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Original Article| Volume 16, ISSUE 5, P565-572, September 2017

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Pulmonary surfactant dysfunction in pediatric cystic fibrosis: Mechanisms and reversal with a lipid-sequestering drug

Open ArchivePublished:June 07, 2017DOI:https://doi.org/10.1016/j.jcf.2017.04.015

      Abstract

      Background

      Airway surfactant is impaired in cystic fibrosis (CF) and associated with declines in pulmonary function. We hypothesized that surfactant dysfunction in CF is due to an excess of cholesterol with an interaction with oxidation.

      Methods

      Surfactant was extracted from bronchial lavage fluid from children with CF and surface tension, and lipid content, inflammatory cells and microbial flora were determined. Dysfunctional surfactant samples were re-tested with a lipid-sequestering agent, methyl-β-cyclodextrin (MβCD).

      Results

      CF surfactant samples were unable to sustain a normal low surface tension. MβCD restored surfactant function in a majority of samples.Mechanistic studies showed that the dysfunction was due to a combination of elevated cholesterol and an interaction with oxidized phospholipids and their pro-inflammatory hydrolysis products.

      Conclusion

      We confirm that CF patients have impaired airway surfactant function which could be restored with MβCD. These findings have implications for improving lung function and mitigating inflammation in patients with CF.

      Keywords

      1. Introduction

      Pulmonary surfactant is a protein-lipid mixture secreted by type-II alveolar epithelial cells which spread as a continuous film at the air–liquid interface and extends from the alveoli to the pharynx [
      • Zuo Y.Y.
      • Veldhuizen R.A.W.
      • Neumann A.W.
      • Petersen N.O.
      • Possmayer F.
      Current perspectives in pulmonary surfactant -inhibition, enhancement and evaluation.
      ]. This continuous film maintains a surface tension gradient reaching near zero in the alveoli [
      • Schoel W.M.
      • Schürch S.
      • Goerke J.
      The captive bubble method for the evaluation of pulmonary surfactant: surface tension, area, and volume calculations.
      ] to the trachea wherethe surface tension is approximately 32 dynes per cm [
      • Im Hof V.
      • Gehr P.
      • Gerber V.
      • Lee M.
      • Schürch S.
      In vivo determination of surface tension in the horse trachea and in vitro model studies.
      ]. Airway surfactant is a major factor that maintains small airway patency. Inflammation in distal airways with impaired surfactant function is thought to be a primary mechanism for small airway collapse in cystic fibrosis (CF)[
      • Griese M.
      • Essl R.
      • Schmidt R.
      • Rietschel E.
      • Ratjen F.
      • Ballmann M.
      • et al.
      Pulmonary surfactant, lung function, and endobronchial inflammation in cystic fibrosis.
      ]. In vitro testing of pediatric CF surfactant samples, obtained largely from small airways, showed that the ability of the surfactant to maintain patency of a capillary tube was markedly reduced [
      • Griese M.
      • Essl R.
      • Schmidt R.
      • Rietschel E.
      • Ratjen F.
      • Ballmann M.
      • et al.
      Pulmonary surfactant, lung function, and endobronchial inflammation in cystic fibrosis.
      ], a finding that may account for a significant degree of the airflow obstruction in CF, particularly in pediatric patients before development of fixed airway structural damage. Impairment of lung function in CF is also related to other factors including plasma phosphatidylcholine, liver phospholipid homeostasis and nutrition [
      • Grothe J.
      • Riethmüller J.
      • Tschürtz S.M.
      • Raith M.
      • Pynn C.J.
      • Stoll D.
      • et al.
      Plasma phosphatidylcholine alterations in cystic fibrosis patients: impaired metabolism and correlation with lung function and inflammation.
      ].
      The current study explores the mechanisms that contribute to surfactant dysfunction in pediatric patients with CF and an approach to treat the abnormality. We compared CF patients toa group of lung-healthy children. We show that the basic abnormality in CF lung surfactant involves elevated surfactant cholesterol (likely genetically determined), with a further deleterious interaction between cholesterol and oxidized unsaturated phospholipids (likely due to infection). All of these abnormalities were reversed by methyl-β-cyclodextrin (MβCD).

      2. Methods and materials

      2.1 Patient population

      The patient population was obtained from the Cystic Fibrosis Clinic at the Alberta Children's Hospital (Calgary, AB, Canada) where bronchial lavage fluid (BLF) is part of patient care. A total of 50 CF patients were enrolled in the study. Twenty-six of the BLF samples contained sufficient surfactant volume for functional analysis. BLFs were also obtained from 9 patients without CF, with a variety of conditions requiring investigative bronchoscopy including foreign body removal, laryngomalacia, hemoptysis and suspected immotile cilia syndrome. These subjects, called lung-healthy controls, had no evidence of current respiratory infection. Ethical approval required that only surplus BLF was used; consequently, most of the samples were small, many with insufficient surface active material for functional testing. A flow chart for the patient groups with sufficient sample for at least one test is shown in Fig. 1. Details of bronchoscopy and BLF procedure are included in online supplementary materials, Fig. S1.
      Fig. 1
      Fig. 1Flow chart of patient selection. BLF = bronchial lavage Fluid and CF = Cystic Fibrosis. Not all samples had sufficient sample to conducts all of the tests (see Supplemental Table T1).
      Approval of the research protocol was obtained from theUniversity of Calgary Child Health Scientific Review Committee/Conjoint Health Research Ethics Board (Calgary, AB, Canada).

