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. Author manuscript; available in PMC: 2013 Jul 1.
Published in final edited form as: J Immunol. 2012 May 25;189(1):304–311. doi: 10.4049/jimmunol.1103196

Cathelicidin Related Antimicrobial Peptide is Required for Effective Lung Mucosal Immunity in Gram-negative Bacterial Pneumonia

Melissa A Kovach *, Megan N Ballinger *, Michael W Newstead *, Xianying Zeng *, Urvashi Bhan *, Fu-shin Yu †, Bethany B Moore *, Richard L Gallo ‡, Theodore J Standiford *
PMCID: PMC3566644  NIHMSID: NIHMS375380  PMID: 22634613

Abstract

Cathelicidins are a family of endogenous antimicrobial peptides that exert diverse immune functions, including both direct bacterial killing and immunomodulatory effects. In this study, we examined the contribution of the murine cathelicidin, CRAMP, to innate mucosal immunity in a mouse model of Gram-negative pneumonia. CRAMP expression is induced in the lung in response to infection with Klebsiella pneumoniae. Mice deficient in the gene encoding CRAMP (Cnlp−/−) demonstrate impaired lung bacterial clearance, increased bacterial dissemination and reduced survival in response to i.t. K. pneumoniae administration. Neutrophil influx into the alveolar space during K. pneumoniae infection was delayed early but increased by 48 hrs in CRAMP-deficient mice, which was associated with enhanced expression of inflammatory cytokines and increased lung injury. Bone marrow chimera experiments indicated that CRAMP derived from bone marrow cells rather than structural cells was responsible for antimicrobial effects in the lung. Additionally, CRAMP exerted bactericidal activity against K. pneumoniae in vitro. Similar defects in lung bacterial clearance and delayed early neutrophil influx were observed in CRAMP-deficient mice infected with Pseudomonas aeruginosa, although this did not result in increased bacterial dissemination, increased lung injury, or changes in lethality. Taken together, our findings demonstrate that CRAMP is an important contributor to effective host mucosal immunity in the lung in response to Gram-negative bacterial pneumonia.

Keywords: Antimicrobial peptides, Klebsiella pneumonia, Pseudomonas aeruginosa, acute lung injury

Introduction

Hospital-acquired pneumonia is a common nosocomial infection, and is a leading cause of death among hospital acquired infections(1–3). Mortality rate in patients with nosocomial pneumonia have been reported as high as 33–50%. Gram-negative pathogens, such as Pseudomonas aeruginosa, are a common cause of hospital acquired pneumonia, particularly in mechanically ventilated patients. Klebsiella pneumoniae is another important Gram-negative bacterium with a rising prevalence as a nosocomial pathogen(4, 5). Nosocomial Klebsiella pneumonia now accounts for approximately 10% of all hospital acquired pneumonias, including a growing number of carbapenemase-producing strains(5, 6). Due to the increasing rate of antibiotic resistant pathogens, new therapeutic options to aid in prevention and treatment of nosocomial infections are needed.

The lung is continuously bombarded by a vast array of inhaled microbial pathogens. To combat these infectious insults, the respiratory tract is armed with diverse mechanisms of innate mucosal immunity, including expression of antimicrobial peptides. Prominent among these antimicrobial peptides are the cathelicidin family. Cathelicidins are a family of cationic endogenous antimicrobial peptides that are synthesized as pre-pro-peptides(7, 8). These molecules are characterized by an N-terminal pre-pro region with a high degree of homology amongst diverse mammalian species, and a far more heterogeneous C-terminal antimicrobial domain(9). Upon stimulation, the C-terminus is proteolytically cleaved. Cathelicidins are constitutively expressed by neutrophils(10), but are induced in epithelial cells, such as keratinocytes, intestinal epithelium, and lung epithelial cells(11–13). Additionally, cathelicidins can be expressed by other myeloid-derived cells, such as macrophages and lymphocytes(14, 15). The murine cathelicidin, CRAMP is encoded by the gene Cnlp on mouse chromosome 9(16).

