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Antimicrobial Activity of PLUNC Protects against Pseudomonas aeruginosa Infection Lina Lukinskiene, Yang Liu, Susan D. Reynolds, Chad Steele, Barry R. Stripp, George D. Leikauf, Jay K. Kolls and This information is current as Y. Peter Di of October 2, 2021. J Immunol 2011; 187:382-390; Prepublished online 1 June 2011; doi: 10.4049/jimmunol.1001769 http://www.jimmunol.org/content/187/1/382 Downloaded from

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Antimicrobial Activity of PLUNC Protects against Pseudomonas aeruginosa Infection

Lina Lukinskiene,* Yang Liu,* Susan D. Reynolds,† Chad Steele,‡ Barry R. Stripp,x George D. Leikauf,* Jay K. Kolls,{ and Y. Peter Di*

Epithelial antimicrobial activity may protect the lung against inhaled pathogens. The bactericidal/permeability-increasing protein family has demonstrated antimicrobial activity in vitro. PLUNC (palate, lung, and nasal epithelium associated) is a 25-kDa secreted protein that shares homology with bactericidal/permeability-increasing proteins and is expressed in nasopharyngeal and respiratory epithelium. The objective of this study was to determine whether PLUNC can limit Pseudomonas aeruginosa infection in mice. Transgenic mice (Scgb1a1-hPLUNC) were generated in which human PLUNC (hPLUNC) was directed to the airway epithelium with the Scgb1a1 promoter. The hPLUNC protein (hPLUNC) was detected in the epithelium throughout the trachea and bronchial airways and in bronchoalveolar lavage fluid. Bronchoalveolar lavage fluid from transgenic mice exhibited higher antibacterial Downloaded from activity than that from wild type littermates in vitro. After in vivo P. aeruginosa challenge, Scgb1a1-hPLUNC transgenic mice displayed enhanced bacterial clearance. This was accompanied by a decrease in neutrophil infiltration and cytokine levels. More importantly, the overexpressed hPLUNC in Scgb1a1-hPLUNC transgenic mouse airway significantly enhanced mouse survival against P. aeruginosa-induced respiratory infection. These data indicate that PLUNC is a novel antibacterial protein that likely plays a critical role in airway epithelium-mediated innate immune response. The Journal of Immunology, 2011, 187: 382–390.