      2.2 Cellular and microbiological analysis of BLF

      Routine cell differentials and microbial culture were determined by Calgary Laboratory Services (CLS). Surplus BLF was centrifuged and live cells imaged with a Richardson RTM-3 highresolution (120 nm optical resolution) light microscope, with fluorescence capability [
      • Clark S.R.
      • Ma A.C.
      • Tavener S.A.
      • Mcdonald B.
      • Goodarzi Z.
      • Kelly M.M.
      • et al.
      Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood.
      ].A portion of the cellular pellet was fixed in 2.5% glutaraldehyde and processed routinely for transmission electron microscopy and examined in a Hitachi H7650 electron microscope.

      2.3 Biochemical characterization of BLF

      An aliquot of the supernatant from the BLF was used to assay for lipid and protein content.Details of the methodology are included in the online supplementary materials.

      2.4 Surface activity assessment

      From each clinical sample, given the young age of this pediatric population, approximately 1 ml of BLF supernatant was available for surfactant isolation. Duplicate samples from the same patient were pooled when available. Samples were centrifuged at 400g for 10 min at 4 °C to separate the supernatant from the cellular components and cell debris. The supernatants were then centrifuged at 40,000g for 30 min at 4 °C to isolate the highly surface active large aggregate (LA) fraction, as previously described [
      • Veldhuizen R.A.
      • Inchley K.
      • Hearn S.A.
      • Lewis J.F.
      • Possmayer F.
      Degradation of surfactant-associated protein B (SP-B) during in vitro conversion of large to small surfactant aggregates.
      ]. Care was taken to remove as much of the supernatant as possible and the remaining highly concentrated portion of lung surfactant was used for surface tension measurements.
      Surface activity of surfactant was determined with a computer-controlled captive bubble surfactometer (CBS) [
      • Schoel W.M.
      • Schürch S.
      • Goerke J.
      The captive bubble method for the evaluation of pulmonary surfactant: surface tension, area, and volume calculations.
      ,
      • Gunasekara L.
      • Schoel W.M.
      • Schürch S.
      • Amrein M.W.
      A comparative study of mechanisms of surfactant inhibition.
      ]. To evaluate the effect of methyl-β-cyclodextrin (MβCD), powdered MβCD (Sigma Aldrich, Catalogue-Nr. C4555) was dissolved in HEPES buffer to a final concentration of 40 mg/ml and added to the CBS chamber prior to the addition of surfactant.

      2.5 Model surfactant studies

      2.5.1 Materials

      Bovine Lipid Extract Surfactant (BLES) was donated by BLESBiochemicals Inc. (London, ON, Canada). 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) and bovine heart cardiolipin (CL, predominantly 1′,3′-Bis[1,2-dilinoleoyl-sn-glycero-3-phospho]-sn-glycerol) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). All lipids were stored at −20 °C under nitrogen. Surfactant mixtures were stored at 4 °C under nitrogen and used within three days. Buffers containing HEPES were stored in the dark at 4 °C to avoid generating H2O2.

      2.5.2 In vitro oxidation

      BLES was exposed to hydroxyl radicals generated from Fenton-like chemistry for 24 h to produce oxidized BLES (oxBLES) as described by Manzanares et al. [
      • Manzanares D.
      • Rodriguez-Capote K.
      • Liu S.
      • Haines T.
      • Ramos Y.
      • Zhao L.
      • et al.
      Modification of tryptophan and methionine residues is implicated in the oxidative inactivation of surfactant protein B.
      ]. PLPC and CL were exposed to identical oxidizing conditions, and are indicated as oxPLPC and oxCL. Oxidation of surfactant phospholipids was confirmed by measuring the formation of secondary lipoperoxidation products malondialdehyde (MDA) and 4-hydroxyalkenal (4-HAE) (BIOXYTECH LPO-586, OxisResearch, Burlingame, CA, USA). oxBLES contained significantly more MDA and 4-HAE compared to control BLES (6.69 ± 1.16 and 2.69 ± 0.63 nmol MDA and 4-HAE/mg phospholipid, respectively, n = 6, p = 0.014).

