Surfactant Phospholipids Research Articles

Recent progress on surfactant protein A: cellular function in lung and kidney disease development.

Pulmonary surfactant is a heterogeneous active surface complex made up of lipids and proteins. The major glycoprotein in surfactant is surfactant protein A (SP-A), which is released into the alveolar lumen from cytoplasmic lamellar bodies in type II alveolar epithelial cells. SP-A is involved in phospholipid absorption. SP-A together with other surfactant proteins and phospholipids prevent alveolar collapse during respiration by decreasing the surface tension of the air-liquid interface. Additionally, SP-A interacts with pathogens to prevent their propagation and regulate host immune responses. Studies in human and animal models have shown that deficiencies or mutations in surfactant components result in various lung or kidney pathologies, suggesting a role for SP-A in the development of lung and kidney diseases. In this mini-review, we discuss the current understanding of SP-A functions, recent findings of its dysfunction in specific lung and kidney pathologies, and how SP-A has been used as a biomarker to detect the outcome of lung diseases.

Molecular dynamics exploring of atmosphere components interacting with lung surfactant phospholipid bilayers

Sulfur dioxide (SO2), nitrogen oxide (NO2) and ozone (O3) in the atmosphere are significantly correlated with various respiratory and cardiovascular diseases. High doses of each of these gases or a mixture can change the physical and chemical properties of the lung membrane, thus leading to an increased pulmonary vascular permeability and structural failure of the alveolar cell membrane. In the present study, detailed molecular dynamic (MD) modeling was applied to investigate the effects of SO2, NO2, O3 and mixtures of these gases on the dipalmitoyl phosphatidylcholine (DPPC) phospholipid bilayer. The results showed that several key physical properties, including the mass density, lipid ordering parameter, lipid diffusion, and electrostatic potential of the cell membrane, have been changed by the binding of different compounds. This resulted in significant variations and more disorder in the DPPC bilayer. The multiple analyses of membrane properties proved the toxicity of NO2, O3, and SO2 to the DPPC bilayer, providing a theoretical basis for the experimental phenomenon that SO2, NO2 and O3 can cause lung cell apoptosis. For the single systems, the damage to DPPC bilayer caused by O3 was more serious than NO2 and SO2. More importantly, the MD simulations using the mixtures of SO2, NO2, and O3 showed a much greater decline of membrane fluidity and the aggravation of membrane damage than the single systems, indicating a synergistic effect when NO2, SO2, and O3 coexisted in the atmosphere, which could lead to much more severe damage and greater toxicities to the lung.

Impact of high tidal volume ventilation on surfactant metabolism and lung injury in infant rats.

The poorly understood tolerance toward high tidal volume (VT) ventilation observed in critically ill children and age-equivalent animal models may be explained by surfactant homeostasis. The aim of our prospective animal study was to test whether high VT with adequate positive end-expiratory pressure (PEEP) is associated with surfactant de novo synthesis and secretion, leading to improved lung function, and whether extreme mechanical ventilation affects intracellular lamellar body formation and exocytosis. Rats (14 days old) were allocated to five groups: nonventilated controls, PEEP 5 cmH2O with VT of 8, 16, and 24 mL/kg, and PEEP 1 cmH2O with VT 24 mL/kg. Following 6 h of ventilation, lung function, surfactant proteins and phospholipids, and lamellar bodies were assessed by forced oscillation technique, quantitative real-time polymerase chain reaction, mass spectrometry, immunohistochemistry, and transmission electron microscopy. High VT (24 mL/kg) with PEEP of 5 cmH2O improved respiratory system mechanics and was not associated with lung injury, elevated surfactant protein expression, or surfactant phospholipid content. Extreme ventilation with VT 24 mL/kg and PEEP 1 cmH2O produced a mild inflammatory response and correlated with higher surfactant phospholipid concentrations in bronchoalveolar lavage fluid without affecting lamellar body count and morphology. Elevated phospholipid concentrations in the potentially most injurious strategy (VT 24 mL/kg, PEEP 1 cmH2O) need further evaluation and might reflect accumulation of biophysically inactive small aggregates. In conclusion, our data confirm the resilience of infant rats toward high VT-induced lung injury and challenge the relevance of surfactant synthesis, storage, and secretion as protective factors.