he ability of the host to avoid infection depends largely Based on predicted structural homology, palate, lung, and nasal http://www.jimmunol.org/ on mechanisms of innate immunity. This rapid-response epithelium associated (PLUNC) has been added to the BPI protein T system acts efficiently without prior exposure to a patho- family. PLUNC protein is expressed specifically in the nasopha- gen (1) and is initiated when bacterial products are detected. ryngeal and respiratory epithelium (6–8). The PLUNC genes are LPS, a cell-wall component of Gram-negative bacteria, is an ag- clustered on human chromosome 20 and encode eight different onist for innate immune response through activation of the TLR4 proteins that share some predicted structural similarity (9). signaling cascade. Exposure to LPS results in a production of PLUNC or short PLUNC protein 1 (SPLUNC1) is the first iden- proinflammatory and anti-inflammatory mediators by myeloid tified PLUNC protein that has multiple alternative names in- lineage and other cell types including epithelial cells. In humans, cluding secretory protein in upper respiratory tracts, lung-specific by guest on October 2, 2021 bactericidal/permeability-increasing protein (BPI) and LPS-binding protein X, and nasopharyngeal carcinoma-related protein. protein (LBP) can bind LPS and modulate the host response All BPI protein family members are defined by a similarly to Gram-negative bacterial infections. Although BPI and LBP predicted three-dimensional structure with a single predicted di- belong to the same protein family (i.e., BPI protein family), their sulphide bond and a conserved exon structure (7). However, the functions are antagonistic: LBP exhibits proinflammatory activity, amino acid homology among members of the BPI protein family whereas BPI displays anti-inflammatory activity and direct bac- is typically ∼15–30% (10). The biological function of PLUNC is tericidal action (2, 3). Two additional lipid transfer LPS-binding not known, but structural similarities to BPI family proteins sug- plasma proteins, cholesteryl ester transfer protein (CETP) and gest that PLUNC may be involved in defense responses in the phospholipid transfer protein (PLTP), are included in the BPI airways. Chu et al. (11) reported that human and mouse bronchial protein family based on predicted structural similarity (4, 5). epithelial cells expressed PLUNC (hSPLUNC1 and mSPlunc1), and that exogenously applied PLUNC decreased Mycoplasma pneumoniae levels and lessened IL-8 production in vitro. Con- *Department of Environmental and Occupational Health, University of Pittsburgh, † versely, PLUNC small interfering RNA enhanced M. pneumoniae Pittsburgh, PA 15260; Department of Pediatrics, National Jewish Health, Denver, CO 80206; ‡Division of Pulmonary Medicine, University of Alabama, Birmingham, growth and IL-8 production. IL-13 significantly decreased x AL 35201; Division of Pulmonary, Allergy and Critical Care, Duke University PLUNC and M. pneumoniae clearance in epithelial cell cultures, Medical Center, Durham, NC 27710; and {Department of Genetics, Louisiana State University Health Science Center, New Orleans, LA 70112 suggesting that decreased PLUNC expression during allergic responses may contribute to asthma pathobiology. Zhou et al. (12) Received for publication July 27, 2010. Accepted for publication April 22, 2011. found that PLUNC protein binds LPS and inhibits the growth of This work was supported by Public Health Service grants from the National Institutes of Health (ES011033 and HL091938 to Y.P.D.) and the American Heart Association- Pseudomonas aeruginosa in vitro. Garcia-Caballero et al. (13) Pennsylvania Delaware Affiliate (Grant 0365327U to Y.P.D.). reported that PLUNC may serve as an airway surface liquid vol- Address correspondence and reprint requests to Dr. Y. Peter Di, Department of ume sensor by regulating epithelial sodium ion-channel activity. Environmental and Occupational Health, University of Pittsburgh, 100 Technology Gakhar et al. (14) demonstrated that PLUNC has surfactant ac- Drive, Room 322, Pittsburgh, PA 15219. E-mail address: [email protected] tivity, and that this activity may account for the low surface ten- Abbreviations used in this article: BAL, bronchoalveolar lavage; BALF, bronchoalveolar lavage fluid; BPI, bactericidal/permeability-increasing protein; GUS-B, b- sion of airway secretions. What was lacking in these studies is glucuronidase; hPLUNC, human PLUNC; LB, Luria-Bertani; LBP, LPS-binding pro- a demonstration that PLUNC has antimicrobial activity in vivo, tein; PLUNC, palate, lung, and nasal epithelium associated; qRT-PCR, quantitative and that additional PLUNC may be protective during infection. To RT-PCR; SPLUNC1, short PLUNC protein 1; TG, transgene; WT, wild type. determine whether PLUNC can limit P. aeruginosa infection in Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 the lung, transgenic mice (Scgb1a1-hPLUNC) were generated in www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001769 The Journal of Immunology 383 which human PLUNC (hPLUNC) was directed to the airway aliquots at 280˚C in 20% glycerol/Luria-Bertani (LB) broth. For each epithelium with the Scgb1a1 promoter and these mice were experiment, an aliquot of bacteria was thawed, inoculated into 10 ml LB, challenged with P. aeruginosa. and incubated (6 h; 37˚C) with shaking. An aliquot was then diluted 1:100 into 100 ml LB broth and incubated (16 h; 37˚C). Bacteria were washed twice and resuspended in 10 ml PBS containing 10 mM magnesium Materials and Methods chloride. Generation of Scgb1a1-hPLUNC transgenic mice CFU assay Constitutive expression of hPLUNC protein in the mouse respiratory epithelium was achieved through generation of mice harboring a transgene The number of CFU was determined by serial dilution and quantitative (TG) composed of the hPLUNC cDNA under the transcriptional control of culture on LB agar plates. P. aeruginosa was resuspended in PBS, and the ∼ ∼ 3 11 the mouse Scgb1a1 (also known as Clara cell secretory protein, or CCSP) OD (OD470) was adjusted to 0.74, 2 10 bacteria/ml. Five serial 10- promoter (Fig. 1A). Total human airway epithelial cell RNA was used as fold dilutions in PBS were prepared immediately before use. Duplicate a template for RT-PCR amplification of PLUNC cDNA (0.78 kb). The bacterial samples were mixed with 50 ml PBS, or bronchoalveolar lavage forward primer included the protein translational start codon and 12 fluid (BALF) from either transgenic or WT control mice were prepared and nucleotides of flanking sequence (59-ATA AGA ATG CGG CCG CCT placed on ice. An equal volume of the 2 3 105 bacteria/ml solution was AAG AGC AAA GAT GTT TC-39), whereas the reverse primer covered added to the wells. Samples were mixed and incubated (2 h; 37˚C) with the translational stop site (59-ATA AGA ATG CGG CCG CAC CTT GAT shaking. Three 50-ml aliquots with different concentrations (no dilution, 5- GAC AAA CTG-39). This cDNA coded for the active and mature PLUNC fold dilution, and 10-fold dilution) from each well were plated on LB agar protein. The PCR product was gel purified, cloned