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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Jul 15, 2011.
Published in final edited form as:
PMCID: PMC3038689
NIHMSID: NIHMS254196

Flagellin Stimulates Protective Lung Mucosal Immunity: Role of Cathelicidin Related Antimicrobial Peptide (CRAMP)

Abstract

Toll like receptors are required for generation of protective lung mucosal immune responses against microbial pathogens. In this study, we evaluated the effect of the TLR5 ligand flagellin on stimulation of antibacterial mucosal immunity in a lethal murine Pseudomonas aeruginosa (PA) pneumonia model. The intranasal pretreatment of mice with purified PA flagellin induced strong protection against intratracheal PA-induced lethality, which was attributable to markedly improved bacterial clearance, reduced dissemination, and decreased alveolar permeability. The protective effects of flagellin on survival required TLR5, and was observed even in the absence of neutrophils. Flagellin induced strong induction of innate genes, most notably the antimicrobial peptide cathelicidin related antimicrobial peptide (CRAMP). Finally, flagellin-induced protection was partially abrogated in CRAMP deficient mice. Our findings illustrate the profound stimulatory effect of flagellin on lung mucosal innate immunity, a response that might be exploited therapeutically to prevent the development of Gram-negative bacterial infection of the respiratory tract.

Keywords: Mucosal immunity, flagellin, Toll-like receptors, antimicrobial peptides

Introduction

Pseudomonas aeruginosa (PA) is an aerobic gram-negative bacterium that is the second most common cause of pneumonia in hospitalized patients, with mortality rates as high as 60–90% in mechanically ventilated with pneumonia due to P. aeruginosa pneumonia 1(1, 2). Lethality in PA pneumonia is caused by the propensity of these patients to develop bacteremia, septic shock, multiple organ failure, and lung injury as compared to patients with pneumonia due to other bacterial pathogens (14).

Toll-like receptors (TLRs) are a family of type I transmembrane receptors that respond to pathogen-associated molecular patterns (PAMPs) expressed by a diverse group of infectious microorganisms, resulting in activation of the host’s immune system (57). Most PA strains express flagella, which primarily consists of the protein flagellin (8). Flagellin is recognized by and activates several pathogen recognition receptors, including TLR5, TLR2 and Ipaf, a component of the NOD/inflammasome pathway (915). In the lung, flagellin can induce neutrophil accumulation, an effect that is dependent on TLR5 expression by lung structural cells rather than bone marrow-derived cells (16). In addition to mediating neutrophil influx, flagellin can activate a broad array of protective innate responses. For instance, the i.p. administration of purified flagellin protected mice from lethal intestinal Salmonella infection, rotavirus induced colitis and bacterial corneal infection (1719). Recently, the repeated intranasal administration of flagellin has been shown to rescue TLR2/4 double deficient mice challenged with non-flagellated PA (20). Mechanism of protection in these models has not been defined, but is felt to be partially due to stimulation of chemokines that facilitated the recruitment of inflammatory cells (17). Flagellin has also been shown to be protective in several non-infectious models, including chemical-induced colitis and radiation pneumonitis (17, 21).

An important component of innate immunity of the respiratory tract is the release of molecules with antimicrobial activity at the mucosal surface. The two best characterized families of cationic antimicrobial peptides are defensins and cathelicidins (22, 23). Cathelicidins are proteins that contain a highly conserved pre-pro region at the N terminus, referred to as the cathelin domain, and substantial heterogeneity at the C terminal domain (2325). These peptides are stored intracellularly as inactive propeptide precursors that are proteolytically cleaved to active peptides upon stimulation (26). The single known human cathelicidin, hCAP-18, is cleaved by proteinase 3 to form the active peptide LL-37. The murine homologue, CRAMP, is encoded by the gene Cnlp (27). Cathelicidins are constitutively expressed in high levels by neutrophils (28). They are also inducibly expressed in response to infection and injury by epithelial cells at mucosal surfaces (2931). Cathelicidin peptides exert bactericidal activity against a broad range of both Gram-negative and Gram-positive organisms, including PA. As compared to wildtype (WT) controls, Cnlp−/− mice display increased susceptibility to several Gram-positive and Gram-negative bacterial infections (3234). The in-vivo contribution of cathelicidins to lung mucosal immunity is not well characterized. However, the forced transgenic expression of LL-37 restored the killing of PA and S. aureus by bronchial epithelial cells isolated from patients with cystic fibrosis, and the in-vivo pulmonary transgenic expression of LL-37 in mice challenged with PA simultaneously reduced lung bacterial burden and reduced inflammation (31, 35). In addition to direct bactericidal properties, cathelicidins exert unique immunomodulatory effects, including binding to anionic molecules such as LPS, resulting in reduced endotoxin immunotoxicity (3638).

