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The Journal of Immunology, 2004, 173: 5148-5155.
Copyright © 2004 by The American Association of Immunologists

CpG Oligodeoxynucleotides Stimulate Protective Innate Immunity against Pulmonary Klebsiella Infection1

Jane C. Deng*, Thomas A. Moore*, Michael W. Newstead*, Xianying Zeng*, Arthur M. Krieg{dagger} and Theodore J. Standiford2,*

* Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109; and {dagger} Coley Pharmaceutical Group, Wellesley, MA 02481


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bacterial pneumonia is a leading cause of mortality in the United States. Innate immune responses, including type-1 cytokine production, are critical to the effective clearance of bacterial pathogens from the lung. Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotide motifs (CpG ODN), which mimic the effects of bacterial DNA, have been shown to enhance type-1 cytokine responses during infection due to intracellular pathogens, resulting in enhanced microbial clearance. The role of CpG ODN in modulating protective innate immunity against extracellular pathogens is unknown. Using a murine model of Gram-negative pneumonia, we found that CpG ODN administration stimulated protective immunity against Klebsiella pneumoniae. Specifically, intratracheal (i.t.) administration of CpG ODN (30 µg) 48 h before i.t. K. pneumoniae challenge resulted in increased survival, compared with animals pretreated with control ODN or saline. Pretreatment with CpG ODN resulted in enhanced bacterial clearance in lung and blood, and higher numbers of pulmonary neutrophils, NKT cells, {gamma}{delta}-T cells, and activated NK1.1+ cells and {gamma}{delta}-T lymphocytes during infection. Furthermore, pretreatment with CpG ODN enhanced the production of TNF-{alpha}, and type-1 cytokines, including IL-12, IFN-{gamma}, and the IFN-{gamma}-dependent ELR– CXC chemokines IFN-{gamma}-inducible protein-10 and monokine induced by IFN-{gamma} in response to Klebsiella challenge, compared with control mice. These findings indicate that i.t. administration of CpG ODN can stimulate multiple components of innate immunity in the lung, and may form the basis for novel therapies directed at enhancing protective immune responses to severe bacterial infections of the lung.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Over the past decade, the discovery of TLRs in mammals has greatly enhanced our understanding of how innate immunity is activated upon encountering a microbial pathogen. Ligands for TLRs have been found to elicit potent inflammatory responses. In particular, ligands for TLR9, including bacterial DNA and synthetic oligodeoxynucleotides (ODN)3 with unmethylated CpG motifs, have been demonstrated to have significant therapeutic potential for enhancing immune responses, particularly in the context of intracellular infections, cancer immunotherapy, and vaccine development (reviewed in Refs. 1 and 2).

Bacterial DNA, in comparison with mammalian DNA, contains a higher proportion of unmethylated CpG dinucleotide motifs. These motifs are believed to be the primary source of its immunogenicity (3). Synthetic oligonucleotides containing unmethylated CpG dinucleotide motifs (CpG ODN) are capable of mimicking the immunostimulatory effects of bacterial DNA on B cells, monocytes/macrophages, NK cells, and dendritic cells (1, 2, 3, 4, 5, 6, 7, 8, 9). CpG ODN can directly activate cells expressing TLR9 (e.g., plasmacytoid dendritic cells, B cells, and macrophages), whereas activation of other leukocytes populations (including NK cells) occurs through CpG-induced production of endogenous molecules such as TNF-{alpha}, IL-12, and type-1 IFNs. Certain bacterial DNA preparations have been shown to enhance NK cell lytic activity and IFN-{gamma} production, whereas B cell proliferation and Ab production are the predominant effects of other preparations (4, 5, 6, 7, 8). Like bacterial DNA, structural features of CpG ODN can determine the types of immune responses elicited. For example, A-type CpG ODN (also referred to as D-type ODN) induces type-I IFN production, particularly IFN-{alpha} from plasmacytoid dendritic cells, which in turn promotes NK cell activation and the maturation of monocytes into dendritic cells (10, 11). B-type CpG ODN (also referred to as K-type ODN) are activators of B cells and humoral immunity but can have stimulatory effects on monocyte and dendritic cells as well (1, 2, 3, 9, 12). Disparity in immune effects of various CpG ODN molecules appears to be dictated, in part, by differences in uptake and intracellular localization of internalized CpG ODN (13). In addition to these effects, bacterial DNA and CpG ODN have been shown to induce NF-{kappa}B activation and cytokine expression in murine macrophages (1, 8, 14, 15, 16, 17). Finally, systemic administration of CpG ODN promotes type-1 immune responses, characterized by enhanced IL-12 and IFN-{gamma} production (18, 19, 20, 21). Thus, CpG ODN can enhance both innate and humoral immune responses.