Secreted Phospholipase A2 Group X Acts as an Adjuvant for Type 2 Inflammation, Leading to an Allergen-Specific Immune Response in the Lung.

Secreted phospholipase A2 (sPLA2) enzymes release free fatty acids, including arachidonic acid, and generate lysophospholipids from phospholipids, including membrane phospholipids from cells and bacteria and surfactant phospholipids. We have shown that an endogenous enzyme sPLA2 group X (sPLA2-X) is elevated in the airways of asthmatics and that mice lacking the sPLA2-X gene (Pla2g10) display attenuated airway hyperresponsiveness, innate and adaptive immune responses, and type 2 cytokine production in a model of airway sensitization and challenge using a complete allergen that induces endogenous adjuvant activity. This complete allergen also induces the expression of sPLA2-X/Pla2g10 In the periphery, an sPLA2 found in bee venom (bee venom PLA2) administered with the incomplete Ag OVA leads to an Ag-specific immune response. In this study, we demonstrate that both bee venom PLA2 and murine sPLA2-X have adjuvant activity, leading to a type 2 immune response in the lung with features of airway hyperresponsiveness and Ag-specific type 2 airway inflammation following peripheral sensitization and subsequent airway challenge with OVA. Further, the adjuvant effects of sPLA2-X that result in the type 2-biased OVA-specific adaptive immune response in the lung were dependent upon the catalytic activity of the enzyme, as a catalytically inactive mutant form of sPLA2-X does not elicit the adaptive component of the immune response, although other components of the immune response were induced by the inactive enzyme, suggesting receptor-mediated effects. Our results demonstrate that exogenous and endogenous sPLA2s play an important role in peripheral sensitization, resulting in airway responses to inhaled Ags.

Surfactant-secreted phospholipase A2 interplay and respiratory outcome in preterm neonates.

Secreted phospholipase A2 hydrolyzes surfactant phospholipids and is crucial for the inflammatory cascade; preterm neonates are treated with exogenous surfactant, but the interaction between surfactant and phospholipase is unknown. We hypothesize that this interplay is complex and the enzyme plays a relevant role in neonates needing surfactant replacement. We aimed to: 1) identify phospholipases A2 isoforms expressed in preterm lung; 2) study the enzyme role on surfactant retreatment and function and the effect of exogenous surfactant on the enzyme system; and 3) verify whether phospholipase A2 is linked to respiratory outcomes. In bronchoalveolar lavages of preterm neonates, we measured enzyme activity (alone or with inhibitors), enzyme subtypes, surfactant protein-A, and inflammatory mediators. Surfactant function and phospholipid profile were also tested. Urea ratio was used to obtain epithelial lining fluid concentrations. Follow-up data were prospectively collected. Subtype-IIA is the main phospholipase isoform in preterm lung, although subtype-IB may be significantly expressed. Neonates needing surfactant retreatment have higher enzyme activity (P = 0.021) and inflammatory mediators (P always ≤ 0.001) and lower amounts of phospholipids (P always < 0.05). Enzyme activity was inversely correlated to surfactant adsorption (ρ = -0.6; P = 0.008; adjusted P = 0.009), total phospholipids (ρ = -0.475; P = 0.05), and phosphatidylcholine (ρ = -0.622; P = 0.017). Exogenous surfactant significantly reduced global phospholipase activity (P < 0.001) and subtype-IIA (P = 0.005) and increased dioleoylphosphatidylglycerol (P < 0.001) and surfactant adsorption (P < 0.001). Enzyme activity correlated with duration of ventilation (ρ = 0.679, P = 0.005; adjusted P = 0.04) and respiratory morbidity score at 12 mo postnatal age (τ-b = 0.349, P = 0.037; adjusted P = 0.043) but was not associated with mortality, bronchopulmonary dysplasia, or other long-term respiratory outcomes.