In this study, we evaluated the effect of flagellin on protective lung mucosal immune responses in a lethal murine PA pneumonia model. The intranasal delivery of purified PA flagellin induced strong protective immunity against PA, which required, in part, the antimicrobial peptide CRAMP. Flagellin also prevented lung injury, which was associated with reduced expression of caspase-3 in lung during pneumonia.

Methods

Reagents

Anti-CRAMP antibodies used in Western immunoblotting were purchased from Genzyme. Purified recombinant murine CRAMP was obtained from Mary O’Riordan at the University of Michigan. For neutrophil depletion, we treated mice with RB6-8C5 mAb. RB6-8C5 us a rat anti-mouse monoclonal antibody directed against Ly-6G (Gr-1). The antibody was produced by TSD Bioservices (Germantown, NY) by the i.p. injection of hybridoma RB6-8C5 into nude mice and collection of acities. Administration of 200 μg i.p. per mouse results in peripheral blood neutropenia (<50 PMN/μl) by days 1 and 3 post administration, with return of peripheral counts to pretreatment levels by day 5 (Tsai).

Animals

SPF C57Bl/6 mice (age- and sex-matched) were purchased from Jackson Labs. Cnlp−/− mouse breeding pairs were obtained from R. Gallo (UCSD) and TLR5−/− mouse breeding pairs were purchased from A. Aldeman, Institute of Systems Biology, Seattle, WA. Breeding pairs of TLR4Lps-d mutant mice (C3H/HeJ bred onto a B6 genetic background) were obtained from Jackson laboratories. All mouse strains will be housed in SPF conditions within the animal care facility (ULAM) until the day of sacrifice.

Isolation of PA flagellin

Purified flagellin was isolated as described previously (18, 19). Briefly, the PA strain PA01 suspension was blended to remove flagellin from the cells. The homogenate was then centrifuged at 12,000g at 4°C for 20 minutes to separate cells from supernatant. The supernatant was then mixed with ammonium sulfate in 5% increments. The differentially saturated supernatants were centrifuged at 12,000g at 4°C for 30 minutes to remove the insoluble materials from solution. The 15% and 20% insoluble fractions that contained the greatest amount of flagellin with the least amount of contaminants, were dissolved in 50 mM sodium phosphate buffer (pH 8.0) and dialyzed against the same buffer.28 The flagellin-containing sample was applied to HiTrap DEAEtmFF column, washed with 50 mM sodium phosphate buffer (pH 8.0) and then eluted with 0.6 M NaCl and 50 mM sodium phosphate buffer (pH 8.0) with AKTA prime chromatography. The protein-containing fractions were collected, concentrated on filters (Amicon Centriplus YM-3; Millipore, Bedford, MA), and applied to 1 mL prepacked gel affinity columns (Detoxi-Gel Affinity Pak; Pierce, Rockford, IL) to remove LPS. The amount of LPS was determined with a quantitative limulus amebocyte lysate kit (QCL-1000; BioWhittaker, Walkersville, MD). The amount of LPS in the flagellin samples after the two steps of chromatography was 2.7 endotoxin units (EU)/mg protein (0.0027 EU/μg protein). Identity of flagellin was confirmed by immunoblot analysis with rabbit anti-PA flagellum B antiserum.

Bacterial preparation

Pseudomonas aeruginosa strain 19660 (ATCC) was used in our studies. Bacteria were grown overnight in Difco nutrient broth (BD) at 37°C while constantly shaken. The concentration of bacteria in broth was determinedby measuring the absorbance at 600 nm, and then plotting the OD on a standard curve generated by known CFU values. The bacteriaculture was then diluted to the desired concentration.