using the pCR2.1- plates. Plates were inverted and incubated overnight at 37˚C. Colonies TOPO cloning system (Invitrogen, Carlsbad, CA), and sequenced. A 9.6- were counted and the number of colonies per plate determined. For Ab kb Scgb1a1 genomic fragment in pUC19 (kindly supplied by Dr. Magnus neutralization studies, mouse BALF samples (50 ml) were preincubated Nord, Karolinska Institute, Stockholm, Sweden) was modified by site- (1 h; 23˚C) with 7.5 ml normal rabbit serum or with anti-PLUNC serum directed mutagenesis to include an NotI site upstream of the Scgb1a1 with gentle agitation, and CFU were calculated. Downloaded from translational start site and resulted in generation of pNotI-9.Scgb1a1.In contrast with the short (2.1-kb) Scgb1a1 promoter, the 9.6-kb construct In vivo exposure of mice to P. aeruginosa included ∼3 and 2 kb of 59- and 39 gene-flanking sequence, respectively. Sex-matched 6- to 8 wk-old WT and transgenic mice were coexposed to P. The hPLUNC cDNA was cloned into the NotI site of pNotI-9.6ScgbB1a1 aeruginosa aerosol using an inhalation exposure system (model A42X; and the construct verified by restriction enzyme digestion and sequencing. Glas-Col, Terre Haute, IN) as previously described (16). Mice were placed The 10.38-kb Scgb1a1-hPLUNC cDNA fragment was isolated by digestion in a compartmentalized mesh basket (5 chambers, 20 mice/chamber ca- with SphI and transgenic generated by microinjection into FVB/N mouse pacity) and exposed (45 min), followed by cloud decay (15 min) and de- http://www.jimmunol.org/ oocytes. One of 19 offspring was positive for the TG, as assessed by PCR contamination (5 min; UV irradiation). Preliminary studies demonstrated genotyping and confirmed by Southern blot analyses of tail DNA. Sub- that exposure to 1010 bacteria/ml for 45 min resulted in a bacterial de- sequently, genotype was determined by PCR. The endogenous Scgb1a1 position of ∼2 3 106 CFU/lung immediately after exposure (5 independent gene and the Scgb1a1-hPLUNC TG were distinguished as 450- and 545-bp experiments, n = 5–6/experimental group, 1.6–2.5 3 106, median and amplicons using a mouse Scgb1a1 promoter-specific forward primer (59- 25th–75th percentile). For survival test, female 6- to 8-wk-old WT and GTT GGC AAG TCT ACA GTT GC-39), a PLUNC coding region forward transgenic mice were infected with an intratracheal instillation of P. aer- primer (59-GAC GTC AGT GAT TCC TGG CC-39), and in combination uginosa at a concentration of 1 3 109 CFU/mouse. with a Scgb1a1 intron 1-specific reverse primer (59-GAA AGA GAC CCT GGG CAC TCA-39). BAL and cell differential counts Animal husbandry At 4 or 24 h after aerosol exposure, mice (5–6 mice/group) were anes- by guest on October 2, 2021 The Scgb1a1-hPLUNC TG was maintained in a hemizygous state by thetized with 2.5% tribromoethanol (Avertin). The trachea was cannulated, breeding with wild type (WT) FVB/N mice. Mendelian transmission fre- the lungs were lavaged (1 ml PBS twice), and the BALF samples pooled quencies were observed, and no overt consequences of the TG on growth, (pool 1). The lungs were lavaged an additional five times with 1 ml PBS breeding, or survival were noted. Mice were maintained in a specific and the recovered fluid was pooled (pool 2). Cells from the two pools were 3 pathogen-free status in 12-h light/dark cycle conditions. All procedures recovered through centrifugation at 300 g and resuspended in 0.5 ml were conducted using mice 8–12 wk of age maintained in ventilated PBS. A 50-ml aliquot was stained with an equal volume of 0.4% trypan microisolator cages housed in an American Association for Accredita- blue (Invitrogen, San Diego, CA) and cells counted with a hemocytometer. tion of Laboratory Animal Care-accredited animal facility. Protocols and An additional aliquot was placed onto glass microscope slides (Shanon studies involving animals were conducted in accordance with National Cytospin; Thermo Fisher, Pittsburgh, PA), stained with Diff-Quick, and Institutes of Health guidelines and approved by Institutional Animal Care cell differential determined microscopically. Protein concentration in pool and Use Committee at the University of Pittsburgh. 1 was determined by DC Protein assay using BSA standards (Bio-Rad, Hercules, CA). Real-time PCR analysis Bacterial clearance Total RNA was isolated from various tissues of WT and transgenic mice by a single-step acid guanidinium thiocyanate extraction method (15). Quan- Bacterial deposition was determined by harvesting the right lungs imme- titative RT-PCR (qRT-PCR) (ABI7700; Applied Biosystems, Foster City, diately postinfection (n = 4 mice/group). Bacterial clearance was assayed 4 CA) was performed using human-specific PLUNC primers (forward: 59- and 24 h postinfection (n = 5–6 mice/group). The right lungs or spleens TTC AGG GCA ACG TGT GCC-39; reverse: 59-TAG TCC GTG GAT were placed into 1 ml sterile PBS and kept on ice before homogenization. CAG CAT GTT AAC A-39; probe: 59-/56-FAM/CTG GTC AAT GAG Six serial 10-fold dilutions were prepared and 50-ml aliquots plated on LB GTT CTC AGA GGC TTG G TAMTph/-39) or mouse-specific plunc agar plates. Each dilution was plated in triplicate. Plates were inverted and primers (forward: 59-TGG GAT TCT CAG CGG TTT GGA TGT-39; re- incubated overnight at 37˚C. The number of viable bacteria in the lung and verse: 59-TCA GCC AAG ATA GCC TTC CTT CCT-39; probe: 59-/56- spleen was determined and expressed as CFU per lung. FAM/CAC CCT GGT GCA CAA CAT TGC TGA AT/TAMTph/-39). Validation tests were performed to confirm equivalent PCR efficiencies for Immunohistochemistry the target genes. Test and calibrator lung RNAs (Ambion, Austin, TX) were Lungs were inflation fixed in situ with 4% paraformaldehyde (10 cm H O; reverse transcribed using a Superscript III kit (Invitrogen), and PCR was 2 10 min) with chest cavity open. The lower right lobe was embedded in amplified as follows: 95˚C for 12 min, 40 cycles; 95˚C for 15 s; 60˚C for paraffin and 5-mm sections prepared. Sections were cleared with xylenes, 1 min. Three replicates were used to calculate the average cycle threshold hydrated to water, and quenched in 3% H O (10 min). Ag retrieval was b 2 2 for the transcript of interest and for a transcript for normalization ( -glu- performed in 10 mM citrate buffer (20 min; 100˚C; pH 6.0). Sections were curonidase [GUS-B]; Assays on Demand; Applied Biosystems). Relative blocked in 0.03% casein in 0.05% PBS/Tween 20 solution (1 h) and in- DD mRNA abundance was calculated by the cycle threshold (Ct) method. cubated (18 h; 4˚C) with hPLUNC Ab (R&D Systems, Minneapolis, MN). Gram-negative bacteria P. aeruginosa Normal goat serum was used as a control. After extensive washing with PBS, sections were incubated (2 h; 23˚C) with biotinylated donkey anti- The P. aeruginosa strain (PAO1, ATCC BAA-47) was used for all goat Ab (Pierce Biotechnology, Rockford, IL) diluted in blocking solution. experiments. P. aeruginosa obtained from a single colony was stored in Sections were washed (30 min; 23˚C) and incubated with streptavidin-HRP 384 ANTIMICROBIAL ACTIVITY OF PLUNC AGAINST P. AERUGINOSA

