· Web viewThe exposure of surfactant to oxidants brings about multiple changes in the overall...
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Online Supplemental: Pulmonary Surfactant Dysfunction in Pediatric Cystic Fibrosis: Mechanisms and Reversal with a Lipid-Sequestering Drug
Lasantha Gunasekara*a,b, Mustafa Al-Saiedy*b,c, Francis Green*†b,d, Ryan Pratt*a,b, Candice Bjornsone, Ailian Yangb, W. Michael Schoela, Ian Mitchelle, Mary Brindlef, Mark Montgomerye, Elizabeth Keysd, John Dennisg, Grishma Shresthad, and Matthias Amrein†a,b
aDepartment of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta, Canada, bSnyder Institute of Chronic Diseases, University of Calgary, Calgary, Alberta, Canada,
cDepartment of Cardiovascular & Respiratory Sciences, University of Calgary, Calgary, Alberta, Canada dDepartment of Pathology & Laboratory Medicine, University of Calgary, Calgary, Alberta, Canada,
ePediatric Cystic Fibrosis Clinic, Alberta Children’s Hospital, Calgary, Alberta, Canada, fDepartment of Surgery, Alberta Children’s Hospital, Calgary, Alberta, Canada,
gSolAeroMed Inc., Calgary, Alberta, Canada.
Corresponding authors: †Francis Green, MD, 3280 Hospital Drive N.W., Calgary, Alberta T2N 4Z6. Tel: 1-403-220-4514; fax: 1-403-270-8928; E-mail: [email protected]. †Matthias. W. Amrein, PhD, 3280 Hospital Drive N.W., Calgary, Alberta T2N 4Z6. Tel: 1-403-210-8177; fax: 1-403-210-8192; E-
mail: [email protected]
*These authors have contributed equally
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Supplementary Methods and Results
Patient Procedures: Bronchial lavage Fluid (BLF), Routine Clinical Testing, and use of
Surplus BLF for Research
No changes to the routine clinical management of the patients were required for this
research. The children were lightly anaesthetized and a flexible bronchoscope inserted through
the larynx. After photographs of the major airways were obtained the left lingula and right
middle lobe bronchi were sequentially lavaged with normal saline (1 ml/kg) to a maximum of 20
ml. The aliquots were immediately aspirated with approximately 50% recovery. The procedure
took approximately 30-40 minutes from induction of anesthesia to recovery room (15 minutes
for induction, 10-15 minutes for bronchoscopy, and 10-15 minutes to arouse). Following
submitting samples for routine testing by microbiology and cytology, the remainder, which is
normally discarded, was used for this study. There was insufficient material to study samples
from the separate lung sites. Therefore all of the surplus BLF samples were combined for each
subject. Figure S1 shows the path of the different aliquots in this study.
In view of the limited material we established a priority route for testing. Priority was
given to functional surfactant analysis followed by treatment with MβCD and lastly biochemical
analysis of BLF/large aggregate fraction (Figure 1 in the manuscript).
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Biochemical characterization of BLF
Cell-free BLF was obtained as described above and assessed for protein content using a
bicinchoninic acid (BCA) assay (Cat #: RK-36490-00, Thermo Scientific, Rockford, IL, USA).
Sodium dodecyl sulfate (SDS) was added at a final concentration of 2% to minimize interference
from lipids. The large and small aggregate fractions were isolated as described above and lipid
extracted according to the method of Bligh and Dyer (1). Phosphate was liberated from
phospholipids by ashing according to the method of Ames and detected using a colorimetric
assay (Cat #: DIPI-500, BioAssay Systems, Hayward, CA, USA). In the BLF samples, total
lipid-soluble phosphate was calculated as the sum of phospholipids in the cell-free supernatant
after ultracentrifugation (i.e., the small aggregate fraction) and phospholipids in the cell-free
pellet after ultracentrifugation (i.e., the large aggregate fraction), assuming an average
phospholipid weight of 740 g/mol. The percent weight of LA phospholipids is therefore equal to
the ratio of lipid-soluble large aggregate phosphate divided by the sum of lipid-soluble large
aggregate phosphate and lipid-soluble small aggregate phosphate multiplied by 100.