Although many of the immunostimulatory effects of CpG ODN have been shown to be beneficial against intracellular pathogens, these molecules have not previously been demonstrated to stimulate protective immunity against extracellular pathogens. Furthermore, few studies have been performed using CpG ODN in the treatment of respiratory infections (22, 23, 24). Thus, using a murine model of Klebsiella pneumonia, we investigated whether administration of CpG ODN, in particular an A-type CpG ODN, would have beneficial effects on innate host defense in the lung against a clinically important extracellular pathogen, Klebsiella pneumoniae.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female specific pathogen-free, 6- to 8-wk-old C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). For the experiments involving IFN-{gamma} knockout (IFN-{gamma}–/–) animals, age- and sex-matched wild-type C57BL/6J and IFN-{gamma}–/– female mice on a C57BL/6J background were purchased from The Jackson Laboratory. All animals were housed in specific pathogen-free conditions within the University of Michigan animal care facility (Ann Arbor, MI) until the day of sacrifice.

Bacterial preparation and intratracheal (i.t.) inoculation

K. pneumoniae strain 43816, serotype 2 (American Type Culture Collection, Manassas, VA) was used in our studies. Klebsiella was grown overnight in tryptic soy broth (Difco, Detroit, MI) at 37°C. The concentration of bacteria in broth was determined by measuring the absorbance at 600 nm, and then plotting the OD on a standard curve generated by known CFU values. The bacteria culture was then diluted to the desired concentration.

Animals 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.

CpG ODN

Active and control CpG ODN were synthesized on a phosphodiester backbone by Oligos Etc. (Wilsonville, OR). The active CpG ODN contained two CpG motifs (underlined) and had the sequence 5'-TCCATGACGTTCCTGACGTT-3', whereas the C and G of the first CpG motif were reversed in the control peptide (underlined) and had the sequence 5'-TCCATGAGCTTCCTGAGTCT-3'. Control or CpG ODN (30 µg) were reconstituted in 30 µl of sterile water or saline. Control groups in the experiments described received either vehicle alone or control ODN. All ODN were free of endotoxin and protein contamination.

Reagents

Anti-IFN-{gamma}, -IL-12, -TNF-{alpha}, -IFN-{gamma}-inducible protein-10 (IP-10), and -monokine induced by IFN-{gamma} (MIG) Abs used in the ELISA were obtained from R&D Systems (Minneapolis, MN). Murine IFN-{alpha} was measured using a kit purchased from R&D Systems. Rat anti-mouse IFN-{beta} mAb (7F-D3) used for a customized ELISA was purchased from Seikagaku America (Falmouth, MA).

Whole-lung homogenization for CFU and cytokine analysis

At designated time points, the mice were euthanized by CO2 inhalation. Before lung removal, the pulmonary vasculature was perfused by infusing 1 ml of PBS containing 5 mM EDTA into the right ventricle. Whole lungs were removed, taking care to dissect away lymph nodes. The lungs were then homogenized in 1 ml of PBS with protease inhibitor (Boehringer Mannheim, Indianapolis, IN). Homogenates were then serially diluted 1/5 in PBS and plated on blood agar to determine lung CFU. The remaining homogenates were sonicated, and then centrifuged at 1400 x g for 15 min. Supernatants were collected, passed through a 0.45-µm-pore-sized filter, and then stored at –20°C for assessment of cytokine levels.