Pulmonary surfactant phosphatidylcholines induce immunological adaptation of alveolar macrophages

Pulmonary surfactant plays an important role in lung surface tension, defense against invading pathogens, and immune response. Furthermore, alveolar macrophages (AM) that comprise the front line of immune defense against inhaled microorganisms are covered by a layer of pulmonary fluid. Phosphatidylcholines (PCs), including unsaturated lipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), are the most prevalent phospholipids in pulmonary surfactant. POPC reacts with ozone to produce 1-palmitoyl-2-(9-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC), a soluble mediator that initiates an inflammatory reaction in the lungs. However, the modulatory effects of POPC and PONPC on biology and activity of AM remain inconclusive. The exposure of AM (cell line AMJ2-C11) to POPC and PONPC was not directly related to the production of inflammatory mediators. However, AM, pre-incubated with POPC or PONPC, showed enhanced response after lipopolysaccharide (LPS) stimulation, and increased the production of nitric oxide and cytokines. This phenomenon was also observed for classical-polarized macrophages (M1). This increment on the production of inflammatory mediators was not associated with macrophage polarization, but with up-regulation of Tlr4 and Myd88 gene expression, which was in accordance with the adaptation of immune cells. This observation was confirmed by the histone acetylation epigenetic pathway. In contrast to the priming effect of POPC on AM activity, a harmful immune response, induced on incubation with PONPC, improved prostaglandin E2 (PGE2) formation, resulting in diminished bacterial phagocytosis. Additionally, PONPC induced production of CXCL1/KC, which potentially mediates neutrophil recruitment and enhances tissue inflammation. These results disclosed another dynamic mechanism by which pulmonary surfactant lipids (natural or oxidized) primed macrophage activity, thus affecting lung host defense.

Oxidative Stress and Endothelial Dysfunction in the Development of Simulated Radio-Induced Lung Damage and their Correction with Pulmonary Surfactant

Radiation exposure to the chest organs used in the complex treatment of neoplasms is often accompanied by the development of radiation pneumonitis, fibrosis and respiratory failure.The aim of the study was to evaluate oxidative stress and endothelial dysfunction in radio-induced lung damage and possibility of their correction with a pulmonary surfactant preparation.Material and methods. Lung tissue samples of 82 male Wistar rats were investigated in the study. Single local irradiation of animals was performed with the X-ray RUM-17 unit at a dose 12 Gy. The quantitative content and fractional composition of phospholipids of pulmonary surfactant were determined in the study. Oxidative stress was detected using Raman spectroscopy, endothelial dysfunction was detected immunohistochemically.Results. Oxidative stress and endothelial dysfunction were registered starting from the first day of the experiment, and manifested by an increased content of free radicals and a decrease in endothelial function indices supported by precise spectroscopic, immunohistochemical and electron microscopic methods. With application of Surfactant-BL preparation oxidative stress manifestations were eliminated to the beginning of the intermediate stage of the experiment, and were preserved without correction until its completion.Conclusions. Three-fold intratracheal administration of a pulmonary surfactant preparation implements an antioxidant, immunomodulatory and compensating effect attenuating the alterative effect of oxidative stress and its initiating impact on the development of endothelial dysfunction. Native surfactant having a pronounced effect on all components of the lung tissue including alveolocytes of the first and second types and endothelial cells of the capillaries of the lung results in the correction of endothelial dysfunction.

Reduced Surfactant Contributes to Increased Lung Stiffness Induced by Rapid Inspiratory Flow.

The mechanism of fast inspiratory flow rate (VI') induced lung injury is unclear. As fast VI' increases hysteresis, a measure of surface tension at the air-liquid interface, surfactant release or function may be important. This experimental study examines the contribution of impaired surfactant release or function to dynamic-VILI. Isolated perfused lungs from male Sprague Dawley rats were randomly allocated to four groups: a long or short inspiratory time (Ti = 0.5s; slow VI' or Ti = 0.1s; fast VI') at PEEP of 2 or 10 cmH2O. Tidal volume was constant (7ml/kg), with f = 60 breath/min. Forced impedance mechanics (tissue elastance (Htis), tissue resistance (Gtis) and airway resistance (Raw) were measured at 30, 60 and 90min following which the lung was lavaged for surfactant phospholipids (PL) and disaturated PL (DSP). Fast VI' resulted in a stiffer lung. Concurrently, PL and DSP were decreased in both tubular myelin rich and poor fractions. Phospholipid decreases were similar with PEEP. In a subsequent cohort, laser confocal microscopy-based assessment demonstrated increased cellular injury with increased VI' at both 30 and 90min ventilation. Rapid VI' may contribute to ventilator induced lung injury (VILI) through reduced surfactant release and/or more rapid reuptake despite unchanged tidal stretch.