Biochemical analyses of BLF supernatants (Table T3) shows the differences in large
aggregate phospholipids between groups were not statistically significant. Cholesterol and
cholesteryl esters were detected using a colorimetric assay according to manufacturer’s
instructions (Cat #: A12216, Invitrogen, Eugene, OR, USA). Total BLF protein was significantly
(p=0.0287) lower in the lung-healthy control compared to the CF patients (Table T3).
Surface activity assessment
A small (~0.05 mL) bubble was introduced into the chamber, and a transparent capillary
used to deposit ~1 µL of concentrated surfactant near the bubble allowing it to spread to the air-
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buffer interface. The bubble image was captured by a video camera (Pulnix TM 7 CN), digitized
and recorded for later analysis. After an initial 5 min adsorption (film formation) period, the
bubble was rapidly expanded. Following these conditioning steps, two quasi-static and two
dynamic cycle series were performed. Interfacial area and surface tension were calculated using
the bubble height and diameter (2).
Comparison between CF subjects with sufficient surfactant and insufficient surfactant for
analysis
Fisher’s test was used to determine if there were any differences between subjects with
insufficient Surfactant (IS) and Sufficient Surfactant (SS) for analysis. Analysis included gender,
exacerbations, upper respiratory tract infections (URI), colony forming units (CFU) positive,
positive culture of non-oropharyngeal organisms, and airway disease severity as assessed by the
bronchoscopist . Any value ≥103 CFU was considered to be CFU positive. Additionally, a two-
tailed t test was done on square root transformed age data.
In order to determine if there were any differences between IS and SS subjects, a two-
way analysis of variance was carried out on square root transformed data. Tukey’s pairwise
comparison was used for post hoc analysis. Both of the transformations were done to meet the
assumption of normality. A p value < 0.05 was considered significant. Table T1 shows summary
characteristics of the study population including the p values following analyses. All of the
statistical analyses were done in R Statistical Software Version 3.2.0 GUI 1.65. Additionally,
Table T2 details the spectrum of genotypes and microbiology in the CF children.
There were significantly more females in the Insufficient Surfactant group compared to
the Sufficient Surfactant group (p=0.02). There were no significant differences in any of the
other parameters between the two groups other than the percent of polymorphonuclear
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leukocytes in the BLF. There were significantly larger proportions of polymorphonuclear
leukocytes in the BLF of patients with insufficient surfactant compared to those with sufficient
surfactant (p=0.02). There were no differences in polymorphonuclear leukocyte composition of
BLF within genders between IS and SS groups; that is to say that the females in the insufficient
surfactant group did not have significantly more polymorphonuclear leukocytes compared to the
females in the sufficient surfactant group. Similarly, males in the insufficient surfactant did not
have significantly more polymorphonuclear leukocytes in the BLF compared to the males in the
sufficient surfactant group.
High Resolution live cell microscopy and Electron Microscopy of BLF cells
High-resolution dark field microscopy (RTM-3 microscopy) of live cells from the BLF
cells showed large numbers of neutrophils in the CF cases. The alveolar macrophages in CF
(figure S2A & S2B) cases show lipid overload. These macrophages are enlarged with rounded
cytoplasmic profiles with reduced cell membrane and cytoplasmic motility (see supplemental
videos SV1 and SV2). Cells showing apoptosis with release of lamellar material were also
commonly seen (Figure S2B). Electron microscopy confirmed that the lipid material was
osmiophilic and within lysosomes, with the characteristic lamellar arrangement of surfactant
(Figure S2C & S2D) (3).
Surface tension-interfacial area isotherms
One quasi-static and two dynamic cycles were repeated after completing an initial,
conditioning series of quasi-static cycling. During quasi-static cycling, the bubble size was first
reduced and then enlarged by altering the internal volume of the chamber in a stepwise fashion.