Cathelicidins exert antibacterial activity against both Gram-positive and Gram-negative bacteria via electrostatic interactions with the bacterial cell membrane(13, 17–21). Their activity is reduced in high salt concentrations, as well as in the presence of other anionic molecules such as f-actin, airway mucus, and DNA(22, 23). In addition to bactericidal activity, cathelicidins exert a number of immunomodulatory effects, including LPS binding and neutralization, chemotaxis of immune cells, and stimulating the release of inflammatory cytokines(24–29). It has also recently been discovered that they mediate lung epithelial cell stiffness and transepithelial permeability(19). In comparison with wild type mice, Cnlp−/− mice demonstrate increased susceptibility to both Gram-positive and Gram-negative mucosal infections(30). The in vivo contribution of cathelicidins to lung mucosal immunity has not been characterized. However, transgenic expression of LL-37 restored the killing of P. aeruginosa and Staphylococcus aureus by bronchial epithelial cells isolated from patients with cystic fibrosis(31), and the in vivo pulmonary transgenic expression of LL-37 in mice challenged with P. aeruginosa simultaneously reduced lung bacterial burden and reduced inflammation(32).

In this study, we sought to characterize the effects of the murine cathelicidin, CRAMP, in two models of Gram-negative bacterial pneumonia. Our findings indicate that CRAMP is required for protective immunity against both K. pneumoniae and P. aeruginosa, and that CRAMP may exert important immunomodulatory effects that regulate lung injury and bacterial dissemination.

Materials and Methods

Animals

SPF C57BL/6 mice (age- and sex-matched) were purchased from The Jackson Laboratory (Bar Harbor, ME). Cnlp−/− mouse breeding pairs (33) bred on a C57BL/6 genetic background were obtained from Richard Gallo (University of California, San Diego, San Diego, CA). All mouse strains were housed in specific pathogen-free conditions within the animal care facility (Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI) until the day of sacrifice. Animal studies were reviewed and approved by the University Committee on the Use and Care of Animals (University of Michigan, Ann Arbor, MI).

Intratracheal inoculation

For intratracheal (i.t.) injection, mice were anesthetized with an intraperitoneal (i.p.) ketamine and xylazine mixture. Under sterile conditions, the trachea was exposed, and a 30 µl inoculum was administered via a sterile 27-gauge needle. The skin incision was then closed with surgical staples.

Reagents

Anti-CRAMP Abs used in Western immunoblotting were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Purified synthetic murine CRAMP (H–GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ-OH) was synthesized by Chi Scientific (Maynard, MA).

Bacterial preparation

Klebsiella pneumoniae strain 43816, serotype 2 and Pseudomonas aeruginosa strain 19660 (ATCC; Manassas, VA) were used in our studies. K. pneumoniae was grown overnight in trypticase soy broth (BD; Franklin Lakes, NJ) at 37°C. P. aeruginosa was grown overnight in Difco nutrient broth (BD) at 37°C while constantly shaken. 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 bacterial culture was then diluted to the desired in-assay concentration.

Murine alveolar epithelial cell isolation

Primary alveolar epithelial cells (AECs) from WT and Cnlp−/− mice were isolated as previously described(34). Briefly, mice were heparinized and euthanized. They were then exsanguinated and the lungs were perfused with saline solution. The lungs were filled with Dispase (1–2 ml; Worthington Biochemical; Lakewood, NJ), followed by 0.45 ml low-melting point agarose and placed in 2 ml Dispase. Lungs were incubated at 24°C for 45 min and then lung parenchymal tissue was separated 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 incubated with anti-CD32 and anti-CD45 Abs. Cells were then incubated with streptavidin-coated magnetic particles and positive bone marrow-derived cells were removed 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(33).

Lung macrophage isolation

Lung macrophages (both alveolar and interstitial macrophages) were isolated from dispersed lung digest cells by adherence purification as previously described(35).

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(35). Briefly, total cellular RNA from the frozen lungs were isolated, reversed transcribed into cDNA, and then amplified using specific primers for Cnlp with β-actin serving as a control. Specific thermal cycling parameters used with the TaqMan One-Step RT-PCR Master Mix Reagents kit are as follows: 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 untreated control cells or whole lung. All experiments were performed in duplicate.

