TLR9 Is Required for Protective Innate
Immunity in Gram-Negative Bacterial
Pneumonia: Role of Dendritic Cells
This information is current as
of June 18, 2017.
Urvashi Bhan, Nicholas W. Lukacs, John J. Osterholzer,
Michael W. Newstead, Xianying Zeng, Thomas A. Moore,
Tracy R. McMillan, Arthur M. Krieg, Shizuo Akira and
Theodore J. Standiford
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 179:3937-3946; ;
doi: 10.4049/jimmunol.179.6.3937
http://www.jimmunol.org/content/179/6/3937
The Journal of Immunology
TLR9 Is Required for Protective Innate Immunity in
Gram-Negative Bacterial Pneumonia: Role of Dendritic Cells
Urvashi Bhan,* Nicholas W. Lukacs,† John J. Osterholzer,* Michael W. Newstead,*
Xianying Zeng,* Thomas A. Moore,* Tracy R. McMillan,* Arthur M. Krieg,‡ Shizuo Akira,§
and Theodore J. Standiford1*
I
nnate 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 (1–5). 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 – 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-␥ in a non-Ag specific fashion by lung macrophages, NK cells, NK T cells, and ␥␦ T cells. Moreover, DC
produce chemokines that facilitate the recruitment and/or activation of specific leukocyte populations that contribute to the innate
response (9 –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 in*Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine
and †Department of Pathology, University of Michigan Medical Center, Ann Arbor,
MI 48109; ‡Coley Pharmaceutical Group, Wellesley, MA 02481; and §Department of
Host Defense, Research Institute for Microbial Defenses, Osaka University, Osaka,
Japan
Received for publication January 30, 2007. Accepted for publication July 3, 2007.
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: [email protected]
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.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
fectious 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-␬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 Gramnegative 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. 22–26). 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 (27–31). 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 (32–34). Stimulation of immune cells
with synthetic CpG motifs or microbial DNA results in a variety
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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 (TLR9ⴚ/ⴚ) 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 TLR9ⴚ/ⴚ mice resulted in reduced lung DC accumulation and maturation as well as impaired
activation of lung macrophages, NK cells, and ␣␤ and ␥␦ 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 TLR9ⴚ/ⴚ mice administered intratracheally reconstituted antibacterial immunity in TLR9ⴚ/ⴚ 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. The Journal of Immunology, 2007, 179: 3937–3946.
3938
Materials and Methods
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 ␮l 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 ␮l 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 ␮l) 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 ⫻ g for 10 min. Supernatants were collected, passed through a
0.45-␮m 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
␮g/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 ⫻ 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 FITCor PE-labeled Abs: anti-␣␤ TCR, anti-␥␦ TCR, anti-DX5, anti-CD11c,
anti-MHC class II, anti-Gr1, anti-CD40, anti-CD80, anti-CD86, and antiCD69 (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 CD45positive “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.
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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– 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-␥ production while inhibiting
type 2 immune responses (47–56). 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 Gramnegative 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.
TLR9 IN Klebsiella PNEUMONIA
The Journal of Immunology
3939
Murine cytokine ELISA
Murine TNF-␣, CXCL10/IFN-inducible protein 10 (IP-10), IFN-␥, 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
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 ⫻ 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.
autofluorescent in the uninfected state, and no specific staining for
TLR9 was detected during infection.
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
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
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 – 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 ⫻ 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 TLR9expressing 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
FIGURE 2. Survival in WT and TLR9⫺/⫺ mice after i.t. administration
of K. pneumoniae (Kp) at a dose of 5 ⫻ 103 (upper panel) or 5 ⫻ 102
(lower panel); n ⫽ 10 animals per group. ⴱ, p ⬍ 0.05 as compared with K.