      2.5.3 Surfactant preparation

      To ensure accurate mixing, additional lipids were added to lipid extracts of BLES or oxBLES as organic solutions. The mixtures were then dried under nitrogen and suspended in CBS buffer (140 mM NaCl, 10 mM HEPES, 2.5 mM CaCl2, pH 6.9) to a BLES phospholipid concentration of 27 mg/mL. The biochemical characterization of BLF is described in more detail in the supplemental file. Total phospholipid concentration was 96.8 ± 6.8% (n = 6) after lipid extraction and 98.6 ± 11.1% (n = 11) after oxidation. BLES contained 2.6% cholesterol species (w/w phospholipids) as determined using an enzymatic assay for cholesterol and cholesteryl esters (Amplex Red Cholesterol Assay, Invitrogen, Eugene, OR, USA). In a subset of experiments, water-soluble products of phospholipid hydrolysis were removed by high-speed centrifugation of dilute surfactant mixtures (40,000g × 30 min × 4 °C). After high-speed centrifugation, the concentration of phospholipids in oxBLES was 61.9 ± 9.7% that of unoxidized BLES (n = 3, p = 0.034).

      2.6 Statistical analysis

      All CBS experiments are reported as mean ± SEM (n = 4–7). Each unique lipid mixture was prepared independently at least twice. Analysis of variance (SPSS 19.0) between two groups was followed by Tukey's HSD for multiple comparisons. Given the small sample sizes, non-parametricMann-WhitneyU tests were performed on biochemical assays of BLF samples to compare the control group to the CF group.
      Qualitative patient demographic data was analyzed using Fisher's exact test using GraphPad Prism 6.0. A Student's t-test was used to analyze data between only two groups.
      Fisher's test was also used to determine if there were any differences between CF patients with insufficient surfactant and sufficient surfactant for analysis (Supplementary Table T1).

      3. Results

      3.1 Patient demographic information

      Table 1 shows the demographic information on the patients with sufficient BLF for at least one test. There were no significant differences in age or sex between the CF and lung-healthy groups, although there were more males than females within both groups. Very few of the CF patients (4/26), were lavaged during an exacerbation of their lung disease. However, 15/26 had ≥103 CFU/ml of BLF, a level considered indicative of active infection in this patient population. None of the lung-healthy control cases had clinical or laboratory evidence of active airway infection. Demographic information for CF patients with sufficient and insufficient surfactant for analysis is included in the online supplement (Supplementary Table T1). There were significantly more females and a higherproportion of leukocytes in the BLF in patients with insufficient surfactant (p = 0.02), but no significant differences in terms of age, infection status, or airway disease severity. A broad spectrum of genotypes was seen in the CF population (Supplementary Table T2).
      Table 1Demographic information for CF, and lung-healthy control subjects.
      CF(n = 26)Lung-healthy control (n = 9)
      Age(median, range)3.85(1.03–12.13)6.10(1.68–14.68)
      Sex(male/female)19/77/2
      103 CFU
      CFU: Colony-forming units.
      /mL bacterial/fungal organisms in BLF
      15/260/9
      p=0.0043, compared to CF patients, Fisher's exact test.
      Exacerbation
      Determined by the clinician on day of study.


      (yes)
      4/260/9
      Bronchoscopic appearances(normal/mild-moderate/severe)4/20/27/2/0
      a CFU: Colony-forming units.
      b Determined by the clinician on day of study.
      low asterisklow asterisk p = 0.0043, compared to CF patients, Fisher's exact test.

      3.2 Microbiology of BLF

      A wide range of potentially pathogenic fungal and bacterial organisms were identified (Supplementary Table T2).Several patients had multiple pathogenic bacteria in their BLF. Table T2 also shows the results of the BLF cultures and number of colony-forming units for the predominant isolates in the CF patients. Viral cultures/tests were not performed.

      3.3 Cellular analysis of BLF

      The percentage of polymorphonuclear leukocytes in the BLF was significantly greater in the CF (p = 0.044) group compared to lung-healthy control subjects (Fig. 2A ). Cystic fibrosis patients with ≥103 CFU/mL had significantly greater (p = 0.013) percent of polymorphonuclear leukocytes compared to CF patients without clinical or laboratory evidence of infection (<103 CFU/mL), (Fig. 2B). Further, the alveolar macrophages in CF were enlarged as a result of lipid overload (Supplementary Figs. S2A and S2B)·These macrophages had reduced external and cytoplasmic motility compared to lung-healthy control cells (Supplementary video V1A, Supplementary video V1B). Electron microscopy confirmed that the lipid material was within lysosomes with a lamellar arrangement characteristic of surfactant (Supplementary Figs. S2C and S2D).
      Fig. 2
      Fig. 2(A) Mean (SEM) of % polymorphonuclear leukocytes in the 2 groups. The proportion of neutrophils in CF samples were significantly greater than lung-healthy controls (*p ≤ 0.05) (B) Neutrophils were also significantly increased in CF patients with active infection compared to those without infection (*p ≤ 0.05).

