Interplay Between Pulmonary Membrane Properties and Lung Disease: A Study of Seven Bottlenose Dolphins
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eLife Assessment
The study presents data on the possible connection of respiratory pathologies like pneumonia in a cohort of dolphins with altered composition and concomitant perturbed biophysical properties of pulmonary surfactant complexes. Overall, it is a valuable contribution that could be of interest to scientists in the field. However, the study as it is appears somewhat incomplete and additional clarification and discussions are required in order to explain a few methodological questions that may limit the impact of the work considerably.
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Abstract
Pneumonia is the leading cause of morbidity and mortality of bottlenose dolphins Tursiops truncatus. We investigate a series of rare and opportunistic samples of pulmonary surfactant membranes (PSMs) extracted from lungs of seven dolphins in the care of the U.S. Navy Marine Mammal Program. We found a striking correlation between PSM structure, lipidome, and mechanical properties with the severity of lung injury. Specifically, lipidomics reveals exacerbated contents of cardiolipins, confirming a result obtained for terrestrial mammals afflicted by experimental pneumonia. Employing a battery of X-ray scattering, atomic force, and electron microscopy, we evaluate how the altered lipid composition impairs the structural integrity of the PSM and leads to dehydration and enhanced rigidity. Our findings demonstrate that the function of pulmonary surfactant membranes goes far beyond lowering alveolar surface tension, regulating their biochemical and biophysical properties with lung pathology progression. This knowledge will be useful to the development of future diagnostics and therapeutics of respiratory diseases targeting lung membranes.
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eLife Assessment
The study presents data on the possible connection of respiratory pathologies like pneumonia in a cohort of dolphins with altered composition and concomitant perturbed biophysical properties of pulmonary surfactant complexes. Overall, it is a valuable contribution that could be of interest to scientists in the field. However, the study as it is appears somewhat incomplete and additional clarification and discussions are required in order to explain a few methodological questions that may limit the impact of the work considerably.
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Reviewer #1 (Public review):
Summary:
This paper describes a number of alterations in pulmonary surfactant recovered from bottlenosed dolphins. Although the sample consists of only seven diseased and two control animals, due to the difficulty in obtaining these animals, this is considered adequate. However, conclusions must be considered in view of this small sample size. The authors employ a number of sophisticated techniques to show differences in the composition and in the structure of bilayers formed by these two surfactant samples
Strengths:
The availability of these samples makes this study quite original. The authors apply mass spectroscopy to observe an increase of an acidic phospholipid and in the level of plasmalogens in the diseased (i.e. pneumonia) aquatic animals. They suggest these increases contribute to hampered function …
Reviewer #1 (Public review):
Summary:
This paper describes a number of alterations in pulmonary surfactant recovered from bottlenosed dolphins. Although the sample consists of only seven diseased and two control animals, due to the difficulty in obtaining these animals, this is considered adequate. However, conclusions must be considered in view of this small sample size. The authors employ a number of sophisticated techniques to show differences in the composition and in the structure of bilayers formed by these two surfactant samples
Strengths:
The availability of these samples makes this study quite original. The authors apply mass spectroscopy to observe an increase of an acidic phospholipid and in the level of plasmalogens in the diseased (i.e. pneumonia) aquatic animals. They suggest these increases contribute to hampered function in vivo. They show alterations in lipid bilayers formed from lipid extracts of these surfactants by electron microscopy, by Atomic Force Microscopy and by small and wide-angle X-ray scattering -SAXS/WAXS. They have previously shown that adding small amounts of cardiolin to the clinical surfactant BLES results in altered bilayer structure, consistent with the current study.
Weaknesses:
It seems surprising to me that the small changes in cardiolipin can alter surfactant function i.e., reducing surface tension to near zero. As it happens, no surfactant function tests monitoring the reduction in surface tension were conducted. This would add a great deal to the paper. Further, the paper would benefit greatly from the inclusion of a table listing the lipid composition of surfactant recovered from diseased and normal animals and comparing this to the composition of BLES, a clinical surfactant. Finally, there is a possibility that the minor lipid identified by mass spec is the lysosomal marker, bis-(monoacylglcerol)phosphate rather than the metachronal marker, cardiolipin.
