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Lack of Whey Acidic Protein (WAP) Four-Disulfide Core Domain Protease Inhibitor 2 (WFDC2) Causes Neonatal Death from Respiratory

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Lack of Whey Acidic Protein (WAP) Four-Disulfide Core Domain Protease Inhibitor 2 (WFDC2) Causes Neonatal Death from Respiratory © 2019. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2019) 12, dmm040139. doi:10.1242/dmm.040139 RESEARCH ARTICLE Lack of whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (WFDC2) causes neonatal death from respiratory failure in mice Kuniko Nakajima1, Michio Ono1,UrošRadović1, Selma Dizdarević1, Shin-ichi Tomizawa1, Kazushige Kuroha1, Go Nagamatsu2, Ikue Hoshi1, Risa Matsunaga1, Takayuki Shirakawa1,*, Takeyuki Kurosawa3, Yasunari Miyazaki4, Masahide Seki5, Yutaka Suzuki5, Haruhiko Koseki6, Masataka Nakamura7, Toshio Suda8,9 and Kazuyuki Ohbo1,‡ ABSTRACT INTRODUCTION Respiratory failure is a life-threatening problem for pre-term and term During the transition from fetal to neonatal life, the cardio-respiratory infants, yet many causes remain unknown. Here, we present evidence system undergoes a drastic and sudden change: placental circulation that whey acidic protein (WAP) four-disulfide core domain protease switches to a pulmonary circulation and the respiratory system goes inhibitor 2 (Wfdc2), a protease inhibitor previously unrecognized in from being fluid filled to air filled. Malfunctions of this switch can respiratory disease, may be a causal factor in infant respiratory failure. cause respiratory failure (Reuter et al., 2014), in particular in premature Wfdc2 transcripts are detected in the embryonic lung and analysis of a infants in whom the immature lung tissues have produced inadequate Wfdc2-GFP knock-in mouse line shows that both basal and club cells, mucosal fluid (Reuter et al., 2014). The components of this fluid are and type II alveolar epithelial cells (AECIIs), express Wfdc2 neonatally. produced by respiratory epithelial cells. There are at least eight different Wfdc2-null-mutant mice display progressive atelectasis after birth with types of these cells, which form the primary barrier between air and a lethal phenotype. Mutant lungs have multiple defects, including lung tissue (Hogan et al., 2014; Montoro et al., 2018; Plasschaert et al., impaired cilia and the absence of mature club cells from the tracheo- 2018; Rock and Hogan, 2011; Treutlein et al., 2014). In the airways, bronchial airways, and malformed lamellar bodies in AECIIs. RNA the epithelium consists of basal stem cells, and ciliated, club, goblet sequencing shows significant activation of a pro-inflammatory and neuroendocrine cells, as well as other rare cell types, including pathway, but with low-quantity infiltration of mononuclear cells in the newly described ionocytes (Montoro et al., 2018; Plasschaert et al., lung. These data demonstrate that Wfdc2 function is vitally important 2018). The goblet and club cells secrete mucins and glycoproteins that for lung aeration at birth and that gene deficiency likely causes failure of have bactericidal and anti-inflammatory functions; the ciliated cells the lung mucosal barrier. move the mucus and entrap particles out of the lungs; and the ionocytes, which highly express cystic fibrosis transmembrane KEY WORDS: Cilia, Surfactant, Atelectasis, Respiratory disease conductance regulator (CFTR), likely play a role in regulating fluid secretion (Montoro et al., 2018; Plasschaert et al., 2018). In the alveolar region, the type II epithelial cells (AECIIs) produce important surfactants that consist of lipids [e.g. phosphatidylcholine (PC), 1Department of Histology and Cell Biology, Yokohama City University, School of phosphatidylglycerol (PG)] and proteins (e.g. SP-A, -B, -C, -D), which Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan. 2Department are stored in specialized organelles known as lamellar bodies of Stem Cell Biology, Kyushu University, Faculty of Medical Sciences, 3-1-1, Maidashi, Higashi-ku, Fukuoka City, 812-8582, Japan. 3Department of Respiratory (Chakraborty and Kotecha, 2013; Lopez-Rodriguez and Pérez-Gil, Medicine, Toho University, School of Medicine, 5-21-16, Ohmorinishi, Ohta-ku, 2014; Olmeda et al., 2017; Pérez-Gil, 2008; Rooney et al., 1994; Tokyo, Japan. 4Department of Respiratory Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. 5Department of Whitsett and Alenghat, 2015). Surfactants form thin films that reduce Computational Biology and Medical Sciences, Graduate School of Frontier surface tension, open up the alveolar space, and are critical for both Sciences, The University of Tokyo, Chiba, 277-8562, Japan. 6Laboratory for preventing collapse of the airways (atelectasis) and for contributing to Developmental Genetics, RIKEN Center for Integrative Medical Sciences, 1-7-22, Tsurumi-ku, Yokohama, 230-0045, Japan. 