(Pierce Biotechnology) in PBS. Ag–Ab complexes were detected with Expression of hPLUNC protein in mouse airways was assessed Metal Enhanced DAB solution (Pierce Biotechnology). Tissue was coun- by immunohistochemical staining of paraffin-embedded trachea terstained with Mayer’s hematoxylin solution (Sigma-Aldrich, St. Louis, and lung sections from WT control and transgenic (Scgb1a1- MO). hPLUNC, TG) littermates using an Ab that detects both hPLUNC Dual immunofluorescence and mouse Plunc (mPlunc). Expression of endogenous mPlunc Cells coexpressing PLUNC and SCGB1A1 were identified using dual- was detected in the tracheal and bronchial epithelium (data not immunofluorescence techniques. Primary Abs were goat anti-PLUNC shown) but was absent in bronchiolar cells and alveolar epithelial (R&D Systems) and rabbit anti-SCGB1A1 (17). Secondary Abs (Invi- cells of WT control mice (Fig. 2A,2C). In transgenic Scgb1a1- trogen) were used in the following combinations: Alexa Fluor 488 donkey hPLUNC mice, PLUNC was detected in tracheal (data not shown), anti-goat IgG for PLUNC and Alexa Fluor 594 donkey anti-rabbit IgG for SCGB1A1. All secondary Abs were used at a dilution of 1:500. Stained bronchial, and bronchiolar epithelial cells (Fig. 2B,2D). The sections were mounted with Fluoromount-G containing 2 mg/ml DAPI distribution of SCGB1A1 protein was not altered (Fig. 2E,2F). (Sigma). Images of representative fields were acquired with a Provis AX70 It has been demonstrated that PLUNC is secreted into lumen of microscope (Olympus, Center Valley, PA) equipped with a DAPI/Texas human airway and can be detected in sputum (6). To determine Red dual optical excitation filter cube and an FITC optical excitation fil- whether hPLUNC is similarly secreted in transgenic mice, we ter cube (Olympus) and a Spot RT color digital camera (Diagnostic In- strument, Sterling Heights, MI). Images were processed with Image-Pro used Western blot analysis to examine BALF and lung homoge- Plus software (Media Cybernetics, Bethesda, MD). nate from WT and TG littermates. SCGB1A1 protein, also known as Clara cell secretory protein (CCSP), was used as a loading SDS-PAGE and Western blot analysis control for secreted BALF proteins and GAPDH was used to BALF (25 ml), lung homogenized in PBS (20 mg), or recombinant PLUNC normalize tissue proteins in lung homogenates. mPlunc is not (rPLUNC, 0.1 mg; R&D Systems) were diluted with sample loading buffer, normally expressed in intrapulmonary airway epithelium, and very Downloaded from heated (5 min; 95˚C), and separated on a NuPAGE 12% Bis-Tris Gel (Invitrogen). Electrophoresis was performed at 70 V (20 min) and con- little protein could be detected in lung homogenate from control tinued at 140 V (80 min). For Western blot analysis, proteins were elec- littermates. Total PLUNC protein (mouse and human) in BALF troblotted onto nitrocellulose membrane (Pierce Biotechnology) in transfer was $10-fold greater in transgenic mice as compared with control buffer containing 25 mM Tris base, 0.2 M glycine, 20% methanol (pH littermates (Fig. 3). These results indicate that hPLUNC was ∼ 8.5). Membranes were blocked with 5% nonfat dry milk in TBST (1 h; highly expressed in intrapulmonary airways, and that it was ef- 23˚C), washed thrice with TBST (5 min), and incubated (18 h; 4˚C) with http://www.jimmunol.org/ goat anti-PLUNC Ab (1:1000 in blocking solution; R&D Systems). fectively secreted into the luminal space. Membranes were washed thrice with TBST and incubated (1 h; 23˚C) with mouse anti–goat-HRP Ab (1:10,000; Pierce Biotechnology). After three Enhanced antibacterial activity in BALF from washes with TBST, the membranes were developed using SuperSignal Scgb1a1-hPLUNC transgenic mice West Pico Chemiluminescent Substrate (Pierce Biotechnology). Densi- tometry was performed and ImageQuant software (GE Healthcare, Pis- To determine whether BALF from transgenic TG mice had in- cataway, NJ) was used to quantify PLUNC protein levels. creased antibacterial activity, we assessed P. aeruginosa (PAO1, ATCC BAA-47) colony formation in BALF obtained from WT Cytokine assay control and Scgb1a1-hPLUNC transgenic littermates. CFU de- Cytokine levels in BAL were quantified using mouse Cytokine Multiplex creased 2.9-fold in cultures treated with BALF from transgenic by guest on October 2, 2021 Panel assay (Bio-Rad). The expression of IL-1b, IL-2, IL-3, IL-4, IL-5, mice compared with that from control littermates (Fig. 4). Pre- IL-6, IL-10, IL-12(p40), IL-17, IFN-g, TNF-a, GM-CSF, KC, and MIP-1b immune normal rabbit serum IgG or rabbit anti-PLUNC IgG were was analyzed using the Luminex assay system. Standard recombinant protein solution was used to generate standard curves. Anti-cytokine Ab- preincubated with BALF and antibacterial assay was performed to conjugated xMAP beads and standard or test BALF proteins were added determine the contribution of hPLUNC and mouse PLUNC to to wells of a 96-well filter plate. The plate was incubated with shaking at this bactericidal activity. Ab neutralization by neutralizing anti- room temperature for 30 min, washed thrice with Bio-Plex wash buffer, PLUNC IgG decreased total bactericidal activity of WT control and 25 ml detection Ab was added. After incubation (30 min; 23˚C), the plate was washed and streptavidin solution added to each well. The plate BALF, indicating that endogenous mouse Plunc was responsible was incubated (10 min) with shaking, washed, and the beads resuspended for a portion of the activity (Fig. 4). In addition, the bactericidal in Bio-Plex assay buffer A. The plate was agitated (30 s) and absorbance activity of Ab-treated Scgb1a1-hPLUNC BALF was not different determined with a Bio-Plex Suspension Array System. Absolute cytokine from Ab-treated BALF from WT control littermates. These results concentrations were calculated from the standard curve for each cytokine. demonstrated that mPlunc is active in the airway, and that the Data analysis additional hPLUNC is secreted to and is active in the airway lumen in Scgb1a1-hPLUNC transgenic mice. Data are expressed as mean 6 SD. Statistical comparisons between the groups of mice were made using ANOVA (continuous and normally dis- mPlunc enhanced host defense in mouse lung against inhaled tributed data) or Poisson regression (discontinuous data). A p value ,0.05 was considered to be statistically significant. Determinations of the sig- P. aeruginosa nificance of the differences in survival outcomes between Scgb1a1- To evaluate the effect of inhaled P. aeruginosa on mPlunc ex- hPLUNC TG mice and WT control littermates after P. aeruginosa infection were calculated by Kaplan–Meier survival curve comparisons and pression, we assessed lung mRNA levels by qRT-PCR analysis. the p values derived from a log-rank test. PAO1 bacterial exposure significantly increased Plunc expression at both 6 (5.1-fold; p = 0.0002) and 24 h (2.5-fold; p = 0.021) postexposure (Fig. 5), but the induction of Plunc at 6 h post- Results exposure was higher than 24 h postexposure. To determine the Expression of hPLUNC protein in transgenic mouse functional significance of increased plunc in mouse lung after A schematic diagram of PLUNC TG construct using Scgb1a1 bacterial infection, we pretreated the mice with specific anti- promoter is represented in Fig. 1A. The tissue specificity of PLUNC Ab to neutralize the antimicrobial activity of PLUNC hPLUNC transcript expression was assessed by qRT-PCR analysis in mouse airways. The Ab concentration at either 10 or 25 mg did (Fig. 1B) and was detected exclusively in trachea and lung from not show any noticeable difference in affecting bacterial suscep- transgenic mice. This distribution paralleled that of SCGB1A1 tibility. However, a pretreatment on mice with either concentration mRNA and was consistent with previously reported specificity of of neutralizing Ab significantly increased the mouse susceptibility the mouse Scgb1a1 promoter. to PAO1-induced bacterial infection when compared with the The Journal of Immunology 385 Downloaded from