Mechanically ventilated patients exhibit decreased particle flow in exhaled breath as compared to normal breathing patients.

IntroductionIn this cohort study, we evaluated whether the particles in exhaled air (PExA) device can be used in conjunction with mechanical ventilation during surgery. The PExA device consists of an optical particle counter and an impactor that collects particles in exhaled air. Our aim was to establish the feasibility of the PExA device in combination with mechanical ventilation (MV) during surgery and if collected particles could be analysed. Patients with and without nonsmall cell lung cancer (NSCLC) undergoing lung surgery were compared to normal breathing (NB) patients with NSCLC.MethodsA total of 32 patients were included, 17 patients with NSCLC (MV-NSCLC), nine patients without NSCLC (MV-C) and six patients with NSCLC and not intubated (NB). The PEx samples were analysed for the most common phospholipids in surfactant using liquid-chromatography-mass-spectrometry (LCMS).ResultsMV-NSCLC and MV-C had significantly lower numbers of particles exhaled per minute (particle flow rate; PFR) compared to NB. MV-NSCLC and MV-C also had a siginificantly lower amount of phospholipids in PEx when compared to NB. MV-NSCLC had a significantly lower amount of surfactant A compared to NB.ConclusionWe have established the feasibility of the PExA device. Particles could be collected and analysed. We observed lower PFR from MV compared to NB. High PFR during MV may be due to more frequent opening and closing of the airways, known to be harmful to the lung. Online use of the PExA device might be used to monitor and personalise settings for mechanical ventilation to lower the risk of lung damage.

Морфогенез ателектазов легких при экспериментальном радиоиндуцированном воздействии

Introduction. Pulmonary atelectasis that occur in the chest organs exposed to radiation is often accompanied by the development of radiation alveolitis, fibrosis, and respiratory failure. Different forms of alveolitis cause severe disturbance of the structure of alveolocytes. However, the effect of radiation on the vascular compartment of pulmonary tissue, bronchioles and alveolocytes, and intercellular structures in the formation of atelectasis was not studied in detail. The importance of quantitative and qualitative changes in pulmonary surfactant (PS) in the development of atelectasis was understudied. The aim of the work was to explore the development of pulmonary atelectasis in rats after radiation exposure. Materials and methods. The study was carried out on 60 male Wistar rats aged 8-9 weeks, body weight 200-220 g. Single local irradiation of animals under Nembutal anesthesia (40 mg / kg weight) was carried out by the RUM-17 apparatus (voltage 200 kV, current 15 mA, filters 0.5 mm Cu + 1.0 mm Al, skin-focal length 25 cm) at a dose of 12 Gy. Phospholipids (PL) in pulmonary surfactant were quantitatively determined in lavage fluid extracted from the rat lung. The material was examined histologically (hematoxylin and eosin, alcian blue, picrofuchsin according to Van Gieson) and under transmission electron microscope. The thickness of the epithelial lining of bronchioles, the average diameter of the nuclei of epithelial cells of bronchioles and the thickness of the interalveolar partitions were calculated morphometrically on control samples at different time periods after exposure to ionizing radiation. Results. Atelectasis occurred at the early stages of the experiment is surfactant-dependent, which is confirmed by the decreased amount of PS and PL in lavage fluid. The formation of atelectasis in later periods is caused by the obturation, narrowing of the lumen of the bronchi and bronchioles due to thickening of the epithelial lining and pronounced bronchial bronchoconstriction. Conclusion. Understanding the mechanisms of development of atelectasis is necessary in order to prevent the pathology described above and reduce the risk of complications. Keywords: atelectasis, distelectasis, morphometry, radiation fibrosis, radiation alveolitis

Structural and Functional Determinants of Rodent and Human Surfactant Protein A: A Synthesis of Binding and Computational Data.

