyen
TLR9-Dependent Activation of Dendritic Cells by DNA from Leishmania major Favors Th1 Cell Development and the Resolution of Lesions
J. Immunol., February 1, 2009; 182(3): 1386 - 1396.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. Pene, B. Zuber, E. Courtine, C. Rousseau, F. Ouaaz, J. Toubiana, A. Tazi, J.-P. Mira, and J.-D. Chiche
Dendritic Cells Modulate Lung Response to Pseudomonas aeruginosa in a Murine Model of Sepsis-Induced Immune Dysfunction
J. Immunol., December 15, 2008; 181(12): 8513 - 8520.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
H. Sjolinder, T. H. Mogensen, M. Kilian, A.-B. Jonsson, and S. R. Paludan
Important Role for Toll-Like Receptor 9 in Host Defense against Meningococcal Sepsis
Infect. Immun., November 1, 2008; 76(11): 5421 - 5428.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
U. Bhan, G. Trujillo, K. Lyn-Kew, M. W. Newstead, X. Zeng, C. M. Hogaboam, A. M. Krieg, and T. J. Standiford
Toll-Like Receptor 9 Regulates the Lung Macrophage Phenotype and Host Immunity in Murine Pneumonia Caused by Legionella pneumophila
Infect. Immun., July 1, 2008; 76(7): 2895 - 2904.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
G. Plitas, B. M. Burt, H. M. Nguyen, Z. M. Bamboat, and R. P. DeMatteo
Toll-like receptor 9 inhibition reduces mortality in polymicrobial sepsis
J. Exp. Med., June 9, 2008; 205(6): 1277 - 1283.
[Abstract] [Full Text] [PDF]

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

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS