4. Discussion
This study confirms profound dysfunction of surfactant obtained from the airways of pediatric CF patients [
[4]- 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 [
[2]- 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 [
[16]- 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 [
[4]- 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 [
[14]- 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 [
[17]- 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 [
[18]- 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 [
[19]- 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 [
[20]- 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[
[4]- 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 [
[1]- 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[
[12]- 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 [
[13]- 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 [
10- 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.
,
11- 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 [
[21]- 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[
19- 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.
,
22Fatty acid metabolism in cystic fibrosis.
,
23- 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 [
[19]- 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 [
[23]- 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 [
18- 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.
,
24- 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 [
[25]- 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 [
[26]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 [
[26]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[
[27]- 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[
28- Griese M.
- Birrer P.
- Demirsoy A.
Pulmonary surfactant in cystic fibrosis.
,
29- 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 [
[29]- 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 [
[11]- 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 [
[11]- 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 [
[30]- 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.
Article info
Publication history
Published online: June 07, 2017
Accepted:
April 25,
2017
Received in revised form:
March 31,
2017
Received:
December 20,
2016
Footnotes
☆Author contributions: Drs Gunasekara, Schoel, Al-Saiedy, Pratt, and Yang performed the experiments, data acquisition and analysis, and drafting of the manuscript. DrsAmrein and Green formulated the scientific question, designed the study, developed the methodology, drafted, edited the manuscript and provided final approval. Drs Bjornson, Brindle, Mitchell, and Montgomery recruited subjects, obtained consents, provided clinical input, conducted lung lavages, collected the research samples and contributed to editing and drafting the manuscript. Drs.Bjornson and Montgomery also provided genotype information on the CF patients. Ms. Keys performed the live cell and electron microscopy studies on patient samples, wrote the relevant methodology and contributed to the draft manuscript. Ms. Shrestha provided statistical support, data analysis, and revisions of the manuscript. Dr. Dennis provided data and expertise on methods for delivering cyclodextrins to patients.
☆☆All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.
Copyright
© 2017 European Cystic Fibrosis Society. Published by Elsevier B.V.