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Reviewer #2 (Public review):
Summary:
In this manuscript, Porras-Gómez et al. analyse the lipid composition and biophysical properties of pulmonary surfactant obtained by bronchoalveolar lavage (BAL) from a group of bottlenose dolphins (Tursiops truncatus), including two healthy individuals and five affected by pneumonia. Through lipidomic analysis, the authors report an exacerbated presence of cardiolipin species in the BAL lipid extracts from diseased dolphins compared to healthy ones. Structural analyses using electron microscopy, atomic force microscopy, and X-ray scattering on rehydrated membrane samples reveal that lipids from diseased animals form membranes with a more pronounced Lβ phase and reduced fluidity. Moreover, the membranes from affected lungs appear more interconnected and less hydrated, as indicated by the X-ray …
Reviewer #2 (Public review):
Summary:
In this manuscript, Porras-Gómez et al. analyse the lipid composition and biophysical properties of pulmonary surfactant obtained by bronchoalveolar lavage (BAL) from a group of bottlenose dolphins (Tursiops truncatus), including two healthy individuals and five affected by pneumonia. Through lipidomic analysis, the authors report an exacerbated presence of cardiolipin species in the BAL lipid extracts from diseased dolphins compared to healthy ones. Structural analyses using electron microscopy, atomic force microscopy, and X-ray scattering on rehydrated membrane samples reveal that lipids from diseased animals form membranes with a more pronounced Lβ phase and reduced fluidity. Moreover, the membranes from affected lungs appear more interconnected and less hydrated, as indicated by the X-ray scattering data. These findings provide valuable and convincing insights into how pulmonary disease alters the lipid composition and structural properties of surfactant in diving mammals, and may have broader implications for understanding surfactant dysfunction in marine mammals.
Strengths:
The study is well designed, and the experimental techniques were applied in a logical and coherent manner. The results are thoroughly analysed and discussed, and the manuscript is clearly written and well organized, making it both easy to follow and scientifically robust. Although the number of samples is limited, the rarity and logistical challenges of obtaining bronchoalveolar lavage material, particularly from animals affected by respiratory disease, make this study especially valuable and relevant.
Weaknesses:
In my opinion, the main issue lies in the treatment of the samples. Pulmonary surfactant is a lipoprotein complex produced by type II pneumocytes of the alveolar epithelium in the form of compact and highly dehydrated structures known as tubular myelin. Once secreted, these structures unfold and, upon contact with the air-liquid interface, form an interfacial monolayer connected to surfactant membranes in the subphase, thereby facilitating respiratory dynamics throughout the breathing cycle.
When bronchoalveolar lavages are treated using the Bligh and Dyer method to extract the hydrophobic fraction of these samples, the structural complexity of the surfactant is disrupted, and this organization cannot be completely restored once the lipids are rehydrated. Although these extracts contain the hydrophobic proteins SP-B and SP-C, the hydrophilic protein SP-A may play an essential role in the formation of pulmonary surfactant structures. It is well established that SP-A is crucial for the formation of tubular myelin, an intermediate structure between the lamellar bodies newly secreted by type II cells and the interfacial surfactant layers.
Moreover, and more importantly, bronchoalveolar lavage fluid may contain cells, tissue debris, and even bacteria that can alter the lipid composition of the samples used in the study after extraction by the Bligh and Dyer method. For this reason, most studies include a density gradient centrifugation step to isolate the surfactant membranes. Consequently, the samples used may be contaminated with phospholipids originating from other cells, such as macrophages, pneumocytes, or bacterial cells, particularly in lavages obtained from diseased animals.
Although the techniques employed provide valuable information about the behaviour of surfactant membranes and allow certain inferences regarding their functionality, no functional studies of these samples have been conducted using methods such as the constrained drop surfactometer or the captive bubble surfactometer. The observed alterations do not necessarily demonstrate that surfactant modulates its properties, as claimed by the authors, but rather indicate that it is altered by the presence of other lipids.
The spin-coating technique used to form lipid films for analysis by atomic force microscopy is not the most suitable approach to reproduce the structures generated by pulmonary surfactant. However, the results obtained may still provide valuable insights into the biophysical behaviour of its components. The analysis of lung tissue shown in Supplementary Figure S3 presents the same limitation, as the samples were embedded in a cutting compound, and the measurements may have been taken from different regions of the tissue. Therefore, it cannot be ensured that the analysed structures correspond to those generated by pulmonary surfactant.
The finding that the structures formed in samples obtained from diseased animals are more tightly packed and dehydrated than those derived from the surfactant of healthy animals contrasts with the notion that the high efficiency of lamellar bodies in generating interfacial structures is related to their high degree of packing and dehydration. The formation of these structures involves the participation of the ABCA3 protein, which pumps phospholipids into the interior of lamellar bodies, and SP-B, which facilitates the formation of close membrane contacts.
While the results are interesting from a comparative perspective, the implications for surfactant performance and respiratory dynamics should be interpreted with caution.
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Reviewer #3 (Public review):
In this manuscript, the authors present data on the supposed composition of pulmonary surfactant obtained from bronchoalveolar lavages (BALs) of a small cohort of dolphins, a group of them suffering from pneumonia. The lipid compositional differences of the sample group are consistent with the different pathological situations of the specimens, suggesting that differences in surfactant composition are somehow associated (as a cause or as a consequence) with the particular pathophysiological contexts. It is particularly remarkable that an increase in cardiolipins and plasmalogens appears as an abnormal composition in pathological surfactants. The study is completed by analyzing the differences in membrane properties (order, packing, phase) of abnormal versus "control" membranes, concluding that pneumonia in …
Reviewer #3 (Public review):
In this manuscript, the authors present data on the supposed composition of pulmonary surfactant obtained from bronchoalveolar lavages (BALs) of a small cohort of dolphins, a group of them suffering from pneumonia. The lipid compositional differences of the sample group are consistent with the different pathological situations of the specimens, suggesting that differences in surfactant composition are somehow associated (as a cause or as a consequence) with the particular pathophysiological contexts. It is particularly remarkable that an increase in cardiolipins and plasmalogens appears as an abnormal composition in pathological surfactants. The study is completed by analyzing the differences in membrane properties (order, packing, phase) of abnormal versus "control" membranes, concluding that pneumonia in dolphins is associated with a significant alteration of surfactant membranes that become more rigid, packed and thicker than those in surfactant from animals with no lung disease.
