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Flagellin Stimulates Protective Lung Mucosal Immunity: Role of -Related Antimicrobial

This information is current as Fu-shin Yu, Matthew D. Cornicelli, Melissa A. Kovach, of September 28, 2021. Michael W. Newstead, Xianying Zeng, Ashok Kumar, Nan Gao, Sang Gi Yoon, Richard L. Gallo and Theodore J. Standiford J Immunol 2010; 185:1142-1149; Prepublished online 21

June 2010; Downloaded from doi: 10.4049/jimmunol.1000509 http://www.jimmunol.org/content/185/2/1142

Supplementary http://www.jimmunol.org/content/suppl/2010/06/18/jimmunol.100050 http://www.jimmunol.org/ Material 9.DC1 References This article cites 49 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/185/2/1142.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Flagellin Stimulates Protective Lung Mucosal Immunity: Role of Cathelicidin-Related Antimicrobial Peptide

Fu-shin Yu,*,†,‡ Matthew D. Cornicelli,x Melissa A. Kovach,x Michael W. Newstead,x Xianying Zeng,x Ashok Kumar,*,†,‡ Nan Gao,*,†,‡ Sang Gi Yoon,*,†,‡ Richard L. Gallo,{ and Theodore J. Standifordx

TLRs are required for generation of protective lung mucosal immune responses against microbial pathogens. In this study, we eval- uated the effect of the TLR5 ligand flagellin on stimulation of antibacterial mucosal immunity in a lethal murine pneumonia model. The intranasal pretreatment of mice with purified P. aeruginosa flagellin induced strong protection against intratracheal P. aeruginosa-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 were observed even in the absence of . Flagellin induced strong induction of innate , most notably the antimicrobial peptide Downloaded from cathelicidin-related antimicrobial peptide. Finally, flagellin-induced protection was partially abrogated in cathelicidin-related antimicrobial peptide-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. The Journal of Immunology, 2010, 185: 1142–1149. http://www.jimmunol.org/ seudomonas aeruginosa is an aerobic Gram-negative bac- derived cells (17). In addition to mediating influx, fla- terium that is the second most common cause of pneumo- gellin can activate a broad array of protective innate responses. For P nia in hospitalized patients, with mortality rates as high as instance, the i.p. administration of purified flagellin protected mice 60–90% in mechanically ventilated patients with pneumonia due to from lethal intestinal infection, rotavirus-induced co- P. aeruginosa pneumonia (1–3). Lethality in P. aeruginosa pneu- litis, and bacterial corneal infection (18–20). Recently, the repeated monia is caused by the propensity of these patients to develop intranasal (i.n.) administration of flagellin has been shown to res- bacteremia, septic shock, multiple organ failure, and lung injury, cue TLR2/4 double-deficient mice challenged with nonflagellated as compared with patients with pneumonia due to other bacterial P. aeruginosa (21). Mechanism of protection in these models has pathogens (1, 2, 4, 5). not been defined, but is believed to be partially due to stimulation by guest on September 28, 2021 TLRs are a family of type I transmembrane receptors that respond of that facilitated the recruitment of inflammatory cells to pathogen-associated molecular patterns expressed by a diverse (18). Flagellin has also been shown to be protective in several group of infectious microorganisms, resulting in activation of the noninfectious models, including chemical-induced colitis and radi- host’s (6–8). Most P. aeruginosa strains express ation pneumonitis (18, 22). flagella, which primarily consists of the protein flagellin (9). Fla- An important component of innate immunity of the respiratory gellin is recognized by and activates several pathogen recognition tract is the release of molecules with antimicrobial activity at the receptors, including TLR5, TLR2, and Ipaf, a component of the mucosal surface. The two best characterized families of cationic NOD/inflammasome pathway (10–16). In the lungs, flagellin can antimicrobial are and (23, 24). induce neutrophil accumulation, an effect that is dependent on Cathelicidins are proteins that contain a highly conserved prepro TLR5 expression by lung structural cells rather than bone marrow- region at the N terminus, referred to as the cathelin domain, and substantial heterogeneity at the C-terminal domain (24–26). These peptides are stored intracellularly as inactive propeptide precur- *Department of Ophthalmology, †Department of Microbiology, and ‡Department of sors that are proteolytically cleaved to active peptides on stimu- x Immunology, Wayne State University, Detroit, MI 48201; Division of Pulmonary lation (27). The single known human cathelicidin, hCAP-18, is and Critical Care Medicine, Department of Medicine, University of Michigan Med- ical Center, Ann Arbor, MI 48109; and {Division of Dermatology, Department of cleaved by to form the active peptide LL-37. The Medicine, University of California San Diego, San Diego, CA 92612 murine homolog, cathelicidin-related antimicrobial peptide Received for publication February 