Peripheral blood CFU

Blood was collected in a heparinized syringe from the right ventricle at designated time points, serially diluted 1/2 with PBS, and plated on blood agar to determine blood CFU.

Total lung leukocyte isolation and cytospins

Total lung leukocytes were isolated as previously described (25). Briefly, lung tissue was minced to a fine slurry in 15 ml of digestion buffer (RPMI 1640, 5% FCS, collagenase 1 mg/ml (Boehringer-Mannheim, Chicago, IL), and DNase (30 µg/ml; Sigma-Aldrich, St. Louis, MO)). Lung slurries were enzymatically digested for 30 min at 37°C. 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 for flow analysis. Cell counts and viability were determined on a hemacytometer using trypan blue exclusion. Cytocentrifugation slides (Cytospin 2; Shandon, Pittsburgh, PA) were prepared from lung digest leukocyte suspensions and stained with Diff-Quik (Dade Behring, Newark, DE) for cell differential.

Isolation and RT-PCR amplification of lung mRNA

Whole lung was harvested at designated time points, immediately snap frozen in liquid nitrogen, and then stored at –70°C for RNA extraction. Total cellular RNA was isolated from frozen lungs, reverse transcribed into cDNA, and amplified as previously described (26). The primers (Sigma Genosys, The Woodlands, TX) used were as follows: murine IP-10, 5'-ATCATCCCTGCGAGCCTATC-3' (forward), 5'-GAACTGACGAGCCTGAGCTA-3' (reverse); murine MIG, 5'-ACATTCTCGGACTTCACTCCA-3' (forward), 5'-CTAGGCAGGTTTGATCTCCGT-3' (reverse); and {beta}-actin, 5'-CTTCTACAATGAGCTGCGTGTG-3' (forward), 5'-GATTCCATACCCAAGAAGGAAGG-3' (reverse). After amplification, the samples were separated on a 2% agarose gel containing 0.003% ethidium bromide. Bands were visualized and photographed using UV transillumination.

Murine cytokine ELISAs

Murine cytokines were quantitated using a modification of a double ligand method as previously described (27). Standards were 0.5-log dilutions of recombinant cytokine from 1 pg/ml to 100 ng/ml. The ELISAs did not cross-react with other cytokines.

Multiparameter flow cytometric analysis

Total lung leukocytes were isolated as described above. Using FITC- or PE-labeled Abs (BD Pharmingen, San Diego, CA), isolated leukocytes were then stained with the following: anti-CD4, anti-CD8, anti-{beta}-TCR ({alpha}{beta}-T cell marker), anti-{gamma}{delta}-TCR ({gamma}{delta}-T cell marker), anti-NK1.1 (NK cell marker), and anti-CD69 Abs. In addition, cells were stained with anti-CD45-Tricolor (Caltag Laboratories, South San Francisco, CA) to distinguish leukocytes from nonleukocytes. Cells were collected on a FACSCalibur cytometer (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences). NK cells and T cell subsets were analyzed after gating on CD45+ lymphocyte-sized cells, and then examining for FL-1 and FL-2 fluorescence expression.

Statistical analysis

Survival curves were compared using the log-rank test. For other data, statistical significance was determined using the unpaired t test. All calculations were performed using the Prism 3.0 software program for Windows (GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of CpG ODN administration on survival in murine Klebsiella pneumonia

To determine the effect of CpG ODN administration on survival in mice infected with K. pneumoniae, animals were administered CpG ODN, control ODN, or vehicle alone i.t., followed 48 h later by the i.t. administration of K. pneumoniae (5 x 103 CFU), and then assessed for survival. In preliminary experiments, it was determined that the maximal benefit of CpG ODN was observed at an i.t. dose of 30 µg per animal. In these studies, Klebsiella-infected animals pretreated with vehicle had a long-term survival of ~20%. In contrast, survival of mice pretreated with CpG ODN was enhanced at both early and late time points following i.t. Klebsiella (Fig. 1). Mice pretreated with control ODN also tended to have improved survival when compared with animals pretreated with vehicle alone; however, this difference was not statistically significant (p > 0.05). Importantly, no survival benefit (in fact, a trend toward decreased survival) was observed in mice that were administered CpG ODN i.p., compared with control-infected animals (data not shown).



