Beyond the Interface: Improved Pulmonary Surfactant-Assisted Drug Delivery through Surface-Associated Structures.

Pulmonary surfactant (PS) has been proposed as an efficient drug delivery vehicle for inhaled therapies. Its ability to adsorb and spread interfacially and transport different drugs associated with it has been studied mainly by different surface balance designs, typically interconnecting various compartments by interfacial paper bridges, mimicking in vitro the respiratory air-liquid interface. It has been demonstrated that only a monomolecular surface layer of PS/drug is able to cross this bridge. However, surfactant films are typically organized as multi-layered structures associated with the interface. The aim of this work was to explore the contribution of surface-associated structures to the spreading of PS and the transport of drugs. We have designed a novel vehiculization balance in which donor and recipient compartments are connected by a whole three-dimensional layer of liquid and not only by an interfacial bridge. By combining different surfactant formulations and liposomes with a fluorescent lipid dye and a model hydrophobic drug, budesonide (BUD), we observed that the use of the bridge significantly reduced the transfer of lipids and drug through the air-liquid interface in comparison to what can be spread through a fully open interfacial liquid layer. We conclude that three-dimensional structures connected to the surfactant interfacial film can provide an important additional contribution to interfacial delivery, as they are able to transport significant amounts of lipids and drugs during surfactant spreading.

Increased Alveolar Heparan Sulphate and Reduced Pulmonary Surfactant Amount and Function in the Mucopolysaccharidosis IIIA Mouse.

Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disease with significant neurological and skeletal pathologies. Respiratory dysfunction is a secondary pathology contributing to mortality in MPS IIIA patients. Pulmonary surfactant is crucial to optimal lung function and has not been investigated in MPS IIIA. We measured heparan sulphate (HS), lipids and surfactant proteins (SP) in pulmonary tissue and bronchoalveolar lavage fluid (BALF), and surfactant activity in healthy and diseased mice (20 weeks of age). Heparan sulphate, ganglioside GM3 and bis(monoacylglycero)phosphate (BMP) were increased in MPS IIIA lung tissue. There was an increase in HS and a decrease in BMP and cholesteryl esters (CE) in MPS IIIA BALF. Phospholipid composition remained unchanged, but BALF total phospholipids were reduced (49.70%) in MPS IIIA. There was a reduction in SP-A, -C and -D mRNA, SP-D protein in tissue and SP-A, -C and -D protein in BALF of MPS IIIA mice. Captive bubble surfactometry showed an increase in minimum and maximum surface tension and percent surface area compression, as well as a higher compressibility and hysteresis in MPS IIIA surfactant upon dynamic cycling. Collectively these biochemical and biophysical changes in alveolar surfactant are likely to be detrimental to lung function in MPS IIIA.

Surfactant Protein B Promotes Cytosolic SiRNA Delivery by Adopting a Virus-like Mechanism of Action.

RNA therapeutics are poised to revolutionize medicine. To unlock the full potential of RNA drugs, safe and efficient (nano)formulations to deliver them inside target cells are required. Endosomal sequestration of nanocarriers represents a major bottleneck in nucleic acid delivery. Gaining more detailed information on the intracellular behavior of RNA nanocarriers is crucial to rationally develop delivery systems with improved therapeutic efficiency. Surfactant protein B (SP-B) is a key component of pulmonary surfactant (PS), essential for mammalian breathing. In contrast to the general belief that PS should be regarded as a barrier for inhaled nanomedicines, we recently discovered the ability of SP-B to promote gene silencing by siRNA-loaded and lipid-coated nanogels. However, the mechanisms governing this process are poorly understood. The major objective of this work was to obtain mechanistic insights into the SP-B-mediated cellular delivery of siRNA. To this end, we combined siRNA knockdown experiments, confocal microscopy, and focused ion beam scanning electron microscopy imaging in an in vitro non-small-cell lung carcinoma model with lipid mixing assays on vesicles that mimic the composition of (intra)cellular membranes. Our work highlights a strong correlation between SP-B-mediated fusion with anionic endosomal membranes and cytosolic siRNA delivery, a mode of action resembling that of certain viruses and virus-derived cell-penetrating peptides. Building on these gained insights, we optimized the SP-B proteolipid composition, which dramatically improved delivery efficiency. Altogether, our work provides a mechanistic understanding of SP-B-induced perturbation of intracellular membranes, offering opportunities to fuel the rational design of SP-B-inspired RNA nanoformulations for inhalation therapy.