In general terms, the data provided are of interest as they somehow offer a framework of effects that may extend what is known about alterations of composition, biophysical properties and functional performance of pulmonary surfactant as a consequence of respiratory pathologies. A collection of pertinent biophysical methodologies (fluorescence, X-ray scattering, AFM) have been applied to complete a full characterization of membrane properties in the different samples.
However, they way the samples have been processed, i.e. by making organic extracts of hydrophobic (lipid and protein) components before surfactant membranes have been purified or at least, separated from bulk lavage, open the question of how much of the altered composition is actually occurring in surfactant or comes from other membranes (from cells, bacteria) that have been completely intermixed as a consequence of the organic extraction. Without an appropriate surfactant membrane obtention, the results of the study should be taken with caution and await confirmation. Specific questions that need to be considered include:
(1) As said, the direct organic extract of BAL samples ends in a full mix of lipid and protein components that in origin could be part of different membranes, either from different surfactant assemblies, or even from pulmonary cells or membrane debris, or microorganisms, collected within the lavage. Obtaining conclusions about the structure and properties of membranes artefactually reconstituted from such lipid and protein mixtures is far from correct.
It is mentioned that "subsequentially" to the organic extraction, the samples were subjected to ultracentrifugation to separate debris and membrane cells. I do not see what the ultracentrifugation is going to change if it is done after the organic extraction. It should have been done before the extraction, for the organic solvents to solubilize exclusively the large, and relatively light, surfactant membrane complexes.
On the other hand, the ulterior reconstitution of the obtained full lipid mixture surely ends in membrane assemblies whose compositional distribution and organization may differ significantly from those in the original membranes.
Taking all this into account, statements such as "These aggregate forms reproduce the expected membrane microstructures observed in native alveolar hypophase" or "pulmonary membranes can be successfully extracted and reconstituted from BALs of Navy dolphins" are simply not true and should be rephrased.
One can understand that the limitation of material may make it difficult to obtain first the purified surfactant membranes and then their organic extract. However, the limitation should be acknowledged to make the readers clear that the actual compositional effects caused in surfactant by pneumonia need confirmation.
(2) In some of the experiments, i.e. in the AFM characterization, supported membranes were prepared by the spray-dry method applied to organic solutions. Again, the spray-dry of organic lipid solutions ends in a lipid dispersion that may be very far from the real organization of the lipids in actual surfactant membranes.
(3) When stated that phospholipid concentrations are greater in BAL from pinnipeds than in humans, how has the actual concentration been determined? BAL volumes are typically subjected to large variations depending on the conditions used to obtain the lavage (including volume of saline instilled, level of atelectasia in the lung tissue, presence of inflammation and edema, etc). If total amounts of phospholipids in BAL are to be compared, certain normalization procedures should be applied, such as for instance, with respect to the urea concentration in serum.
(4) All the differences regarding membrane phase and lipid order/packing have been interpreted in terms of the potential coexistence of Lbeta (gel)/Lalpha (liquid crystalline) phases. However, it has been well established that in lipid systems containing cholesterol, such as pulmonary surfactant, phase coexistence can actually be of the type liquid-ordered (Lo)/liquid-disordered (Ld), very different in terms of mobility and true molecular order. Why do the authors consider that Lbeta is the phase observed in the surfactant membranes they have reconstituted? The presence of round-shaped domains seems to indicate that a liquid/liquid phase segregation is actually occurring.
(5) In the same line as the previous comment, the authors state that SAXS shows that bovine-extracted pulmonary membranes exhibit a coexistence of two lamellar phases, one rich in unsaturated lipids and one in saturated lipids. SAXS and WAXS cannot provide compositional information, but structural parameters such as membrane thickness, or molecular order. This should be clarified.
(6) It is mentioned that the surfactant monolayer at the air-liquid interface is interconnected to tubular membranous structures (tubular myelin, TM). It is true that TM, when present, appears interconnected with the interface. However, it is widely recognized that there are many other structures connected with the interfacial film, including multilamellar membrane arrays or reservoirs that have not been mentioned here. Furthermore, TM is not required for surfactant function, because it is absent, for instance, in mice lacking expression of surfactant protein SP-A, which can breathe perfectly.
(7) In the Discussion, the authors mention that "...after squeeze-out, the excluded multilayers remain closely associated with the interfacial monolayer rather than escaping into the subphase". The authors may like to complete this discussion by specifying that the stable association of excluded assemblies with the interfacial film is actually possible thanks to the surfactant proteins.
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