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. Whole lung homogenates were prepared by grinding tissue in 1 ml RIPA buffer) with a Tissue Tearor (Cole-Parmer; Vernon Hills, IL) for approximately 20 s. 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 anti-CRAMP Abs, blots were incubated with a secondary Ab linked to HRP and bands visualized using ECL (SuperSignal West Pico Substrate, Pierce Biotechnology; Rockford, IL).

Whole lung, spleen, and blood CFU determination

At designated time points, the mice were euthanized by CO2 asphyxiation. The thoracic cavity was opened under sterile conditions and the pulmonary vasculature was perfused with 1 ml sterile PBS containing 5 mM EDTA into the right ventricle. Whole lungs and spleen were removed, taking care to dissect away lymph nodes. The organs were then homogenized separately in 1 ml PBS with protease inhibitor (Boehringer Mannheim, Indianapolis, IN). Homogenates were serially diluted 1:5 in PBS and plated on nutrient agar (BD; Franklin Lakes, NJ) to determine CFU. Whole blood was aspirated into heparinized syringes from the right ventricle at designated time points, serially diluted 1:5 with sterile PBS, and plated.

Bronchoalveolar lavage

Bronchoalveolar lavage (BAL) was performed for collection of BAL fluid (BALF) as previously described(35). Briefly, the trachea was exposed and intubated using 1.7-mm outer diameter polyethylene tubing. PBS containing 5mM EDTA was instilled into the trachea in 3 – 1 ml aliquots and aspirated by syringe suctioning. Approximately 90% of BALF was retrieved.

BALF leukocyte analysis

BALF was centrifuged at 1800 rpm at 4°C for 10 min. Supernatants were removed and reserved for other separate experiments. Cell pellets were resuspended in 250 µl GIBCO® RPMI medium (Invitrogen; Carlsbad, CA). 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.

Murine ELISAs for cytokine measurement

Cell-free BALF supernatants were analyzed for TNFα, IL-17, MIP-2, and KC using mouse DuoSet ELISA kits (R&D Systems; Minneapolis, MN) employing a modified double ligand method.

Murine ELISA for albumin measurement

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

Bone Marrow Transplantation

Bone marrow transplantation (BMT) was performed as previously described(36). Donor mice were euthanized via CO2 asphyxiation and the hind legs were removed. Bone marrow cells were harvested from donor mice and re-suspended in serum-free medium (SFM; DMEM, 0.1% BSA, 1% penicillin-streptomycin, 1% L-glutamine, and 0.1% amphotericin B). Recipient mice received 13.5 Gy of total body irradiation (TBI; orthovoltage x-ray source) split in two fractions, 3 h apart. Bone marrow cells (5 × 106) were administered by tail vein injection into TBI recipient mice. All experiments with BMT mice were performed 5 wk post-BMT when mice were fully donor-cell reconstituted.

In vitro bacterial killing assay

A modified colony-counting assay was performed using K. pneumoniae culture(37). Briefly, bacteria were diluted to a concentration of 5 × 104 in 100 µl in 96-well plates. Cultures were incubated with various concentrations of synthetic murine CRAMP at 37°C for 2 hours, then were serially diluted 1:5 with sterile PBS and plated on nutrient agar plates. Each condition was carried out in triplicate.

Statistical Analysis

Survival curves were compared using the log rank (Mantel-Cox) test. Statistical significance was determined using a two-tailed unpaired t test for figs. 1, 2B, 3–4, and 6–8. Fig. 5 was analyzed using one-way ANOVA. All calculations were performed using GraphPad Prism 5.0 software for Windows (GraphPad Software, San Diego, CA).

Figure 1. Effect of intrapulmonary K. pneumoniae challenge on expression of CRAMP.

Figure 1

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(A) Wild type mice were challenged with i.t. K. pneumoniae. CRAMP expression at 0 hr, 6 hr, 24 hr, and 48 hr post-infection were assessed by real-time PCR and Western immunoblotting with densitometric analysis of whole lung homogenates (* p < 0.01, † p < 0.001 compared to control). Values are shown as mean ± SEM. mRNA and protein densitometry signify 4 mice/group, combined from two independent experiments. Western blotting is representative of 3 different mice/group. (B) AECs and pulmonary macrophages (Mac) were isolated from wild type mouse lungs. Cells were cultured in the presence of purified heat-killed K. pneumoniae (10:1 bacteria:cells). mRNA expression of CRAMP increases following exposure to bacteria in both cell types (* p < 0.01, † p < 0.001 compared to control time point). Values are expressed as mean ± SEM, and represent 3 wells/group, combined from 2 separate experiments.