During dynamic cycling, the bubble volume was rapidly and continuously changed over the
same volume range for 20 cycles at a rate of 20 cycles/min. Surface tension-interfacial area
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isotherms for dynamic cycles for CF (Figure S3A and S3B) and lung healthy (Figure S3C)
samples are shown in Figure S3.
Figure S3A from a CF patient shows a high minimum surface tension and an extended
plateau in the surface tension-interfacial area isotherm indicating continuous film collapse upon
compression (Film compressibility (C = (∆A / ∆γ) / A, where A = surface area and γ = surface
tension)), a property of dysfunctional surfactant (4,5). Two CF samples reached surface tensions
below 2 mN/m at the start of dynamic cycling, one of these is shown in Figure S3B. However,
relatively large area reductions (60-65%) were required to reach these minimum surface
tensions. Surfactant function severely deteriorated towards the end of dynamic cycling for these
two samples in terms of minimum surface tension, indicating film instability during repeated
compression-expansion. A lung healthy control sample isotherm is shown in Figure S3C for
comparison. These show minimal hysteresis, indicating that these films compressed and
expanded elastically. In summary, we report consistent near-zero surface tensions for control
samples and high minimum surface tensions (>15 mN/m) for CF cases.
Dose-dependent inhibition of BLES surfactant by addition of LPC or an FFA (oleic acid)
To determine the effect of polyunsaturated phospholipids in mediating surfactant
dysfunction in the presence of oxidation, we individually oxidized two normal constituents of
surfactant, PLPC (oxPLPC) and cardiolipin (oxCL) before adding them to native BLES,
resulting in substantial deterioration in surface activity only in the presence of cholesterol
(Figure S4).
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Additionally, figure S5 shows minimum surface tension of dynamic cycle 20. The
addition of LPC or FFA to native BLES caused a dose-dependent inhibition of the ability of
surfactant to stably reach low minimum surface tensions. Although the addition of both FFA and
LPC resulted in significant impairment of surfactant function, FFA appears to have higher
inhibitory impact than LPC on surfactant minimum surface tension. The exposure of surfactant
to oxidants brings about multiple changes in the overall functional capabilities of the surfactant
film. Oxidation results in greater production of conjugated dienes and phospholipid
fragmentation products, mainly LPC and FFAs (6–8). We show that these products inhibit
surfactant function (Figure S6). Additionally, we examined whether surfactant inhibition by lipid
hydrolysis products is dose-dependent. We added three different concentrations of an FFA (oleic
acid) and LPC (lyso-phosphatidylcholine) to native BLES 15%, 30%, or 50% (w/w) and
examined film function via dynamic mode of CBS.
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References:
1. Bligh EG, Dyer WJ. Extraction of Lipids in Solution by the Method of Bligh & Dyer. Can J Biochem Physiol [Internet]. 1959; 2(37):911–7.
2. Schoel WM, Schürch S, Goerke J. The captive bubble method for the evaluation of pulmonary surfactant: surface tension, area, and volume calculations. BBA - Gen Subj. 1994;1200(3):281–90.
3. Kazachkov M, Muhlebach M. Lipid-laden macrophage index and inflammation in bronchoalveolar lavage fluids in children. Eur Respir [Internet]. 2001.
4. Gunasekara L, Schoel WM, Schürch S, Amrein MW. A comparative study of mechanisms of surfactant inhibition. Biochim Biophys Acta - Biomembr. 2008;1778(2):433–44.
5. Gunasekara L, Schürch S, Schoel WM, Nag K, Leonenko Z, Haufs M, et al. Pulmonary surfactant function is abolished by an elevated proportion of cholesterol. Biochim Biophys Acta - Mol Cell Biol Lipids. 2005;1737(1):27–35.
6. Markart P, Ruppert C, Wygrecka M, Colaris T, Dahal B, Walmrath D, et al. ACUTE RESPIRATORY DISTRESS SYNDROME Patients with ARDS show improvement but not normalisation of alveolar surface activity with surfactant treatment: putative role of neutral lipids. Thorax. 2007;62:588–94.