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  		FIGURE 1. Survival following pretreatment with i.t. CpG ODN in Klebsiella-infected animals. Animals were administered i.t. CpG ODN, control ODN (Ctl ODN), or vehicle 2 days before i.t. Klebsiella, and assessed for survival through day 12 following infection. **, p ≤ 0.01 by two-tailed log-rank test compared with each of the other groups (20 animals per group; composite results from three separate experiments).

 
To determine the timing and duration of protective effects, mice were administered CpG ODN either concomitant with, or 2, 3, or 6 days before K. pneumoniae administration. No significant survival benefit was observed in animals given i.t. CpG ODN (30 µg) concomitant with or 6 days before challenge with K. pneumoniae (Fig. 2). In contrast, survival was significantly improved in animals that received CpG ODN 2 days before Klebsiella administration (p < 0.01), whereas partial protection was suggested by 3-day pretreatment with CpG ODN (p > 0.05).



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  		FIGURE 2. Survival of Klebsiella-infected animals following pretreatment with i.t. CpG ODN at various time points. Animals were administered i.t. CpG ODN at 6, 3, 2, and 0 days or i.t. vehicle at 2 days before i.t. Klebsiella. Survival was assessed through day 12 following infection. **, p < 0.01 by two-tailed log-rank test compared with vehicle-treated animals (10 animals per group).

 
Effect of CpG ODN administration on bacterial clearance in murine Klebsiella pneumonia

Experiments were performed to determine whether the beneficial effect of CpG ODN was attributable to improved bacterial clearance and decreased dissemination. Because no significant survival differences were observed between vehicle- and control ODN-pretreated animals, subsequent studies were performed using either vehicle or control ODN as a control group. In these studies, mice were treated with CpG ODN (30 µg) i.t. or vehicle, followed 2 days later by K. pneumoniae (5 x 103 CFU), and then bacterial burden in lung and blood was determined 1 and 3 days later. Pretreatment with CpG ODN resulted in 22- and 17-fold reduction in K. pneumoniae CFU in lung at days 1 and 3 postinfection, respectively (p < 0.01) (Fig. 3, top panel). Similarly, pretreatment with CpG ODN resulted in a significant reduction in K. pneumoniae CFU in blood, compared with that observed in control-infected animals (p < 0.05) (Fig. 3, bottom panel).



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  		FIGURE 3. Bacterial clearance of K. pneumoniae after i.t. CpG ODN pretreatment. Animals were administered i.t. CpG ODN or vehicle, followed by i.t. Klebsiella 48 h later. Lungs and blood were harvested at 24 and 72 h after Klebsiella administration. Top, Lung CFU; bottom, blood CFU. Bacterial CFU is shown in log10 ± SEM and composited from two different experiments. *, p < 0.05, and **, p < 0.01 compared with i.t. vehicle (experimental n = 15–20 per group).

 
Effect of CpG ODN administration on lung leukocyte influx in murine Klebsiella pneumonia

We next determined whether pretreatment with CpG ODN enhanced bacterial clearance in murine Klebsiella pneumonia by augmenting the influx of cells required for effective antibacterial host defense. To address this, mice were administered either CpG ODN or equal volumes of vehicle i.t. 48 h before K. pneumoniae challenge, and then lungs were harvested at 24 and 48 h postinfection and total leukocyte populations were quantitated by lung digestion. The 24- and 48-h time points were chosen, because near-maximal influx of leukocytes was observed at these time points post Klebsiella administration. As shown in Fig. 4, the i.t. administration of K. pneumoniae resulted in a substantial increase in the number of total leukocytes, neutrophils, and mononuclear cells in whole-lung digest, compared with uninfected controls. Importantly, animals pretreated with CpG had a significant early increase (24 h) in the numbers of neutrophils (upper panel) and mononuclear cells (lower panel), compared with infected control animals. By 48 h post Klebsiella challenge, there was no significant difference in the number of lung neutrophils or mononuclear cells in infected animals pretreated with CpG ODN, compared with infected control animals.