      3.4 Biochemical characterization of BLF samples

      Biochemical analyses of BLF supernatants from a subset of subjects with sufficient material for analysis are shown in Supplementary Table T3. Cholesterol was elevated in CF patients (13.00 ± 1.4 wt%) compared to lung-healthy controls (4.96 ± 0.70, p = 0.008). Cell-freeBLF protein was also elevated in CF patients (486.48 ± 73.90 μg/mL) compared to lung-healthy controls (181.97 ± 53.08 μg/mL, p = 0.0287). There was no significant differences in LA phospholipid content (p = 0.0607).

      3.5 Surface activity assessment

      Minimum surface tension during dynamic compression for the patient groups are shown in Fig. 3A . Only 2 of the CF samples achieved normal (<3 mN/m) minimum surface tension following dynamic compression. The other CF samples had minimum surface tensions ranging from 14 to 18 mN/m.
      Fig. 3
      Fig. 3(A) Mean (SEM) minimum surface tensions following dynamic cycling. Lung-healthy controls achieved low (normal) surface tensions. CF patient samples showed severe impairment in surfactant function. (B) Mean (SEM) minimum surface tension obtained during dynamic compression-expansion cycles of CF samples before and after methyl-β-cyclodextrin treatment (MβCD 40 mg/mL).*** p ≤ 0.001.
      We previously showed that surfactant dysfunction can be reversed with MβCD if cholesterol is decreased relative to surfactant phospholipids [
      • Gunasekara L.C.
      • Pratt R.M.
      • Schoel W.M.
      • Gosche S.
      • Prenner E.J.
      • Amrein M.W.
      Methyl-β-cyclodextrin restores the structure and function of pulmonary surfactant films impaired by cholesterol.
      ,
      • Vockeroth D.
      • Gunasekara L.
      • Amrein M.
      • Possmayer F.
      • Lewis J.F.
      • RAW Veldhuizen
      Role of cholesterol in the biophysical dysfunction of surfactant in ventilator-induced lung injury.
      ]. Furthermore, cholesterol has been shown elevated in BLF[
      • Gilljam H.
      • Andersson O.
      • Ellin A.
      • Robertson B.
      • Strandvik B.
      Composition and surface properties of the bronchial lipids in adult patients with cystic fibrosis.
      ] and tracheo-bronchial secretions in CF[
      • Slomiany A.
      • Murty V.L.N.
      • Aono M.
      • Snyder C.E.
      • Herp A.
      • Slomiany B.L.
      Lipid composition of tracheobronchial secretions from normal individuals and patients with cystic fibrosis.
      ], a finding substantiated in our small sample. Sixteen of the CF samples were retested in a buffer containing MβCD. This showed that minimum surface tension during dynamic compression in the presence of MβCD was significantly decreased (p < 0.001) compared to untreated samples (Fig. 3B).
      Film compressibility as expressed in the surface tension-interfacial area isotherms was also abnormal for the CF cases compared to the lung-healthy controls. Representative figures for two CF and lung-healthy controls are shown in online supplementary Figs. S3A and B.
      In summary, we report consistent near-zero surface tensionsfor lung-healthy control samples and elevated minimum surface tensions (>12 mN/m) for a majority of CF cases. After MβCD treatment, a majority of CF samples resembled controlsurfactant in terms of minimum surface tension and film stability.