Intranasal (i.n.) or intratracheal (i.t.) inoculation

Mice were anesthetized with an i.p. ketamine and xylazinemixture. For i.n. administration of flagellin or vehicle, 10 μl were administered to each nostril. For i.t. inoculation of PA, 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.

Murine AEC isolation

Primary alveolar epithelial cells from WT and mutant mice were isolated as previously published (39). Briefly, after mice were heparinized and euthanized, they were exsanguinated and lungs perfused with saline. The lungs were filled with Dispase (1–2 ml; Worthington), followed by 0.45ml of low-melting point agarose and placed in 2ml of Dispase. Lungs were incubated at 24°C for 45 min, then lung tissue teased away from the airways and minced in DMEM with 0.1% DNase. Lung minces were filtered through 100-, 43-, and 15-mm nylon mesh filters. Cells were collected by centrifugation and then incubated with anti-CD32 and anti-CD45 antibodies. Cells are then incubated with streptavidin-coated magnetic particles and positive bone-marrow derived cells were collected on a magnetic column. The negative cells were collected and mesenchymal cells removed by adherence purification overnight. We have shown that these type II cells are 96% pure by intermediate filament staining (41).

Lung macrophage isolation

Lung macrophages (consisting of both alveolar and interstitial macrophages) were isolated from dispersed lung digest cells by adherence purification as previously described (40).

Whole lung homogenization for CFU determination

At designated time points, the mice were euthanized by CO2 inhalation. Before lung removal, the pulmonary vasculature was perfusedby infusing 1 ml of PBS containing 5 mM EDTA into the rightventricle. 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 platedon blood agar to determine lung CFU.

Peripheral blood CFU determination

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

Bronchoalveolar lavage (BAL)

BAL was performed for collection of BALF as previously described (40). Briefly, the trachea was exposed and intubated using a 1.7-mm-outer-diameter polyethylene catheter. BAL was performed by instilling PBS containing 5 mM EDTA in 1-ml aliquots. A total of 3 mls PBS was instilled per mouse, with 90% of lavage fluid retrieved.

Total lung leukocyte preparation by lung digestion

Lungs were removed from euthanized animal and leukocytes prepared as previously described (40). Briefly, lungs were minced with scissors to fine slurry in 15 ml of digestion buffer [RPMI/10%fetal calf serum/1mg/ml collagenase (Boehringer Mannheim Biochemical)/30 μg/ml DNAse, (Sigma) per lung. Lung slurries were enzymatically digested for 30 minutes 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 40% 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.

Real-Time Quantitative RT-PCR

Measurement of gene expression was performed utilizing the ABI Prism 7000 Sequence Detection System (Applied Biosystem, Foster City, CA) as previously described (40). Briefly, total cellular RNA from the frozen lungs were isolated, reversed transcribed into cDNA, and then amplified using specific primers for mTNF-α, MIP-2, IL-17, IL-22, β-defensin-3, CRAMP, and caspase-3 with β-actin serving as a control. Specific thermal cycling parameters used with the TaqMan OneStep 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 sec, annealing/extension at 60°C for 1 min. Relative quantitation of cytokine mRNA levels was plotted as fold-change compared to untreated control cells or whole lung. All experiments were performed in duplicate.

Murine ELISA for albumin measurement

Albumin (Albumin Quantification Kit; Bethyl Laboratories, Montgomery, TX, USA) for lung permeability assessment were quantified using a modified double ligand method.

Western immunoblotting

Whole cell lysates were obtained by treating cells with RIPA buffer (1% w/w NP-40, 1% w/v sodium deoxycholate, 0.1% w/v SDS, 0.15 M NaCl, 0.01 M sodium phosphate, 2 mM EDTA, and 50 mM sodium fluoride) plus protease and phosphatase inhibitors. Protein concentrations were determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA). Samples were electrophoresed in 4–12% gradient SDS-PAGE gels, transferred to nitrocellulose and blocked with 5% skim milk in PBS. After incubation with primary antibodies, blots were incubated with secondary antibody linked to horseradish peroxidase and bands visualized using enhanced chemiluminescence (SuperSignal West Pico Substrate, Pierce Biotechnology, Inc., Rockford, IL).

