Alveolar Liquid Research Articles

Prediction of shear stress imposed on alveolar epithelium of healthy and diseased lungs.

Lung alveoli are modeled as spherical caps, lined internally by a thin surfactant-laden liquid film, and the periodic wall shear stress exerted along the epithelium during small-amplitude radial oscillations of their wall is computed. A novel set of boundary conditions, applied at the rim, reveals the dominant role of Marangoni stresses. These stresses develop along the air/liquid interface due to spatial gradients of interfacial surfactant concentration and are transported to the wall by the action of viscosity. The effect of a variety of geometric and functional characteristics, including rim interstitial thickness, alveolar opening angle and liquid film thickness and viscosity, is interrogated, and the results are discussed in relation to the onset and evolution of acute and chronic lung diseases, such as asthmatic attacks, pulmonary emphysema and pulmonary fibrosis.

Rescue of alveolar wall liquid secretion blocks fatal lung injury due to influenza-staphylococcal coinfection.

Secondary lung infection by inhaled Staphylococcus aureus (SA) is a common and lethal event for individuals infected with influenza A virus (IAV). How IAV disrupts host defense to promote SA infection in lung alveoli, where fatal lung injury occurs, is not known. We addressed this issue using real-time determinations of alveolar responses to IAV in live, intact, perfused lungs. Our findings show that IAV infection blocked defensive alveolar wall liquid (AWL) secretion and induced airspace liquid absorption, thereby reversing normal alveolar liquid dynamics and inhibiting alveolar clearance of inhaled SA. Loss of AWL secretion resulted from inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel in the alveolar epithelium, and airspace liquid absorption was caused by stimulation of the alveolar epithelial Na+ channel (ENaC). Loss of AWL secretion promoted alveolar stabilization of inhaled SA, but rescue of AWL secretion protected against alveolar SA stabilization and fatal SA-induced lung injury in IAV-infected mice. These findings reveal a central role for AWL secretion in alveolar defense against inhaled SA and identify AWL inhibition as a critical mechanism of IAV lung pathogenesis. AWL rescue may represent a new therapeutic approach for IAV-SA coinfection.

한국어 자연 발화 코퍼스에서 10대 화자들의 단어 내 자음 탈락 현상

Jungsun Kim. 2022. Word-Internal Consonant Deletion: Evidence from Teenagers in Spontaneous Seoul Korean Speech. Studies in Modern Grammar 115, 187-205. This study investigated word-internal consonant deletion for teenagers in spontaneous Seoul Korean speech. The dataset of speakers for this study was excerpted from the Korean Corpus of Spontaneous Speech (Yun et al. 2015). For the first part of the study, the factors triggering word-internal consonant deletion were examined by the number of syllables, syllable positions, syllable structures. For the second part of the study, the types of consonants deleted were examined on a basis of the production of individual speakers.
 The results showed that the proportions of deletion for a velar stop [k], an alveolar nasal [n], and an alveolar liquid [r] were likely to be higher between vowels for two-and three-syllable words. The deletion proportion of a velar stop was prevalent among individual speakers. In addition, the proportion of consonant deletion (i.e., [k], [n], [r]) for teenagers was compared to the one for speakers of 20s (Kim et al. 2016). Regarding [k], [n], and [r], younger speakers (i.e., teenagers) showed higher proportion for consonant deletion than older speakers (i.e., twenties). Therefore, this study implies that the consonant deletion of teenagers may be related to the development of articulatory strategy in spontaneous speech.

Application value of metagenomic next-generation sequencing for infectious pathogens in patients receiving allogeneic hematopoietic stem cell transplantation

Objective: To investigate the application value of Metagenomic Next-Generation sequencing (mNGS) in infectious patients after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Methods: Patients suspected with local or systemic infections were retrospectively included after allo-HSCT in our department from April 2019 to November 2020. Pathogenic microorganisms were tested by mNGS in samples from peripheral blood, cerebrospinal fluid, alveolar lavage Liquid, abscess, etc. Other diagnostic methods such as bacterial/fungal culture, viral PCR detection were simultaneously explored comparing with mNGS results. Results: A total of 112 samples in 83 patients were detected by mNGS, and 34 pathogenic microorganisms were determined. Among these positive samples, 11 strains of bacteria (17 times) with the most common Escherichia coli (4/17) were reported. There were 7 strains of fungi (10 times) detected with primary Candida albicans (7/29). Although arvovirus 30.2% (39/129) were predominantly detected, its diagnostic relevance with infections was not definite. Other pathogenic viruses including cytomegalovirus (CMV) 25.6% (33/129) and Epstein Barr virus (EBV) 14.0% (18/129)were of significance. Comparing with golden diagnostic criteria, the sensitivity of mNGS was 86.5%, and specificity was 45.0%. Regarding single pathogen infection, the consistency of mNGS and conventional methods was 82.9% (29/35), while it was 16/17 in combination infections. Conclusion: mNGS could be a potential method to determine pathogens in patients suspected with infections after allo-HSCT.