Structure and activity of human surfactant protein D from different natural sources.

Surfactant protein D (SP-D) is a C-type lectin that participates in the innate immune defense of lungs. It binds pathogens through its carbohydrate recognition domain in a calcium-dependent manner. Human surfactant protein D (hSP-D) has been routinely obtained from bronchoalveolar lavage of patients suffering from pulmonary alveolar proteinosis (PAP) and from amniotic fluid (AF). As a consequence of the disease, hSP-D obtained from PAP is found in higher amounts and is mainly composed of higher order oligomeric forms. However, PAP-hSP-D has never been directly compared with nonpathological human protein in terms of structure and biological activity. Moreover, the quantitative distribution of the different hSP-D oligomeric forms in human protein obtained from a natural source has never been evaluated. In this work, we have determined the quantitative distribution of AF-hSP-D oligomers, characterized the sugars attached through the N-glycosylation site of the protein, and compared the activity of hSP-D from AF and PAP with respect to their ability to bind and agglutinate bacteria. We have found that fuzzy balls (40%) are the most abundant oligomeric form in AF-hSP-D, very closely followed by dodecamers (33%), with both together constituting 73% of the protein mass. The glycan attached to the N-glycosylation site was found to be composed of fucose, galactose, sialic acid, and N-acetylglucosamine. Finally, in the functional assays performed, hSP-D obtained from PAP showed higher potency, probably as a consequence of its higher proportion of large oligomers compared with hSP-D from AF.

Functional characterization of the different oligomeric forms of human surfactant protein SP-D.

Surfactant Protein D (SP-D) is a collectin protein that participates in the innate immune defense of the lungs. SP-D mediates the clearance of invading microorganisms by opsonization, aggregation or direct killing, which are lately removed by macrophages. SP-D is found as a mixture of trimers, hexamers, dodecamers and higher order oligomers, "fuzzy balls". However, it is unknown whether there are differences between these oligomeric forms in functions, activity or potency. In the present work, we have obtained fractions enriched in trimers, hexamers and fuzzy balls of full-length recombinant human (rh) SP-D by size exclusion chromatography, in a sufficient amount to perform functional assays. We have evaluated the differences in protein lectin-dependent activity relative to aggregation and binding to E. coli, one of the ligands of SP-D in vivo. Fuzzy balls are the most active oligomeric form in terms of binding and aggregation of bacteria, achieving 2-fold binding higher than hexamers and 50% bacteria aggregation at very short times. Hexamers, recently described as a defined oligomeric form of the protein, have never been isolated or tested in terms of protein activity. rhSP-D hexamers efficiently bind and aggregate bacteria, achieving 50-60% aggregation at final time point and high protein concentrations. Nevertheless, trimers are not able to aggregate bacteria, although they bind to them. Therefore, SP-D potency, in functions that relay on the C-lectin activity of the protein, is proportional to the oligomeric state of the protein.

Surfactant Injury in the Early Phase of Severe Meconium Aspiration Syndrome.