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 3.0software program for Windows (GraphPad Software, San Diego, CA).

Results

The i.n. administration of flagellin markedly improves survival in mice challenged with PA i.t

Previous studies suggest that flagellin can stimulate protective immunity in non-pulmonary infection models, although the mechanism of protection is unknown (1821). To define the effect of flagellin in lung bacterial infection, we first pre-treated C57Bl/6 mice with graded concentrations of endotoxin-free flagellin (250 ng-1 μg purified from PA strain PA01 in 10 μl saline) or vehicle i.n., then administered a lethal dose of PA strain 19660 (7–8×105 CFU) 48 hrs later. As shown in Figure 1A, pretreatment with flagellin resulted in a striking dose-dependent increase in survival, with nearly 90% of animals pretreated with 1 μg of flagellin i.n. surviving, whereas no survival was observed in animals pretreated with vehicle followed by PA (p<0.05). To define the temporal window of protection, mice were treated with flagellin (1 μg) 48 or 24 hrs prior to PA, or concomitant with the i.t. administration of PA. Pretreatment with flagellin at 48 or 24 hrs prior to PA stimulated equally strong protection (Figure 1B). Importantly, administration of flagellin concomitant with PA induced partial protection (45% survival), albeit less than that observed with pretreatment. The protective effects of flagellin were not attributable to LPS contamination, as flagellin induced a high degree of protection in mice with defective TLR4 signaling (TLR4Lps-d), as compared to saline pretreated animals (1C). To define whether the protection afforded by flagellin required TLR5, wildtype mice and TLR5−/− mice bred on a B6 background (obtained from A. Aldeman, Institute of Systems Biology, Seattle, WA) were pretreated with 1 μg flagellin i.n., followed 24 hrs later by the i.t. administration of PA. Flagellin pretreatment provided significant survival benefit in the PA-infected wildtype mice but not in TLR5−/− mice (Figure 1D).

Figure 1
Effect of i.n. flagellin administration on survival post PA challenge. A. Dose-dependent effect of flagellin on survival (n=10–15 per group, combined from two separate experiments). Animals were pretreated with vehicle or flagellin 24 hrs prior ...

The i.n. administration of flagellin improves lung bacterial clearance and decreases dissemination in mice challenged with PA i.t

To determine the mechanism by which flagellin induces protection, we first examined the effect of flagellin administration on bacterial clearance in the lung and dissemination of bacteria to the bloodstream and distant organs. Mice were pretreated with flagellin or vehicle i.n., PA administered 24 hrs later, then PA CFU in lung, blood and spleen determined 8 and 24 hrs after PA. As shown in Figure 2A, challenge with PA at a dose of 8×105 CFU resulted in substantial numbers of bacteria in lung by 8 hrs, with the development of high grade bacteremia and seeding of spleen by 24 hrs. Impressively, flagellin administration dramatically reduced bacterial burden in lung at both 8 and 24 hrs post PA administration (>100 and >1000-fold reduction in CFU, respectively, p<0.01). Moreover, pretreatment with flagellin decreased blood PA CFU by >1000 fold and completely eliminated splenic seeding.

Figure 2
Effect of i.n pretreatment with flagellin on bacterial CFU, alveolar permeability, and expression of caspase-3 mRNA at various times post i.t. PA administration. A. WT mice were administered flagellin (1 μg) i.n. 24 hrs prior to i.t. PA administration ...

Flagellin has been shown to promote protective effects on non-respiratory epithelium (21, 36). To determine if pretreatment with flagellin reduced lung injury in response to PA administration, animals were administered vehicle or flagellin (1 μg) i.n., followed 24 hrs later by the i.t. administration of PA (5×105 CFU). Bronchoalveolar lavage was performed at 6 and 24 hrs post PA challenge, and alveolar permeability determined by quantitation of bronchoalveolar lavage fluid (BALF) albumin levels. As shown in Figure 2B, left panel), BALF albumin levels were not different in PA challenged mice pretreated with either flagellin or vehicle at 6 hrs post PA. However, BALF albumin levels in vehicle/PA treated mice were markedly increased at 24 hrs, whereas albumin levels were not elevated at 24 hrs in mice pretreated with flagellin. Moreover, we observed significant reduction in caspase-3 mRNA expression in PA-infected animals pretreated with flagellin, as compared to control animals (2B, right panel).