Figure 2. Effect of CRAMP deletion on survival, lung bacterial clearance, and bacterial dissemination following K. pneumoniae infection.

Figure 2

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(A) Wild type mice or Cnlp−/− mice were infected with K. pneumoniae (5 × 103 CFU; n=10 per group) and observed for survival (* p < 0.01 compared to WT). Survival curves are representative of 2 separate experiments with 8–10 animals/group. (B) Wild type or Cnlp−/− mice were challenged with 5×103 CFU i.t. of K. pneumoniae. CFU were assessed at 48 hours post-infection from whole blood, as well as lung and spleen homogenates. († p < 0.001 compared to WT; * p < 0.01 compared to WT). Values are expressed as mean ± SEM, and represents 4–6 animals per group, combined from 2 separate experiments.

Figure 3. Effect of CRAMP deletion on inflammatory cell influx after K. pneumoniae infection.

Figure 3

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Wild type or Cnlp−/− mice were challenged with i.t. K. pneumoniae (5×103 CFU). Total leukocyte cell counts and differentials were measured from BAL fluid at 24 and 48hrs post-infection (* p < 0.05 compared to WT at the same time point). Values are expressed as mean ± SEM, and represent 4–6 animals/group, combined from two separate experiments.

Figure 4. Effect of CRAMP on lung inflammation and lung injury in response to K. pneumoniae infection.

Figure 4

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Wild type or Cnlp−/− mice were challenged with i.t. K. pneumoniae (5×103 CFU). Values are expressed as mean ± SEM and represent 4–6 animals/group, combined from two separate experiments. (A) BAL fluid was analyzed at 0 and 48 hrs after infection for TNFα, IL-17, MIP-2, and KC (* p < 0.05 compared to WT at the same time point). (B) BAL fluid was also analyzed for albumin as a surrogate for epithelial permeability (* p < 0.01 compared to WT at the same time point).

Figure 6. Bactericidal activity of CRAMP against K. pneumoniae.

Figure 6

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K. pneumoniae (5×104 CFU/ml) was incubated in TSB broth at 37°C in the presence or absence of synthetic CRAMP (0.1–50 µM). CFU were quantitated 2 hrs later and expressed as percent of untreated control (* p < 0.05 compared to control). Values represent mean ± SEM of 3 wells/dose, and are representative of 3 separate experiments.

Figure 8. Effects of CRAMP in a P. aeruginosa pneumonia model.

Figure 8

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(A) Wild type or Cnlp−/− mice were challenged with i.t. P. aeruginosa (5×105 CFU). BAL fluid total leukocyte and neutrophil cell counts were assessed at 0, 24, and 48 hours after infection (* p < 0.01 compared to WT at the same time point). Values are expressed as mean ± SEM and represent 5 animals/group, combined from three separate experiments. (B) Wild type or Cnlp−/− mice were challenged with i.t. P. aeruginosa (3×105 CFU). CFU were assessed at 24 hrs post-infection from whole blood, and lung and spleen homogenates (* p < 0.05 compared to WT). Values are expressed as mean ± SEM and represent 6 animals/group, combined from three separate experiments. (C) WT or Cnlp−/− mice were challenged with i.t. P. aeruginosa representing an LD80 for WT (5×105 CFU; n= 10 per group) and observed for survival over a 5 day period. In a separate experiment WT and Cnlp−/− mice were given an LD20 inoculum i.t (1 × 105 CFU; n = 6 per group) and observed for 5 days. The LD80 survival curve is representative of 10 mice/group, combined from three different experiments. The LD20 curve is representative of 6 mice/group and demonstrates a single experiment. (D) WT or Cnlp−/− mice were challenged with i.t. P. aeruginosa (3×105 CFU). BAL fluid was analyzed at 0, 24, and 48 hrs after infection for TNFα, IL-17, MIP-2, and KC (* p < 0.05 compared to WT at the same time point). Values are expressed as mean ± SEM and represent 5 animals/group, combined from two separate experiments.