Alveolar Responses to Influenza Lung Infection Determined by Confocal Imaging of Live Lungs

One‐third of patients with severe lung infection by influenza A virus (IAV) develop secondary lung infection by inhaled S. aureus(SA), leading to mortality in 30% of cases. It is unclear how IAV promotes secondary SA infection in lung alveoli, where fluid barrier dysfunction causes fatal acute lung injury (ALI). Our objective is to define alveolar responses to IAV that promote SA infection, leading to SA‐induced alveolar damage and mortality. Our hypothesis is IAV lung infection blocks alveolar wall liquid (AWL) secretion, a homeostatic alveolar function that defends against alveolar stabilization of inhaled particles. AWL secretion depends on function of the alveolar epithelial cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. Our methods include optical imaging of live, intact mouse lungs and mouse survival determinations. Mice were untreated or intranasally instilled with IAV (2000 PFU, A/PR/8/34). We excised, inflated, and blood‐perfused the lungs at 24h, then viewed live alveoli by confocal microscopy. Alveolar airspaces were microinstilled separately with: (A) the fluorescent cytosolic dye, calcein‐AM; (B) the fluorescent AWL tracer, tetramethylrhodamine‐conjugated dextran (70 kD); (C) the CFTR potentiator, ivacaftor; and (D) the SA toxin, alpha hemolysin (Hla). For survival studies, IAV‐infected mice were intranasally instilled with SA (USA300, 1x10^8 CFU) at 24h, then observed for 72h. Mice were given intraperitoneal injections of ivacaftor or vehicle at 4, 24, and 48h after SA instillation. Analysis was by t test (n=3) and log rank. Our results follow. Calcein fluorescence indicated the alveolar epithelium of IAV‐infected lungs was viable. In alveoli of untreated lungs, airspace dextran fluorescence decreased over 1h, indicating dextran was progressively diluted by AWL secretion. However, in IAV‐infected lungs, dextran fluorescence was steady (P<0.05), indicating IAV blocked AWL secretion. Since alveolar ivacaftor pretreatment restored dextran loss (P=NS versus untreated), we interpret IAV blocked AWL secretion in a CFTR‐dependent manner, and CFTR potentiation rescued AWL secretion. Hla microinstillation into alveoli of IAV‐infected lungs caused loss of epithelial calcein fluorescence, but alveolar pretreatment with ivacaftor blocked the loss (P<0.05). We interpret that rescue of AWL secretion protected against Hla‐induced alveolar damage, probably by promoting Hla clearance and disrupting Hla‐epithelial interactions. IAV‐infected mice treated with ivacaftor were protected from SA‐induced mortality (60% and 0% in vehicle‐ versus ivacaftor‐treated mice, respectively; P<0.05; N=10 each). We conclude that IAV disrupted AWL secretion, causing retention of airspace contents and augmenting Hla‐induced alveolar damage. Rescue of AWL secretion blocked Hla‐induced alveolar damage and increased survival in IAV‐SA coinfected mice. We propose strategies that rescue AWL secretion may represent a new therapeutic approach for ALI caused by IAV‐SA coinfection.

Gene Therapy Potential for Genetic Disorders of Surfactant Dysfunction.

Pulmonary surfactant is critically important to prevent atelectasis by lowering the surface tension of the alveolar lining liquid. While respiratory distress syndrome (RDS) is common in premature infants, severe RDS in term and late preterm infants suggests an underlying genetic etiology. Pathogenic variants in the genes encoding key components of pulmonary surfactant including surfactant protein B (SP-B, SFTPB gene), surfactant protein C (SP-C, SFTPC gene), and the ATP-Binding Cassette transporter A3 (ABCA3, ABCA3 gene) result in severe neonatal RDS or childhood interstitial lung disease (chILD). These proteins play essential roles in pulmonary surfactant biogenesis and are expressed in alveolar epithelial type II cells (AEC2), the progenitor cell of the alveolar epithelium. SP-B deficiency most commonly presents in the neonatal period with severe RDS and requires lung transplantation for survival. SFTPC mutations act in an autosomal dominant fashion and more commonly presents with chILD or idiopathic pulmonary fibrosis than neonatal RDS. ABCA3 deficiency often presents as neonatal RDS or chILD. Gene therapy is a promising option to treat monogenic lung diseases. Successes and challenges in developing gene therapies for genetic disorders of surfactant dysfunction include viral vector design and tropism for target cell types. In this review, we explore adeno-associated virus (AAV), lentiviral, and adenoviral (Ad)-based vectors as delivery vehicles. Both gene addition and gene editing strategies are compared to best design treatments for lung diseases resulting from pathogenic variants in the SFTPB, SFTPC, and ABCA3 genes.