The i.n. administration of flagellin does not substantially alter lung leukocyte influx in mice challenged with PA i.t., and flagellin-induced protection did not require PMN

A possible mechanism of improved bacterial clearance in flagellin-treated mice is by altering the recruitment of innate phagocytic cells. To address this, we quantitated the number of lung PMN and macrophages (both resident and exudates macrophages) in lung digests of vehicle- and flagellin-pretreated mice before and after PA administration. As compared to vehicle-treated animals, flagellin alone induced a modest but statistically significant increase in the influx of PMN but not macrophages at 24 hrs post administration (Figure 3, panel A). However, no differences in numbers of lung PMN, macrophages, or total leukocytes were observed between flagellin and vehicle pretreated groups at 12 or 24 hrs post PA administration. In fact, the number of lung PMN tended to be lower in the flagellin-pretreated group 24 hrs post PA. These studies suggest that flagellin-mediated protection occurred in the absence of large changes in numbers of infiltrating phagocytic cells. These results were confirmed histologically, as flagellin administration resulted in a modest influx of inflammatory cells at 24 hrs (3B, upper right panel). Lung inflammation was most prominent in PA-infected mice pretreated with vehicle (lower left panel), especially when compared to that observed in flagellin-pretreated mice 24 hrs after PA (lower right panel).

Figure 3
Influx of PMN and macrophages in lungs of mice after i.n. administration of flagellin and subsequent PA challenge. Mice were pretreated with flagellin or vehicle, then i.t. administered 5×105 CFU PA. A. Lung digest leukocyte populations quantitated ...

To definitively exclude a role for neutrophils in flagellin-induced protection, mice were depleted of PMN by the i.p. administration of rat anti-mouse anti-Ly-6G antibody (200 μg/animal), given concomitant with either i.n. flagellin or vehicle, followed 24 hrs later by the i.t. administration of PA (5×104 CFU). The inoculum of PA in this experiment was reduced due to enhanced susceptibility to bacterial pneumonia in neutrophil-depleted animals. As shown in Figure 3C, pretreatment of neutrophil-depleted mice with flagellin resulted in a >100, >1000, and >400 fold reduction in lung, blood and spleen CFU at 24 hrs post bacterial administration, as compared to vehicle-pretreated mice challenged with PA.

The i.n. administration of flagellin induces the expression of innate host defense genes in lung

To further explore the mechanism of protection in flagellin-treated animals, we assessed the expression of innate genes that participate in lung antibacterial host defense by quantitative PCR. As shown in Figure 4, the i.n. administration of flagellin resulted in an early induction of TNF-α mRNA (23-fold increase) and IL-17 message (14-fold increase for both) in whole lung by 6 hrs, with a decline in expression by 24 hrs, whereas peak mRNA expression of the neutrophil active chemokine macrophage inflammatory protein-2 (MIP-2/CXCL1) and IL-22 occurred at 24 hrs post flagellin. Flagellin also strongly induced expression of genes encoding antimicrobial peptides. In particular, CRAMP was the most abundantly expressed gene, with a >40-fold induction at 6 hrs, and persistently high expression out to 24 hrs. β-defensin-3 was also induced in response to flagellin, albeit to a considerably lesser degree and in a delayed fashion relative to CRAMP.

Figure 4
Levels of murine TNF-α, MIP-2, IL-17, IL-22, β–defensin 3 and CRAMP mRNA in lung after i.n. flagellin administration. Lungs were harvested at times indicated post flagellin (1 μg), homogenates prepared, and cytokine mRNA ...