15, 2010. Accepted for publication May 13, 2010. (CRAMP), is encoded by the Cnlp (28). Cathelicidins are This work was supported by National Institutes of Health/National Heart, Lung, and constitutively expressed in high levels by neutrophils (29). They Blood Institute Grants HL97546 and HL25243 (to T.J.S.) and National Institutes of are also inducibly expressed in response to infection and injury by Health/National Eye Institute Grants EY10869 and EY 17960 (to F.S.Y.). epithelial cells at mucosal surfaces (30–32). Cathelicidin peptides Address correspondence and reprint requests to Dr. Theodore J. Standiford, Division exert bactericidal activity against a broad range of both Gram- of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, 109 Zina Pitcher Place, 4065 BSRB, Ann Arbor, MI 48109-2200. E-mail address: negative and Gram-positive organisms, including P. aeruginosa. [email protected] As compared with wild-type (WT) controls, Cnlp2/2 mice display The online version of this article contains supplemental material. increased susceptibility to several Gram-positive and Gram- Abbreviations used in this paper: AEC, alveolar epithelial cell; BAL, bronchoalveolar negative bacterial infections (33–35). The in vivo contribution of lavage; BALF, BAL fluid; CRAMP, cathelicidin-related antimicrobial peptide; i.n., cathelicidins to lung mucosal immunity is not well characterized. intranasal; i.t., intratracheal; PMN, polymorphonuclear neutrophil; WT, wild-type. However, the forced transgenic expression of LL-37 restored the Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 killing of P. aeruginosa and by bronchial www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000509 The Journal of Immunology 1143 epithelial cells isolated from patients with cystic fibrosis, and the The i.n. or intratracheal inoculation in vivo pulmonary transgenic expression of LL-37 in mice chal- Mice were anesthetized with an i.p. ketamine and xylazine mixture. For i.n. lenged with P. aeruginosa simultaneously reduced lung bacterial administration of flagellin or vehicle, 10 ml were administered to each burden and reduced inflammation (32, 36). In addition to direct nostril. For intratracheal (i.t.) inoculation of P. aeruginosa, the trachea was bactericidal properties, cathelicidins exert unique immunomodu- exposed, and 30 ml inoculum was administered via a sterile 26-gauge latory effects, including binding to anionic molecules, such as needle. The incision was closed using surgical staples. LPS, resulting in reduced endotoxin immunotoxicity (37–39). Murine alveolar epithelial cell isolation In this study, we evaluated the effect of flagellin on protective lung mucosal immune responses in a lethal murine P. aeruginosa pneu- Primary alveolar epithelial cells (AECs) from WT and mutant mice were isolated as previously published (42). Briefly, after mice were heparinized monia model. The intranasal (i.n.) delivery of purified P. aeruginosa and euthanized, they were exsanguinated and lungs perfused with saline flagellin induced strong protective immunity against P. aeruginosa, solution. The lungs were filled with Dispase (1–2 ml; Worthington Bio- which required, in part, the antimicrobial peptide CRAMP. Flagellin chemical, Lakewood, NJ), followed by 0.45 ml low-melting point agarose also prevented lung injury, which was associated with reduced ex- and placed in 2 ml Dispase. Lungs were incubated at 24˚C for 45 min and then lung tissue was teased away from the airways and minced in DMEM pression of caspase-3 in lung during pneumonia. with 0.1% DNase. Lung minces were filtered through 100-, 43-, and 15-mm nylon mesh filters. Cells were collected by centrifugation and then Materials and Methods incubated with anti-CD32 and anti-CD45 Abs. Cells are then incubated Reagents with streptavidin-coated magnetic particles and positive bone marrow- derived cells were collected on a magnetic column. The negative cells Anti-CRAMP Abs used in Western immunoblotting were purchased from were collected and mesenchymal cells removed by adherence purification Genzyme (Cambridge, MA). Purified recombinant murine CRAMP was overnight. We have shown that these type II cells are 96% pure by in- Downloaded from obtained from Mary O’Riordan at the University of Michigan (Ann Arbor, termediate filament staining (41). MI). For neutrophil depletion, we treated mice with RB6-8C5 mAb. RB6- 8C5 us a rat anti-mouse mAb directed against Ly-6G (Gr-1). The Ab was Lung isolation produced by TSD Bioservices (Germantown, NY) by the i.p. injection of Lung (consisting of both alveolar and interstitial macro- hybridoma RB6-8C5 into nude mice and collection of acities. Administra- m , phages) were isolated from dispersed lung digest cells by adherence pu- tion of 200 g i.p. per mouse results in peripheral blood neutropenia ( 50 rification as previously described (43). polymorphonuclear neutrophils [PMNs]/ml) by days 1 and 3 postadminis- http://www.jimmunol.org/ tration, with return of peripheral counts to pretreatment levels by day 5 (40). Whole lung homogenization for CFU determination

