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 References This article cites 37 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/187/1/382.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 respira- tory 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, bronchoal- veolar 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 ep- ithelium 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