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A Role for Whey Acidic Protein Four-Disulfide-Core 12 (WFDC12)

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A Role for Whey Acidic Protein Four-Disulfide-Core 12 (WFDC12) Thorax Online First, published on March 13, 2015 as 10.1136/thoraxjnl-2014-206488 Critical care ORIGINAL ARTICLE Thorax: first published as 10.1136/thoraxjnl-2014-206488 on 13 March 2015. Downloaded from A role for whey acidic protein four-disulfide-core 12 (WFDC12) in the regulation of the inflammatory response in the lung Arlene M A Glasgow,1 Donna M Small,1 Aaron Scott,1 Denise T McLean,1 Nicolas Camper,1 Umar Hamid,1 Shauna Hegarty,2 Dhruv Parekh,3 Cecilia O’Kane,1 Fionnuala T Lundy,1 Paul McNally,4 J Stuart Elborn,1 Danny F McAuley,1 Sinéad Weldon,1 Clifford C Taggart1 ▸ Additional material is ABSTRACT published online only. To view Introduction Secretory leucocyte protease inhibitor Key messages please visit the journal online fi (http://dx.doi.org/10.1136/ and ela n are members of the whey acidic protein thoraxjnl-2014-206488) (WAP), or WAP four disulfide-core (WFDC), family of proteins and have multiple contributions to innate 1 What is the key question? Centre for Infection and defence including inhibition of neutrophil serine ▸ Does whey acidic protein four-disulfide-core 12 Immunity, Queen’s University fl Belfast, Belfast, UK proteases and inhibition of the in ammatory response to (WFDC12) possess innate defence properties 2Department of Pathology, lipopolysaccharide (LPS). This study aimed to explore similar to other WFDC proteins in the lung such Royal Victoria Hospital, Belfast, potential activities of WFDC12, a previously as secretory leucocyte protease inhibitor and UK fi 3 uncharacterised WFDC protein expressed in the lung. ela n? College of Medical and Dental Methods Recombinant expression and purification of Sciences, University of WFDC12 were optimised in Escherichia coli. What is the bottom line? Birmingham, Birmingham, UK ▸ 4Our Lady’s Children’s Hospital Antiprotease, antibacterial and immunomodulatory We have functionally characterised a member Crumlin, Dublin, Ireland activities of recombinant WFDC12 were evaluated and of the WFDC family of proteins, WFDC12, and levels of endogenous WFDC12 protein were have shown it to possess antiprotease and Correspondence to characterised by immunostaining and ELISA. anti-inflammatory activities and to be present Dr Sinéad Weldon, Centre for at elevated levels in the respiratory tract of Infection and Immunity, Health Results Recombinant WFDC12 inhibited cathepsin G, Sciences Building, Queen’s but not elastase or proteinase-3 activity. Monocytic cells patients with acute respiratory distress University Belfast, 97 Lisburn pretreated with recombinant WFDC12 before LPS syndrome. Road, Belfast BT9 7AE, UK; stimulation produced significantly lower levels of the http://thorax.bmj.com/ [email protected] fl Why read on? pro-in ammatory cytokines interleukin-8 and monocyte ▸ Given its properties we speculate that WFDC12 SW and CCT are joint senior chemotactic protein-1 compared with cells stimulated may play a role in protecting lung tissue during authors. with LPS alone. Recombinant WFDC12 became inflammation and infection. conjugated to fibronectin in a transglutaminase-mediated Received 24 October 2014 Revised 9 January 2015 reaction and retained antiprotease activity. In vivo Accepted 10 February 2015 WFDC12 expression was confirmed by immunostaining of human lung tissue sections. WFDC12 levels in human fl innate immune response via their antiprotease, anti- on September 29, 2021 by guest. Protected copyright. bronchoalveolar lavage uid from healthy and lung- microbial and anti-inflammatory activities. As a injured patients were quantitatively compared, showing result, they have each been involved in clinical WFDC12 to be elevated in both patients with acute trials as potential therapeutic agents to aid the respiratory distress syndrome and healthy subjects treated control of inflammation.23 with LPS, relative to healthy controls. The small molecular weight proteins SLPI and Conclusions Together, these results suggest a role for elafin are found constitutively expressed in lung this lesser known WFDC protein in the regulation of fl epithelia and other mucosal surfaces, demonstrating lung in ammation. upregulation in response to a range of proinflam- matory stimuli such as bacterial lipopolysaccharide (LPS), neutrophil elastase, interleukin (IL)-1β and tumour necrosis factor α.4 They are also produced INTRODUCTION by inflammatory cells such as neutrophils and Members of the whey acidic protein (WAP), or macrophages.4 WAP four-disulfide-core (WFDC), family of human SLPI and elafin each function as inhibitors of proteins are defined by the presence of one or mul- elastase, with elafin also inhibiting proteinase-3.4 tiple WAP domains—a40–50 amino acid sequence SLPI shows no