FIGURE 1. Generation and assessment of hPLUNC gene expression in Scgb1a1-PLUNC transgenic mice. A, TG construct. B, Real-time TaqMan RT- PCR analysis of hPLUNC mRNA abundance in tissues from WT and transgenic mice. Relative expression was determined by the DDCt method using mouse GUS-B RNA as a control (mean 6 SD; n = 10). hPLUNC was highly expressed in intrapulmonary airways of transgenic mice.

control mice that were pretreated with nonspeciﬁc IgG (Fig. 6). and deposition and clearance were monitored. The aerosolized http://www.jimmunol.org/ There was an at least 3-fold increase in bacterial burden of mouse exposure protocol resulted in a deposition of 2.26 6 0.39 3 106 lung when mice were pretreated with the neutralizing Ab. These CFU/lung P. aeruginosa and was consistent among exposures. data further supported the important role of PLUNC in host de- Immediately after exposure, bacterial deposition was equivalent fense against P. aeruginosa-induced lung infection. between transgenic mice and WT control littermates (Fig. 7). This exposure concentration enabled us to examine PLUNC-mediated Improved bacterial clearance in Scgb1a1-hPLUNC transgenic antimicrobial activity after a reversible respiratory infection. Be- mice against inhaled P. aeruginosa cause a whole-body exposure method was used, a systemic bac- To determine whether hPLUNC expression could alter bacterial infection in vivo, WT control and Scgb1a1-hPLUNC transgenic by guest on October 2, 2021 littermates were coexposed to saline or P. aeruginosa aerosols,

FIGURE 3. Expression and secretion of hPLUNC is significantly increased in Scgb1a1-PLUNC transgenic mice. A, Western blot analysis was used to assess protein expression of PLUNC in BAL and total lung ho- FIGURE 2. hPLUNC protein is expressed in bronchiolar airway epi- mogenate from 6-wk-old WT and Scgb1a1-PLUNC transgenic (TG) mice. thelial cells in Scgb1a1-PLUNC transgenic mice. Cellular localization of Proteins were separated by SDS-PAGE and blotted onto PVDF mem- PLUNC protein in mice was assessed by immunohistochemistry (A, B: branes. Blots were incubated with anti-PLUNC Ab, which detected both diaminobenzidine staining) and immunofluorescence microscopy (C, D: mouse PLUNC and hPLUNC. Recombinant protein (rPLUNC) was used Alexa 488; E, F: Alexa 594) on lung sections of wild-type (A, C, E) and as a positive control. GAPDH was used as a loading control for lung transgenic (B, D, F) mice using anti-PLUNC Ab (A–D) and anti- homogenates. Mouse SCGB1A1 was used as a control for blots of BAL SCGB1A1 Ab (E, F). Signal was not detected when parallel sections from samples. B, Quantitative results that represent PLUNC protein expression transgenic mice were incubated with preimmune goat serum (data not levels detected by Western blot analysis after normalization with GAPDH shown). PLUNC was detected in airway epithelial cells (arrowheads) of (lung) or SCGB1A1 (BAL). *p , 0.05 and **p , 0.05 when the trans- transgenic but not WT mice. Original magnification 3400 (A, B) and genic mouse group is compared with the WT mouse group with each re- 3100 (C–F). spective sample type (mean 6 SD; n = 8). 386 ANTIMICROBIAL ACTIVITY OF PLUNC AGAINST P. AERUGINOSA

FIGURE 4. BALF from transgenic mice exhibits enhanced antimicrobial activity. BALF from un- challenged WT and TG littermates (ﬁrst and second bars, respectively) was mixed with P. aeruginosa (PAO1) as described in Materials and Methods and CFU were assessed. Neutralizing Ab against PLUNC was preincubated with WT or TG BALF (third and fourth bars, respectively) for 1 h before mixing with bacteria and CFU counts were assessed. Results are the mean and SD from three independent experiments (n = 8 for each experiment). *p , 0.05, **p , 0.05, and ***p , 0.05 when the transgenic mouse group is compared with the control WT mouse group.