7. Hite RD, Seeds MC, Jacinto RB, Grier BL, Waite BM, Bass DA. Lysophospholipid and fatty acid inhibition of pulmonary surfactant: Non-enzymatic models of phospholipase A2 surfactant hydrolysis. Biochim Biophys Acta - Biomembr [Internet]. 2005; 1720(1–2):14–21.
8. Davidson KG, Bersten AD, Barr HA, Dowling KD, Nicholas TE, Doyle IR. Lung function, permeability, and surfactant composition in oleic acid-induced acute lung injury in rats. Am J Physiol Lung Cell Mol Physiol [Internet]. 2000;279(6):L1091-102.
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Supplementary Tables and Figures
Supplementary Table T1: Characteristics of the study population: Patients with Insufficient Surfactant (IS) and Sufficient Surfactant (SS).
IS SS
p value(n=24) (n=26)
Sex (M/F) 9/15 19/7 0.02*Age (median, range) 5.8, (1.2-16.0) 3.85, (1.0-12.1) 0.14Polymorphonuclear leukocytes (%)‡ 36.8 ± 5.7 20.0 ± 3.5 0.02*⁺Exacerbations (Y/N), (% of subjects) 5/16, (23.8) 4/22, (15.4) 0.49⁺Upper Respiratory Infection (Y/N), (% of subjects) 2/15, (11.8) 3/20, (13.0) 1.00⁺Colony Forming Unit Positive (Y/N), (% of subjects) 9/14, (39.1) 14/12, (53.8) 0.39⁺Known Pathogens Cultured (Y/N), (% of subjects) 16/5, (76.2) 16/10, (61.5) 0.35
Airway disease severity (% of subjects) 0.22Normal 36.8 16.0Mild 47.4 48.0Moderate-Severe 15.8 36.0
Abbreviations: F = female; M = male⁺some missing information on subjects.‡Data expressed as mean ( standard error).*Significant (p value < 0.05)
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Supplementary Table T2: Genotypes and microbiology for the cystic fibrosis children.
Case Age at Lavage Allele 1 Allele 2 Culture Results* Colony Forming Units
(CFU/mL)1 6.78 ΔF508 ΔF508 HI 104
2 1.76 E60X 1138insG ORPF 03 5.55 ΔF508 ΔF508 Yeast >104
4 3.50 M1101K M1101K HI, SA 104
5 5.40 ΔF508 ΔF508 NG 06 1.27 M1101K M1101K PA 104
7 7.39 ΔF508 ΔF508 GNB 104
8 11.25 ΔF508 M1101K PA 104
9 2.29 M1101K M1101K HI 104
10 12.13 ΔF508 M1101K NG 011 2.24 ΔF508 ΔF508 KN, AB, SM 104
12 1.03 ΔF508 2184delA MC, GNB 104
13 7.17 ΔF508 I506L HI, SA 104
14 6.04 E60X 1138insG ORPF 015 2.10 2143delT 2184insA ORPF 016 9.34 ΔF508 ΔF508 PA >107
17 5.62 M1101K M1101K HI >103
18 11.25 ΔF508 W1282X NG 019 9.90 ΔF508 G458V AX <103
20 3.35 ΔF508 G458V NG 021 1.67 ΔF508 ΔF508 ORPF <103
22 4.20 G551D ΔF508 EC, SM 104
23 1.90 ΔF508 A445E CA, PS, CS 104
24 1.30 N1303K N1303K ORPF 103
25 2.00 ΔF508 A455E ORPF 026 1.10 ΔF508 ΔF508 ORPF 0
*Legend: AB = Acinetobacter Baumannii, AX = Achromobacter Xylosoxidans, CA = Candida Albicans, CS = Chrysosporium Species, EC = Escherichia Coli, GNB = Gram Negative Bacteria, HI = Haemophilus Influenzae, KN = Klebsiella Pneumoniae, MC = Moraxella Catarrhalis, NG = No Growth, ORPF = oropharangeal flora, PA = Pseudomonas Aeruginosa, PS = Penicillium Species, SA = Staphylococcus Aureus, SM = Strenotophomonas Maltophilia
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Microbiology (Bacterial /Fungal Cultures)
Cytology (Manual Cell Differentials)
Fresh BLF
Electron Microscopy
Research Samples: surfactant assays, lipid biochemistry, and vital microscopy
Routine Testing
Supplementary Table T3: Biochemical analysis of BLF fluid
CF patients
(n = 5)
Lung-healthy control patients
(n = 4)
Total cell-free BLF protein (μg/mL)
486.48 ± 73.90 181.97 ± 53.08*
Large aggregate phospholipids (wt % total lipid) 90.07 ± 1.54 78.30 ± 5.39
Cholesterol: LA phospholipids (wt %)
13.00 ± 1.44 4.96 ± 0.70**
Data presented as mean ± SEM (*p ≤ 0.05, ** p ≤ 0.01 compared to CF patients, Mann-Whitney U test)
Supplementary Figure S1: Path of bronchial(BLFF) aliquots in study.