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  		FIGURE 4. Effect of i.t. CpG ODN pretreatment on lung leukocyte populations during Klebsiella infection. Animals were inoculated with i.t. Klebsiella 2 days after administration of i.t. CpG ODN or vehicle. On days 1 and 2 after infection, cell differentials of isolated whole-lung leukocytes were determined from Klebsiella-infected animals. Uninfected, untreated animals were used as a separate control group. Neutrophil counts are shown in the upper panel, whereas mononuclear cell counts are shown in the lower panel. *, p < 0.05, compared with infected mice pretreated with vehicle. A total of five to eight animals per group, composited from two separate experiments.

 
Effect of CpG ODN administration on the influx of specific T cell and NK cell populations in murine Klebsiella pneumonia

To determine whether CpG ODN administration altered the influx and/or activation of selected T cell and NK cell populations, animals were pretreated with CpG ODN or vehicle 48 h before K. pneumoniae administration, and then the presence of specific T and NK cell populations was determined by flow cytometry. Pretreatment with CpG ODN resulted in a trend toward increased number of total NK cells (NK 1.1+, TCR–) and {alpha}{beta}-T cells ({alpha}{beta}-TCR+) in Klebsiella-infected mice, compared with vehicle-pretreated mice with pneumonia, although these differences did not reach the level of statistical significance (Fig. 5A). In contrast, there was a significant increase in the number of total {gamma}{delta}-T cells ({gamma}{delta}-TCR+) and NKT cells (NK1.1+, {alpha}{beta}/{gamma}{delta}-TCR+) in infected animals pretreated with CpG ODN i.t., compared with Klebsiella-challenged animals pretreated with vehicle or uninfected mice (Fig. 5B).



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  		FIGURE 5. Effect of i.t. CpG ODN pretreatment on pulmonary NK and T lymphocyte populations during Klebsiella infection. Two days after i.t. CpG ODN or saline administration, animals were inoculated with i.t. Klebsiella. Two days after infection, animals were sacrificed and lungs were harvested. Lymphocyte populations were determined by flow cytometry. *, p < 0.05 compared with other groups (data representative of two separate experiments).

 
We next determined the effect of intrapulmonary CpG ODN on the accumulation of activated NK cells and T cells, as determined by cell surface expression of the activational marker CD69. As compared with uninfected controls, we observed an increase in the accumulation of CD69+NK1.1+ cells and {gamma}{delta}-T cells and a trend toward increased numbers of CD69+ {alpha}{beta}-T cells in the lungs of infected control animals (Fig. 6). Moreover, a further 2- to 2.5-fold increase in the number of CD69+NK1.1+ cells, {alpha}{beta}-T cells, and {gamma}{delta}-T cells was detected in CpG ODN-pretreated infected animals over that observed in Klebsiella-infected controls.



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  		FIGURE 6. Effect of i.t. CpG ODN pretreatment on activation of NK and T lymphocyte populations in the lung during Klebsiella infection. Animals were administered i.t. CpG ODN or saline, followed by i.t. Klebsiella 2 days later. Lung leukocytes were then isolated on day 2 after infection and stained for the expression of CD69 and {alpha}{beta}-TCR, {gamma}{delta}-TCR, or NK1.1. Flow cytometric analysis was performed after gating for CD45+ lymphocyte-sized populations. *, p < 0.05 compared with other groups (data representative of two separate experiments).

 
Effect of CpG administration on the expression of chemotactic and activating cytokines in uninfected and Klebsiella-infected mice

The previous studies indicated that the intrapulmonary administration of CpG ODN resulted in an early increase in neutrophils, as well as an accumulation of {gamma}{delta}-T cells, NKT cells, and activated NK and T cell populations in mice infected with K. pneumoniae. Given that CpG has been shown in other model systems to induce type-1 cytokines and the ELR– CXC chemokines IP-10 and MIG (which induce recruitment and activation of {alpha}{beta}- and {gamma}{delta}-T cells, NK, and NKT cells) (28), we measured TNF-{alpha} and type-1 cytokines/chemokines in the lungs of animals treated with CpG or control ODN alone. Treatment with CpG, but not control ODN in uninfected mice resulted in a modest but detectable induction of TNF-{alpha}, IP-10, and MIG mRNA expression in lung homogenates, maximal at 2–3 days post i.t. administration (data not shown). However, we did not observe any significant increases in the levels of TNF-{alpha}, type-1 cytokines (IL-12, IFN-{gamma}) or chemokine (IP-10, MIG) protein in the lungs of uninfected animals after CpG ODN administration (data not shown).