The i.n. administration of flagellin ± PA induces the expression of CRAMP protein in lung

Given that CRAMP was the most robustly expressed gene in response to purified flagellin, we next sought to determine the induction of CRAMP protein in response to flagellin alone and in PA infected mice pretreated with flagellin i.n. As determined by Western blot analysis, minimal CRAMP was detected in lung at baseline (Figure 5, panel A). Flagellin administration resulted in a >5-fold increase in lung levels of the pro-peptide form of CRAMP (20 kDa). Challenge with PA in vehicle-pretreated mice also resulted in an increase lung CRAMP expression, although the induction of CRAMP was greatest in PA-infected mice pretreated with flagellin. Importantly, flagellin-induced expression of CRAMP was abrogated in TLR5−/− mice as compared to that observed in WT animals (Figure 5B).

Figure 5
Expression of CRAMP protein in lung after flagellin ± PA administration. Panel A is Western blot analysis (upper panel) and corrected densitometry (lower panel) showing CRAMP induction 6 and 24 hrs after i.n. flagellin (top row) and 24 hrs after ...

Flagellin induces expression of CRAMP by MLE-12 alveolar epithelial cells, primary AEC, and lung macrophages

In preliminary studies, immunohistochemical analysis indicated that PMN, alveolar macrophages, and alveolar epithelial cells expressed CRAMP post PA administration, and expression by these cells was greatest in animals pretreated i.n. with flagellin (data not shown). To more definitely establish the cellular sources of CRAMP in response to flagellin, we isolated primary mouse AEC and pulmonary macrophages recovered from whole lung digests as previously described (39). In addition, we utilized MLE-12 cells, a transformed murine alveolar epithelial cell line that retains many features of type II alveolar epithelial cells (41). Treatment with flagellin strongly stimulated the time-dependent expression of CRAMP mRNA, with an 8-, 50- and 35-fold increase in message observed in AEC, MLE-12, and lung macrophages at 24 hrs, respectively, compared to untreated cells (Figure 6, panel A).

Figure 6
CRAMP mRNA expression by MLE-12, AEC, and lung macrophages (panel A) after incubation with flagellin 100 ng/ml. Values shown represent mean fold increase over unstimulated macrophages. N=4 per condition per time point, *p<0.05 as compared to unstimulated ...

We next performed Western blot analysis to detect the presence of CRAMP in cell lysates from flagellin-stimulated MLE-12 and primary murine AEC. Minimal CRAMP was detected at baseline in unstimulated MLE-12 or primary AEC. Treatment of cells with purified flagellin resulted in an increase in CRAMP by 4 hrs in stimulated MLE-12, and 8 hrs in primary AEC (Figure 6, panel B).

Contribution of CRAMP to flagellin-induced protection in PA pneumonia

Our earlier studies clearly identified flagellin as a major inducer of CRAMP, raising the possibility that CRAMP might mediate, in part, the protective effects of flagellin on antibacterial host responses. In preliminary studies, we found that incubation of murine recombinant CRAMP with PA resulted in suppression of bacterial growth, with bacterostatic activity observed at concentrations of 1 μM and above (Supplemental Figure 1). To determine the contribution of CRAMP to flagellin protective effects, we utilized mice deficient in the gene encoding CRAMP (Cnlp−/− mice). Cnlp−/− and WT C57Bl/6 mice were pretreated with flagellin (1 μg) or vehicle i.n., then administered PA at a dose of 2–3×105 CFU, and PA CFU quantitated in lung and blood 24 hrs later (Figure 7, panel A). A lower PA inoculum was chosen in these experiments due to concerns for excessive mortality in CRAMP-deficient mice. As compared to vehicle-treated WT infected mice, bacterial CFU in lung was approximately 6.5-fold greater in lungs of Cnlp mutant mice (p=0.05). More impressively, there was markedly increased dissemination of PA in Cnlp−/− mice, with >20-fold higher bacterial counts in blood of knockout mice (p<0.05). As compared to WT mice pretreated with vehicle, flagellin pretreated mice had a significant reduction in lung CFU and no dissemination to blood. In contrast, no reduction in lung CFU was observed in Cnlp−/− mice pretreated with flagellin, as compared to Cnlp−/− mice not receiving flagellin, although a trend toward reduction in blood CFU was observed in flagellin/PA-infected Cnlp−/− mice.