Surfactant protein A (SP-A) provides surfactant stability, first line host defense, and lung homeostasis by binding surfactant phospholipids, pathogens, alveolar macrophages (AMs), and epithelial cells. Non-primates express one SP-A protein whereas humans express two: SP-A1 and SP-A2 with core intra- and inter-species differences in the collagen-like domain. Here, we used macrophages and solid phase binding assays to discern structural correlates of rat (r) and human (h) SP-A function. Binding assays using recombinant rSP-A expressed in insect cells showed that lack of proline hydroxylation, truncations of amino-terminal oligomerization domains, and site-directed serine (S) or alanine (A) mutagenesis of cysteine 6 (C6S), glutamate 195 (E195A), and glutamate 171 (E171A) in the carbohydrate recognition domain (CRD) all impaired SP-A binding. Replacement of arginine 197 with alanine found in hSP-A (R197A), however, restored the binding of hydroxyproline-deficient rSP-A to the SP-A receptor SP-R210 similar to native rat and human SP-A. In silico calculation of Ca++ coordination bond length and solvent accessibility surface area revealed that the “humanized” R197A substitution alters topology and solvent accessibility of the Ca++ coordination residues of the CRD domain. Binding assays in mouse AMs that were exposed to either endogenous SP-A or hSP-A1 (6A2) and hSP-A2 (1A0) isoforms in vivo revealed that mouse SP-A is a functional hybrid of hSP-A1 and hSP-A2 in regulating SP-A receptor occupancy and binding affinity. Binding assays using neonatal and adult human AMs indicates that the interaction of SP-A1 and SP-A2 with AMs is developmentally regulated. Furthermore, our data indicate that the auxiliary ion coordination loop encompassing the conserved E171 residue may comprise a conserved site of interaction with macrophages, and SP-R210 specifically, that merits further investigation to discern conserved and divergent SP-A functions between species. In summary, our findings support the notion that complex structural adaptation of SP-A regulate conserved and species specific AM functions in vertebrates.

Lavage lipidomics signatures in children with cystic fibrosis and protracted bacterial bronchitis

BackgroundBalanced composition of a well-functioning pulmonary surfactant is crucial and essential for normal breathing. Here, we explored whether the composition of lipids recovered by broncho-alveolar lavage (BAL) in children with cystic fibrosis (CF) differ from children with protracted bacterial bronchitis (PBB) and controls. We wanted to differentiate, if alterations are primarily caused by the disease process or secondary due to an increased amount of cell-membrane lipids derived from inflammatory cells. MethodsComprehensive lipidomics profiles of BAL fluid from children diagnosed with CF, PBB and controls were generated by electrospray ionization tandem mass spectrometry analysis. BAL cell differential and numbers were examined. Results55 children (37 patients with CF, 8 children with PBB and 10 controls) were included in this study. Results showed comparable total quantities of lipids in all groups. Phospholipids were the major lipid fraction and similar in all groups, whereas the fractions of cholesteryl esters were less and of free cholesterol were increased in children with CF. Among the phospholipids, patients with CF had higher proportion of the non-surfactant membrane-lipids in the classes phosphatidylethanolamine based plasmalogens (PE P), phosphatidylethanolmine (PE) and phosphatidylserine (PS), but a lower proportion of phosphatidylcholine (PC) compared to healthy controls. No such changes were identified in the BAL fluid of children diagnosed with PBB. No differences were observed for the surfactant lipids dipalmitoyl-phosphatidylcholin (PC 32:0) and phosphatidylglycerol (PG). ConclusionsIn CF patients with neutrophilic airway inflammation the lipid composition for surfactant phospholipid components were unchanged, whereas alteration in lipid profile were characteristic for those found in membranes of inflammatory cells. We suspect that the changes in CF were caused by the prolonged inflammation in contrast to a relatively short standing process in PBB.

Cationic Nanoliposomes Are Efficiently Taken up by Alveolar Macrophages but Have Little Access to Dendritic Cells and Interstitial Macrophages in the Normal and CpG-Stimulated Lungs.