pneumoniae-infected WT mice. Balb, BALB/c.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
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-␣, 5⬘-CAGCCGATGGGTTGTACCTT-3⬘ (forward), 5⬘-TGTGGGT
GAGGAGCACGTAGT-3⬘ (reverse), and 5⬘-TCCCAGGTTCTCTTCAAG
GGACAAGGC-3⬘ (probe); CXCL10/murine IP-10, 5⬘-CCAGTGAGAA
TGAGGGCCATA-3⬘ (forward), 5⬘-CTCAACACGTGGGCAGGAT-3⬘
(reverse), and 5⬘-FAM-TTTGGGCATCATCTTCCTGGA-TAMRA-3⬘
(TaqMan probe); murine IL-12 p40, 5⬘-AGACCCTGCCCATTGAACT
G-3⬘ (forward), 5⬘-GAAGCTGGTGCTGTAGTTCTCATATT-3⬘ (reverse), and 5⬘-CGTTGGAAGCACGGCAGCAGAA-3⬘ (probe); MCP1/CCL2, 5⬘-GGCTCAGCCAGATGCAGTTAAC-3⬘ (forward), 5⬘-CCT
ACTCATTGGGATCATCTTGCT-3⬘ (reverse), and 5⬘-CCCCACTCA
CCTGCTGCTACTCATTCAC-3⬘ (reverse); IFN-␥, ⬘-CTGCGGCCTA
GCTCTGAGA-3⬘ (forward), 5⬘-CAGCCAGAAACAGCCATGAG-3⬘
(reverse), and 5⬘-CACACTGCATCTTGGCTTTGCAGCTTCTA-3⬘ (probe);
murine iNOS, 5⬘-CCCTCCTGATCTTGTGTTGGA-3⬘ (forward), 5⬘-CAAC
CCGAGCTCCTGGAA-3⬘ (reverse), and 5⬘-TGACCATGGAGCATCCC
AAGTACGAGT-3⬘ (probe); and murine ␤-actin, 5⬘-CCGTGAAAAG
ATGACCCAGATC-3⬘ (forward), 5⬘-CACAGCCTGGATGGCTACG
T-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.
3940
TLR9 IN Klebsiella PNEUMONIA
either an LD30 (5 ⫻ 102 CFU) or an LD80 (5 ⫻ 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 ⫻ 103
CFU; upper panel) or a lower dose (5 ⫻ 102 CFU; lower panel) of
K. pneumoniae.
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 ⫻ 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).
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 ⬎1,000fold 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).
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 ⫻ 103 CFU of K. pneumoniae
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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 ⫻ 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. ⴱ, 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. ⴱ, 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. ⴱ, p ⬍ 0.01 as compared with macrophages isolated from the lungs of infected WT mice.
Balb, BALB/c.
The Journal of Immunology
3941
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 ⫻ 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. ⴱ, 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 ⫻ 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.
ⴱ, p ⬍ 0.01 as compared with K. pneumoniaeinfected WT mice. Balb, BALB/c.
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 per-
centage 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).
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 ␣␤ T cells as well as
a significant and substantial decrease in the number of DX5⫹, ␣␤,
and ␥␦ 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
Table I. Leukocyte population in lung digest from BALB/c and TLR9-deficient mice 48 h after i.t. K. pneumoniae administrationa
Total no. of cells
Polymorphonuclear cells
Monocyte/macrophage
DX5⫹ cells
DX5⫹CD69⫹ cells
␣␤ TCR⫹ cells
␣␤ TCR⫹CD69⫹ cells
␥␦ TCR⫹CD69⫹ cells
BALB/c
BALB/c/Kp
TLR9⫺/⫺
TLR9⫺/⫺/Kp
1.3 ⫾ 0.3 ⫻ 107
1.3 ⫾ 0.1 ⫻ 106
12 ⫾ 0.1 ⫻ 106
2.7 ⫾ 0.2 ⫻ 105
1.1 ⫾ 0.1 ⫻ 105
1.3 ⫾ 0.1 ⫻ 105
2.5 ⫾ 0.1 ⫻ 104
0.9 ⫾ 0.1 ⫻ 104
1.9 ⫾ 0.3 ⫻ 107
5.9 ⫾ 0.6 ⫻ 106
3.7 ⫾ 0.5 ⫻ 106
4.9 ⫾ 0.1 ⫻ 105
3.1 ⫾ 0.1 ⫻ 105
2.7 ⫾ 0.1 ⫻ 105
12 ⫾ 0.1 ⫻ 104
2.4 ⫾ 0.2 ⫻ 104
1.4 ⫾ 0.4 ⫻ 107
1.5 ⫾ 0.1 ⫻ 106
12 ⫾ 0.1 ⫻ 106
2.9 ⫾ 0.3 ⫻ 105
0.7 ⫾ 0.2 ⫻ 105
1.1 ⫾ 0.1 ⫻ 105
2.1 ⫾ 0.1 ⫻ 104
0.6 ⫾ 0.1 ⫻ 104
1.7 ⫾ 0.2 ⫻ 107
5.5 ⫾ 0.5 ⫻ 106
4.2 ⫾ 0.3 ⫻ 106
3.5 ⫾ 0.2 ⫻ 105
1.0 ⫾ 0.1 ⫻ 105*
1.8 ⫾ 0.2 ⫻ 105
5.7 ⫾ 0.1 ⫻ 104*
0.7 ⫾ 0.1 ⫻ 104*
a
n ⫽ 4 for uninfected mice and n ⫽ 6 for Klebsiella (Kp)-infected mice combined from two separate experiments.