No in vivo data are available regarding the effect of meconium on human surfactant in the early stages of severe meconium aspiration syndrome (MAS). In the present study, we sought to characterize the changes in surfactant composition, function, and structure during the early phase of meconium injury. We designed a translational prospective cohort study of nonbronchoscopic BAL of neonates with severe MAS (n = 14) or no lung disease (n = 18). Surfactant lipids were analyzed by liquid chromatography-high-resolution mass spectrometry. Secretory phospholipase A2 subtypes IB, V, and X and SP-A (surfactant protein A) were assayed by ELISA. SP-B and SP-C were analyzed by Western blotting under both nonreducing and reducing conditions. Surfactant function was assessed by adsorption test and captive bubble surfactometry, and lung aeration was evaluated by semiquantitative lung ultrasound. Surfactant nanostructure was studied using cryo-EM and atomic force microscopy. Several changes in phospholipid subclasses were detected during MAS. Lysophosphatidylcholine species released by phospholipase A2 hydrolysis were increased. SP-B and SP-C were significantly increased together with some shorter immature forms of SP-B. Surfactant function was impaired and correlated with poor lung aeration. Surfactant nanostructure was significantly damaged in terms of vesicle size, tridimensional complexity, and compactness. Various alterations of surfactant phospholipids and proteins were detected in the early phase of severe meconium aspiration and were due to hydrolysis and inflammation and a defensive response. This impairs both surfactant structure and function, finally resulting in reduced lung aeration. These findings support the development of new surfactant protection and antiinflammatory strategies for severe MAS.

In Vitro Functional and Structural Characterization of A Synthetic Clinical Pulmonary Surfactant with Enhanced Resistance to Inhibition.

CHF5633 is a novel synthetic clinical pulmonary surfactant preparation composed by two phospholipid species, dipalmitoyl phosphatidylcholine (DPPC) and palmitoyloleoyl phosphatidylglycerol (POPG), and synthetic analogues of the hydrophobic surfactant proteins SP-B and SP-C. In this study, the interfacial properties of CHF5633 in the absence and in the presence of inhibitory serum proteins have been assessed in comparison with a native surfactant purified from porcine lungs and with poractant alpha, a widely used clinical surfactant preparation. The study of the spreading properties of CHF5633 in a Wilhelmy balance, its ability to adsorb and accumulate at air-liquid interfaces as revealed by a multiwell fluorescence assay, and its dynamic behavior under breathing-like compression-expansion cycling in a Captive Bubble Surfactometer (CBS), all revealed that CHF5633 exhibits a good behavior to reduce and sustain surface tensions to values below 5 mN/m. CHF5633 shows somehow slower initial interfacial adsorption than native surfactant or poractant alpha, but a better resistance to inhibition by serum proteins than the animal-derived clinical surfactant, comparable to that of the full native surfactant complex. Interfacial CHF5633 films formed in a Langmuir-Blodgett balance coupled with epifluorescence microscopy revealed similar propensity to segregate condensed lipid domains under compression than films made by native porcine surfactant or poractant alpha. This ability of CHF5633 to segregate condensed lipid phases can be related with a marked thermotropic transition from ordered to disordered membrane phases as exhibited by differential scanning calorimetry (DSC) of CHF5633 suspensions, occurring at similar temperatures but with higher associated enthalpy than that shown by poractant alpha. The good interfacial behavior of CHF5633 tested under physiologically meaningful conditions in vitro and its higher resistance to inactivation by serum proteins, together with its standardized and well-defined composition, makes it a particularly useful therapeutic preparation to be applied in situations associated with lung inflammation and edema, alone or in combined strategies to exploit surfactant-facilitated drug delivery.

Pulmonary Surfactant and Drug Delivery: An Interface-Assisted Carrier to Deliver Surfactant Protein SP-D Into the Airways.

This work is focused on the potential use of pulmonary surfactant to deliver full-length recombinant human surfactant protein SP-D (rhSP-D) using the respiratory air-liquid interface as a shuttle. Surfactant protein D (SP-D) is a collectin protein present in the pulmonary surfactant (PS) system, involved in innate immune defense and surfactant homeostasis. It has been recently suggested as a potential therapeutic to alleviate inflammatory responses and lung diseases in preterm infants suffering from respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD). However, none of the current clinical surfactants used for surfactant replacement therapy (SRT) to treat RDS contain SP-D. The interaction of SP-D with surfactant components, the potential of PS as a respiratory drug delivery system and the possibility to produce recombinant versions of human SP-D, brings the possibility of delivering clinical surfactants supplemented with SP-D. Here, we used an in vitro setup that somehow emulates the respiratory air-liquid interface to explore this novel approach. It consists in two different compartments connected with a hydrated paper bridge forming a continuous interface. We firstly analyzed the adsorption and spreading of rhSP-D alone from one compartment to another over the air-liquid interface, observing low interfacial activity. Then, we studied the interfacial spreading of the protein co-administered with PS, both at different time periods or as a mixed formulation, and which oligomeric forms of rhSP-D better traveled associated with PS. The results presented here demonstrated that PS may transport rhSP-D long distances over air-liquid interfaces, either as a mixed formulation or separately in a close window time, opening the doors to empower the current clinical surfactants and SRT.