The purpose of this study was to assess whether cationic nanoliposomes could address tumor vaccines to dendritic cells in the lungs in vivo. Nanoliposomes were prepared using a cationic lipid, dimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol) or dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the most abundant phospholipid in lung surfactant. The liposomes presented a size below 175 nm and they effectively entrapped tumor antigens, an oligodeoxynucletotide containing CpG motifs (CpG) and the fluorescent dye calcein used as a tracer. Although the liposomes could permanently entrap a large fraction of the actives, they could not sustain their release in vitro. Liposomes made of DOTAP were safe to respiratory cells in vitro, while liposomes composed of DC-cholesterol were cytotoxic. DOTAP nanoliposomes were mainly taken up by alveolar macrophages following delivery to the lungs in mice. Few dendritic cells took up the liposomes, and interstitial macrophages did not take up liposomal calcein more than they took up soluble calcein. Stimulation of the innate immune system using liposomal CpG strongly enhanced uptake of calcein liposomes by all phagocytes in the lungs. Although a small percentage of dendritic cells took up the nanoliposomes, alveolar macrophages represented a major barrier to dendritic cell access in the lungs.

Assessing the Interactions of Auristatin Derivatives with Mixed Phospholipid-Sodium Dodecyl Sulfate Aggregate Dispersions.

The aim of this study was to assess what properties of the pseudostationary phases in electrokinetic capillary chromatography affect the interactions between monomethyl auristatin E (MMAE) and hydrophilically modified structural analogues thereof with various lipophilic phases. MMAE is a widely used cytotoxic agent in antibody–drug conjugates (ADC), which are used as selective biopharmaceutical drugs in the treatment of cancers. MMAE and its derivatives are highly lipophilic, yet they fail to interact with biomimicking phosphatidylcholine–phosphatidylserine liposomes. To reveal what properties affect the interaction of the auristatin derivatives with cell plasma membrane-mimicking vesicles, capillary electrokinetic chromatography was used with four different types of micellar and vesicular pseudostationary phases: pure vesicles, mixed vesicles, mixed micelles, and pure micelles. Vesicular phases were composed of pure phospholipids [dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC)] and phospholipid–surfactant mixtures [sodium dodecyl sulfate, (SDS) with DMPC and DLPC] while the micellar phases comprised pure surfactant (SDS) and surfactant–phospholipid mixtures (SDS–DMPC and SDS–DLPC). In addition, differential scanning calorimetry and dynamic light scattering were used to monitor the aggregate composition. Our data shows that the interaction between hydrophobic auristatin derivatives and hydrophobic pseudostationary phases critically depends on the type, size, and hydrogen bonding capability of the pseudostationary phases.

Effect of cooling on lung secretory phospholipase A2 activity in vitro, ex vivo, and in vivo.

Hypothermia can modify surfactant composition and function. Secretory phospholipase A2 (sPLA2) hydrolyses surfactant phospholipids and is important in the pathobiology of several critical respiratory disorders. We hypothesize that sPLA2 activity might be influenced by the temperature partially explaining surfactant changes. This study aims to evaluate comprehensively the effect of hypothermia on sPLA2 activity. We measured sPLA2 activity at different temperatures, alone or combined with bile acids, in vitro (incubating human recombinant sPLA2-IIA and porcine sPLA2-IB), ex vivo (by cooling bronchoalveolar lavage samples from neonates with respiratory distress syndrome or no lung disease), and in vivo (using lavage samples obtained before and after 72 h of whole body cooling in neonates with hypoxic-ischemic encephalopathy). We also measured concentrations of various sPLA2 subtypes and natural sPLA2 inhibitors in in vivo cooled samples. Results were corrected for protein content and dilution. In vitro cooling did not show any effect of hypothermia on sPLA2. Ex vivo cooling did not alter total sPLA2 activity, and the addition of bile acids increased sPLA2 activity irrespective of the temperature and the type of sampled patient. In vivo hypothermia reduced median sPLA2 activity from 16.6 [15.2-106.7] IU/mg to 3.3 [2.7-8.5] IU/mg ( P = 0.026) and mean sPLA2-IIA from 1.1 (0.8) pg/μg to 0.6 (0.4) pg/μg ( P = 0.047), whereas dioleylphosphatidylglycerol increased from 8.3 (3.9)% to 12.8 (5.1)% ( P = 0.02). Whole body hypothermia decreases in vivo global sPLA2 activity in bronchoalveolar lavage fluids through the reduction of sPLA2-IIA and increment of dioleylphosphatidylglycerol. This effect is absent during in vitro or ex vivo hypothermia.