Figure 7
Bacterial clearance (A) and survival (B) in WT and Cnlp−/− mice with or without pretreated with flagellin. Mice were pretreated with flagellin (1 μg) i.n. or saline, challenged with PA (2–3×105 CFU), then lungs ...

To further determine the relative contribution of CRAMP to the protective effects of flagellin, we pretreated WT and Cnlp−/− mice with flagellin (1 μg) i.n. followed by administration of PA (7–08×105 CFU), then assessed survival. As shown in Figure 7B, all control PA infected WT and Cnlp−/− mice died after challenge with this inoculum of PA. As observed previously, no mortality was observed in PA infected WT mice pretreated with flagellin. By comparison, only one third of Cnlp−/− survived even when pretreated with flagellin.

Discussion

Protective immunity against bacterial pathogens of the lung requires the generation of robust but tightly controlled innate immune responses, which is mediated, in part, by toll like receptors (TLR). Our studies indicate that the compartmentalized administration of flagellin can augment host immunity against PA, which is associated with enhanced bacterial clearance from the lung, reduced bacterial dissemination, and protection against PA-induced lung injury.

The mechanism by which flagellin activates protective immune reponses has not been completely characterized. The early protection induced by flagellin (within 24 hrs post administration) indicates stimulation of innate, rather than adaptive immunity. Moreover, protection occurred in the absence of significant changes in the recruitment of PMN or exudate macrophages. The effects of flagellin appear to be primarily mediated by TLR5, as we observed substantial loss of flagellin-mediated protection in TLR5−/− mice. This observation is consistent with the observations of others (913) and suggests that other PRRs, including TLR2 and the Ipaf/NOD pathway are less relevant in the generation of protective immunity in responses to exogenous PA flagellin (14, 15). Importantly, our data indicates that flagellin stimulatory effects in our model are maintained in mice with defective TLR4 signaling (TLR4Lps-d), confirming that the effects of flagellin are not mediated by LPS contamination. Flagellin induced potent protection against PA strain 19660, which is a cytotoxic strain expressing type III secreted exotoxins and flagellin. In addition, flagellin-induced broad protection against both flagellated and non-flagellated isogenic mutant PA strains (PAK and PAKΔfilC, provided by A. Prince, Columbia University) and the more virulent encapsulated Gram-negative bacteria Klebsiella pneumoniae (data not shown). Our findings are consistent with immunostimulatory properties of flagellin against diverse microbial pathogens, including lethal intestinal Salmonella infection, rotavirus induced colitis and bacterial corneal infection (3234), and provide further support for induction of innate rather than specific immunity.

There are several possible mechanisms accounting for enhanced lung PA clearance. Firstly, flagellin induced an influx of PMN into the lung airspace by 24 hrs after administration. However, the influx of PMN was relatively modest at the time of bacterial challenge, and flagellin pre-treatment induced strong protection even in animals depleted of PMN. Collectively, this data indicates that flagellin effects on PMN influx is unlikely to be a major contributor to enhancement of bacterial clearance. An early induction of innate cytokine and chemokine genes, including TNF-α, MIP-2, IL-17, and IL-22 was also observed in flagellin-pre-treated animals. Finally, flagellin is a potent inducer of cationic antimicrobial peptides, including β-defensin-2 and especially CRAMP. CRAMP exerts direct bactericidal effects against both Gram-positive and Gram-negative organisms, including PA (42). We found reduced lung bacterial clearance and increased dissemination in PA-infected CRAMP deficient mice. Importantly, flagellin-induced protective effects on bacterial clearance and survival were significantly diminished in Cnlp/ mice relative to WT animals, indicating that CRAMP was a dominant mediator of protection in response to flagellin. We cannot exclude the possibility that the induction of CRAMP in flagellin-treated mice is indirect due to induction of other regulatory genes such as IL-22. However, since IL-22 has not been previously been shown to be expressed by alveolar epithelial cells, flagellin-induced CRAMP expression in these cells in-vitro strongly argues in support of a direct stimulatory effect.