      3.6 Model surfactant studies

      Excess cholesterol (approx. 10–20% w/w) alone has been shown to impair otherwise normal surfactant, a defect reversible by MβCD [
      • Gunasekara L.C.
      • Pratt R.M.
      • Schoel W.M.
      • Gosche S.
      • Prenner E.J.
      • Amrein M.W.
      Methyl-β-cyclodextrin restores the structure and function of pulmonary surfactant films impaired by cholesterol.
      ]. Excess cholesterol alone could account for surfactant dysfunction in CF. We found a mean level of cholesterol (~13%), a level sufficient to cause surfactant dysfunction. One of the CF samples had a normal (8.4%) cholesterol level, but with MβCD-reversible surfactant dysfunction. In view of the inflammatory milieu of the CF lung, we wished to determine if factors, in addition to cholesterol, were involved in the dysfunction. Model studies were therefore carried out to test whether oxidative damage, similar to that present in the inflamed lung [
      • Manzanares D.
      • Rodriguez-Capote K.
      • Liu S.
      • Haines T.
      • Ramos Y.
      • Zhao L.
      • et al.
      Modification of tryptophan and methionine residues is implicated in the oxidative inactivation of surfactant protein B.
      ], is involved in the surfactant dysfunction and to determine if there is an interaction between oxidation and cholesterol.
      A prominent target of reactive oxygen species (ROS)-induced oxidation is polyunsaturated phospholipids, constituting ≃10% of total phospholipids in surfactant [
      • Lam B.C.C.
      • Ng Y.K.
      • Wong K.Y.
      Randomized trial comparing two natural surfactants (Survanta vs. bLES) for treatment of neonatal respiratory distress syndrome.
      ]. Thus, we first exposed a clinical surfactant, BLES, to hydroxyl radicals produced by the Fenton reaction (oxBLES) as a model of the oxidative environment of the inflamed lung [
      • Manzanares D.
      • Rodriguez-Capote K.
      • Liu S.
      • Haines T.
      • Ramos Y.
      • Zhao L.
      • et al.
      Modification of tryptophan and methionine residues is implicated in the oxidative inactivation of surfactant protein B.
      ]. ROS-induced oxidation of native BLES (from which cholesterol is largely removed during manufacturing (14)) did not significantly degrade surfactant performance (Fig. 4A ). However, oxBLES became dysfunctional after reestablishing physiological levels (5–10%) of cholesterol (Fig. 4A). MβCD treatment of this preparation restored normal function (p = 0.0079) (Fig. 4B), revealing a mechanism whereby oxidative damage of surfactant requires cholesterol to manifest. Separate experiments revealed that adding oxidized polyunsaturated phospholipids (PLPC and CL) to native BLES resulted in impairment of surface activity only in the presence of cholesterol (Supplementary Fig. S4).
      Fig. 4
      Fig. 4(A) Minimum surface tension during CBS dynamic-cycles of BLES and oxBLES with 0%(white), 5%(gray), or 10%(black) w/w added cholesterol. (B)Minimum surface tension during dynamic-cycles of oxBLES +10% w/w cholesterol in control CBS buffer or buffer containing 40 mg/ml MβCD. (*p ≤ 0.05,**p ≤ 0.01,*** p ≤ 0.001). (N = 5).
      Oxidative stress is responsible for the excessive release of pro-inflammatory mediators in CF[
      • Griese M.
      • Essl R.
      • Schmidt R.
      • Rietschel E.
      • Ratjen F.
      • Ballmann M.
      • et al.
      Pulmonary surfactant, lung function, and endobronchial inflammation in cystic fibrosis.
      ], including phospholipid hydrolysis products. Free fatty acids (FFA) and lysophosphatidylcholine (LPC), are generated in considerable quantities (~15% w/w) upon ROS exposure and are capable of surfactant inhibition [
      • Zuo Y.Y.
      • Veldhuizen R.A.W.
      • Neumann A.W.
      • Petersen N.O.
      • Possmayer F.
      Current perspectives in pulmonary surfactant -inhibition, enhancement and evaluation.
      ,
      • Manzanares D.
      • Rodriguez-Capote K.
      • Liu S.
      • Haines T.
      • Ramos Y.
      • Zhao L.
      • et al.
      Modification of tryptophan and methionine residues is implicated in the oxidative inactivation of surfactant protein B.
      ].
      The addition of LPC or FFA to native BLES caused a dose-dependent inhibition (Supplementary Fig. S5) of the surfactant to reach low minimum surface tensions (Supplementary Fig. S6). Surface activity inhibition was achieved, albeit to a far lesser extent than that mediated by cholesterol, at levels reasonably expected in oxidized surfactant. Furthermore, LPC or FFA mediated surfactant inhibition was reversed by MβCD, even in the relative absence of cholesterol (Supplementary Fig. S6). This finding likely reflects the capacity of MβCD to sequester non-steroidal lipids in addition to cholesterol, though perhaps with lesser affinity [
      • Brunaldi K.
      • Huang N.
      • Hamilton J.
      Fatty acids are rapidly delivered to and extracted from membranes by methyl-β-cyclodextrin.
      ]. These model studies indicate that surfactant oxidation products, including but not limited to FFAs and lyso-phospholipids are also involved in MβCD-reversible surfactant dysfunction.