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Supplementary Figure S2: Light micrographs of fresh alveolar macrophages from CF children
(A, B). Numerous large lipid-containing lysosomes are seen in their cytoplasm. Electron
micrographs of alveolar macrophages from CF (C) and control (D) children. Numerous large,
abnormal lipid-containing lamellar bodies are seen only within the lysosomes of the CF
macrophage. Bar = 2µm.
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Supplementary Figure S3. Dynamic cycle surface tension-area isotherms for representative
samples of an included (A) CF samples, (B) a partially functional CF sample, and (C) a lung
healthy control sample.
Supplementary Figure S4. (A) Minimum (circles) and maximum (triangles) surface tensions
during dynamic compression-expansion cycles of BLES containing 20% w/w oxPLPC without
(white) or with (black) 10% w/w cholesterol added. (B) Minimum and maximum surface
tensions during dynamic compression-expansion cycles of BLES containing 20% w/w oxCL
without or with 10% w/w cholesterol added.
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A B C
0
10
20
30
40
50
60
70
0 5 10 15 20
Cycle number
Sur
face
tens
ion
(mN
/m)
0
10
20
30
40
50
60
70
0 5 10 15 20
Cycle number
Sur
face
tens
ion
(mN
/m)
A B
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0 20 40 600
5
10
15
20
***
*
Amount added (% w/w)
Min
imum
Surf
ace
Tens
ion
(mN
/m)
Supplementary Figure S5. Minimum surface tensions during dynamic cycle 20 with healthy
BLES containing 0%, 15%, 30%, or 50% w/w oleic acid (squares), or LPC (circles). Both
hydrolysis products showed dose-dependent surfactant inhibition, with greater inhibition shown
by oleic acid compared to LPC. * p < 0.05, *** p < 0.001 compared to native (0%) BLES.
Supplementary Figure S6. Minimum surface tension during dynamic cycle 20 with BLES
containing 27 mg/ml BLES in control CBS buffer (checkers bars), 50% w/w FFA (Oleic acid) or
LPC in control CBS buffer (horizontal bars) or in buffer containing 40 mg/ml MβCD (solid
black bars). (** p ≤ 0.01, *** p ≤ 0.001). BLES with FFA or LPC shows marked impairment,
which is repaired to normal functionality in the presence of MβCD. Cholesterol was present at
very low concentrations (~2.1%) in these experiments.
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Video microscopy of CF macrophages
Video files V1A and V1B below show alveolar macrophages engorged with excess lipid
material, some of which has a lamellar appearance. In addition, the cytoplasm of both cells
contains large amounts of exogenous material which stained for DNA using Hoechst blue (not
shown in the video). There is marked asymmetry of cytoplasmic motility in both cells, some
areas of the cytoplasm are completely immotile. The cell membrane of the cell shown in video 1
is relatively inactive. The cell membrane in video two is more motile.
Supplementary video V1A: Alveolar Macrophage engorged with excess lipid material
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Supplementary video V1B: Asymmetric cytoplasmic motility of alveolar macrophage
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