Figure 5. Relative contribution of CRAMP expression from marrow-derived cells vs. epithelial cells on lung bacterial clearance.

Figure 5

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Bone marrow chimeras were generated using wild type and Cnlp−/− mice. Mice were then challenged with i.t. K. pneumoniae (1 × 103 CFU). As observed before, in non-transplanted mice, there were significantly higher CFU in the lung at 48 hours after infection in Cnlp−/− mice (data not shown). CFU were assessed in lung homogenates at 48 hours after K. pneumoniae administration in each of the transplanted groups (* p < 0.05; † p < 0.01). Values represent mean ± SEM of 5 animals/group, and represent a single experiment.

Results

CRAMP is induced in response to Klebsiella infection of the respiratory tract

To better understand the role of CRAMP during Gram-negative infection, we first sought to determine whether CRAMP was induced after i.t. challenge with K. pneumoniae. We examined whole lungs of infected mice (n = 3 per time point) by real time qRT-PCR and western blot at 6, 24, and 48 hours after infection (Fig. 1A). There was a time-dependent increase in CRAMP mRNA in lung, which was maximal by 48 hours post K. pneumoniae administration (p < 0.01). There was also a corresponding induction of CRAMP protein in infected mice, as compared to whole lung from uninfected controls (p < 0.001).

To define potential cellular sources of CRAMP expression by lung cells, we analyzed CRAMP induction in vitro in isolated primary lung macrophages and alveolar epithelial cells (AEC) following exposure to heat-killed Klebsiella (Fig. 1B). Expression of CRAMP mRNA was significantly increased in both cell types, maximal by 24 hours after exposure (p < 0.01).

Survival and bacterial clearance are reduced in Cnlp−/− mice post i.t. administration of K. pneumoniae

Given that CRAMP was expressed during the evolution of Klebsiella pneumonia, we next examined the function of CRAMP in bacterial pneumonia. Wild type and mice lacking the gene encoding CRAMP (Cnlp−/− mice) were inoculated with 5 × 103 CFU K. pneumoniae and survival assessed to 9 days. As shown in Figure 2A, survival was 50% in WT infected mice, whereas Cnlp−/− mice died earlier and no mice survived past 6 days (p < 0.01 compared to WT mice).

To determine if reduced survival in Cnlp−/− mice was associated with changes in bacterial clearance, Klebsiella CFU were quantified from whole lung, blood, and spleen at 48 hours after i.t. infection (Fig. 2B). In lung, there was 37-fold increase in bacterial CFU in Cnlp−/− mice, as compared to WT (p < 0.001). Likewise, bacterial counts were higher in blood (14-fold change, p < 0.01) and spleen homogenates (45-fold change, p < 0.001) in Cnlp−/− mice.

Enhanced lung inflammation/injury in Cnlp−/− mice post i.t. K. pneumoniae administration

Cathelicidins have been proposed to exert direct chemotactic effects on inflammatory leukocytes, including PMN. To determine whether this effect contributes to the phenotypic difference observed in Cnlp−/− mice, we examined inflammatory cell counts in WT and Cnlp−/− mice following i.t. K. pneumoniae administration (Fig. 3). At 24 hours after infection, there was increase in total BAL cells and BAL neutrophils in WT mice. While there was no difference in total leukocyte counts, there was a small yet statistically significant decrease in neutrophils in the Cnlp−/− mice at 24 hours (p < 0.05). By 48 hours after infection, the increase in both total cells and numbers of neutrophils was considerably greater in the Cnlp−/− mice as compared to their WT counterparts (p < 0.05).

Inflammatory cytokine levels in whole lung during infection were analyzed at 48 hours post bacterial challenge (Fig. 4A). There was significantly more MIP-2, KC, and IL-17 in Cnlp−/− mice as compared to wild type (p < 0.05 for all cytokines). We noted an increase in TNFα in Cnlp−/− mice, however this change did not meet the level of statistical significance.