Effect of Whole Body Hypothermia on Surfactant Function When Amniotic Fluid Is Meconium Stained.

We provide the first complete biophysical study of surfactant by captive bubble surfactometry from a neonate with meconium-stained amniotic fluid under controlled whole body hypothermia. Surfactant function improves after 48 hours of hypothermia, as surfactant cholesterol decreases. These findings partially explain positive effect of hypothermia on respiratory outcomes during meconium aspiration syndrome. A larger study including several neonates with or without lung disease is being conducted to better define the effect of hypothermia on surfactant function.

SP-D attenuates LPS-induced formation of human neutrophil extracellular traps (NETs), protecting pulmonary surfactant inactivation by NETs.

An exacerbated amount of neutrophil extracellular traps (NETs) can cause dysfunction of systems during inflammation. However, host proteins and factors that suppress NET formation (NETosis) are not clearly identified. Here we show that an innate immune collectin, pulmonary surfactant protein-D (SP-D), attenuates lipopolysaccharide (LPS)-mediated NETosis in human neutrophils by binding to LPS. SP-D deficiency in mice (Sftpd-/-) leads to excess NET formation in the lungs during LPS-mediated inflammation. In the absence of SP-D, NETs inhibit the surface-active properties of lung surfactant, essential to prevent the collapse of alveoli, the air breathing structures of the lungs. SP-D reverses NET-mediated inhibition of surfactant and restores the biophysical properties of surfactant. To the best of our knowledge, this study establishes for the first time that (i) SP-D suppresses LPS-mediated NETosis, (ii) NETs inhibit pulmonary surfactant function in the absence of SP-D, and (iii) SP-D can restore NET-mediated inhibition of the surfactant system.

Nanocarrier Lipid Composition Modulates the Impact of Pulmonary Surfactant Protein B (SP-B) on Cellular Delivery of siRNA.

Two decades since the discovery of the RNA interference (RNAi) pathway, we are now witnessing the approval of the first RNAi-based treatments with small interfering RNA (siRNA) drugs. Nevertheless, the widespread use of siRNA is limited by various extra- and intracellular barriers, requiring its encapsulation in a suitable (nanosized) delivery system. On the intracellular level, the endosomal membrane is a major barrier following endocytosis of siRNA-loaded nanoparticles in target cells and innovative materials to promote cytosolic siRNA delivery are highly sought after. We previously identified the endogenous lung surfactant protein B (SP-B) as siRNA delivery enhancer when reconstituted in (proteo) lipid-coated nanogels. It is known that the surface-active function of SP-B in the lung is influenced by the lipid composition of the lung surfactant. Here, we investigated the role of the lipid component on the siRNA delivery-promoting activity of SP-B proteolipid-coated nanogels in more detail. Our results clearly indicate that SP-B prefers fluid membranes with cholesterol not exceeding physiological levels. In addition, SP-B retains its activity in the presence of different classes of anionic lipids. In contrast, comparable fractions of SP-B did not promote the siRNA delivery potential of DOTAP:DOPE cationic liposomes. Finally, we demonstrate that the beneficial effect of lung surfactant on siRNA delivery is not limited to lung-related cell types, providing broader therapeutic opportunities in other tissues as well.

Inhibition and counterinhibition of Surfacen, a clinical lung surfactant of natural origin.