Animals At designated time points, the mice were euthanized by CO2 inhalation. Before lung removal, the pulmonary vasculature was perfused by infusing SPF C57BL/6 mice (age- and sex-matched) were purchased from The Jack- 2 2 1 ml PBS containing 5 mM EDTA into the right ventricle. Whole lungs Cnlp / son Laboratory (Bar Harbor, ME). mouse breeding pairs (41) were were removed, taking care to dissect away lymph nodes. The lungs were obtained from R. Gallo (University of California, San Diego, San Diego, 2 2 then homogenized in 1 ml PBS with protease inhibitor (Boehringer CA) and TLR5 / mouse breeding pairs were purchased from A. Alde- Mannheim, Indianapolis, IN). Homogenates were then serially diluted 1:5 man, Institute of Systems , Seattle, WA. Breeding pairs of in PBS and plated on blood agar to determine lung CFUs. TLR4Lps-d mutant mice (C3H/HeJ bred onto a B6 genetic background) were obtained from The Jackson Laboratory. All mouse strains were Peripheral blood CFU determination by guest on September 28, 2021 housed in specific pathogen-free conditions within the animal care facility (Unit for Laboratory Animal Medicine, University of Michigan, Ann Ar- Blood was collected in a heparinized syringe from the right ventricle at bor, MI) until the day of sacrifice. designated time points, serially diluted 1:2 with PBS, and plated on blood agar to determine blood CFUs. Isolation of P. aeruginosa flagellin Bronchoalveolar lavage Purified flagellin was isolated as described previously (19, 20). Briefly, the P. aeruginosa strain P. aeruginosa 01 suspension was blended to remove Brochoalveolar lavage (BAL) was performed for collection of BAL fluid flagellin from the cells. The homogenate was then centrifuged at 12,000 3 (BALF) as previously described (43). Briefly, the trachea was exposed and g at 4˚C for 20 min to separate cells from supernatant. The supernatant was intubated using a 1.7-mm outer diameter polyethylene catheter. BAL was then mixed with ammonium sulfate in 5% increments. The differentially performed by instilling PBS containing 5 mM EDTA in 1 ml aliquots. A saturated supernatants were centrifuged at 12,000 3 g at 4˚C for 30 min to total of 3 ml PBS was instilled per mouse, with 90% of BALF retrieved. remove the insoluble materials from solution. The 15 and 20% insoluble fractions that contained the greatest amount of flagellin with the least Total lung leukocyte preparation by lung digestion amount of contaminants were dissolved in 50 mM sodium phosphate Lungs were removed from euthanized animals and leukocytes prepared as buffer (pH 8.0) and dialyzed against the same buffer (28). The flagellin- previously described (43). Briefly, lungs were minced with scissors to fine containing sample was applied to HiTrap DEAEtmFF column, washed slurry in 15 ml digestion buffer (RPMI 1640/10% FCS/1 mg/ml with 50 mM sodium phosphate buffer (pH 8.0) and then eluted with 0.6 [Boehringer Mannheim Biochemical]/30 mg/ml DNAse, [Sigma-Aldrich]) M NaCl and 50 mM sodium phosphate buffer (pH 8.0) with AKTA prime per lung. Lung slurries were enzymatically digested for 30 min at 37˚C. Any chromatography. The protein-containing fractions were collected, concen- undigested fragments were further dispersed by drawing the solution up and trated on filters (Amicon Centriplus YM-3; Millipore, Bedford, MA), and down through the bore of a 10-ml syringe. The total lung cell suspension was applied to 1 ml prepacked gel affinity columns (Detoxi-Gel Affinity Pak; pelleted, resuspended, and spun through a 40% Percoll gradient to enrich for Pierce Biotechnology, Rockford, IL) to remove LPS. The amount of LPS leukocytes. Cell counts and viability were determined using Trypan blue was determined with a quantitative limulus amebocyte lysate kit (QCL- exclusion counting on a hemacytometer. Cytospin slides were prepared and 1000; BioWhittaker, Walkersville, MD). The amount of LPS in the flagel- stained with a modified Wright-Giemsa stain. lin samples after the two steps of chromatography was 2.7 endotoxin unit/mg protein (0.0027 endotoxin unit/mg protein). Identity of flagellin Real-time quantitative RT-PCR was confirmed by immunoblot analysis with rabbit anti-P. aeruginosa flagellum B antiserum. Measurement of was performed utilizing the ABI Prism 7000 Sequence Detection System (Applied Biosystem, Foster City, CA) as Bacterial preparation previously described (43). Briefly, total cellular RNA from the frozen lungs were isolated, reversed transcribed into cDNA, and then amplified using P. aeruginosa strain 19660 (ATCC) was used in our studies. Bacteria were specific primers for mTNF-a, MIP-2, IL-17, IL-22, b- 3, CRAMP, grown overnight in Difco nutrient broth (BD) at 37˚C while constantly and caspase-3 with b-actin serving as a control. Specific thermal cycling shaken. The concentration of bacteria in broth was determined by mea- parameters used with the TaqMan One-Step RT-PCR Master Mix Reagents suring the absorbance at 600 nm, and then plotting the OD on a standard kit included 30 min at 48˚C, 10 min at 95˚C, and 40 cycles involving de- curve generated by known CFU values. The bacteria culture was then naturation at 95˚C for 15 s, annealing/extension at 60˚C for 1 min. Relative diluted to the desired concentration. quantitation of mRNA levels was plotted as fold-change compared 1144 FLAGELLIN STIMULATES PROTECTIVE IMMUNITY with untreated control cells or whole lung. All experiments were performed survival was observed in animals pretreated with vehicle, followed in duplicate. by P. aeruginosa (p , 0.05). To define the temporal window of m Murine ELISA for albumin measurement protection, mice were treated with flagellin (1 g) 48 or 24 h prior to P. aeruginosa, or concomitant with the i.t. administration of Albumin (Albumin Quantification Kit; Bethyl Laboratories, Montgomery, P. aeruginosa. Pretreatment with flagellin at 48 or 24 h prior to TX) for lung permeability assessment were quantified using a modified P. aeruginosa stimulated equally strong protection (Fig. 1B). Im- double ligand method. portantly, administration of flagellin concomitant with P. aerugi- Western immunoblotting nosa induced partial protection (45% survival), albeit less than that observed with pretreatment. The protective effects of flagellin were 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, not attributable to LPS contamination, as flagellin induced a high 0.01 M sodium phosphate, 2 mM EDTA, and 50 mM sodium fluoride) plus degree of protection in mice with defective TLR4 signaling protease and phosphatase inhibitors. Protein concentrations were deter- (TLR4Lps-d), as compared with saline solution-pretreated animals mined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, (Fig. 1C). To define whether the protection afforded by flagellin CA). Samples were electrophoresed in 4–12% gradient SDS-PAGE gels, 2/2 transferred to nitrocellulose and blocked with 5% skim milk in PBS. After required TLR5, WT mice and TLR5 mice bred on a B6 back- incubation with primary Abs, blots were incubated with secondary Ab ground (obtained from A. Aldeman, Institute of Systems Biology, linked to HRP and bands visualized using ECL (SuperSignal West Pico Seattle, WA) were pretreated with 1 mg flagellin i.n., followed 24 h Substrate, Pierce Biotechnology). later by the i.t. administration of P. aeruginosa. Flagellin pretreat- ment provided significant survival benefit in the P. aeruginosa- Statistical analysis 2 2 infected WT mice but not in TLR5 / mice (Fig. 1D). Downloaded from Survival curves were compared using the log-rank test. For other data, sta- tistical significance was determined using the unpaired t test. All calcula- The i.n. administration of flagellin improves lung bacterial tions were performed using the Prism 3.0 software program for Windows clearance and decreases dissemination in mice challenged (GraphPad Software, San Diego, CA). with P. aeruginosa i.t. To determine the mechanism by which flagellin induces protection, Results