In addition, we examined the albumin content in BAL fluid from infected wild type and Cnlp−/− mice as a surrogate for injury to the alveolar capillary membrane (Fig. 4B). Using this inoculum of K. pneumoniae in WT mice, we observed no increase in BAL albumin levels as compared to uninfected control mice. By comparison, we observed an increase in BAL albumin levels in infected Cnlp−/− mice at 48 hours post bacterial challenge, and there was significantly more albumin in Cnlp−/− mice compared to wild type (p < 0.05) at that time point.

Bone marrow cell-derived expression of CRAMP is responsible for the Cnlp−/− phenotype

We have shown that CRAMP is induced in response to Klebsiella infection, and that this protein is expressed by both cells of myeloid original and alveolar epithelial cells. To address which cell type is responsible for the expression of CRAMP required for effective bacterial clearance, we generated bone marrow chimeras of wild type and Cnlp−/− mice (Fig. 5). Wild type bone marrow cells transplanted into wild type mice (WT→ WT) and Cnlp−/− marrow into Cnlp−/− mice (Cnlp−/− → Cnlp−/− ) resulted in lung bacterial clearance similar to non-transplanted controls (WT and Cnlp−/− mice, respectively, data not shown). When wild type marrow cells were transplanted into Cnlp−/− mice (WT → Cnlp−/−), bacterial clearance in the lung was identical to that of transplanted WT→ WT mice, and was significantly less impaired than either Cnlp−/− marrow transplanted into wild type mice (Cnlp−/− → WT) or Cnlp−/− → Cnlp−/− (p < 0.01 for both comparisons). However, Cnlp−/− marrow transplanted into wild type mice (Cnlp−/− → WT) demonstrated significantly impaired lung bacterial clearance, comparable to that of transplanted Cnlp−/− mice (Cnlp−/− → Cnlp−/−).

CRAMP exerts direct bactericidal effects against K. pneumoniae in vitro

Among the many putative functions of cathelicidins, these molecules are known to exert direct bactericidal effects on a wide range of microbial pathogens. However, effects of CRAMP on heavily encapsulated bacteria are less well described. Similar to effects on other Gram-negative and Gram-positive organisms, we observed an increase in bacterial killing when K. pneumoniae was incubated with varying doses of synthetically generated CRAMP (Fig. 6), with bactericidal effects plateauing at a CRAMP concentration of 1 µM and above (p < 0.05).

CRAMP contributes to mucosal immunity in murine Pseudomonas aeruginosa pneumonia

We next assessed the importance of CRAMP in another relevant Gram-negative bacterial infection of the respiratory tract, P. aeruginosa. The i.t. administration of P. aeruginosa (105 CFU) resulted in upregulation of CRAMP mRNA and protein (Fig. 7). As compared to Klebsiella administration, the increase in expression began earlier, with a progressive rise in both mRNA and protein at 6 and 24 hours after infection (p < 0.01 for mRNA and p < 0.05 for protein at both time points compared to control). mRNA and protein dropped to basal levels of expression by 48 hours after infection.

Figure 7. Expression of CRAMP in a P. aeruginosa pneumonia model.

Figure 7

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Wild type mice were challenged with i.t. P. aeruginosa (1 × 106 CFU). CRAMP expression at 0 hr, 6 hrs, 24 hrs, and 48 hrs post-infection were assessed by real-time qRT-PCR and Western immunoblotting (* p < 0.01; † p < 0.05 compared to control). mRNA and protein densitometry values are expressed as mean ± SEM and represent 2–3 animals/group, combined from 2 separate experiments. Western blotting shows 2 animals/group, and is representative of 2 separate experiments.