inhibition of proteinase-3 but To cite: Glasgow AMA, containing eight conserved cysteine residues that instead is active against cathepsin G, trypsin and Small DM, Scott A, et al. fi 1 4 Thorax Published Online fold to form four disul de bonds. The most chymotrypsin. Both proteins are bactericidal First: [please include Day studied members of this group of proteins are against Gram negative and Gram positive species of Month Year] doi:10.1136/ secretory leucocyte protease inhibitor (SLPI) and bacteria56and display notable antifungal46and thoraxjnl-2014-206488 elafin, both of which contribute significantly to the antiviral effects.78SLPI and elafin also play Glasgow AMA, et al. Thorax 2015;0:1–7. doi:10.1136/thoraxjnl-2014-206488 1 Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd (& BTS) under licence. Critical care important immunomodulatory roles through their ability to Cross-linking of rWFDC12 to fibronectin and evaluation of Thorax: first published as 10.1136/thoraxjnl-2014-206488 on 13 March 2015. Downloaded from reduce the host response to LPS. This is understood to occur by antiprotease activity of fibronectin-bound rWFDC12 – both extracellular and intracellular mechanisms.9 12 Transglutamination reactions were based on previously described The role of most other WFDC proteins remains largely methods,16 17 with minor modifications using rWFDC12 and unknown. Our focus here is on WFDC12 (also known as fibronectin (see online supplement). WAP2). Without the predicted signal peptide, the mature 88 amino acid protein has an estimated molecular weight of 9.7 kDa and an isoelectric point of 5.77.13 It has an N-terminal Immunostaining of human tissue from an ex vivo lung WAP domain and a C-terminal region showing no significant perfusion model homology with any currently recognised motifs.13 The Lungs harvested from potential donors which were found to be WFDC12 gene is found highly expressed in lung, skin, prostate unsuitable for transplantation were obtained from International and oesophageal tissue.13 While the murine homologue protein Institute for Advancement of Medicine. Ethical approval for the SWAM2 has been shown to have antibacterial properties,14 use of these samples was obtained from the Queen’s University there has been no data on the function of human WFDC12 Belfast School of Medicine, Dentistry and Biomedical Science reported in the literature. Research Ethics Committee (Reference Number 12.17). The The primary aim of this research was to investigate if lungs were perfused ex vivo based on previously described WFDC12 harboured host defence properties akin to SLPI and methods.18 Briefly, the lungs were initially rewarmed over a elafin. Recombinant expression and purification of WFDC12 in period of 1 h by perfusion of the pulmonary artery using Escherichia coli were first optimised, followed by evaluation of Dulbecco’s modified Eagle’s medium (high glucose, 4.5 g/L) the protein’s antiprotease, antibacterial and immunomodulatory containing 5% albumin, which was contained in a reservoir sur- actions. Recombinant WFDC12 (rWFDC12) was found to rounded by a heated water bath (38°C). The lungs were gently inhibit activity of the serine protease cathepsin G. No antibac- inflated with CPAP (10 cmH2O, 95% O2,5%CO2). Fresh terial ability was observed, but rWFDC12 inhibited whole human blood (anticoagulated using acid citrate dextrose) LPS-induced cytokine production in THP-1 monocytic cells. was added to the perfusate once physiological temperatures Similar to SLPI and elafin, rWFDC12 was found to be capable were reached, and LPS (6 mg) was then introduced intrabronchi- of binding the extracellular matrix protein, fibronectin, and ally into a preselected lobe via a PE catheter (BD, 240 tubing). retained its antiprotease activity. Finally, we show WFDC12 Experimentation ceased 4 h after LPS instillation, upon which expression in human lung tissue via immunohistochemistry and tissue samples for histology were taken from both non-LPS quantify WFDC12 levels in supernatants from LPS-treated (control) and LPS-perfused lobes. Immunostaining methods for THP-1 cells and bronchoalveolar lavage fluid (BALF) from a WFDC12 and selected cell markers (epithelial cells: pan cyto- range of patient cohorts. keratin, macrophages: CD68, neutrophils: neutrophil elastase) are detailed in the online supplement. METHODS Recombinant WFDC12 expression and purification Human BALF collection and measurement of WFDC12 http://thorax.bmj.com/ The mature WFDC12 cDNA sequence was cloned by Gene Art WFDC12 levels were evaluated in BALF samples obtained from (Life Technologies, Paisley, UK) into a pQE30 vector (Qiagen, 0 a number of different clinical trials: Samples from patients West Sussex, UK) with a six histidine coding sequence 5 to the within 48 h of acute respiratory distress syndrome (ARDS) onset cloning region. His-tagged
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