Anti-inflammatory and antioxidant activity of the hot water-soluble polysaccharides from Anacyclus pyrethrum (L.) Lag. roots

Ethnophamacological relevancethe roots of Anacyclus pyrethrum (L.) Lag. (Family: Asteraceae) are used in Algeria to treat respiratory infections, to cure chronic head and nostrils catarrh, and to clear the brain by stimulating the free flow of nasal mucous. They contain a high quantity of hot water-soluble polysaccharides. Aims of the studyThe study aims to evaluate the antioxidant and anti-inflammatory activity of polysaccharides extracted from Anacyclus pyrethrum roots (APPS). Materials and methodsThe APPS were extracted using boiling water, separated from proteins by the Sevag method then precipitated with 90% ethanol. The antioxidant effect of crude APPS was evaluated using FRAP assay. To investigate the anti-inflammatory potential, mice were treated with crude polysaccharides (25, 50, and 100 mg/kg, i.p.) for 3 days (14th, 15th, and 16th day of the experimentation). Respiratory inflammation was induced by HDM (House Dust Mite), mice were sensitized intranasally with 25 μg HDM suspended in 10 μl NaCl (5 μl/nostril) on days 0 and 7 then challenged with 5 μg HDM on days 14, 15, and 16. Mice were sacrificed 24 h after the last challenge. The number of immune cells in the blood in NL (Nasal Liquid) and in BAL (Broncho Alveolar Liquid) was enumerated, the spleen was removed to calculate the relative spleen weight and to count splénocytes, lungs histopathological examination was carried out to confirm the protective effect of APPS. Structural characterization of APPS was identified using FTIR (Fourier-Transform Infrared Spectroscopy) and SEM (Scanning Electron Microscopy). ResultsThe crude APPS possessed reducing power. In vivo assay, treatment with APPS causes a decrease in the number of blood leucocytes at all doses on the one hand, and in the relative spleen weight and splénocytes number on the other hand except at the dose of 50 mg/kg in which an enhancement of the number of splénocytes and immune cells in NL and BAL was significant. The histopathological examination showed clear protection of lung tissue damaged by HDM, after treatment with APPS mainly, at the dose of 50 mg/kg. ConclusionOur data clearly showed antioxidant and anti-inflammatory activity of APPS on HDM-challenged mice induced lungs inflammation by equilibrating the inflammatory reaction mostly, with an optimal dose of 50 mg/kg.

Intravenous sulforhodamine B reduces alveolar surface tension, improves oxygenation, and reduces ventilation injury in a respiratory distress model.

In the neonatal respiratory distress syndrome (NRDS) and acute respiratory distress syndrome (ARDS), mechanical ventilation supports gas exchange but can cause ventilation-induced lung injury (VILI) that contributes to high mortality. Further, surface tension, T, should be elevated and VILI is proportional to T. Surfactant therapy is effective in NRDS but not ARDS. Sulforhodamine B (SRB) is a potential alternative T-lowering therapeutic. In anesthetized male rats, we injure the lungs with 15 min of 42 mL/kg tidal volume, VT, and zero end-expiratory pressure ventilation. Then, over 4 h, we support the rats with protective ventilation-VT of 6 mL/kg with positive end-expiratory pressure. At the start of the support period, we administer intravenous non-T-altering fluorescein (targeting 27 µM in plasma) without or with therapeutic SRB (10 nM). Throughout the support period, we increase inspired oxygen fraction, as necessary, to maintain >90% arterial oxygen saturation. At the end of the support period, we euthanize the rat; sample systemic venous blood for injury marker ELISAs; excise the lungs; combine confocal microscopy and servo-nulling pressure measurement to determine T in situ in the lungs; image fluorescein in alveolar liquid to assess local permeability; and determine lavage protein content and wet-to-dry ratio (W/D) to assess global permeability. Lungs exhibit focal injury. Surface tension is elevated 72% throughout control lungs and in uninjured regions of SRB-treated lungs, but normal in injured regions of treated lungs. SRB administration improves oxygenation, reduces W/D, and reduces plasma injury markers. Intravenous SRB holds promise as a therapy for respiratory distress.NEW & NOTEWORTHY Sulforhodmaine B lowers T in alveolar edema liquid. Given the problematic intratracheal delivery of surfactant therapy for ARDS, intravenous SRB might constitute an alternative therapeutic. In a lung injury model, we find that intravenously administered SRB crosses the injured alveolar-capillary barrier thus reduces T specifically in injured lung regions; improves oxygenation; and reduces the degree of further lung injury. Intravenous SRB administration might help respiratory distress patients, including those with the novel coronavirus, avoid mechanical ventilation or, once ventilated, survive.