teremia was also assessed by determining the CFU in spleen ho- Decreased inflammatory cell infiltration in Scgb1a1-hPLUNC mogenates 24 h postinfection. Less than 500 CFU were detected transgenic mouse lung after inhaled P. aeruginosa Downloaded from in spleens of mice, and no statistical difference was detected be- BALF total cell count (2–4 3 105 cells/lung) or differentials tween the transgenic mice as compared with their control litter- ($99% macrophages) from unexposed Scgb1a1-hPLUNC trans- mates. genic mice were not different from those from WT control mice. Four hours postexposure, WT control mice became slightly Four hours after bacterial exposure, the total inflammatory cell lethargic and demonstrated signs of infection (e.g., ruffed fur and counts in BALF increased in both TG and WT control mice, but http://www.jimmunol.org/ hunched back), whereas Scgb1a1-hPLUNC transgenic littermates the increase was higher in WT mice than in TG mice. The in- displayed more active behavior and little observable signs of increased macrophage number between WT (3.1 6 1.4 3 106; n = ∼ , fection. The bacterial clearance was 3-fold greater (p 0.05) 18) and TG (3.6 6 1.6 3 106; n = 18) mice was not statistically in lung homogenates from Scgb1a1-hPLUNC transgenic mice different, but the total neutrophils in WT control mice (26.1 6 6 3 5 (2.31 0.41 10 CFU/lung/ml) as compared with that from 2.8 3 106; n = 18) were slightly greater (∼1.44-fold; p , 0.05) 6 3 5 WT control mice (6.83 0.57 10 CFU/lung/ml) (Fig. 7A). At than from Scgb1a1-hPLUNC transgenic mice (18.1 6 2.3 3 106; 24 h after exposure, WT control and Scgb1a1-hPLUNC trans- n = 18) (Fig. 7B). However, 24 h postexposure, the total neutro- genic mice exhibited similar activity levels. However, bacteria phils were .2-fold (p , 0.01) greater in BALF from WT control clearance was ∼15-fold greater (p , 0.05) in lung homogenates 6 3 6 mice (16.2 2.0 10 ; n = 18) as compared with BALF from by guest on October 2, 2021 6 3 4 from Scgb1a1-hPLUNC transgenic mice (2.6 0.29 10 CFU/ Scgb1a1-hPLUNC transgenic mice (6.90 6 0.6 3 106; n = 18) 6 lung/ml) as compared with that from WT control mice (3.9 (Fig. 7B). There were no significant differences in the total mac- 3 5 0.34 10 CFU/lung/ml) (Fig. 7A). Thus, control mice had rophage number between TG and WT mice at 24 h after PAO1- cleared 80%, whereas transgenic mice had cleared 98% of the induced bacterial infection. These observations were supported by initial bacterial burden. These data suggest that PLUNC acts early in infection and promotes efficient clearance of the initial bacterial inoculum.

FIGURE 6. Pretreatment with anti-PLUNC Ab increases susceptibility FIGURE 5. Exposure to P. aeruginosa increases the mRNA expression of WT mice to bacterial infection. WT FVB/N mice were intranasally of mPlunc. Murine Plunc expression was analyzed 6 and 24 h after instilled (10 and 25 mg/mouse) with control rabbit IgG or neutralizing aerosolized exposure of P. aeruginosa PAO1 strain to FVB/N WT mice, mouse anti-PLUNC Ab (nAb) 2 h before exposure to ∼2 3 106 CFU/lung and all data are expressed as fold induction over saline-exposed mice for aerosolized P. aeruginosa (PAO1). The number of bacteria in lung time point at 6 h postexposure. Levels of mPlunc were quantiﬁed in lung homogenates was determined 24 h after bacterial exposure. Anti-PLUNC homogenates by real-time RT-PCR and determined by the DDCt method Ab treatment resulted in a signiﬁcant increase in CFU counts compared using mouse GUS-B RNA as a control. Results are mean 6 SEM from two with IgG control. Results are mean 6 SEM from two experiments; n =5 experiments; n = 6–8 mice for each group. *p , 0.001 and **p , 0.05 mice for each group. *p , 0.05 and **p , 0.05 when two different when the PAO1-exposed mouse group is compared with the saline-exposed neutralization Ab concentrations were compared with their respective IgG mouse group at 6 and 24 h postexposure. control group. The Journal of Immunology 387

FIGURE 7. Scgb1a1-PLUNC transgenic mice are resistant to P. aeruginosa-induced lung infection. Age-matched WT and TG littermates were infected with ∼2 3 106 CFU P. aeruginosa (PAO1) per lung by inhalation. A, CFU in lung homogenates were determined at the indicated recovery time points. *p , 0.01 for WT to TG comparisons. B, Total neutrophils in BALF from WT compared with TG mice 4 and 24 h after bacterial exposure (*p , 0.05). Total neutrophils were signiﬁcantly lower in TG mice 24 h postinfection (**p , 0.01). Results are mean 6 SD (n = 5–6) from three independent experiments. histological analysis of lung tissue. Lungs from uninfected WT hPLUNC transgenic mice as compared with that from WT control control mice (Fig. 8A) were not different from those from mice (Fig. 9A). At 24 h postexposure, the difference was greater Scgb1a1-hPLUNC transgenic mice (Fig. 8C). However, 24 h after and additional cytokines including IL-6 (Fig. 9B) and chemotactic