we first examined the effect of flagellin administration on bacterial http://www.jimmunol.org/ The i.n. administration of flagellin markedly improves survival clearance in the lung and dissemination of bacteria to the blood- in mice challenged with P. aeruginosa i.t stream and distant organs. Mice were pretreated with flagellin or Previous studies suggest that flagellin can stimulate protective vehicle i.n., P. aeruginosa administered 24 h later, then P. aerugi- immunity in nonpulmonary infection models, although the mech- nosa CFUs in lung, blood, and spleen were determined 8 and 24 h anism of protection is unknown (18–21). To define the effect of after P. aeruginosa. As shown in Fig. 2A, challenge with P. aeru- flagellin in lung bacterial infection, we first pretreated ginosa at a dose of 8 3 105 CFU resulted in substantial numbers of C57BL/6 mice with graded concentrations of endotoxin-free fla- bacteria in lung by 8 h, with the development of high-grade bac- gellin (250 ng–1 mg purified from P. aeruginosa strain P. aerugi- teremia and seeding of spleen by 24 h. Impressively, flagellin ad- nosa 01 in 10 ml saline solution) or vehicle i.n., then administered ministration dramatically reduced bacterial burden in lung at both 8 by guest on September 28, 2021 a lethal dose of P. aeruginosa strain 19660 (7–8 3 105 CFU) 48 h and 24 h post-P.aeruginosa administration (.100- and .1000-fold later. As shown in Fig. 1A, pretreatment with flagellin resulted in reduction in CFUs, respectively, p , 0.01). Moreover, pretreatment a striking dose-dependent increase in survival, with nearly 90% of with flagellin decreased blood P. aeruginosa CFUs by .1000-fold animals pretreated with 1 mg flagellin i.n. surviving, whereas, no and completely eliminated splenic seeding.

FIGURE 1. Effect of i.n. flagellin administration on survival post-P. aeruginosa 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 h prior to P. aeruginosa administration. B, Effect of varying times of flagellin (1 mg) administration on survival (n = 10–15 per group, combined from two separate experiments). C, Effect of flagellin pretreatment (24 h prior to P. aeruginosa) on survival in WTand TLR4 mutant mice (TLR4Lps-d), n = 5 per group. D, Effect of i.n. administration of flagellin or vehicle 24 h pre-P. aeruginosa challenge on survival in WT and TLR52/2 mice, n = 5 per group. pp , 0.05 as compared with infected animals pretreated with vehicle. The Journal of Immunology 1145