7Human Gene Sciences Center, Tokyo the host defense strategy (Chakraborty and Kotecha, 2013; Rooney Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, et al., 1994; Whitsett and Alenghat, 2015; Whitsett et al., 2010). Japan. 8Cancer Science Institute of Singapore, National Singapore University In addition to mucins and surfactants, the epithelial cells of the Centre for Translational Medicine, 14 Medical Drive, #12-01, Singapore 117599. 9International Research Center for Medical Sciences, Kumamoto University, 2-2-1 lung secrete numerous proteases and protease inhibitors – such as Honjo, Chuo-ku, Kumamoto, 860-0811, Japan. serine proteases, matrix metalloproteinases (MMPs), serine protease *Present address: Mitsubishi Tanabe Pharma, Yokohama office, 1000, Kamoshida- – cho, Aoba-ku, Yokohama, 227-0033, Japan. inhibitors and tissue inhibitor of metalloproteinases (TIMPs) that together form the protease/protease-inhibitor web (Löffek et al., ‡Author for correspondence ([email protected]) 2011; Abboud, 2012; Meyer and Jaspers, 2015). These proteases and M.O., 0000-0001-9054-5298; S.T., 0000-0002-8291-6623; G.N., 0000-0002- protease inhibitors are deeply involved in the pathophysiology of 4901-8514; T.K., 0000-0002-8640-8853; Y.M., 0000-0002-9073-9815; Y.S., 0000- neonatal respiratory distress syndrome (RDS) as well as other lung 0002-7559-5139; H.K., 0000-0001-8424-5854; M.N., 0000-0002-9797-0673; T.S., diseases such as emphysema, cystic fibrosis, infection and pulmonary 0000-0001-7540-1771; K.O., 0000-0001-5319-7319 fibrosis (Meyer and Jaspers, 2015; Taggart et al., 2017). RDS is This is an Open Access article distributed under the terms of the Creative Commons Attribution mainly caused by pulmonary surfactant deficiency in premature License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. infants, and the lungs of RDS patients are diffusely atelectatic (Bhandari, 2014; Reuter et al., 2014). A subpopulation of RDS Received 12 April 2019; Accepted 11 September 2019 infants who show limited responses to surfactant may go on to Disease Models & Mechanisms 1 RESEARCH ARTICLE Disease Models & Mechanisms (2019) 12, dmm040139. doi:10.1242/dmm.040139 develop chronic neonatal lung injury known as bronchopulmonary (P)1.5. Transcripts were already expressed at E11.5 and were strongly dysplasia (BPD), which involves failure of alveogenesis and upregulated at P1.5 (Fig. 1A). Analysis of lungs 6-8 h after caesarean vasculogenesis, and the risk of developing long-term morbidities section at E18.5 revealed that mRNA expression rose significantly (Bhandari, 2014; Dumpa and Bhandari, 2018). Inflammation is a after respiration began (Fig. 1A). To track the lung epithelial cells that cornerstone in the transition from RDS to BPD (Bhandari, 2014); its produce WFDC2, we generated knock-in mouse lines driving either causes include hyperoxia and mechanical stress from ventilation, but GFP or lacZ from the Wfdc2 locus (Fig. S1A-D). In agreement with proteases such as neutrophil elastase (NE) have also been shown to the mRNA expression data, Wfdc2-GFP embryos showed signal from play crucial roles in the induction of IL-1α and TNFβ (Bhandari, E14.5 (the pseudoglandular stage) in the proximal region of the 2014). However, the failure of α-antitrypsin therapy trials in human bronchial tubes. The GFP-positive cells were located in the mesial infants implies that other proteases and/or protease inhibitors may part of the Sox2-positive proximal region (Fig. 1B), and few, if any, participate in the pathogenesis of both RDS and BPD, although a were seen in the distal, Sox9-positive, region (Fig. 1C). possibility remains that drug delivery efficiency to the affected areas Mice heterozygous for the GFP knock-in allele (Wfdc2GFP/+) were might need to be improved (Beam et al., 2014). indistinguishable in lifespan and phenotype from wild-type One of the serine protease inhibitor families predicted to function littermates (Table 1). By contrast, the frequency of Wfdc2GFP/GFP in the innate immune system in the lung is the evolutionarily mice followed Mendel’s laws until birth, but thereafter declined, and conserved group of whey acidic protein (WAP) four-disulfide core none survived post-weaning (Table 1). Wfdc2GFP/GFP neonatal mice (FDC) proteins (WFDC protein family; Bingle and Vyakarnam, became cyanotic immediately after delivery (Movie 1), and all died 2008). The WFDC signature – eight conserved cysteine residues within 10 days (Table 2), suggesting the cause of death was either linked by four disulfide bonds – is often found in antimicrobial and heart failure or respiratory failure. Hearts from Wfdc2GFP/GFP mice did antifungal molecules (Jia et al., 2017; Scaletta et al., 2017), and not show any abnormality in volume or structure (Fig. S1E).
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