      4. Discussion

      This study confirms profound dysfunction of surfactant obtained from the airways of pediatric CF patients [
      • Griese M.
      • Essl R.
      • Schmidt R.
      • Rietschel E.
      • Ratjen F.
      • Ballmann M.
      • et al.
      Pulmonary surfactant, lung function, and endobronchial inflammation in cystic fibrosis.
      ]. We followed an experimental protocol optimized for sub-microgram samples of surfactant [
      • Schoel W.M.
      • Schürch S.
      • Goerke J.
      The captive bubble method for the evaluation of pulmonary surfactant: surface tension, area, and volume calculations.
      ], appropriate for pediatric studies, that allowed us to differentiate between dysfunctional CF surfactant (minimum surface tension: >10 mN/m) and surfactant from lung-healthy controls (close to 1 mN/m) with high precision.
      High minimum surface tensions observed with CF surfactant samples may not be an indication of dysfunctional surfactant within all compartments of the lung, as otherwise lung mechanics would be more severely affected than reported [
      • Linnane B.M.
      • Hall G.L.
      • Nolan G.
      • Brennan S.
      • Stick S.M.
      • Sly P.D.
      • et al.
      Lung function in infants with cystic fibrosis diagnosed by newborn screening.
      ]. In CF, the small airways are the region of the lung most severely affected by inflammation and infection [
      • Griese M.
      • Essl R.
      • Schmidt R.
      • Rietschel E.
      • Ratjen F.
      • Ballmann M.
      • et al.
      Pulmonary surfactant, lung function, and endobronchial inflammation in cystic fibrosis.
      ]. The diagnostic lavage used in the current study was designed primarily to sample airway secretions.
      Dysfunctional surfactant is unable to sustain the low surface tension of the functional film. In the healthy lung, the surfactant film is purged of degraded surfactant components by uptake, degradation, and recycling by alveolar macrophages and type-II lung epithelial cells [
      • Lam B.C.C.
      • Ng Y.K.
      • Wong K.Y.
      Randomized trial comparing two natural surfactants (Survanta vs. bLES) for treatment of neonatal respiratory distress syndrome.
      ]. The live cell images in this study revealed overloading of macrophages with lysosomal lamellar material and evidence of apoptosis. The surfactant nature of the lysosomal material was confirmed by electron microscopy. Increased lysosomal lipids in alveolar macrophages from pediatric CF patients is consistent with previous reports [
      • Kazachkov M.
      • Muhlebach M.
      Lipid-laden macrophage index and inflammation in bronchoalveolar lavage fluids in children.
      ]. In our study, the CF alveolar macrophages also exhibited decreased intracellular and extracellular motility which may be a factor contributing to an impaired ability of CF alveolar macrophages to phagocytose and kill bacteria [
      • Simonin-Le Jeune K.
      • Le Jeune A.
      • Jouneau S.
      • Belleguic C.
      • Roux P.F.
      • Jaguin M.
      • et al.
      Impaired functions of macrophage from cystic fibrosis patients: CD11b, TLR-5decrease and sCD14, inflammatory cytokines increase.
      ]. In addition, CF alveolar macrophages overloaded with lipid exhibit impaired cholesterol handling [
      • White N.M.
      • Jiang D.
      • Burgess J.D.
      • Bederman I.R.
      • Previs S.F.
      • Kelley T.J.
      Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis.
      ] and produce excessive inflammatory cytokines [
      • Lubamba B.A.
      • Jones L.C.
      • O'Neal W.K.
      • Boucher R.C.
      • Ribeiro C.M.P.
      X-box–binding protein 1 and innate immune responses of human cystic fibrosis alveolar macrophages.
      ]. Abnormalities of the hydrophilic surfactant proteins, SP-A and SP-D, with important innate immune functions, have also been reported in CF[
      • Griese M.
      • Essl R.
      • Schmidt R.
      • Rietschel E.
      • Ratjen F.
      • Ballmann M.
      • et al.
      Pulmonary surfactant, lung function, and endobronchial inflammation in cystic fibrosis.
      ]. Thus abnormalities of surfactant in CF are broad based.
      The current study focused on the role of neutral lipids, primarily cholesterol, as well as its interaction with phospholipid oxidation products, a factor previously not studied for bronchiolitis. The cholesterol content of normal surfactant is reported to be on the order of 5–10% by weight [
      • Zuo Y.Y.
      • Veldhuizen R.A.W.
      • Neumann A.W.
      • Petersen N.O.
      • Possmayer F.
      Current perspectives in pulmonary surfactant -inhibition, enhancement and evaluation.
      ]. In a small subsample we showed a significant, approximately two to three-fold, increase incholesterol by weight % in CF surfactant compared to lung-healthy controls. Other investigators have shown increased cholesterol in BLF[
      • Gilljam H.
      • Andersson O.
      • Ellin A.
      • Robertson B.
      • Strandvik B.
      Composition and surface properties of the bronchial lipids in adult patients with cystic fibrosis.
      ] and tracheobronchial secretions [
      • Slomiany A.
      • Murty V.L.N.
      • Aono M.
      • Snyder C.E.
      • Herp A.
      • Slomiany B.L.
      Lipid composition of tracheobronchial secretions from normal individuals and patients with cystic fibrosis.
      ] from CF patients. Elevated cholesterol alone is a potent inhibitor of surfactant function [
      • Gunasekara L.C.
      • Pratt R.M.
      • Schoel W.M.
      • Gosche S.
      • Prenner E.J.
      • Amrein M.W.
      Methyl-β-cyclodextrin restores the structure and function of pulmonary surfactant films impaired by cholesterol.
      ,
      • Vockeroth D.
      • Gunasekara L.
      • Amrein M.
      • Possmayer F.
      • Lewis J.F.