To determine whether priming with intrapulmonary CpG augmented the expression of important type-1 cytokines/chemokines in mice during active lung bacterial infection, animals were pretreated with either control or CpG ODN i.t. 48 h before K. pneumoniae administration, and then lungs were harvested at 1, 2, and 4 days postinfection and assessed for the production of TNF-{alpha}, and type-1 cytokines and chemokines. Importantly, pretreatment with CpG ODN resulted in an early increase in lung TNF-{alpha} levels (24 h), compared with that observed in animals pretreated with control ODN (Fig. 7). Pretreatment with CpG also resulted in increases in IL-12 levels, which were significantly higher at 4 days (p < 0.05) and trended toward increased IL-12 levels noted at 2 days post Klebsiella administration. Contrasting the changes in IL-12 expression, pretreatment with CpG ODN resulted in maximal and significant increases in production of IFN-{gamma} and the IFN-{gamma}-inducible chemokines IP-10 and MIG in whole-lung homogenates at 2 days post Klebsiella challenge, compared with that observed in control ODN-treated mice (p < 0.05 for all cytokines). Because type-1 IFNs can also induce ELR– CXC chemokines (in particular IP-10), we also measured lung IFN-{alpha} and IFN-{beta} levels by ELISA in CpG and control ODN-pretreated mice at various time points post K. pneumoniae administration. Importantly, we did not observe induction of either of these cytokines in either infected or uninfected mice pretreated with control or CpG ODN (data not shown).



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  		FIGURE 7. Cytokine levels in whole-lung homogenates following i.t. Klebsiella. Two days before i.t. Klebsiella inoculation, animals were administered i.t. CpG or Ctl ODN. On days 1, 2, and 4 following infection, cytokine levels in lung homogenates were assessed by ELISA. *, p < 0.05 compared with control (CTL) ODN-treated controls (five to nine animals per group; data are composited from two separate experiments). Solid horizontal line represents cytokine levels in uninfected control animals.

 
Role of IFN-{gamma} in CpG ODN-induced augmentation of bacterial clearance in murine Klebsiella pneumonia

Observations made in this study and by others indicate that CpG ODN is a potent inducer of IFN-{gamma}. To determine whether the stimulatory effect of CpG ODN on lung innate responses in bacterial pneumonia is partially mediated through and requires IFN-{gamma}, we compared the effect of pretreatment with CpG ODN on lung bacterial clearance in mice deficient in IFN-{gamma} (IFN-{gamma}–/–) with that observed in wild-type B6 mice. In wild-type mice, pretreatment with CpG ODN resulted in a ~9-fold reduction in lung K. pneumoniae CFU on day 3 postinfection, compared with vehicle-treated infected animals (Table I). In contrast, pretreatment with CpG ODN resulted in no significant reduction of lung K. pneumoniae CFU in IFN-{gamma}–/– mice, indicating that the protective effect of CpG ODN in bacterial pneumonia requires the endogenous production of IFN-{gamma}. Furthermore, survival studies performed in Klebsiella-infected IFN-{gamma}–/– animals demonstrated that in the absence of IFN-{gamma}, CpG ODN pretreatment did not confer a survival advantage over animals pretreated with vehicle (data not shown).


