Pulmonary Surfactant Phospholipids Research Articles

The anti-inflammatory and antiviral properties of anionic pulmonary surfactant phospholipids.

The pulmonary surfactant system of the lung is a lipid and protein complex, which regulates the biophysical properties of the alveoli to prevent lung collapse and the innate immune system in the lung. Pulmonary surfactant is a lipoprotein complex consisting of 90% phospholipids and 10% protein, by weight. Two minor components of pulmonary surfactant phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), exist at very high concentrations in the extracellular alveolar compartments. We have reported that one of the most dominant molecular species of PG, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and PI inhibit inflammatory responses induced by multiple toll-like receptors (TLR2/1, TLR3, TLR4, and TLR2/6) by interacting with subsets of multiprotein receptor components. These lipids also exert potent antiviral effects against RSV and influenza A, in vitro, by inhibiting virus binding to host cells. POPG and PI inhibit these viral infections in vivo, in multiple animal models. Especially noteworthy, these lipids markedly attenuate SARS-CoV-2 infection including its variants. These lipids are natural compounds that already exist in the lung and, thus, are less likely to cause adverse immune responses by hosts. Collectively, these data demonstrate that POPG and PI have strong potential as novel therapeutics for applications as anti-inflammatory compounds and preventatives, as treatments for broad ranges of RNA respiratory viruses.

The Role of Pulmonary Surfactant Phospholipids in Fibrotic Lung Diseases

Diffuse parenchymal lung diseases (DPLD) or Interstitial lung diseases (ILD) are a heterogeneous group of lung conditions with common characteristics that can progress to fibrosis. Within this group of pneumonias, idiopathic pulmonary fibrosis (IPF) is considered the most common. This disease has no known cause, is devastating and has no cure. Chronic lesion of alveolar type II (ATII) cells represents a key mechanism for the development of IPF. ATII cells are specialized in the biosynthesis and secretion of pulmonary surfactant (PS), a lipid-protein complex that reduces surface tension and minimizes breathing effort. Some differences in PS composition have been reported between patients with idiopathic pulmonary disease and healthy individuals, especially regarding some specific proteins in the PS; however, few reports have been conducted on the lipid components. This review focuses on the mechanisms by which phospholipids (PLs) could be involved in the development of the fibroproliferative response.

The influence of a biomimetic pulmonary surfactant modification on the in vivo fate of nanoparticles in the lung

Direct biomimetic modification of nanoparticles (NPs) with endogenous surfactants is helpful to improve the biocompatibility of NPs and avoid damage to the physiological function of the lung. Therefore, the objective of this study is to investigate the influence of biomimetic endogenous pulmonary surfactant phospholipid modification on the in vivo fate of NPs after lung delivery. Here, two neutral phospholipids (dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylamine (DPPE)) and two negatively charged phospholipids (dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS)) were selected to modify paclitaxel (PTX)-loaded PLGA NPs with different molar ratio. DPPC, DPPE, and DPPG improved mucoadhesion, in contrast, DPPS improved the mucus permeability of the NPs. Neutral DPPC and DPPE reduced, but negatively charged DPPS and DPPG increased the uptake by alveolar macrophages, all types of phospholipid increased the uptake by lung epithelial cells and increased PTX retention in the whole lung. Whereas, DPPC, DPPE, and DPPG promoted PTX retention in bronchoalveolar lavage fluid (BALF), while DPPS promoted PTX absorption in the lung tissue. Only DPPS-PLGA (1:1) NPs remarkably increased PTX systemic exposure. A good correlation between PTX percentage in the supernatant of BALF and PTX concentration in plasma was established, implying PTX entered the system circulation mainly in molecular form. Phospholipid modification had no effect on extrapulmonary organ distribution of PTX. Taken together, our study disclosed that different phospholipid modification can endow the NPs mucoadhesive or mucus penetration and cellular uptake properties, with tunable retention in BALF and absorption in the lung tissue, providing the scientific background for translational nanocarrier design for inhalation as required. Statement of significanceInhaled nanomedicines will inevitably interact with pulmonary surfactant and form “surfactant corona”. However, the contribution of individual pulmonary surfactant phospholipid on the in vivo fate of nanomedicines is still unclear. In this regard, the most abundant pulmonary surfactant phospholipid dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylamine, and dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylserine were selected to modify the paclitaxel loaded PLGA nanoparticles and the effect of these pulmonary surfactant phospholipids on their in vivo fate was investigated. It was demonstrated that different phospholipid modification can endow the nanoparticles mucoadhesive or mucus penetration properties, with tunable retention in bronchoalveolar lavage fluid, alveolar macrophages uptake and absorption in the lung tissue. The present study provided a comprehensive understanding for the role of pulmonary surfactant phospholipid on inhaled nanomedicines.