between flagellin- and vehicle-pretreated groups at 12 or 24 h post-P. aeruginosa administration. In fact, the number of lung PMNs tended to be lower in the flagellin-pretreated group 24 h post-P. aeruginosa. These studies suggest that flagellin-mediated protection occurred in the absence of large changes in numbers of infiltrating phagocytic cells. These results were confirmed histo- logically, as flagellin administration resulted in a modest influx of inflammatory cells at 24 h (Fig. 3B, upper right panel). Lung inflammation was most prominent in P. aeruginosa-infected mice pretreated with vehicle (Fig. 3B, lower left panel), especially when compared with that observed in flagellin-pretreated mice 24 h after P. aeruginosa (Fig. 3B, lower right panel). To definitively exclude a role for neutrophils in flagellin-induced protection, mice were depleted of PMNs by the i.p. administration of rat anti-mouse anti–Ly-6G Ab (200 mg/animal), given concomitant with either i.n. flagellin or vehicle, followed 24 h later by the i.t. FIGURE 2. Effect of i.n pretreatment with flagellin on bacterial CFUs, administration of P. aeruginosa (5 3 104 CFU). The inoculum of alveolar permeability, and expression of caspase-3 mRNA at various times P. aeruginosa in this experiment was reduced due to enhanced post-i.t. P. aeruginosa administration. A, WT mice were administered flagel- susceptibility to bacterial pneumonia in neutrophil-depleted ani- Downloaded from lin (1 mg) i.n. 24 h prior to i.t. P.aeruginosa administration (5–8 3 105 CFU). 6 mals. As shown in Fig. 3C, pretreatment of neutrophil-depleted Values represent mean SEM expressed in log10 from 8–10 animals per . . . group, combined from two separate experiments. pp , 0.05 as compared mice with flagellin resulted in a 100-, 1000-, and 400-fold with animals pretreated with vehicle. Blood CFUs represent number per reduction in lung, blood, and spleen CFUs at 24 h postbacterial milliliter; lung and spleen CFUs represent number per whole organ. B, BALF administration, as compared with vehicle-pretreated mice chal- albumin levels (left panel) and induction of caspase-3 mRNA and protein lenged with P. aeruginosa. expression (right panel) after i.n. flagellin or vehicle administration, followed The i.n. administration of flagellin induces the expression of http://www.jimmunol.org/ by P.aeruginosa challenge. Animals were treated with flagellin i.n., followed innate host defense genes in lung 24 h later by i.t. P.aeruginosa administration. BAL was performed 6 and 24 h after P. aeruginosa challenge. Whole lung caspase-3 mRNA was determined To further explore the mechanism of protection in flagellin-treated by real-time PCR at 6 and 12 h post-P. aeruginosa. Each value for albumin animals, we assessed the expression of innate genes that participate and mRNA represents mean 6 SEM of five mice per data point. pp , 0.05 as in lung antibacterial host defense by quantitative PCR. As shown in compared with vehicle/P. aeruginosa group. Fig. 4, the i.n. administration of flagellin resulted in an early in- duction of TNF-a mRNA (23-fold increase) and IL-17 message (14-fold increase for both) in whole lung by 6 h, with a decline in

expression by 24 h, whereas, peak mRNA expression of the neu- by guest on September 28, 2021 Flagellin has been shown to promote protective effects on non- trophil active MIP-2/CXCL1 and IL-22 occurred at respiratory epithelium (22, 37). To determine whether pretreatment 24 h postflagellin. Flagellin also strongly induced expression of with flagellin reduced lung injury in response to P. aeruginosa ad- genes encoding . In particular, CRAMP was m ministration, animals were administered vehicle or flagellin (1 g) the most abundantly expressed gene, with a .40-fold induction at i.n., followed 24 h later by the i.t. administration of P. aeruginosa 6 h, and persistently high expression out to 24 h. b-defensin 3 was 3 5 (5 10 CFU). BAL was performed at 6 and 24 h post-P.aeruginosa also induced in response to flagellin, albeit to a considerably lesser challenge, and alveolar permeability determined by quantitation of degree and in a delayed fashion relative to CRAMP. BALF albumin levels. As shown in Fig. 2B, left panel, BALF 6 albumin levels were not different in P. aeruginosa-challenged mice The i.n. administration of flagellin P. aeruginosa induces the pretreated with either flagellin or vehicle at 6 h post-P. aeruginosa. expression of CRAMP protein in lung However, BALF albumin levels in vehicle/P. aeruginosa-treated Given that CRAMP was the most robustly expressed gene in re- mice were markedly increased at 24 h, whereas, albumin levels sponse to purified flagellin, we next sought to determine the in- were not elevated at 24 h in mice pretreated with flagellin. More- duction of CRAMP protein in response to flagellin alone and in over, we observed significant reduction in caspase-3 mRNA expres- P. aeruginosa-infected mice pretreated with flagellin i.n. As deter- sion in P. aeruginosa-infected animals pretreated with flagellin, as mined by Western blot analysis, minimal CRAMP was detected compared with control animals (Fig. 2B, right panel). in lung at baseline (Fig. 5A). Flagellin administration resulted in a .5-fold increase in lung levels of the propeptide form of CRAMP The i.n. administration of flagellin does not substantially alter (20 kDa). Challenge with P. aeruginosa in vehicle-pretreated mice lung leukocyte influx in mice challenged with P. aeruginosa i.t., also resulted in an increase lung CRAMP expression, although the and flagellin-induced protection did not require PMNs induction of CRAMP was greatest in P. aeruginosa-infected mice pretreated with flagellin. Importantly, flagellin-induced expression A possible mechanism of improved bacterial clearance in flagellin- of CRAMP was abrogated in TLR52/2 mice compared with that treated mice is by altering the recruitment of innate phagocytic observed in WT animals (Fig. 5B). cells. To address this, we quantitated the number of lung PMNs and macrophages (both resident and exudates macrophages) in lung Flagellin induces expression of CRAMP by MLE-12 AECs, digests of vehicle- and flagellin-pretreated mice before and after primary AECs, and lung macrophages P. aeruginosa administration. As compared with vehicle-treated In preliminary studies, immunohistochemical analysis indicated that animals, flagellin alone induced a modest but statistically signif- PMNs, alveolar macrophages, and AECs expressed CRAMP post- icant increase in the influx of PMNs but not macrophages at 24 h P. aeruginosa administration, and expression by these cells was postadministration (Fig. 3A). However, no differences in numbers greatest in animals pretreated i.n. with flagellin (data not shown). of lung PMNs, macrophages, or total leukocytes were observed To more definitely establish the cellular sources of CRAMP in 1146 FLAGELLIN STIMULATES PROTECTIVE IMMUNITY Downloaded from http://www.jimmunol.org/