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  		Table I. Effect of CpG ODN on lung K. pneumoniae CFU in wild-type B6 and IFN-{gamma}–/– micea

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report that pretreatment of mice with intrapulmonary CpG ODN can induce protective immune responses against i.t. challenge with K. pneumoniae. Others have shown that pretreatment of animals with CpG ODN can confer protective immunity against several intracellular pathogens, including Francisella tularensis, Listeria monocytogenes, Plasmodium yoelii, and Leishmania major (29, 30, 31, 32, 33, 34). CpG ODN have also been found to enhance immune responses against several respiratory pathogens. In a murine model of tuberculosis, i.p. CpG led to enhanced lung and systemic clearance of Mycobacteria tuberculosis and improved survival (23). Moreover, CpG ODN has been found to be effective as a vaccine adjuvant in models of Chlamydia trachomatis mouse pneumonitis (22) and invasive pulmonary aspergillosis (24). To our knowledge, this study is the first to demonstrate that CpG ODN is effective in augmenting host immune responses against an extracellular bacterial pathogen.

Most of the previous studies examining the use of CpG ODN in enhancing antimicrobial immunity have involved systemic administration (22, 23, 30, 31, 32, 33, 34). Only a few studies have used inhaled CpG ODN. For example, the coadministration of intranasal bacillus Calmette-Guérin and CpG ODN was superior to the administration of intranasal bacillus Calmette-Guérin and i.p. CpG ODN in terms of clearance of lung pathogens following aerosol challenge with M. tuberculosis (35). Similarly, intranasal CpG ODN combined with Aspergillus fumigatus recombinant protein protected immunocompromised animals against subsequent A. fumigatus inhalational challenge (24). Our study supports the concept of compartmentalized delivery of CpG ODN in the prevention of a localized infection such as pneumonia, because we found that animals pretreated with i.t. CpG ODN had superior survival compared with animals pretreated with i.p. CpG ODN when subsequently challenged with i.t. Klebsiella. Importantly, although potentially beneficial in systemic infections, deleterious effects can occur when bacterial DNA or CpG ODN is administered in a systemic fashion (3, 21, 36, 37). In our model, the compartmentalized delivery of CpG ODN was well tolerated and may be preferable to systemic delivery methods when protecting against respiratory pathogens.

In bacterial pneumonia, clearance of pathogens is primarily dependent upon a vigorous innate immune response. The present study demonstrates that CpG ODN administration enhances several aspects of the cytokine-mediated innate immunity in the lung. In particular, TNF-{alpha} and the type-1 cytokines IL-12 and IFN-{gamma} are important to the effective clearance of K. pneumoniae and other bacterial pathogens from the lung (38, 39, 40). The i.t. administration of CpG ODN results in an early increase (24 h) in the expression of TNF-{alpha} within the lung, compared with that observed in infected control animals. Moreover, increased pulmonary expression of IL-12 and IFN-{gamma} was observed in CpG-pretreated animals following i.t. Klebsiella inoculation. Finally, the expression of cytokines that are induced by IFN-{gamma}, namely the ELR– CXC chemokines IP-10 and MIG, is enhanced by CpG ODN during Klebsiella pneumonia. Collectively, the presence of these cytokines indicates that CpG ODN administration skews the immune system toward a type-1 phenotype, which is clearly beneficial in host defense against both intracellular and extracellular bacterial pathogens. The protection conferred by CpG ODN in bacterial pneumonia was diminished in IFN-{gamma}-deficient mice, providing further evidence in support of the notion that the beneficial effects of CpG ODN are at least partially mediated through IFN-{gamma}. Given that we failed to observe significant induction of type-1 IFNs, it is most plausible that CpG augments the production of IFN-{gamma} during pneumonia by directly inducing the production of TNF-{alpha}, IL-12, or potentially other IFN-inducing cytokines.