Lipid biophysics and/or soft matter-inspired approach for controlling enveloped virus infectivity.

Proven as a natural barrier against viral infection, pulmonary surfactant phospholipids have a biophysical and immunological role within the respiratory system, acting against microorganisms including viruses. Enveloped viruses have, in common, an outer bilayer membrane that forms the underlying structure for viral membrane proteins to function in an optimal way to ensure infectivity. Perturbating the membrane of viruses using exogenous lipids can be envisioned as a generic way to reduce their infectivity. In this context, the potential of exogenous lipids to be used against enveloped virus infectivity would be indicated by the resulting physical stress imposed to the viral membrane, and conical lipids, i.e. lyso-lipids, would be expected to generate stronger biophysical disturbances. We confirm that when treated with lyso-lipids the infectivity three strains of influenza virus (avian H2N3, equine H3N8 or pandemic human influenza H1N1) is reduced by up to 99% in a cell-based model. By contrast, lipids with a similar head group but two aliphatic chains were less effective (reducing infection by only 40–50%). This work opens a new path to merge concepts from different research fields, i.e. ‘soft matter physics' and virology.

Pulmonary surfactants affinity Pluronic-hybridized liposomes enhance the treatment of drug-resistant lung cancer

For a long time, the incidence and mortality of lung cancer have ranked first among all kinds of cancers, of which the major type is non-small cell lung cancer (NSCLC). Until now, chemotherapy and radiotherapy are still the first choice for patients with advanced or metastatic NSCLC. However, the emergence of multi-drug resistance (MDR) always leads to the failure of chemotherapy and increases cancer-related mortality. In this study, we prepared a Pluronic-hybridized paclitaxel-loaded liposome (PPL), which was used in combination with ambroxol (Ax) to not only resensitize drug-resistant tumor cells, but also increase the preparation retention in the lung. On the one hand, Ax induced the production of pulmonary surfactants (PS) and responsively improved the accumulation of pulmonary surfactants affinity liposomes whose skeleton was exogenous pulmonary surfactant phospholipids DPPC, because of the specific affinity of phospholipids related to pulmonary surfactant proteins. On the other hand, drug-resistant tumor cells were resensitized due to the inhibition of autophagy by Ax and the reduced expression of the drug-resistant protein P-glycoprotein (P-gp) by Pluronic P105. Therefore, we concluded that the combination of PPL and Ax achieved excellent killing tumor effects through multi-path and multi-strategy, having great application prospects in the future.

The Anionic Phospholipids of Bovine Pulmonary Surfactant.

The only known compositional change in the phospholipids (PL) of pulmonary surfactant in response to a physiologic stimulus occurs around the time of birth. In most species, the predominant anionic PL changes from phosphatidylinositol (PtdIns) to phosphatidylglycerol (PtdGro). Because prior studies have shown that the change in the headgroup itself is functionally insignificant, we tested the hypothesis that the PtdIns and PtdGro contain different diacyl pairs. Experiments used electrospray-ionization mass spectrometry to determine the molecular species in PtdIns, PtdGro, and phosphatidylcholine (PtdCho) in surfactant from newborn calves and cows. The profiles for the two anionic PL were distinct. The PtdIns contained long, unsaturated fatty acid chains and no disaturated species. The PtdGro more closely resembled the profile from PtdCho. For each headgroup, the molecular species for calf and cow were similar. The differences between the two anionic PL indicate that the switch from PtdIns to PtdGro during maturation involves more than simple substitution of the headgroup, and suggest that the functional significance of the shift may reflect the different pool of diacyl pairs.