bacterial exposure, lung tissues from WT control mice (Fig. 8B) cytokines, chemokine (C-X-C motif) ligand 1 (also known as KC; Downloaded from contained more areas of focal peribronchial and alveolar neutro- Fig. 9C) and chemokine (C-X-C motif) ligand 2 (also known as philic infiltrates than lungs from Scgb1a1-hPLUNC transgenic MIP-2; Fig. 9D), were significantly lower in BALF from Scgb1a1- littermates (Fig. 8D). hPLUNC transgenic mice as compared with that from WT control mice. We did not observe significant differences in the production Decreased proinflammatory cytokines in Scgb1a1-hPLUNC of IL-2, IL-3, IL-10, and IL-12(p40) in steady-state or P. aeru- transgenic mice after P. aeruginosa exposure ginosa-challenged WT and Scgb1a1-hPLUNC transgenic mice http://www.jimmunol.org/ BALF samples were collected and cytokine concentrations were (data not shown). determined by Bio-Plex assay to determine whether production of inflammatory cytokines varied 4 or 24 h postexposure. Without Enhanced survival in Scgb1a1-hPLUNC transgenic mice after bacterial exposure, BALF cytokine levels from WT control mice P. aeruginosa exposure were not different from those from Scgb1a1-hPLUNC transgenic To determine whether PLUNC affects susceptibility to P. aeru- mice, and most cytokine levels were not detected (data not ginosa-induced lung infection, groups of female 6- to 8 wk-old shown). Postexposure, the spectrum of BALF cytokines detected WT and Scgb1a1-hPLUNC transgenic mice were challenged did not vary by genotype, but the magnitude of the response was through intratracheal instillation with various doses (107,108, and 9 consistently lower in BALF from Scgb1a1-hPLUNC transgenic 10 CFU/mouse) of P. aeruginosa PAO1. We did not observe any by guest on October 2, 2021 mice as compared with that from WT control mice (Fig. 9). At 4 h mortality when concentrations of PAO1 at 107 and 108 CFU/lung postexposure, IL-1b levels were lower in BALF from Scgb1a1- were administered (data not shown). When mice were challenged with 109 CFU/animal of PAO1, Scgb1a1-hPLUNC transgenic mice displayed enhanced survival and decreased lethality (log- rank test, p = 0.001; Fig. 10). All WT mice succumbed to PAO1-induced lung infection within 48 h (mean survival time, 41.6 6 1.3 h), whereas 5 of 10 Scgb1a1-hPLUNC transgenic mice survived the challenge and were still alive 1 wk postinfection. These results indicate that the overexpressed PLUNC is essential to an enhanced survival and resistance to P. aeruginosa infection. Discussion Effective host defense against microbial invasion requires an innate immune system whose response is both rapid and independent of prior exposure (18). Several secreted proteins participate in this innate immune response (19), and data presented in this article indicate that PLUNC is a member of this group. In this study, we successfully expressed hPLUNC protein in the airway epithelium of transgenic mice using the mouse Scgb1a1 promoter. Scgb1a1- hPLUNC transgenic mice expressed hPLUNC mRNA and hPLUNC protein in the airway epithelium and secreted hPLUNC was detected in BALF. Expression of hPLUNC did not alter lung FIGURE 8. Decreased neutrophilic infiltration Scgb1a1-PLUNC mice structure or function, nor did it change the expression and secre- after bacterial exposure. WT and Scgb1a1-PLUNC mice were exposed to tion of the major mouse airway secretory protein SCGB1A1. saline (A, C) or aerosolized P. aeruginosa (B, D). Lung tissues were har- However, hPLUNC was associated with enhanced bacterial killing vested at 24 h after exposure, fixed and H&E-stained for histological evaluation. No histological differences were observed between WT and TG and decreased inflammation after exposure with a major human mice before bacterial exposure. After bacterial exposure, TG mice showed airway pathogen, the Gram-negative bacteria P. aeruginosa less neutrophil infiltration. Original magnification 340. Sections are rep- (PAO1). Our results provide evidence of a role for PLUNC in resentative of at least five mice studied at each time point from each in- mitigation of bacterial infection both in vitro and in vivo. Al- fection. though other pathogens were not tested in the study, the transgenic 388 ANTIMICROBIAL ACTIVITY OF PLUNC AGAINST P. AERUGINOSA Downloaded from http://www.jimmunol.org/

FIGURE 9. Decreased proinflammatory cytokine production in Scgb1a1-PLUNC mice after PAO1 exposure. WT and TG mice were infected with P. aeruginosa as described. Cytokines concentrations in BALF were measured using a Bio-Plex assay and are reported in pg/ml. Values were determined for 4 and 24 h after bacterial exposure. Results from the measurements of IL-1b (A) and MIP2 (D) showed significance between WT and TG mice both 4 h and 24 h after bacterial challenge. Measurements of IL-6 (B) and KC (C) only exhibited statistical significance between WT and TG mice 24 h after bacterial challenge. Striped bars represent WT mice; solid bars represent Scgb1a1-PLUNC TG mice. Results are mean 6 SD from three experiments (n = 5–6 mice). *p , 0.05, **p , 0.05 for WT to TG comparisons at each time point. by guest on October 2, 2021 mice generated in this study represent a useful animal model for PLUNC belongs to the short-peptide subfamily of PLUNC family future investigations to identify PLUNC-mediated antimicrobial proteins and has homology to the N-terminal domains of BPI (23– activity in the airway. 25). Peptides derived from this region of BPI have previously been Several lines of evidence suggest that PLUNC-mediated anti- shown to possess direct bactericidal activity (23, 26). Using a bio- bacterial activity is an important aspect of the innate immune activity assay to test antimicrobial functions of secretory proteins response in the airway. First, PLUNC is structurally related to BPI in BALF, we demonstrated a significant decrease in CFU counts of and LBP, proteins that have been demonstrated to be important to P. aeruginosa when bacteria were coincubated with BALF from host defense against Gram-negative bacteria (20–22). Second, Scgb1a1-hPLUNC transgenic mice. Furthermore, both in vitro and

