Introduction
Since the first description of cystic fibrosis (CF) in 1938 the median age of survival has increased progressively, reaching over 40 years in developed countries [
1]. Progression of CF is characterized by inflammation, viral, and bacterial infection, and deterioration of lung function due to structural changes [
2]. Accordingly, pulmonary tissues in advanced stages show pronounced inflammation resulting in increased pulmonary symptoms and decreased lung function [
3]. Furthermore, infections with distinct bacteria, e.g., Staphylococcus aureus and Pseudomonas aeruginosa as well as general pulmonary bacterial load contribute to airway inflammation and lung damage [
4]. Therefore, many efforts have been made to reduce inflammation and bacterial colonization in children with CF. Previous research has used mice with airway-specific overexpression of the β-subunit of the epithelial Na+ channel (βENaC; encoded by the
Scnn1b gene) showing a CF-like phenotype with increased absorption of Na+ and fluid from the airway lumen, increased mucus concentration, delayed mucus transport and mucus adhesion. In analogy to humans with CF, these mice suffer from airway surface dehydration leading to mucus obstruction, chronic inflammation, reduced bacterial clearance, and emphysema [
5‐
8]. Airway inflammation is marked by transient perinatal recruitment of macrophages and eosinophils and expression of tumor necrosis factor-alpha and IL-13 whereas persistent increases in neutrophils, keratinocyte-derived cytokine, and chitinases have been described [
7].
Another protein with known functions in innate immunity and inflammation and therefore a possible role in dysregulated inflammation during CF is named DMBT1 (deleted in malignant brain tumors 1; alternative names: glycoprotein-340 (gp-340) or salivary agglutinin (SAG)). DMBT1 is a secreted scavenger receptor cysteine-rich (SRCR) protein, of the adult respiratory tract expressed in alveolar type II cells, epithelial cells and associated glands. Steady-state DMBT1 levels appear to be low to moderate in the lung of healthy adults but increased during inflammation and bacterial and viral infection [
9,
10]. Moreover, the percentage of DMBT1-positive type II pneumocytes has been shown to positively correlate with severity of inflammation. In infants pulmonary DMBT1 expression has likewise been observed in epithelial cells and associated glands [
11,
12]. In analogy to adults, DMBT1 expression is upregulated during inflammatory processes in the newborn lung, for example respiratory distress syndrome where DMBT1 is detected in hyaline membranes and inactivates different surfactant preparations in a dose-dependent manner [
11,
12]. Multiple mechanisms of action of DMBT1 have been described: DMBT1 binds to other proteins with functions in innate immunity (e.g., secretory IgA, surfactant protein D, surfactant protein A) [
13], aggregates diverse species of pathogenic bacteria using the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains or glycosylation [
14,
15], functions as a pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands. These findings provide a molecular basis for its broad bacterial-binding specificity and inhibitory effects on lipopolysaccharide-induced Toll-like receptor 4 (TLR4)-mediated nuclear factor kappa B activation [
16,
17]. DMBT1 binds also various viruses (e.g., influenza A virus and human immunodeficiency virus type I, hepatitis B) [
13,
18]. Therefore, DMBT1 is found in the respiratory tract as well in the gastrointestinal tract and in lacrimal fluid to be part of innate immunity and to be part of regulations against inflammation [
18]. DMBT1 has already been examined in several inflammatory diseases like Crohn's disease, active bacteria-related appendicitis and bacterial endocarditis [
17,
19,
20]. Besides inflammation, DMBT1 has functions in angiogenesis and epithelial differentiation illustrating characteristics with important impact in tissue repair [
13,
21].
The aim of this study is to analyze the potential role of DMBT1 in CF. We examined pulmonary DMBT1 expression in patients with CF, the effect of DMBT1 on motility of ciliated respiratory epithelium and the potential therapeutic use of acetylcysteine (ACC) to reduce DMBT1 levels and hence inflammation.
Methods
Immunohistochemistry
Formalin-fixed and paraffin-embedded lung sections of patients with CF were analyzed by immunohistochemistry to detect DMBT1. Post-mortem examinations of patients with CF are very rare since the diagnosis is normally made during lifetime. Therefore, post-mortem lung sections of only one case of CF were available for staining. Additionally, lung tissue of 13 patients with CF (age: 29.26 ± 1.9 years) who had undergone lung transplantation was stained. Two corresponding persons without lung disease were included as control subjects. The study was approved by the responsible ethics committees of the University of Heidelberg (No. 361/2003) and the University of Erlangen-Nürnberg (No. 443_19B). DMBT1 protein expression was detected using the polyclonal antibody anti-DMBT1p84 and a protocol described earlier [
11,
12,
22].