As compared to WT mice, Cnlp−/− mice demonstrate impaired early lung bacterial clearance in response to P. aeruginosa (Fig. 8B, p < 0.05). Specifically, there was an approximately 35-fold increase in bacterial CFU at 24 hours post-infection in Cnlp−/− mice compared to wild type. However, statistically significant differences in lung bacterial clearance were not seen by 48 hours post-infection. Moreover, bacterial dissemination did not differ between WT and Cnlp−/− mice when challenged with P. aeruginosa, as assessed by blood and spleen bacterial counts at any time point. Similar to Klebsiella-infected mice, we observed an early decrease in BAL PMN at 24 hrs post i.t. P. aeruginosa administration in Cnlp−/− mice (Fig. 8A, p < 0.01). Although PMNs were actually increased in Cnlp−/− mice at 48 hrs in Klebsiella-infected mice, there was no difference in BAL PMNs between WT and Cnlp−/− mice at 48 hrs in Pseudomonas-infected mice. We observed significantly increased levels of IL-17, MIP-2, and KC, but not TNFα, in BAL fluid of Cnlp−/− mice as compared to WT at 24 hrs post-infection (Fig. 8D, p < 0.05). By 48 hrs post-infection, cytokine/chemokine levels were undetectable in BALF of WT mice, whereas levels of each of the measured cytokines and chemokines remained significantly elevated in Cnlp−/− mice. Despite the early differences in bacterial burden and cytokine/chemokine expression in lungs, we observed no survival difference between wild type and Cnlp−/− mice when administered either an LD20 or LD80 inoculum of P. aeruginosa (Fig. 8C).

Discussion

Cathelicidins exert a variety of effects that can contribute to innate mucosal immunity. In this study, we have identified an important role for the murine cathelicidin CRAMP in a mouse model of Klebsiella pneumonia. Previous work in our laboratory has shown that CRAMP expression in the lung is significantly upregulated in response to i.n. bacterial flagellin administration(21). Additionally, i.t. Pseudomonas instillation also results in upregulation of CRAMP. Here, we have demonstrated that CRAMP is also induced in the lung following infection with another Gram-negative bacteria, Klebsiella pneumoniae (Fig. 1). While Pseudomonas is a flagellated bacterium, Klebsiella does not express flagellin. Furthermore, we have found that CRAMP is induced in response to both a standard isolate of Pseudomonas as well as an isogenic strain lacking flagellin production (unpublished observations, T. Standiford). Taken together, these observations suggest that, although flagellin is one factor that triggers CRAMP expression, multiple bacterial PAMPs certainly contribute to the induction of CRAMP in the lung.

We have shown that CRAMP can be expressed by both alveolar epithelial cells, as well as pulmonary macrophages in vitro (Fig. 1A). This is consistent with previous studies showing CRAMP expression from dermal, intestinal and bronchial epithelial cells, and from various hematopoietic cells sources, including macrophages and neutrophils(21, 38–40). Although in vitro studies indicate that both cell types express CRAMP upon exposure to Gram-negative bacteria, the relative contribution of myeloid vs. epithelial cell expression to innate immune responses has not been elucidated in lung. In keratinocyte and gastrointestinal bacterial infection models, it appears that non-neutrophil sources of CRAMP were more relevant contributors to mucosal immunity, as the phenotype of Cnlp−/− mice was preserved when neutrophils were depleted. To directly address this question in the lung, we generated bone marrow chimeras of wild type and Cnlp−/− mice, and assessed clearance of K. pneumoniae from lung (Fig. 5). Importantly, Cnlp−/− hematopoietic cells transplanted into a wild type mouse demonstrated clearance defects comparable to non-transplanted Cnlp−/− mice as well as Cnlp−/− marrow transplanted into Cnlp−/− mice. Conversely, wild type hematopoetic cells transplanted into Cnlp−/− mice demonstrate similar bacterial clearance to both non-transplanted wild type mice as well as wild type marrow transplanted into wild type mice. Our results indicate that CRAMP expression from bone marrow derived cells, rather than structural cells (including epithelial cells) is primarily responsible for enhanced mucosal bacterial clearance.