FIGURE 3. Influx of PMNs and macrophages in lungs of mice after i.n. administration of flagellin and subsequent P. aeruginosa challenge. Mice were by guest on September 28, 2021 pretreated with flagellin or vehicle, then i.t. administered 5 3 105 CFU P. aeruginosa. A, Lung digest leukocyte populations quantitated at the time of P. aeruginosa administration and 12 and 24 h post-P. aeruginosa administration. Upper panel is total lung cells, middle panel is macrophages, and lower panel is PMNs. Each value represents mean 6 SEM, four to six mice per value. pp , 0.05 compared with vehicle control group. B, Histology prepared from flagellin- or vehicle-pretreated mice at 24 h after vehicle or flagellin administration or an additional 24 h post-P. aeruginosa administration. Representative of three separate experiments. C, Lung, blood, and spleen P. aeruginosa CFUs (log10) in neutrophil-depleted mice pretreated with either vehicle or flagellin. Organs were harvested 24 h post-P. aeruginosa administration. n = 5 per each group. p , 0.01 as compared with vehicle pretreated mice. response to flagellin, we isolated primary mouse AECs and of 1 mM and above (Supplemental Fig. 1). To determine the con- pulmonary macrophages recovered from whole lung digests as tribution of CRAMP to flagellin protective effects, we used mice previously described (42). In addition, we used MLE-12 cells, a trans- deficient in the gene encoding CRAMP (Cnlp2/2 mice). Cnlp2/2 formed murine AEC line that retains many features of type II AECs and WT C57BL/6 mice were pretreated with flagellin (1 mg) or (44). Treatment with flagellin strongly stimulated the time-dependent vehicle i.n., then administered P. aeruginosa at a dose of 2–3 3 105 expression of CRAMP mRNA, with an 8-, 50-, and 35-fold increase CFU, and P. aeruginosa CFUs quantitated in lung and blood 24 h in message observed in AECs, MLE-12, and lung macrophages at later (Fig. 7A). A lower P. aeruginosa inoculum was chosen in these 24 h, respectively, compared with untreated cells (Fig. 6A). experiments due to concerns for excessive mortality in CRAMP- We next performed Western blot analysis to detect the presence deficient mice. As compared with vehicle-treated WT infected of CRAMP in cell lysates from flagellin-stimulated MLE-12 and mice, bacterial CFUs in lung were ∼6.5-fold greater in lungs of primary murine AECs. Minimal CRAMP was detected at baseline Cnlp mutant mice (p = 0.05). More impressively, there was mark- in unstimulated MLE-12 or primary AECs. Treatment of cells with edly increased dissemination of P. aeruginosa in Cnlp2/2 mice, purified flagellin resulted in an increase in CRAMP by 4 h in stim- with .20-fold higher bacterial counts in blood of knockout mice ulated MLE-12, and 8 h in primary AECs (Fig. 6B). (p , 0.05). As compared with WT mice pretreated with vehicle, flagellin-pretreated mice had a significant reduction in lung CFUs Contribution of CRAMP to flagellin-induced protection in and no dissemination to blood. In contrast, no reduction in lung P. aeruginosa pneumonia CFUs was observed in Cnlp2/2 mice pretreated with flagellin, as 2 2 Our earlier studies clearly identified flagellin as a major inducer of compared with Cnlp / mice not receiving flagellin, although CRAMP, raising the possibility that CRAMP might mediate, in part, a trend toward reduction in blood CFUs was observed in flagel- 2 2 the protective effects of flagellin on antibacterial host responses. In lin/P. aeruginosa-infected Cnlp / mice. preliminary studies, we found that incubation of murine recombi- To further determine the relative contribution of CRAMP to the 2 2 nant CRAMP with P. aeruginosa resulted in suppression of bac- protective effects of flagellin, we pretreated WT and Cnlp / mice terial growth, with bacterostatic activity observed at concentrations with flagellin (1 mg) i.n., followed by administration of P. ae ru gi no sa The Journal of Immunology 1147