Inactivation of pulmonary surfactant by different components such as serum, cholesterol or meconium contributes to severe respiratory pathologies through destabilization and collapse of airspaces. Recent studies have analyzed in detail how the interfacial properties of natural surfactant purified from animal lungs are altered as a consequence of its exposure to serum proteins or meconium-mobilized cholesterol. It has been also demonstrated that pre-exposure of surfactant to polymers such as hyaluronic acid provides resistance to inactivation by multiple inhibitory agents. In the current work, we have extended these studies to the analysis of Surfacen, a clinical surfactant currently in use to rescue premature babies suffering or at risk of respiratory distress due to congenital lack of surfactant. This surfactant is also strongly inhibited by both meconium and serum when tested in the captive bubble surfactometer (CBS) under conditions mimicking respiratory dynamics. As it occurs with native surfactant, Surfacen is markedly protected from inhibition by pre-exposure to hyaluronic acid, confirming that clinical surfactants can be improved to treat pathologies associated with strongly deactivating contexts, such as those associated with lung injury and inflammation. Remarkably, we found that, under physiologically-mimicking conditions, a cholesterol-free clinical surfactant such as Surfacen is less susceptible to inhibition by cholesterol-mobilizing environments than cholesterol-containing natural surfactant, as a consequence of a markedly reduced susceptibility to incorporation of exogenous cholesterol.

Metabolism of a synthetic compared with a natural therapeutic pulmonary surfactant in adult mice.

Secreted pulmonary surfactant phosphatidylcholine (PC) has a complex intra-alveolar metabolism that involves uptake and recycling by alveolar type II epithelial cells, catabolism by alveolar macrophages, and loss up the bronchial tree. We compared the in vivo metabolism of animal-derived poractant alfa (Curosurf) and a synthetic surfactant (CHF5633) in adult male C57BL/6 mice. The mice were dosed intranasally with either surfactant (80 mg/kg body weight) containing universally 13C-labeled dipalmitoyl PC (DPPC) as a tracer. The loss of [U13C]DPPC from bronchoalveolar lavage and lung parenchyma, together with the incorporation of 13C-hydrolysis fragments into new PC molecular species, was monitored by electrospray ionization tandem mass spectrometry. The catabolism of CHF5633 was considerably delayed compared with poractant alfa, the hydrolysis products of which were cleared more rapidly. There was no selective resynthesis of DPPC and, strikingly, acyl remodeling resulted in preferential synthesis of polyunsaturated PC species. In conclusion, both surfactants were metabolized by similar pathways, but the slower catabolism of CHF5633 resulted in longer residence time in the airways and enhanced recycling of its hydrolysis products into new PC species.

Surfactant protein B (SP-B) enhances the cellular siRNA delivery of proteolipid coated nanogels for inhalation therapy.

Despite the many advantages of small interfering RNA (siRNA) inhalation therapy and a growing prevalence of respiratory pathologies, its clinical translation is severely hampered by inefficient intracellular delivery. To this end, we previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). Interestingly, the PS shell was shown to markedly improve particle stability as well as intracellular siRNA delivery in vitro and in vivo. The major aim of this work was to identify the key molecular components of PS responsible for the enhanced siRNA delivery and evaluate how the complexity of the PS coat could be reduced. We identified surfactant protein B (SP-B) as a potent siRNA delivery enhancer when reconstituted in proteolipid coated hydrogel nanocomposites. Improved cytosolic siRNA delivery was achieved by inserting SP-B into a simplified phospholipid mixture prior to nanogel coating. This effect was observed both in vitro (lung epithelial cell line) and in vivo (murine acute lung injury model), albeit that distinct phospholipids were required to achieve these results. Importantly, the developed nanocomposites have a low in vivo toxicity and are efficiently taken up by resident alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Our results demonstrate the potential of the endogenous protein SP-B as an intracellular siRNA delivery enhancer, paving the way for future design of nanoformulations for siRNA inhalation therapy. STATEMENT OF SIGNIFICANCE: Despite the therapeutic potential of small interfering RNA (siRNA) and a growing prevalence of lung diseases for which innovative therapies are needed, a safe and effective siRNA inhalation therapy remains non-existing due to a lack of suitable formulations. We identified surfactant protein B (SP-B) as a potent enhancer of siRNA delivery by proteolipid coated nanogel formulations in vitro in a lung epithelial cell line. The developed nanocomposites have a low in vivo toxicity and show a high uptake by alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Importantly, in vivo SP-B is also critical for the developed formulation to obtain a significant silencing of TNFα in a murine LPS-induced acute lung injury model.