The cellular components of CpG ODN-stimulated immunity have not been clearly defined in our model, but several candidate cell populations are likely involved. We observed a more robust early influx of neutrophils in CpG-pretreated animals post bacterial challenge. Neutrophils represent an important phagocytic cell in the clearance of bacterial pathogens from the lung (41). CpG-induced up-regulation of TNF-{alpha} may contribute to increased neutrophil trafficking, either by regulating adhesion molecule expression or stimulating the production of chemotactic cytokines and/or chemokines. Moreover, {gamma}{delta}-T cells, NK cells, and NKT cells are cell populations that play an important role in innate immunity. For instance, NK cells are considered to be the primary source of IFN-{gamma} in the lung early in the course of bacterial infection. In models of bacterial infection, {gamma}{delta}-T cells have also been found to be an early source of IFN-{gamma}, can promote the production of IFN-{gamma} from NK cells, and are required for effective host defense in murine Klebsiella pneumonia (42, 43, 44, 45). Finally, NKT cells can be primed to secrete prodigious quantities of IFN-{gamma} in the setting of infection (46, 47, 48, 49, 50), and these cells have been shown to contribute to innate immunity against pulmonary Streptococcus pneumoniae challenge (51). Importantly, increased numbers of {gamma}{delta}-T cells and NKT cells were found in the lung in Klebsiella-infected animals pretreated with CpG ODN, compared with animals pretreated with control ODN. The accumulation of {gamma}{delta}-T cell and NKT cell populations in the lungs of CpG ODN-pretreated animals may be partially attributable to the enhanced expression of IP-10 and MIG, which are chemoattractants for these cells in vivo and in vitro (28, 52, 53, 54). Furthermore, Klebsiella-infected animals pretreated with CpG ODN demonstrated evidence of increased activation of {gamma}{delta}-T cells and NK1.1+ cells. Thus, the recruitment and/or activation of several immune cell populations likely contribute to improved bacterial clearance and outcome in animals pretreated with CpG ODN.

Previous reports have demonstrated that the immunoprotective effects conferred by CpG ODN last at least 2 wk (30, 32, 34, 55); we found that the beneficial effects of CpG ODN in bacterial pneumonia appeared to be relatively short-lived, because animals pretreated with i.t. CpG ODN 6 days before infection did not have any survival benefit. The most plausible explanation for this discrepancy likely relates to the fact that our ODN was constructed with a phosphodiester backbone, whereas prior studies have used CpG ODN with phosphorothioate-modified backbones, which are more resistant to nuclease degradation (30, 34, 55, 56). Phosphorothioate-modified ODN have longer half-lives in vivo than ODN with phosphodiester backbones (57, 58, 59), which are rapidly degraded by nucleases in serum and cells (60). However, CpG ODN made with a phosphorothioate-modified backbone have more B cell-immunostimulatory effects but less NK cell-activating ability (2, 3, 6). In contrast, CpG ODN with a chimeric backbone (i.e., primarily phosphodiester with phosphorothioate-modified 5' and 3' ends) remain resistant to nuclease degradation, yet demonstrate NK cell-stimulatory properties. Similar to observations made by others (30, 34), we found that beneficial effects of CpG were observed only with pretreatment, rather than concomitant therapy. This can be explained by the need for sufficient time for lung leukocyte priming effects, or alternatively, the CpG ODN used may be less stable within the alveolar microenvironment during active infection. It is plausible that, with the appropriate modifications to our CpG ODN, even greater and longer-lasting immunoprotective effects against bacterial pneumonia may be realized.

In these studies, we demonstrate that CpG ODN modulates multiple aspects of innate immunity, and therefore may play a potentially beneficial role in enhancing host defense in patients who are at high risk for pneumonia. Future studies are needed to investigate different preparations of CpG ODN to determine which ODN types will have optimal pharmacokinetics and beneficial immunostimulatory effects in vivo.


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

1 This research was supported in part by a research grant from the GlaxoSmithKline Pulmonary Fellowship Award Program (to J.C.D.), and Grants U01 AI57264, PO50 HL60289, and HL57243 from the National Heart, Lung, and Blood Institute (to T.J.S.). Back

2 Address correspondence and reprint requests to Dr. Theodore J. Standiford, University of Michigan Medical Center, Division of Pulmonary and Critical Care Medicine, 1150 West Medical Center Drive, Medical Science Research Building III 6301, Ann Arbor, MI 48109-0642. E-mail address: tstandif{at}umich.edu Back

3 Abbreviations used in this paper: ODN, oligodeoxynucleotide; i.t., intratracheal; IP-10, IFN-{gamma}-inducible protein-10; MIG, monokine induced by IFN-{gamma}. Back

Received for publication February 26, 2004. Accepted for publication August 10, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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