Surfactant phospholipids act as molecular switches for premature induction of quorum sensing-dependent virulence in Pseudomonas aeruginosa

ABSTRACT The virulence behaviors of many Gram-negative bacterial pathogens are governed by quorum-sensing (QS), a hierarchical system of gene regulation that relies on population density by producing and detecting extracellular signaling molecules. Although extensively studied under in vitro conditions, adaptation of QS system to physiologically relevant host environment is not fully understood. In this study, we investigated the influence of lung environment on the regulation of Pseudomonas aeruginosa virulence factors by QS in a mouse model of acute pneumonia. When cultured under laboratory conditions in lysogeny broth, wild-type P. aeruginosa strain PAO1 began to express QS-regulated virulence factors elastase B (LasB) and rhamnolipids (RhlA) during transition from late-exponential into stationary growth phase. In contrast, during acute pneumonia as well as when cultured in mouse bronchial alveolar lavage fluids (BALF), exponential phase PAO1 bacteria at low population density prematurely expressed QS regulatory genes lasI-lasR and rhlI-rhlR and their downstream virulence genes lasB and rhlA. Further analysis indicated that surfactant phospholipids were the primary components within BALF that induced the synthesis of N-(3-oxododecanoyl)-L-homoserine lactone (C12-HSL), which triggered premature expression of LasB and RhlA. Both phenol extraction and phospholipase A2 digestion abolished the ability of mouse BALF to promote LasB and RhlA expression. In contrast, provision of the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) restored the expression of both virulence factors. Collectively, our study demonstrates P. aeruginosa modulates its QS to coordinate the expression of virulence factors during acute pneumonia by recognizing pulmonary surfactant phospholipids.

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.

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

Phospholipid Regulation of Inflammatory Processes and Viral Infection

Gas exchange between the external environment and erythrocyte hemoglobin takes place within the alveolar compartment of the lung, which contains an extracellular, phospholipid‐rich complex known as pulmonary surfactant. The major phospholipid classes of pulmonary surfactant consist of phosphatidylcholine (~70%), phosphatidylglycerol (PG) (~10%) and phosphatidylinositol (PI) (~2%). Dipalmitoyl‐phosphatidylcholine plays an essential role in reducing surface tension within the alveoli during respiration, but the roles played by phosphatidylglycerol (PG) and phosphatidylinositol (PI) remained enigmatic for decades. PG and PI are uniquely secreted by alveolar epithelial cells, and their concentrations in this extracellular compartment greatly exceed that of any other tissue. Unexpectedly, studies of the innate immune Toll‐like receptors (TLRs) have revealed that PG and PI are potent antagonists of the activation of multiple TLRs including TLRs 1,2,3,4 and 6. The lipids bind to the receptors and prevent their activation by cognate ligands, and consequently prevent downstream activation of inflammatory signaling pathways, and release of inflammatory mediators such as interleukins 6 and 8, interferon‐lambda, eicosanoids and TNF‐alpha. In addition to suppressing activation of TLRs, the lipids also inhibit infection by major respiratory viruses including respiratory syncytial virus (RSV) and influenza A virus (IAV). Both PG and PI prevent the viral infections in cultured cells, and in vivo in mice. PG also completely abrogates lethal challenge of mice with pandemic H1N1‐IAV. Additional studies with ferrets further demonstrate protection against pandemic H1N1‐IAV. The lipids act as decoy receptors for both RSV and IAV, and block virus attachment to epithelial cell surfaces. We have constructed a library of 25 analogs of POPG and PI, which vary in both the structure of the polar headgroup and their hydrophobic moieties. Some of these analogs exhibit improved pharmacological properties compared with PG and PI. These studies demonstrate regulation of innate immune processes by pulmonary surfactant phospholipids, and also reveal their potential use for suppressing inflammatory processes in the lung and inhibiting establishment and spread of respiratory viral infections.Support or Funding InformationSupported by NIH Grants HL 094629, U19‐AI 125357, GM 118819This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tandem mass spectrometry of novel ether-linked phospholipid analogs of anionic pulmonary surfactant phospholipids.

Structural analogs of the bioactive lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were synthesized with a xylitol polar head group and both diacyl and diether radyl groups. Mass spectral characterization of xylitol phospholipids (PX) was carried out using collisional activation and high-resolution mass measurements of positive molecular ion species and compared with the phosphatidylglycerol (PG) analogs. PX were synthesized using a transphosphatidylation reaction catalyzed by phospholipase D and purified by high-performance liquid chromatography (HPLC). Compounds were subjected to electrospray ionization and collision-induced dissociation (CID) was performed using a tandem quadrupole mass spectrometer to generate positive and negative molecular ions. Diether phospholipids were additionally analyzed by high-resolution mass spectrometry as protonated and sodiated molecular species in positive ion mode. Ester-linked PX analogs behaved similarly to PG after collisional activation of [M-H]- . The product ions formed by CID of the diether PG and PX negative ions only revealed information about the head group with no information about the aliphatic chains. In contrast, CID of protonated and sodiated diether phospholipid positive ions revealed reactions corresponding to cleavage of the ether chain, likely occurring by charge-driven reaction mechanisms. Novel PX analogs with diacyl and diether radyl substituents of the glycerol backbone were characterized by tandem mass spectrometry. These unique diether phospholipid analogs enabled exploration of ether cleavage reactions of the positive molecular ion species resulting from collision-induced decomposition. Copyright © 2016 John Wiley & Sons, Ltd.