      • RAW Veldhuizen
      Role of cholesterol in the biophysical dysfunction of surfactant in ventilator-induced lung injury.
      ]. Rodriguez-Capote and coworkers showed that surfactant function was impaired after the exposure of phospholipids to oxidation in the absence of cholesterol [
      • Rodríguez-Capote K.
      • Manzanares D.
      • Haines T.
      • Possmayer F.
      Reactive oxygen species inactivation of surfactant involves structural and functional alterations to surfactant proteins SP-B and SP-C.
      ]. However, we were unable to reproduce the same degree of dysfunction in the absence of cholesterol after exposing clinical surfactant to oxidation, we showed minor derangements in minimum surface tension (< 5 mN/m). This controversy could be associated with differences in surfactant concentration, whereby lower surfactant concentrations were found more susceptible to inhibition (data not shown).
      There is increasing evidence of disturbed cholesterol homeostasis in CF[
      • White N.M.
      • Jiang D.
      • Burgess J.D.
      • Bederman I.R.
      • Previs S.F.
      • Kelley T.J.
      Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis.
      ,
      • Strandvik B.
      Fatty acid metabolism in cystic fibrosis.
      ,
      • Manson M.E.
      • Corey D.A.
      • Bederman I.
      • Burgess J.D.
      • Kelley T.J.
      Regulatory role of β-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells.
      ]. The source of the cholesterol may be airway epithelial cells. Cultured CFTR−/−type-II pneumocytes exhibit elevated intracellular cholesterol and elevated basal release of pro-inflammatory cytokines as a consequence of intracellular cholesterol [
      • White N.M.
      • Jiang D.
      • Burgess J.D.
      • Bederman I.R.
      • Previs S.F.
      • Kelley T.J.
      Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis.
      ]. Three different CF cholesterol phenotypes have been described; accumulation of perinuclear free cholesterol, increased cellular cholesterol membrane content and increased de novo cholesterol synthesis [
      • Manson M.E.
      • Corey D.A.
      • Bederman I.
      • Burgess J.D.
      • Kelley T.J.
      Regulatory role of β-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells.
      ].
      Recent studies indicate that CF has an intrinsically pro-inflammatory phenotype [
      • Simonin-Le Jeune K.
      • Le Jeune A.
      • Jouneau S.
      • Belleguic C.
      • Roux P.F.
      • Jaguin M.
      • et al.
      Impaired functions of macrophage from cystic fibrosis patients: CD11b, TLR-5decrease and sCD14, inflammatory cytokines increase.
      ,
      • Xu Y.
      • Krause A.
      • Hamai H.
      • Harvey B.-G.
      • Worgall T.S.
      • Worgall S.
      Proinflammatory phenotype and increased Caveolin-1 in alveolar macrophages with silenced CFTR mRNA.
      ]. An elevated inflammatory profile in CF BLF in the absence of detectable airway infection has been reported [
      • Armstrong D.S.
      • Hook S.M.
      • Jamsen K.M.
      • Nixon G.M.
      • Carzino R.
      • Carlin J.B.
      • et al.
      Lower airway inflammation in infants with cystic fibrosis detected by newborn screening.
      ]. In our study, the proportion of polymorphonuclear leukocytes was significantly higher in the BLF of CF patients even with no evidence of active infection as compared to lung-healthy controls (Fig. 2B). The pro-inflammatory milieu of the CF lung may be related to increased expression of phospholipases [
      • Strandvik B.
      Fatty acid metabolism in cystic fibrosis.
      ]. Abnormal activity of phospholipases with the release of FFAs, such as arachidonic and oleic acid, LPC, and lysophosphatidic acid (LPA) from phospholipids are thought to play a critical role in the initiation and progression of CF airway disease [
      • Strandvik B.
      Fatty acid metabolism in cystic fibrosis.
      ]. Furthermore, anaerobic bacteria in CF secrete short chain fatty acids that in turn trigger bronchial epithelial cells to release IL-8[
      • Mirković B.
      • Murray M.A.
      • Lavelle G.M.
      • Molloy K.
      • Azim A.A.
      • Gunaratnam C.
      • et al.
      The role of short-chain fatty acids, produced by anaerobic bacteria, in the cystic fibrosis airway.
      ], the key pro-inflammatory cytokine in CF.
      We conducted further in vitro research to identify the cause for the surfactant dysfunction in CF at the molecular level. We showed that the mechanism for the surfactant dysfunction in CF involves two factors; an elevated cholesterol content and an inhibitory effect of oxidized unsaturated phospholipids on cholesterol function.We showed that both abnormalities were corrected in vitro by removing cholesterol from the surfactant with MβCD.
      We also showed that hydrolysis products of phosphatidylcholine; LPCs and FFAs inhibited surfactant function. The dysfunction caused by these agents was also reversed by MβCD even in the relative absence of cholesterol (Supplementary Fig. S6). It would appear that sequestration of hydrolysis products by MβCD has potential to not only correct the surfactant dysfunction but also to reduce the inflammatory effects of these products.
      Our study was limited in that there was insufficient surfactant material available from the clinical samples to permit extensive biochemical testing beyond simple assays of cholesterol, protein, and phospholipids. In many cases, even this limited testing exceeded the quantity of surfactant available. Previous studies on the biochemical composition of surfactant in pediatric CF showed slight differences between surfactant from CF patients and control patients in terms of phospholipid composition, including lysophospholipids, and SP-B and C[
      • Griese M.
      • Birrer P.
      • Demirsoy A.