Multiple cells in the lung express TLR5 and are capable of responding in a TLR5-dependent fashion, including airway and alveolar epithelial cells and alveolar macrophages (11, 14, 16). Interestingly, lung neutrophil accumulation that occurs after the i.n. administration of purified flagellin has been shown to be mediated by structural cells rather than TLR5-mediated myeloid cell responses (16). In-vitro studies suggest that flagellin is a strong inducer of CRAMP in alveolar epithelium, including both alveolar epithelial cell lines (MLE-12) and primary murine AEC. CRAMP has been shown to be expressed at several epithelial surfaces, such as skin, cornea, intestine, and urinary tract (30, 3234, 43). While the human homolog LL-37 is known to be expressed by airway epithelial cells and A549 alveolar epithelial cells (31, 44), the production of CRAMP by primary alveolar epithelial cells has not been previously reported. Epithelial cell-derived CRAMP contributes meaningfully to antibacterial host defense. For example, Cnlp−/− mice display impaired clearance of group A streptococcus in a skin infection model, and this defect in clearance persisted in Cnlp−/− mice depleted of neutrophils (34). Moreover, keratinocytes isolated from Cnlp−/− mice displayed reduced intracellular killing of bacteria relative to WT keratinocytes. Our data showing protection in neutrophil-deficient mice implicates meaningful contributions from cells other than PMN in response to flagellin.

These data indicate that pretreatment with flagellin protected against PA-induced lung injury. While the reduction in lung injury observed could be attributable to improved lung bacterial clearance, a plausible alternative explanation is that flagellin might exert pro-survival effects on alveolar epithelial cells, resulting in improved barrier function of the alveolar capillary membrane and reduced access of bacteria to the bloodstream. Flagellin has previously been shown to drive pro-survival responses in other epithelial cell populations, and can promote protective responses in non-infectious radiation induced lung injury model (17, 21). We have found that treatment of either murine primary AEC or epithelial cell lines promotes resistance to apoptosis and selective induction of anti-apoptotic genes (unpublished observations, T. Standiford). These findings are consistent with effects observed in other epithelial cell populations. We cannot exclude that the effects of flagellin are indirect via induction of other molecules regulating apoptosis. For instance, CRAMP exerts multiple immunomodulatory effects that might influence epithelial integrity, including stimulation of epithelial proliferation and repair, angiogenesis, and cytoprotection via stimulation of IL-10 (29, 30, 36, 45).

Our findings indicate that pre-exposure to flagellin, as compared to concomitant administration, markedly enhanced protective mucosal immunity. A possible explanation for enhanced efficacy is a priming effect on the expression of innate genes by both hematopoetic and structural cells. Alternatively, factors present in the inflammatory milieu may block optimal function of antimicrobial molecules released in response to flagellin. Cathelicidins can bind to polyanions present in the inflammatory milieu, such as F-actin, DNA, and mucous, resulting in can reduce biological activity of these molecules (46, 47). Cathelicidins can also be degraded by proteases elaborated by bacteria within the lung. Despite reduced efficacy, we observed some degree of protection with concomitant administration. Collectively, our results have clearly identified a novel means to stimulate lung innate mucosal immunity that can be exploited therapeutically to prevent serious bacterial infections of the respiratory tract.

Supplementary Material

Supplemental Figure 1

Supplemental Figure 1:

Inhibition of PA growth by mrCRAMP. PA (5×104 CFU/ml) was incubated in Difco nutrient broth at 37°C in the presence or absence of mrCRAMP (0.1–50 μM), then PA CFU quantitated 4 hrs later and expressed as percent of untreated control. Concentration of PA at 4 hrs in vehicle treated positive control was 4×105 CFU/ml. *p<0.05 as compared to untreated control, n = 4 per condition.

Acknowledgments

This grant is supported by NIH/NHLBI grants HL97546 and HL25243 (TJS) and NIH/NEI grants EY10869 and EY 17960 (FSY).

Abbreviations

AM
alveolar macrophages
i.n
intranasal
i.t
intratracheal
PAMPs
pathogen associated molecular patterns
BAL
bronchoalveolar lavage
CRAMP
cathelicidin related antimicrobial peptide
AEC
alveolar epithelial cells
PMN
polymorphonuclear neutrophils

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