ⴱ, p ⬍ 0.05 as compared to K. pneumoniae-infected BALB/c group.
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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.
3942
TLR9 IN Klebsiella PNEUMONIA
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/
FIGURE 6. Levels
of
chemokines,
TNF-␣, 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 ⫻ 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. ⴱ, p ⬍ 0.05 as compared with K. pneumoniae-infected WT mice. Balb, BALB/c.
MIP-1␣ expression and even an increase in CCL20/MIP 3␣
expression in infected TLR9⫺/⫺ mice as compared with the WT
mice (data not shown).
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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 ⫻
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 ⫻ 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.
The Journal of Immunology
3943
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
Uninfected
24 h after Kp
48 h after Kp
Cytokine
BALB/c
TLR9⫺/⫺
BALB/c
TLR9⫺/⫺
BALB/c
TLR9⫺/⫺
TNF-␣
IL-12
IFN-␥
IP-10
MCP-1
45 ⫾ 07
125 ⫾ 50
196 ⫾ 30
30 ⫾ 10
36 ⫾ 11
38 ⫾ 06
126 ⫾ 40
151 ⫾ 10
49 ⫾ 21
22 ⫾ 23
390 ⫾ 70
540 ⫾ 34
2,440 ⫾ 87
421 ⫾ 54
434 ⫾ 48
186 ⫾ 44
263 ⫾ 58*
1,523 ⫾ 89*
207 ⫾ 38*
346 ⫾ 23
527 ⫾ 40
996 ⫾ 40
1,445 ⫾ 30
442 ⫾ 56
571 ⫾ 42
233 ⫾ 29*
5714 ⫾ 3*
1,296 ⫾ 24
293 ⫾ 53*
292 ⫾ 53*
a
Measured by ELISA in picograms per lung; n ⫽ 6 – 8 mice in all groups, results combined from two different experiments.
ⴱ, p ⬍ 0.05 as compared to BALB/c at specific time points.
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.
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 ␮l
with K. pneumoniae (5 ⫻ 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. ⴱ, 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, ⴱ, p ⬍ 0.05 as compared with K. pneumoniae-infected
TLR9⫺/⫺ mice without DC transfer.
Balb, BALB/c.
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 ⫻ 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 ⫻ 106 cells) obtained from WT mice or TLR9⫺/⫺ mice
were administered i.t. concomitantly with the administration of
high-dose K. pneumoniae (5 ⫻ 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.
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Expression of the mRNA of TNF-␣ and the type 1 cytokines and
chemokines IL-12, IFN-␥, 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-␣, IL-12
p40, IFN-␥, 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-␣ 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-␣, IL-12, IFN-␥, 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).
3944
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.
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-␣ in the lung homogenates of K.
pneumoniae-infected TLR9⫺/⫺ mice (Fig. 7C).
Discussion
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-␥-producing cells, including NK cells and ␣␤ and ␥␦ 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 –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-␥ and
TNF-␣, 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
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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 –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
(22–26). 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
TLR9 IN Klebsiella PNEUMONIA
The Journal of Immunology
Disclosures
The authors have no financial conflict of interest.
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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 TLR9deficient 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-␣. 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.
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TLR9 IN Klebsiella PNEUMONIA