L-α-Phosphatidylglycerol Chlorohydrins as Potential Biomarkers for Chlorine Gas Exposure.

Chlorine is a widely available toxic chemical that has been repeatedly used in armed conflict globally. The Organization for the Prohibition of Chemical Weapons (OPCW) have on numerous occasions found "compelling confirmation" that chlorine gas has been used against civilians in northern Syria. However, currently, there are no analytical methods available to unambiguously prove chlorine gas exposure. In this study, we describe the screening for chlorinated biomolecules by the use of mass isotope ratio filters followed by the identification of two biomarkers present in bronchoalveolar lavage fluid (BALF) from chlorine gas exposed mice. The relevance of these markers for human exposure was verified by their presence in in vitro chlorinated human BALF. The biomarkers were detectable for 72 h after exposure and were absent in nonexposed control animals. Furthermore, the biomarkers were not detected in humans diagnosed with chronic respiratory diseases. The potential chlorine specific markers were all chlorohydrins of unsaturated pulmonary surfactant phospholipids; phosphatidylglycerols, and phosphatidylcholines. Mass spectrometry fragmentation characteristics were favorable for the phosphatidylglycerol chlorohydrins, and they were therefore proposed as the best biomarker candidates.

Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit toll-like receptor 2 and 4 signaling

The pulmonary surfactant phospholipid, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), potently inhibits toll-like receptor (TLR)2 and TLR4 signaling from the cell surface of macrophages. Analogs of POPG that vary in polar head group length, hydroxylation, and alkyl branching were synthesized using a phospholipase D-catalyzed transphosphatidylation reaction and a 1-palmitoyl-2-oleoyl phosphatidylcholine substrate. Lipid analogs with C3 and C4 alkyl head group length (POP-propanol and POP-butanol) are less effective than POPG as TLR2 and TLR4 antagonists. However, adding a hydroxyl group at the alkyl chain 3- or 4-position (POP-propanediols or POP-butanediols) greatly increased their inhibitory effects against TLR2 and TLR4. POP-2',2'-dimethylpropanediol is a weak inhibitor of TLR2 and TLR4 activation that results in arachidonic acid release, but an effective inhibitor of TLR4 activation that results in TNF-α production. Addition of an amino group at the alkyl-2 position (POP-2'-aminopropanediol) completely abolished the antagonism of TLRs 2 and 4. Multiple analogs strongly bind to the TLR4 coreceptors, cluster of differentiation 14 (CD14) and myeloid differentiation 2, but competition for di[3-deoxy-D-manno-octulosonyl]-lipid A binding to CD14 is the best predictor of biological activity at the cellular level. Collectively, these findings identify new compounds for antagonizing TLR2 and TLR4 activation and define structural properties of POPG analogs for discriminating between two TLR systems.

Phosphatidylinositol inhibits respiratory syncytial virus infection

Respiratory syncytial virus (RSV) infects nearly all children under age 2, and reinfection occurs throughout life, seriously impacting adults with chronic pulmonary diseases. Recent data demonstrate that the anionic pulmonary surfactant lipid phosphatidylglycerol (PG) exerts a potent antiviral effect against RSV in vitro and in vivo. Phosphatidylinositol (PI) is also an anionic pulmonary surfactant phospholipid, and we tested its antiviral activity. PI liposomes completely suppress interleukin-8 production from BEAS2B epithelial cells challenged with RSV. The presence of PI during viral challenge in vitro reduces infection by a factor of >10(3). PI binds RSV with high affinity, preventing virus attachment to epithelial cells. Intranasal inoculation with PI along with RSV in mice reduces the viral burden 30-fold, eliminates the influx of inflammatory cells, and reduces tissue histopathology. Pharmacological doses of PI persist for >6 h in mouse lung. Pretreatment of mice with PI at 2 h prior to viral infection effectively suppresses inflammation and reduces the viral burden by 85%. These data demonstrate that PI has potent antiviral properties, a long residence time in the extracellular bronchoalveolar compartment, and a significant prophylaxis window. The findings demonstrate PG and PI have complementary roles as intrinsic, innate immune antiviral mediators in the lung.