in the recruitment of PMNs or exudate macrophages. The effects of flagellin appear to be primarily mediated by TLR5, as we observed substantial loss of flagellin-mediated protection in TLR52/2 mice. This observation is consistent with the observations of others (10– 14) and suggests that other pathogen recognition receptors, in- cluding TLR2 and the Ipaf/NOD pathway are less relevant in the generation of protective immunity in responses to exogenous P. aeruginosa flagellin (15, 16). Importantly, our data indicate 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 P. aeruginosa strain 19660, which is a cytotoxic strain expressing type III secreted exotoxins and flagellin. In addition, flagellin-induced broad pro- tection against both flagellated and nonflagellated isogenic mutant a b FIGURE 4. Levels of murine TNF- , MIP-2, IL-17, IL-22, -defensin P. aeruginosa strains (PAK and PAKΔfilC, provided by A. Prince, 3, and CRAMP mRNA in lung after i.n. flagellin administration. Lungs Columbia University, New York City, NY) and the more virulent were harvested at times indicated postflagellin (1 mg), homogenates pre- encapsulated Gram-negative bacteria Klebsiella pneumoniae (data pared, and cytokine mRNA levels determined by real-time PCR. Fold- increase represents change from vehicle control. Each value represents not shown). Our findings are consistent with immunostimulatory Downloaded from mean of four mice per data point. properties of flagellin against diverse microbial pathogens, includ- ing lethal intestinal Salmonella infection, rotavirus-induced coli- tis, and bacterial corneal infection (33–35), and provide further 3 5 (7–8 10 CFU), then assessed survival. As shown in Fig. 7B,all support for induction of innate rather than specific immunity. 2/2 control P. aeruginosa-infected WT and Cnlp mice died after There are several possible mechanisms accounting for enhanced challenge with this inoculum of P. ae ru gi no sa. As observed lung P. aeruginosa clearance. First, flagellin induced an influx of http://www.jimmunol.org/ previously, no mortality was observed in P. aeruginosa-infected PMNs into the lung airspace by 24 h after administration. How- WT mice pretreated with flagellin. By comparison, only one third ever, the influx of PMNs was relatively modest at the time of 2/2 of Cnlp survived even when pretreated with flagellin. bacterial challenge, and flagellin pretreatment induced strong protection even in animals depleted of PMNs. Collectively, these Discussion data indicate that flagellin effects on PMN influx is unlikely to be Protective immunity against bacterial pathogens of the lung a major contributor to enhancement of bacterial clearance. An requires the generation of robust but tightly controlled innate early induction of innate cytokine and chemokine genes, including immune responses, which is mediated, in part, by TLRs. Our TNF-a, MIP-2, IL-17, and IL-22, was also observed in flagellin- studies indicate that the compartmentalized administration of pretreated animals. Finally, flagellin is a potent inducer of cationic by guest on September 28, 2021 flagellin can augment host immunity against P. aeruginosa, which antimicrobial peptides, including b-defensin 2 and especially is associated with enhanced bacterial clearance from the lung, CRAMP. CRAMP exerts direct bactericidal effects against both reduced bacterial dissemination, and protection against P. aeru- Gram-positive and Gram-negative organisms, including P. aerugi- ginosa-induced lung injury. nosa (45). We found reduced lung bacterial clearance and increased The mechanism by which flagellin activates protective immune dissemination in P. aeruginosa-infected CRAMP-deficient mice. reponses has not been completely characterized. The early pro- Importantly, flagellin-induced protective effects on bacterial clear- tection induced by flagellin (within 24 h postadministration) ance and survival were significantly diminished in Cnlp2/2 mice indicates stimulation of innate, rather than adaptive immunity. relative to WT animals, indicating that CRAMP was a dominant Moreover, protection occurred in the absence of significant changes mediator of protection in response to flagellin. We cannot exclude

FIGURE 5. Expression of CRAMP protein in lung after flagellin 6 P. aeruginosa adminis- tration. A is the Western blot analysis (upper panel) and corrected densitometry (lower panel) showing CRAMP induction 6 and 24 h after i.n. flagellin (top row) and 24 h after P. aeruginosa (5 3 105 CFU) in vehicle- or flagellin- pretreated animals (bottom row). Three animals were included in each group. Mean densitome- try is corrected for b-actin. pp , 0.05 as com- pared with vehicle control. B represents expression of CRAMP in lung 24 h after admin- istration of vehicle or flagellin in WT or TLR52/2 mice as assessed by Western blot analysis. Densitometry (lower panel) represents mean of three separate mice, corrected for b-ac- tin. pp , 0.05 as compared with vehicle control. 1148 FLAGELLIN STIMULATES PROTECTIVE IMMUNITY Downloaded from

FIGURE 7. Bacterial clearance (A) and survival (B) in WT and Cnlp2/2 mice with or without pretreated with flagellin. Mice were pretreated with flagellin (1 mg) i.n. or saline solution, challenged with P. aeruginosa (2– 3 3 105 CFU), then lungs and blood collected 24 h after P. aeruginosa.