FIGURE 10. Overexpressed hPLUNC in Scgb1a1-PLUNC mice enhances the survival from PAO1-induced acute pneumonia. WT and TG mice were infected with P. aeruginosa as described at a concentration of 1 3 109 CFU/mouse of PAO1 strain. Survival is represented by Kaplan–Meier survival curves, and there was a statistically significant difference between survival curves of WT and TG groups (p = 0.001, log rank test). The Journal of Immunology 389 in vivo Ab neutralization studies indicated that the increased an- and epithelial cells after exposure to aerosolized P. aeruginosa. tibacterial activity was due to PLUNC. Therefore, the decreased Normally, after recognition of microbial products, TLR-mediated sensitivity in Scgb1a1-hPLUNC transgenic mice may have par- signaling results in the production of TNF-a and IL-1b, two early- tially attributed to the direct killing activity. These data suggested responsive cytokines that regulate subsequent recruitment of neu- that expression of hPLUNC in mouse airways increased mouse trophils (34–36). These substances usually play beneficial roles in resistance to pathogens. the body’s defense systems. However, excess cytokine production Because BPI is normally expressed in inflammatory cells but not may contribute to an exacerbated inflammatory response and could airway epithelial cells, these data may also suggest that critical even contribute to severe inflammation and dysfunction in other aspects of the innate response can be mediated by similar proteins organs if these inflammatory mediators are released excessively that are secreted by different cell types. Moreover, the augmen- into the bloodstream. Therefore, a well-regulated and balanced tation of protection by the additional hPLUNC suggests that production of inflammatory mediators is critical to an effective PLUNC or BPI congeners could provide a novel anti-infection local and systemic host defense. In our studies, proinflammatory therapy. PLUNC-mediated antibacterial activity highlights its cytokines such as IL-1b, IL-6, and TNF-a were nearly undetect- functional significance and supports the notion that PLUNC plays able before bacterial exposure but were significantly increased an important role in innate immunity against respiratory infection. after challenge. Enhanced clearance of inhaled bacteria in Scgb1a1- PLUNC is one of the most abundant secretory proteins in nasal PLUNC mice also correlated with decreased production of pro- drainage, BALF, and in primary epithelial cell cultures (6, 27, 28). inflammatory cytokines (including IL-1b, TNF-a, and IL-6) after The significant amount of PLUNC secreted into the airway lumen bacterial challenge. Another proinflammatory chemokine, KC, has and its antibacterial activity indicated that PLUNC plays a critical been shown to substantially increase the accumulation of neu- role in airway epithelial cell-based innate immunity. The enhanced trophils in the lungs after the intratracheal administration of LPS Downloaded from antibacterial activity in Scgb1a1-PLUNC transgenic mice corre- (37), and we observed significantly lower levels of KC secretion lated with expression of hPLUNC in mouse airways. The mag- in BALF from Scgb1a1-PLUNC transgenic mice when compared nitude of enhanced antibacterial activity (∼3-fold) was similar to with WT control mice after P. aeruginosa exposure. The lower previously reported antibacterial activity for lysozyme, a known amount of KC in BALF of Scgb1a1-PLUNC transgenic mice might antimicrobial protein in airway secretions (29). Because PLUNC also partially account for decreased neutrophil infiltration and later is an abundant component of the antimicrobial fraction of nasal resulted in lesser damage to the lungs in transgenic mice as com- http://www.jimmunol.org/ secretions (30), is present at high levels in airway secretions pared with their WT littermates. Although the decrease in proin- (6, 27, 28, 31, 32), is expressed at levels similar to that of lyso- flammatory cytokine production noted in P. aeruginosa-exposed zyme in tracheobronchial submucosal glands, and has antimicro- Scgb1a1-PLUNC transgenic mice is likely to be a consequence bial activity comparable with that of lysozyme, it is reasonable of antibacterial activities of hPLUNC, it is also possible that the to propose that PLUNC is an important component of the innate lower cytokine levels in Scgb1a1-PLUNC transgenic mice could response to airway pathogens that originates from airway epi- be because of unanticipated roles for hPLUNC in regulation of cy- thelial cells. It has been suggested that PLUNC is a novel airway tokine production in epithelial or inflammatory cells, or both. Fur- secretory protein with a surfactant protein function that modulates ther studies to identify potential bioactivity of PLUNC in association by guest on October 2, 2021 surface tension and inhibits biofilm formation (14). Therefore, one with inflammatory response are worthy of future investigation. of the potential PLUNC-mediated antimicrobial mechanisms may Our data suggest that expression of hPLUNC provides an airway be overexpressed hPLUNC in Scgb1a1-PLUNC transgenic mice epithelial cell-specific protection against opportunistic pathogens that enhanced the mucociliary clearance and disrupted the biofilm such as P. aeruginosa. Although the major difference between formation of P. aeruginosa after PAO1 challenged respiratory transgenic mice and their WT littermates is most likely due to infection. The enhanced survival of Scgb1a1-PLUNC transgenic expression of hPLUNC, we could not rule out the possibility that mice compared with their WT littermates after a high dose of other antimicrobial proteins/peptides may also act synergistically P. aeruginosa challenge further indicates an important antimi- with ectopically expressed hPLUNC to enhance the bacterial crobial activity of PLUNC to P. aeruginosa-induced lung infection killing effect. The enhanced bacterial clearance observed in and supports the notion that PLUNC plays a critical role in airway Scgb1a1-PLUNC transgenic mice may be because of interaction epithelium-mediated innate immune response. of overexpressed PLUNC with other antimicrobial peptides such PLUNC may possess anti-inflammatory activities that are im- as defensins and/or antimicrobial proteins such as lysozyme to portant in modulating airway innate immune response. Our data potentiate its antibacterial activities. demonstrate a lower degree of neutrophil recruitment and less In conclusion, this study demonstrated antibacterial activity of lung inflammation in PAO1-exposed Scgb1a1-PLUNC transgenic PLUNC against airway bacterial pathogens using both in vitro and mice than their WT littermates and suggest that constitutive in vivo approaches. We also demonstrated that Scgb1a1-PLUNC overexpression of hPLUNC in mouse airways resulted in more transgenic mice exhibited enhanced survival and improved re- efficient bacterial killing. The decreased inflammation noted in sistance to P. aeruginosa infection, one of the most common Scgb1a1-PLUNC transgenic mice could have been a direct con- airway infections associated with chronic colonization in cystic sequence of more efficient bacterial clearance. The potential fibrosis patients. Given the emergence of highly resistant bacteria binding of PLUNC to LPS may modulate LPS-mediated in- pathogens and the increasing population of immunocompromised flammatory response. It has been suggested that PLUNC binds patients, the treatment of bacterial infection has and will continue directly to LPS from Escherichia coli (28). A structurally related to be challenging. A better understanding of airway epithelial cell- protein murine parotid secretory protein (PSP), also called initiated host defense may provide an alternative approach to ef- SPLUNC2, has been shown to bind to bacterial membranes (33). ficiently combat airway bacterial infection. Cytokines play an important role in regulation and modulation of immunological and inflammatory processes. Unlike an in vitro Acknowledgments cell culture system that represents a single cell type, the use of We thank Dr. Jonathan Chao for making constructs for the generation of a transgenic mouse model provided an opportunity to identify Scgb1a1-PLUNC transgenic mice and Dr. Thomas Zhao for technical as- coordinated changes in cytokine production between inflammatory sistance in dissecting mice. 390 ANTIMICROBIAL ACTIVITY OF PLUNC AGAINST P. AERUGINOSA

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