Tuning optimum transfection of gemini surfactant-phospholipid-DNA nanoparticles by validated theoretical modeling.

Gemini nanoparticles (NPs) are a family of non-viral gene delivery systems with potential for applications in non-invasive gene therapy. Translation of these non-viral gene delivery systems requires improvement of transfection efficiency (TE) through fine-tuning of their physicochemical properties such as electric charge and exact ratios of their components. Since high-throughput experimental screening of minute differences in NP compositions is not routinely feasible, we have developed a coarse-grained model to quantitatively study the energetics of the formation of gene delivery complexes with cationic gemini surfactants (G) (m-s-m type) and helper lipids (H) (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and DOPE/1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC)), in order to use it as a tool to predict effective compositions. The model is based on the polymorphic structural conformational flip of NPs and incorporates the electrostatic, entropic and elastic energies, to predict the formation energy and stability of different polymorphic structures as a function of the electric charge of cationic surfactants and concentration of constituent helper lipids. Our results show that these two factors are intertwined in determining the behavior of gene delivery vectors. Specifically, we show that increasing H/G lowers free energy per DNA base pair and increases the stability of the complex. At pH 7, low H/G and charge ratio (ρ±), where the lamellar structure is favored, the formation free energy per DNA base pair is between 0 and -14kBT. At higher values of H/G (2-3) and ρ±, where HII and cubic structures are formed, the formation free energy drops down to values ≈-50kBT, indicating the stable existence of these polymorphic structures in the NPs. At pH 5, the structural transformation of NPs in the endosomes to the lamellar/HII structure with free energy values of about -40kBT is beneficial for endosomal escape, and correlates with increased transfection efficiency. The theoretical model is supported by transfection data in A7 astrocytes with a panel of 16-3-16 gemini NPs, which validates the mathematical model and supports the hypothesis that the NP polymorphic phase transition increases transfection efficiency.

Cxxc Finger Protein 1 Positively Regulates GM-CSF-Derived Macrophage Phagocytosis Through Csf2rα-Mediated Signaling.

Macrophages have a defensive function against bacteria through phagocytosis and the secretion of cytokines. Histone modifications play an essential role in macrophage functions. Here, we report that Cxxc finger protein 1 (CFP1), a key component of the SETD1 histone methyltransferase complex, promoted the phagocytic and bactericidal activity of GM-CSF-derived macrophages. CFP1-deficient mice were more susceptible to bacterial infection due to the decreased expression of Csf2rα, a subunit of the GM-CSF receptor essential for inflammation and alveolar macrophage development, through the loss of H3K4 modifications in the promoter of the Csf2rα gene. In addition, the lung tissues of CFP1-deficient mice exhibited spontaneous inflammatory symptoms, including both the infiltration of inflammatory cells and the accumulation of surfactant phospholipids and proteins. Furthermore, we showed that Csf2rα and PU.1 can partially rescue the defects in phagocytosis and in the intracellular killing of bacteria. Collectively, our data highlight the importance of CFP1 in the phagocytic and bactericidal activity of macrophages.

The involvement of GM-CSF deficiencies in parallel pathways of pulmonary alveolar proteinosis and the alcoholic lung.

Chronic alcohol consumption renders the lung more susceptible to infections by disrupting essential alveolar macrophage functions. Emerging evidence suggests that these functional deficits are due, in part, to a suppression of GM-CSF signaling, which is believed to compromise monocyte growth and maturation in the lung. However, in addition to controlling monocyte behaviors, GM-CSF also regulates surfactant homeostasis. For example, mice with targeted deletion of the gene for GM-CSF accumulate large amounts of surfactant phospholipids in their lungs. Moreover, decreased GM-CSF signaling in humans has been linked to the development of pulmonary alveolar proteinosis (PAP), a rare disorder in which surfactant lipids and proteins accumulate in alveolar macrophages and the lung exhibits enhanced susceptibility to infection. Consistent with parallel mechanisms in the PAP and alcoholic lung, we have recently reported that levels of intrapulmonary lipids, specifically triglycerides and free fatty acids, are increased in BAL fluid, whole lung digests and alveolar macrophages of chronically alcohol exposed rats. Additionally, we showed that uptake of saturated fatty acids alone could induce phenotypic and functional changes in alveolar macrophages that mimicked those in the alcohol-exposed rat and human lung. Herein, we discuss the role of GM-CSF in surfactant homeostasis and highlight the evidence that links decreased GM-CSF signaling to alveolar macrophage dysfunction in both the PAP and alcohol-exposed lung. Moreover, we discuss how lipid accumulation itself might contribute to altering alveolar macrophage function and propose how targeting these mechanisms could be employed for reducing the susceptibility to pulmonary infections in alcoholics.