Prenatal administration of all-trans retinoic acid upregulates leptin signaling in hypoplastic rat lungs with experimental congenital diaphragmatic hernia

Pulmonary hypoplasia (PH), characterized by alveolar immaturity, is one of the leading causes of respiratory insufficiency in newborns with congenital diaphragmatic hernia (CDH). Leptin (Lep) and its receptor (Lep-R) play an important role in fetal lung growth by stimulating alveolar differentiation and maturation. Lep and Lep-R are strongly expressed by alveolar cells during the saccular stage of fetal lung development. Lep-deficient mice exhibit decreased alveolarization with reduced pulmonary surfactant phospholipid synthesis, similar to human and nitrofen-induced PH. Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced PH. Recent studies have demonstrated that Lep and Lep-R expression in developing lungs is regulated by ATRA. We hypothesized that prenatal treatment with ATRA increases pulmonary Lep and Lep-R expression in the nitrofen model of CDH-associated PH. Time-mated rats received either 100 mg nitrofen or vehicle via oral-gastric lavage on embryonic day 9.5 (E9.5). Control and nitrofen-exposed dams were randomly assigned to either intraperitoneal ATRA (5 mg/kg/d) or placebo administration on E18.5, E19.5 and E20.5. Fetal lungs were harvested on E21.5, and divided into Control+Placebo, Control+ATRA, Nitrofen+Placebo and Nitrofen+ATRA. Alveolarization was assessed using stereo- and morphometric analysis techniques. Surfactant phospholipid synthesis was analyzed by labeling for surfactant protein B (SP-B). Pulmonary gene expression levels of Lep and Lep-R were determined using quantitative real-time polymerase chain reaction. Immunohistochemical staining for Lep and Lep-R was performed to evaluate alveolar protein expression and localization. In vivo administration of ATRA resulted in significantly increased lung-to-body weight ratio with enhanced radial alveolar count and decreased mean linear intercept compared to placebo treatment. Immunofluorescence analysis demonstrated markedly increased pulmonary SP-B expression in Nitrofen+ATRA compared to Nitrofen+Placebo. Relative mRNA expression of Lep and Lep-R was significantly increased in Nitrofen+ATRA compared to Nitrofen+Placebo. Lep and Lep-R immunoreactivity was markedly increased in interstitial and alveolar epithelial cells of Nitrofen+ATRA compared to Nitrofen+Placebo. Increased Lep and Lep-R expression after prenatal administration of ATRA in nitrofen-induced PH suggests that ATRA may have therapeutic potential in attenuating CDH-associated PH by stimulating alveolarization and de novo surfactant production.

Structural characterization of the pulmonary innate immune protein SPLUNC1 and identification of lipid ligands.

The short palate, lung and nasal epithelial clone 1 (SPLUNC1) protein is a member of the palate, lung, and nasal epithelium clone (PLUNC) family, also known as bactericidal/permeability-increasing (BPI) fold-containing protein, family A, member 1 (BPIFA1). SPLUNC1 is an abundant protein in human airways, but its function remains poorly understood. The lipid ligands of SPLUNC1 as well as other PLUNC family members are largely unknown, although some reports provide evidence that lipopolysaccharide (LPS) could be a lipid ligand. Unlike previous hypotheses, we found significant structural differences between SPLUNC1 and BPI. Recombinant SPLUNC1 produced in HEK 293 cells harbored several molecular species of sphingomyelin and phosphatidylcholine as its ligands. Significantly, in vitro lipid-binding studies failed to demonstrate interactions between SPLUNC1 and LPS, lipoteichoic acid, or polymyxin B. Instead, one of the major and most important pulmonary surfactant phospholipids, dipalmitoylphosphatidylcholine (DPPC), bound to SPLUNC1 with high affinity and specificity. We found that SPLUNC1 could be the first protein receptor for DPPC. These discoveries provide insight into the specific determinants governing the interaction between SPLUNC1 and lipids and also shed light on novel functions that SPLUNC1 and other PLUNC family members perform in host defense.