Given the multiple reported functions of cathelicidins, there are several possible mechanisms to account for the impaired bacterial clearance and reduced survival observed in CRAMP-deficient mice. We have demonstrated that CRAMP has direct bactericidal effects on Klebsiella pneumoniae (Fig. 6). Prior studies have shown similar bactericidal activity of cathelicidins against a variety of bacteria, including Pseudomonas aeruginosa(13, 17–21). Thus, direct bacterial killing is a likely mechanism for bacterial containment. However, cathelicidins are also known to exert other immunomodulatory effects, including binding and inhibition of LPS(26, 27) and stimulation of neutrophil and CD4+ T lymphocyte chemotaxis(28, 29). We observed a modest yet statistically significant impairment in early neutrophil influx into the alveolar space of Cnlp−/− mice infected with either K. pneumoniae (Fig. 3) or P. aeruginosa (Fig. 8A). Analysis of chemokines present in the alveolar space indicates that reduced PMN influx is not due to a deficiency in the neutrophil chemoattractants MIP-2 and KC (Figs. 4A and 8D). It is possible that this delay in neutrophil influx might contribute to impaired bacterial clearance and dissemination. Despite the early delay in neutrophil recruitment, inflammation and lung injury are overall increased in Cnlp−/− mice as compared with wild type by 48 hours after infection in Klebsiella-infected mice (Figs. 3 and 4). There are several potential explanations for enhanced inflammation at this later time point, including a higher bacterial burden driving inflammation, or alternatively the absence of CRAMP effects on LPS sequestration resulting in amplification of LPS-induced inflammatory cytokine/chemokine expression. Additionally, Byfield et al recently demonstrated that the human cathelicidin, LL-37, mediates increased lung epithelial cell stiffness and decreased transepithelial permeability in vitro(19). Thus, the enhanced epithelial permeability observed in Cnlp−/− mice 48 hours after administration of Klebsiella could also be attributable to alterations in epithelial cell cytoprotection or other mechanical properties of the alveolar epithelium. Regardless of mechanism(s), there is appreciable lung injury observed in Klebsiella-infected Cnlp−/− mice, whereas no alveolar leak was noted in WT mice infected with K. pneumoniae. Furthermore, this enhanced lung injury and increased bacterial dissemination in Cnlp−/− mice resulted in decreased survival in the setting of Klebsiella infection.

In this study, we show that CRAMP is required for effective lung mucosal immunity in the setting of Klebsiella pneumonia, as CRAMP-deficient mice displayed impaired bacterial clearance and increased dissemination, increased lung injury, and increased mortality (Figs. 2 and 3). However when we examine the role of CRAMP in a Pseudomonas pneumonia model, the effects are less robust. This does not appear to be attributable to a lack of bactericidal activity, as we and others have previously shown bactericidal activity against P. aeruginosa in vitro in a dose range comparable to that observed with K. pneumoniae. Moreover, we observed that the clearance of P. aeruginosa from lung is impaired in Cnlp−/− mice to a degree similar to that observed in the Klebsiella model at 24 hours post-infection (Fig. 8). Nevertheless, there was no appreciable change in bacterial dissemination, and significant differences in lung bacterial clearance were not apparent at later time points (48 hrs). Despite early reductions in PMN influx in Cnlp−/− mice similar to that observed in our Klebsiella model, by 48 hours after infection there was no difference in BAL PMN counts between wild type and Cnlp−/− mice. Additionally, there were no significant differences between wild type and Cnlp−/− mice in epithelial permeability as assessed by BAL albumin levels in Pseudomonas-infected mice (data not shown). Potential explanations for these discrepancies include the fact that Klebsiella is a more virulent and invasive bacteria than Pseudomonas, and a fully intact innate mucosal system may be required for effective host defense against K. pneumoniae. An alternative explanation is that the fact that Pseudomonas aeruginosa expresses several proteases in abundance. These proteases may degrade and inactivate CRAMP more efficiently than Klebsiella-derived proteolytic enzymes resulting in comparatively reduced biological activity in vivo.

In summary, we show that CRAMP is induced in response to Gram-negative bacterial infections in the lung, and this molecule is required for effective lung bacterial clearance. While further studies are required to define the precise mechanisms of action, our findings suggest a role for cathelicidins which could be exploited therapeutically to improve outcomes in respiratory infections.

Acknowledgements

The authors would like to thank Dr. Mary O’Riordan for the contribution of synthetic murine CRAMP.

This work was supported by National Institutes of Health/National Heart, Lung, and Blood Institute Grants HL97546 and HL25243 (to T.J.S.)

Abbreviations used in this paper

AEC

alveolar epithelial cell

BAL

bronchoalveolar lavage

BALF

BAL fluid

BMT

bone marrow transplant

CRAMP

cathelicidin-related antimicrobial peptide

i.n.

intranasal

i.t.

intratracheal

PMN

polymorphonuclear neutrophil

WT

wild-type

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