Homo- and hetero-oligomerization of hydrophobic pulmonary surfactant proteins SP-B and SP-C in surfactant phospholipid membranes

Pulmonary surfactant is a lipid/protein mixture that reduces surface tension at the respiratory air-water interface in lungs. Among its nonlipidic components are pulmonary surfactant-associated proteins B and C (SP-B and SP-C, respectively). These highly hydrophobic proteins are required for normal pulmonary surfactant function, and whereas past literature works have suggested possible SP-B/SP-C interactions and a reciprocal modulation effect, no direct evidence has been yet identified. In this work, we report an extensive fluorescence spectroscopy study of both intramolecular and intermolecular SP-B and SP-C interactions, using a combination of quenching and FRET steady-state and time-resolved methodologies. These proteins are compartmentalized in full surfactant membranes but not in pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles, in accordance with their previously described preference for liquid disordered phases. From the observed static self-quenching and homo-FRET of BODIPY-FL labeled SP-B, we conclude that this protein forms homoaggregates at low concentration (lipid:protein ratio, 1:1000). Increases in polarization of BODIPY-FL SP-B and steady-state intensity of WT SP-B were observed upon incorporation of under-stoichiometric amounts of WT SP-C. Conversely, Marina Blue-labeled SP-C is quenched by over-stoichiometric amounts of WT SP-B, whereas under-stoichiometric concentrations of the latter actually increase SP-C emission. Time-resolved hetero-FRET from Marina Blue SP-C to BODIPY-FL SP-B confirm distinct protein aggregation behaviors with varying SP-B concentration. Based on these multiple observations, we propose a model for SP-B/SP-C interactions, where SP-C might induce conformational changes on SP-B complexes, affecting its aggregation state. The conclusions inferred from the present work shed light on the synergic functionality of both proteins in the pulmonary surfactant system.

Estimating the contribution of surfactant replacement therapy to the alveolar pool: An in vivo study based on 13 C natural abundance in rabbits.

Variation of the isotopic abundance of selected nutrients and molecules has been used for pharmacological and kinetics studies under the premise that the administered molecule has a different isotopic enrichment from the isotopic background of the recipient subject. The aim of this study is to test the feasibility of assessing the contribution of exogenous surfactant phospholipids to the endogenous alveolar pool in vivo after exogenous surfactant replacement therapy in rabbits. The study consisted in measuring the consistency of 13 C/12 C ratio of disaturated-phosphatidylcholine palmitate (DSPC-PA) in 7 lots of poractant alfa, produced over a year, and among bronchoalveolar lavages of 20 rabbits fed with a standard chow. A pilot study was performed in a rabbit model of lavage-induced surfactant deficiency: 7 control rabbits and 4 treated with exogenous surfactant. The contribution of exogenous surfactant to the alveolar pool was assessed after intra-tracheal administration of 200mg/kg of poractant alfa. The 13 C content of DSPC-PA was measured by isotope ratio mass spectrometry. The mean DSPC-PA 13 C/12 C ratio of the 7 lots of poractant alfa was -18.8‰ with a SD of 0.1‰ (range: -18.9‰; -18.6‰). The mean 13 C/12 C ratio of surfactant DSPC recovered from the lung lavage of 20 rabbits was -28.8±1.2‰ (range: -31.7‰; -25.7‰). The contribution of exogenous surfactant to the total alveolar surfactant could be calculated in the treated rabbits, and it ranged from 83.9% to 89.6%. This pilot study describes a novel method to measure the contribution of the exogenous surfactant to the alveolar pool. This method is based on the natural variation of 13 C, and therefore it does not require the use of chemically synthetized tracers. This method could be useful in human research and especially in surfactant replacement studies in preterm infants.