Keratinocyte Growth Factor and Glucocorticoid Induction of Human Peroxiredoxin 6 Gene Expression Occur by Independent Mechanisms That Are Synergistic

Peroxiredoxin 6 (Prdx6), a 1-cys Prdx has both peroxidase and phospholipase A2 activities, protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. This study determined the mechanism by which keratinocyte growth factor (KGF) and the glucocorticoid analogue, dexamethasone (Dex), induce increased Prdx6 expression. Transcriptional activation by KGF in both A549 lung adenocarcinoma cells and rat lung alveolar epithelial type II (ATII) cells utilizes an antioxidant response element (ARE), located between 357 and 349 nucleotides before the PRDX6 translational start, that is also necessary for upregulation of the human PRDX6 promoter in response to oxidative stress. Activation is mediated by binding of the transcription factor, Nrf2, to the ARE as shown by experiments using siRNA against Nrf2 and by transfecting ATII cells isolated from lungs of Nrf2 null mice. KGF triggers the migration of Nrf2 from cytoplasm to nucleus where it binds to the PRDX6 promoter as shown by chromatin immunoprecipitation assays. Activation of transcription by Dex occurs through a glucocorticoid response element located about 750 nucleotides upstream of the PRDX6 translational start. This study demonstrates that KGF can activate an ARE in a promoter without reactive oxygen species involvement and that KGF and Dex can synergistically activate the PRDX6 promoter and protect cells from oxidative stress. These two different activators work through different DNA elements. Their combined effect on transcription of the reporter gene is synergistic; however, at the protein level, the combined effect is additive and protects cells from oxidative damage.

Phosphatidylglycerol provides short-term prophylaxis against respiratory syncytial virus infection

Respiratory syncytial virus (RSV) causes respiratory tract infections in young children, and significant morbidity and mortality in the elderly, immunosuppressed, and immunocompromised patients and in patients with chronic lung diseases. Recently, we reported that the pulmonary surfactant phospholipid palmitoyl-oleoyl-phosphatidylglycerol (POPG) inhibited RSV infection in vitro and in vivo by blocking viral attachment to epithelial cells. Simultaneous application of POPG along with an RSV challenge to mice markedly attenuated infection and associated inflammatory responses. Based on these findings, we expanded our studies to determine whether POPG is effective for prophylaxis and postinfection treatment for RSV infection. In vitro application of POPG at concentrations of 0.2-1.0 mg/ml at 24 h after RSV infection of HEp-2 cells suppressed interleukin-8 production up to 80% and reduced viral plaque formation by 2-6 log units. In vivo, the turnover of POPG in mice is relatively rapid, making postinfection application impractical. Intranasal administration of POPG (0.8-3.0 mg), 45 min before RSV inoculation in mice reduced viral infection by 1 log unit, suppressed inflammatory cell appearance in the lung, and suppressed virus-elicited interferon-γ production. These findings demonstrate that POPG is effective for short-term protection of mice against subsequent RSV infection and that it has potential for application in humans.

Nanodiscs as a therapeutic delivery agent: inhibition of respiratory syncytial virus infection in the lung

There is increasing interest in the application of nanotechnology to solve the difficult problem of therapeutic administration of pharmaceuticals. Nanodiscs, composed of a stable discoidal lipid bilayer encircled by an amphipathic membrane scaffold protein that is an engineered variant of the human Apo A-I constituent of high-density lipoproteins, have been a successful platform for providing a controlled lipid composition in particles that are especially useful for investigating membrane protein structure and function. In this communication, we demonstrate that nanodiscs are effective in suppressing respiratory syncytial viral (RSV) infection both in vitro and in vivo when self-assembled with the minor pulmonary surfactant phospholipid palmitoyloleoylphosphatidylglycerol (POPG). Preparations of nanodiscs containing POPG (nPOPG) antagonized interleukin-8 production from Beas2B epithelial cells challenged by RSV infection, with an IC50 of 19.3 μg/mL. In quantitative in vitro plaque assays, nPOPG reduced RSV infection by 93%. In vivo, nPOPG suppressed inflammatory cell infiltration into the lung, as well as IFN-γ production in response to RSV challenge. nPOPG also completely suppressed the histopathological changes in lung tissue elicited by RSV and reduced the amount of virus recovered from lung tissue by 96%. The turnover rate of nPOPG was estimated to have a halftime of 60–120 minutes (m), based upon quantification of the recovery of the human Apo A-I constituent. From these data, we conclude that nPOPG is a potent antagonist of RSV infection and its inflammatory sequelae both in vitro and in vivo.