      Pulmonary surfactant in cystic fibrosis.
      ,
      • Mander A.
      • Langton-Hewer S.
      • Bernhard W.
      • Warner J.O.
      • Postle A.D.
      Altered phospholipid composition and aggregate structure of lung surfactant is associated with impaired lung function in young children with respiratory infections.
      ]. We cannot comment with certainty on the role of diglycerides or triglycerides in CF surfactant dysfunction, although our unpublished results suggest that these species would only have an appreciable effect on surfactant function at levels greatly exceeding those found in the aforementioned studies.
      Based on mass spectrometric analysis of large aggregate material collected from CF patients, Mander et al. concluded that the endothelial/epithelial barrier was relatively preserved even in samples collected from patients with active infection, and that the altered lipid profiles seen in CF patients may be due to inflammatory cell membrane debris [
      • Mander A.
      • Langton-Hewer S.
      • Bernhard W.
      • Warner J.O.
      • Postle A.D.
      Altered phospholipid composition and aggregate structure of lung surfactant is associated with impaired lung function in young children with respiratory infections.
      ]. Specifically, they noted an unchanged level of PLPC, reflecting plasma lipidcontamination, and elevated levels of POPC and steroylarachidonylphosphatidylinositol, a lipid found preferentially in neutrophil cell membranes. However, the absolute increase in the levels of these latter species was modest (18.6% vs. 15.5% in control samples, and 16.1% vs. 10.0%, respectively). While our study does not specifically address the source of inhibitory surfactant lipids in CF, it seems possible that some of these species, including cholesterol, are incorporated into surfactant from the cellular debris which is generated during active inflammation and infection in CF. The present study elaborates on previous findings by suggesting an important biophysical inhibitory role for these unsaturated phospholipids, subjected to oxidation, and cholesterol. Further biochemical studies will be necessary to better characterize phospholipid molecular composition and cholesterol handling within the various lipid compartments of the inflamed pulmonary microenvironment. However, in the current study we show that regardless of surfactant contamination, MβCD treatment restored the surfactant activity in vitro.
      Further, we have previously shown ex vivo that MβCD can restore the structure and function of pulmonary surfactant films impaired by cholesterol in ventilator-induced lung injury [
      • Vockeroth D.
      • Gunasekara L.
      • Amrein M.
      • Possmayer F.
      • Lewis J.F.
      • RAW Veldhuizen
      Role of cholesterol in the biophysical dysfunction of surfactant in ventilator-induced lung injury.
      ]. MβCD is a toroid-shaped molecule with a relatively hydrophobic interior. MβCD is therefore able to sequester various hydrophobic molecules, including cholesterol and inflammatory agents such as FFAs and lysophospholipids [
      • Vockeroth D.
      • Gunasekara L.
      • Amrein M.
      • Possmayer F.
      • Lewis J.F.
      • RAW Veldhuizen
      Role of cholesterol in the biophysical dysfunction of surfactant in ventilator-induced lung injury.
      ]. In view of their low toxicity, cyclodextrins have been extensively used in pharmaceutical products and have been shown to improve the delivery of pulmonary drugs [
      • European Medicines Agency
      Background review for cyclodextrins used as excipients -in the context of the revision of the guideline on “excipients in the label and package leaflet of medicinal products for human use” (CPMP/463/00 rev. 1).
      ]. MβCD can be made readily available for inhalation in aerosol form as a dry powder or by nebulization; however these routes of administration for this clinical indication (CF) will require preclinical toxicology assessment and regulatory approvals before administration to humans.
      In conclusion, we confirm that the surface activity of pulmonary surfactant from pediatric CF patients is markedly impaired compared to lung-healthy patient samples. Exposure of CF surfactant to MβCD significantly improved surfactant function in a majority of samples. Finally, we also showed that the pro-inflammatory hydrolysis products of phosphatidylcholine (FFAs and LPC) independently impair surfactant activity in an MβCD-reversible fashion. Lipid-modifying therapies, including cyclodextrins, may therefore offer a potential treatment to restore surfactant function and reduce inflammation in the lungs of children with CF.
      The following are the supplementary data related to this article.

      Funding source

      Source(s) of support in the form of grants, gifts, equipment, and/or drugs: We are grateful to BLES Biochemicals Inc., London, Canada, for generously providing BLES.
      This research has been supported by operating grants from the TAO Foundation (RT753688), Alberta Innovates Health Solutions (AIHS) (20150980), Calgary Laboratory Services (CLS) (73-2144), and the Alberta Lung Association (2008/2009).

      Conflict of interest

      Drs. Francis Green, Matthias Amrein, Lasantha Gunasekara, Ryan Pratt, and John Dennis have shares in SolAeroMed Inc. Drs. Matthias Amrein, Lasantha Gunasekara, and Ryan Pratt have US patent (No. 13/629,474). All other authors have no conflict of interest to disclose.

      Acknowledgements

      We thank Andrea Chiu (MBT) for assistance in researching background bibliography and manuscript formatting. We thank the CF clinics, doctors, nurses, and Lori Fairchild for assistance with sample collection and Dr. Samuel Schürch for his pioneer work in surfactant research.

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