Controlled hypothermia may improve surfactant function in asphyxiated neonates with or without meconium aspiration syndrome.

BACKGROUND: Whole-body hypothermia (WBH) is used to improve neurological outcomes in perinatal asphyxia. Recent studies suggested a beneficial effect of hypothermia for some types of acute respiratory failure. However, no data are available about the biophysical function of human surfactant during WBH. We investigated whether WBH improves surfactant biophysical properties in asphyxiated neonates with or without meconium aspiration syndrome (MAS). METHODS: Non-bronchoscopic bronchoalveolar lavage (BAL) has been collected from 10 asphyxiated neonates (2 with MAS, 8 with no lung disease (NLD)) at different time-points (pre-WBH, 24h, 48h, 72h of WBH and post-WBH). Surfactant was extracted and tested by captive bubble surfactometry (CBS) in triplicate, at 37°C and 33.5°C, through initial adsorption and dynamic compression-expansion cycling. Phosphatidylcholine and cholesterol were assayed using enzymatic methods. Clinical data were recorded in real-time. RESULTS: Minimum surface tension under dynamic testing was significantly improved as assessed at 33.5°C compared with its behavior at 37°C in NLD neonates: the difference was evident after at least 72h of WBH and remained significant at 6h after rewarming (72h: p = 0.009; rewarming: p = 0.040). Similar results were obtained in MAS patients whose surfactant activity improved already at 48h of hypothermia. Total cholesterol showed a trend to increase at the first 24-48h of hypothermia in NLD patients. Conversely, hypothermia seemed to reduce the excess of exogenous cholesterol in MAS surfactant. CONCLUSIONS: Surfactant biophysical properties may improve after 48-72h of WBH in asphyxiated neonates and the improvement is maintained shortly after rewarming. Due to study limitations, further studies are warranted to better clarify the effects of hypothermia on surfactant activity.

Restoring pulmonary surfactant membranes and films at the respiratory surface.

Pulmonary surfactant is a complex of lipids and proteins assembled and secreted by the alveolar epithelium into the thin layer of fluid coating the respiratory surface of lungs. There, surfactant forms interfacial films at the air-water interface, reducing dramatically surface tension and thus stabilizing the air-exposed interface to prevent alveolar collapse along respiratory mechanics. The absence or deficiency of surfactant produces severe lung pathologies. This review describes some of the most important surfactant-related pathologies, which are a cause of high morbidity and mortality in neonates and adults. The review also updates current therapeutic approaches pursuing restoration of surfactant operative films in diseased lungs, mainly through supplementation with exogenous clinical surfactant preparations. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

A Noninvasive Surfactant Adsorption Test Predicting the Need for Surfactant Therapy in Preterm Infants Treated with Continuous Positive Airway Pressure.