Values represent mean 6 SEM on a log10 scale from three to five animals http://www.jimmunol.org/ per group. pp , 0.05 as compared with WT animals without flagellin pretreatment; ppp , 0.05 as compared with WT animals. Blood CFUs FIGURE 6. CRAMP mRNA expression by MLE-12, AECs, and lung represent number per milliliter, lung CFUs represent number per whole macrophages (A) after incubation with flagellin 100 ng/ml. Values shown organ. For survival studies, WT and Cnlp2/2 mice were pretreated with represent mean fold increase over unstimulated macrophages. n = 4 per flagellin (1 mg) i.n. or saline solution, challenged with P. aeruginosa (7– condition per time point. pp , 0.05 as compared with unstimulated cells. 8 3 105 CFU), then survival assessed out to 7 d. n = 6–10 per group. pp , B, Expression of CRAMP protein from unstimulated and flagellin-treated 0.05 as compared with animals without flagellin pretreatment. MLE-12 and AECs by Western blot analysis. Representative of two sep- arate experiments, densitometry (C) represents mean corrected for b-actin.

lung injury observed could be attributable to improved lung bacte- by guest on September 28, 2021 the possibility that the induction of CRAMP in flagellin-treated rial clearance, a plausible alternative explanation is that flagellin mice is indirect due to induction of other regulatory genes, such might exert prosurvival effects on AECs, resulting in improved as IL-22. However, because IL-22 has not been previously been barrier function of the alveolar capillary membrane and reduced shown to be expressed by AECs, flagellin-induced CRAMP expres- access of bacteria to the bloodstream. Flagellin has previously sion in these cells in vitro strongly argues in support of a direct been shown to drive prosurvival responses in other epithelial cell stimulatory effect. populations, and can promote protective responses in a noninfectious Multiple cells in the lung express TLR5 and are capable of radiation-induced lung injury model (18, 22). We have found that responding in a TLR5-dependent fashion, including airway and treatment of either murine primary AECs or epithelial cell lines AECs and alveolar macrophages (12, 15, 17). Interestingly, lung promotes resistance to apoptosis and selective induction of antia- neutrophil accumulation that occurs after the i.n. administration of poptotic genes (T. Standiford, unpublished observations). These purified flagellin has been shown to be mediated by structural cells findings are consistent with effects observed in other epithelial cell rather than TLR5-mediated myeloid cell responses (17). In vitro populations. We cannot exclude that the effects of flagellin are in- studies suggest that flagellin is a strong inducer of CRAMP in direct via induction of other molecules regulating apoptosis. For alveolar epithelium, including both AEC lines (MLE-12) and pri- instance, CRAMP exerts multiple immunomodulatory effects that mary murine AECs. CRAMP has been shown to be expressed at might influence epithelial integrity, including stimulation of epithe- several epithelial surfaces, such as skin, cornea, intestine, and uri- lial proliferation and repair, angiogenesis, and cytoprotection via nary tract (31, 33–35, 46). Although the human homolog LL-37 is stimulation of IL-10 (30, 31, 37, 48). known to be expressed by airway epithelial cells and A549 AECs Our findings indicate that pre-exposure to flagellin, as compared (32, 47), the production of CRAMP by primary AECs has not been with concomitant administration, markedly enhanced protective mu- previously reported. Epithelial cell-derived CRAMP contributes cosal immunity. A possible explanation for enhanced efficacy with meaningfully to antibacterial host defense. For example, Cnlp2/2 pretreatment is a effect on the expression of innate genes mice display impaired clearance of group A Streptococcus in a skin by both hematopoetic and structural cells. Alternatively, factors pres- infection model, and this defect in clearance persisted in Cnlp2/2 ent in the inflammatory milieu may block optimal function of antimi- mice depleted of neutrophils (35). Moreover, isolated crobial molecules released in response to flagellin. Cathelicidins can from Cnlp2/2 mice displayed reduced intracellular killing of bac- bind to polyanions present in the inflammatory milieu, such as F-actin, teria relative to WT keratinocytes. Our data showing protection in DNA, and mucous, resulting in reduced biological activity of these neutrophil-deficient mice implicate meaningful contributions from molecules (49, 50). Cathelicidins can also be degraded by proteases cells other than PMNs in response to flagellin. elaborated by bacteria within the lung. Despite reduced efficacy, we These data indicate that pretreatment with flagellin protected observed some degree of protection with concomitant administration. against P.aeruginosa-induced lung injury. 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