Cell culture and incubation with ACC
Human lung epithelial A549 cells showing the characteristic morphology of type II lung epithelial cells with typical lamellar bodies were used to examine the effect of ACC on DMBT1 expression [
22‐
24]. Briefly, the cells were stably transfected with an expression plasmid encoding the largest (8 kb) DMBT1 variant (DMBT1+ cells) under the control of a constitutive promoter or an empty vector control (DMBT1− cells) [
16]. The cells were cultured in DMEM/F-12 (1:1) + GlutaMAX TM medium (Gibco, Thermo Fisher, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (PAN Biotech, Aidenbach, Germany) and 1% penicillin/streptomycin (Sigma-Aldrich Life Science, Taufkirchen, Germany). Hygromycin B (Carl Roth GmbH + Co. KG, Karlsruhe, Germany; final concentration: 500 μg/ml) was added to keep selection pressure on the cells with inserted plasmids. The DMBT1− and DMBT1+ cells were seeded in 6-well plates. After reaching 90–100% confluence, medium was removed and wells were washed with PBS and incubated in medium without fetal bovine serum for 2 h. Then, ACC (Hexal AG, Holzkirchen, Germany; final concentration: 15 mM) or sodium chloride 0.9% as control was added for another 2 h. The medium was collected and frozen at – 80 °C until determination of DMBT1 concentration by ELISA. Subsequently, the cells were washed again with PBS and medium without fetal bovine serum was added onto the cell layer. After 24 h, medium was removed and frozen for DMBT1 determination.
Quantification of DMBT1 in supernatants of DMBT1− and DMBT1+ A549 cells by ELISA
The DMBT1 concentrations in the supernatants of DMBT1− and DMBT1+ cells were determined by ELISA (Abbexa, Cambridge, UK) according to the manufacturer’s instructions. The ELISA measured DMBT1 concentrations in the range of 0.156 ng/ml to 10 ng/ml. The sensitivity of the ELISA was < 0.055 ng/ml. Supplemental Figure
1 demonstrated the standard curve of the ELISA. The DMBT1 concentrations of A549 cells incubated with sodium chloride 0.9% instead of ACC were assigned as 100% (control).
Motility of ciliated respiratory epithelium
Two nasal swabs with respiratory epithelial cells were collected from each of 10 healthy volunteers (6 males, 4 females) after giving informed consent. The swabs were rotated in a tube with 2 ml of cell culture medium (RPMI 1640, HEPES, Thermo Fisher Scientific, Schwerte, Germany) to separate the respiratory epithelial cells from the swab. Human recombinant DMBT1 (hrDMBT1; final concentration: 0.5 μg/ml) was added to one sample and PBS as control to the second sample of each volunteer. Phase contrast microscopy was used to investigate the motility of the cilia.
Animals
All animal studies were approved by the Regierungspräsidium Karlsruhe, Germany. The generation of ENaC-transgenic mice (line 6608) has been previously described [
6]. The colony was maintained on a mixed genetic background (C57BL/6N × C3H/HeN), and ENaC-transgenic mice were identified by PCR as described [
6,
7]. Wild-type littermates served as control animals. Mice were housed in a specific pathogen-free animal facility and had free access to chow and water. At the age of 6 weeks, mice were sacrificed and lungs were stored as samples to isolate RNA for later expression studies (Applied Biosystems, Darmstadt, Germany).
Quantitative RT-PCR analyses
Single-stranded cDNA synthesis was done with 300 ng of total RNA and oligo-dT primers according to standard procedures. Quantitative RT-PCR experiments were carried out using 5 ng of reverse transcribed RNA per reaction and TaqMan assays-on-demand (Thermo Fisher Scientific, Karlsruhe, Germany) according to the manufacturer’s instructions. All PCR reactions were done in triplicate. Signal detection was performed with the ABI Prism 7900HT Sequence detection system (Thermo Fisher Scientific, Karlsruhe, Germany). Cycle threshold (Ct) values were normalized against Ct values obtained for mouse beta-actin (Mm00607939_s1). All primer assays used during this study are listed in Table
1.
Table 1
Primer assays used for quantitative RT-PCR analysis
Mm00455996_m1 | Dmbt1 | Deleted in malignant brain tumors 1 |
Mm00499170_m1 | Sftpa | Surfactant protein A |
Mm00486060_m1 | Sftpd | Surfactant protein D |
Mm00436945_m1 | Tff1 | Trefoil factor 1 |
Mm00447491_m1 | Tff2 | Trefoil factor 2 (spasmolytic protein 1) |
Mm00445274_m1 | Tlr4 | Toll-like receptor 4 |
Statistics
Statistical analysis was performed with SAS software, release 9.4 (SAS Institute Inc., Cary, NC, USA). To analyze quantitative RT-PCR data, t test (Gosset) and Welch-Satterthwaite t test was applied. To compare DMBT1 concentrations in cell culture media, paired t test was used for pairwise comparisons and t test for independent samples was used for unpaired samples. Variance analysis for paired values was used to analyze cilia motility data. In general, test results with p values less than 0.05 were regarded as statistically significant.
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