OBJECTIVE: To determine the diagnostic accuracy of the surfactant adsorption test (SAT) as a predictor for the need for surfactant replacement therapy in neonates with respiratory distress syndrome (RDS). STUDY DESIGN: Amniotic fluid samples were collected from 41 preterm neonates with RDS treated with continuous positive airway pressure (CPAP) and 15 healthy control term neonates. Purified porcine surfactant served as a further control. Lamellar bodies and lung ultrasound score were also measured in a subset of the neonates treated with CPAP. Surfactant was administered according to the European guidelines, and clinical data were collected prospectively. Surfactant activity was measured as adsorption at the air/liquid interface and given in relative fluorescent units (RFU). RESULTS: Surfactant activity differed among native porcine surfactant (median, 4863 RFU; IQR, 4405-5081 RFU), healthy term neonates (median, 2680 RFU; IQR, 2069-3050 RFU), and preterm neonates with RDS (median, 442 RFU; IQR, 92-920 RFU; P <.0001). The neonates who failed CPAP had lower surfactant activity compared with those who did not fail CPAP (median, 92 RFU; IQR, 0-315 RFU vs 749 RFU; IQR, 360-974 RFU; P = .0002). Differences between groups were more evident beyond 20-30 minutes of fluorescence; the 30-minute time point showed the highest area under the curve (0.84; P <.001) and the best cutoff level (170 RFU; specificity, 72%; sensitivity, 96%) for the prediction of CPAP failure. Surfactant activity at 30 minutes was significantly correlated with lamellar bodies (r = 0.51, P = .006) and lung ultrasound score (r = -0.39, P = .013). CONCLUSION: This technique has the potential to be developed into a fast, simple-to-interpret clinical test. The SAT can reliably identify preterm infants with subsequent CPAP failure and shows promise as a screening test for surfactant replacement in preterm neonates.

Surface Activity as a Crucial Factor of the Biological Actions of Ole e 1, the Main Aeroallergen of Olive Tree (Olea europaea) Pollen.

Aeroallergens are airborne substances-mainly proteins-capable of triggering Th2-immune responses in respiratory allergies. They enter into the body through the upper airways, reaching the mucosa afterward. Mucosae lining at the luminal side consists of an epithelial barrier completely covered by mucus and pulmonary surfactant. Both pulmonary surfactant and plasma membrane of the epithelial cells represent two physiological phospholipid-based barriers where allergens first impact before triggering their biological effects. The interaction of allergens with lipids at relevant physiological surfaces could promote structural changes on the molecule, resulting on a potential modification of its allergenic properties. In this work, we have first described the surface and phospholipid interaction capabilities of the clinically relevant aeroallergen Ole e 1, the main allergen of olive tree pollen. By using epifluorescence microscopy of Langmuir transferred films, we observed that lipid-packed ordered domains may function as a preferential location for allergen to accumulate at the air-liquid interface, an effect that is abolished in the presence of cholestenone. The possible implications of phospholipid-interfacial effects in the modification of allergen structural and functional properties will be discussed.

Surfactant dysfunction during overexpression of TGF-ß1 precedes profibrotic lung remodeling in vivo.

Transforming growth factor-ß1 (TGF-ß1) is involved in regulation of cellular proliferation, differentiation, and fibrogenesis, inducing myofibroblast migration and increasing extracellular matrix synthesis. Here, TGF-ß1 effects on pulmonary structure and function were analyzed. Adenovirus-mediated gene transfer of TGF-ß1 in mice lungs was performed and evaluated by design-based stereology, invasive pulmonary function testing, and detailed analyses of the surfactant system 1 and 2 wk after gene transfer. After 1 wk decreased static compliance was linked with a dramatic alveolar derecruitment without edema formation or increase in the volume of septal wall tissue or collagen fibrils. Abnormally high surface tension correlated with downregulation of surfactant proteins B and C. TTF-1 expression was reduced, and, using PLA (proximity ligand assay) technology, we found Smad3 and TTF-1 forming complexes in vivo, which are normally translocated into the nucleus of the alveolar epithelial type II cells (AE2C) but in the presence of TGF-ß1 remain in the cytoplasm. AE2C show altered morphology, resulting in loss of total apical surface area per lung and polarity. These changes of AE2C were progressive 2 wk after gene transfer and correlated with lung compliance. Although static lung compliance remained low, the volume of septal wall tissue and collagen fibrils increased 2 wk after gene transfer. In this animal model, the primary effect of TGF-ß1 signaling in the lung is downregulation of surfactant proteins, high surface tension, alveolar derecruitment, and mechanical stress, which precede fibrotic tissue remodeling and progressive loss of AE2C polarity. Initial TTF-1 dysfunction is potentially linked to downregulation of surfactant proteins.