P2 Purinergic Signaling in the Distal Lung in Health and Disease.

The distal lung provides an intricate structure for gas exchange in mammalian lungs. Efficient gas exchange depends on the functional integrity of lung alveoli. The cells in the alveolar tissue serve various functions to maintain alveolar structure, integrity and homeostasis. Alveolar epithelial cells secrete pulmonary surfactant, regulate the alveolar surface liquid (ASL) volume and, together with resident and infiltrating immune cells, provide a powerful host-defense system against a multitude of particles, microbes and toxicants. It is well established that all of these cells express purinergic P2 receptors and that purinergic signaling plays important roles in maintaining alveolar homeostasis. Therefore, it is not surprising that purinergic signaling also contributes to development and progression of severe pathological conditions like pulmonary inflammation, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and pulmonary fibrosis. Within this review we focus on the role of P2 purinergic signaling in the distal lung in health and disease. We recapitulate the expression of P2 receptors within the cells in the alveoli, the possible sources of ATP (adenosine triphosphate) within alveoli and the contribution of purinergic signaling to regulation of surfactant secretion, ASL volume and composition, as well as immune homeostasis. Finally, we summarize current knowledge of the role for P2 signaling in infectious pneumonia, ALI/ARDS and idiopathic pulmonary fibrosis (IPF).

Sodium‐coupled neutral amino acid transporter SNAT2 is critical for alveolar fluid transport and resolution of pulmonary edema

ObjectiveIn the intact lung, constant movement of Na+ and Cl− across the epithelial barrier regulates alveolar liquid volume and maintains a balance between fluid secretion and clearance, which is critical for normal lung function. Dysregulation or inhibition of Na+ transport can lead to accumulation of fluid in the airspace resulting in impaired gas exchange and respiratory failure. Previous studies have largely focused on the critical role of the amiloride‐sensitive epithelial sodium channel (ENaC) in alveolar fluid transport, yet activation of ENaC failed to reverse edema formation in clinical trials. Since 40–50% of alveolar fluid clearance is amiloride‐insensitive, sodium channels other than ENaC such as sodium‐coupled neutral amino acid transporter (SNAT, Slc38a2) 2 may provide for new potential therapeutic targets. SNAT2 co‐transports a neutral amino acid along with Na+ and may promote alveolar fluid clearance by mediating epithelial Na+ uptake. Here, we studied the role of SNAT2 in a murine model of lung injury, in isolated perfused lungs (IPL) and in a pulmonary epithelial cell culture System.MethodsFor functional in vitro analyses, L‐alanine transport across NCI‐H441 cells was analyzed by ELISA, with cells cultured in the presence or absence of amino acids, and following treatment with HgCl2, a SNAT inhibitor, or siRNA (control or siSNAT2).In IPL of slc38a2+/− and wildtype mice, edema formation was induced by hydrostatic stress (7 cm H2O) and fluid transport was assessed in the absence or presence of the SNAT2 inhibitor methylaminoisobutyric acid (MeAIB) or SNAT2 substrate L‐alanine in the alveolar instillate. Edema formation was assessed after 30 min of perfusion and ventilation by measurement of wet‐to‐dry lung weight ratio (W/D).For in vivo measurements of edema formation and resolution, acid (HCl) or saline was instilled intratracheally in slc38a2+/− and wildtype mice. After 2 h of mechanical ventilation, lungs were collected for W/D measurement.ResultsIn vitro, L‐alanine transport was significantly decreased in H441 cells treated with HgCl2 or SNAT2‐siRNA as compared to respective controls.In vivo, SNAT2 knockout (slc38a2−/−) mice were found to be sublethal and newborn pups typically succumbed to cyanotic dyspnea.In IPL, lungs of slc38a2+/− showed elevated W/D ratio as compared to wildtype lungs in response to hydrostatic stress and after treatment with the SNAT2 inhibitor MeAIB.Similarly, in HCl‐induced ALI, W/D ratio was increased in slc38a2+/− mice.ConclusionIn its function as Na+ transporter, we propose a crucial role for SNAT2 in alveolar fluid transport. Our results indicate that SNAT2 is functional relevant for alveolar fluid absorption and may contribute to the resolution of pulmonary edema. Hence, activation of SNAT2 may, provide a new therapeutic strategy to counteract and/or reverse formation of pulmonary edema.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tracheal acid or surfactant instillation raises alveolar surface tension.

Whether alveolar liquid surface tension, T, is elevated in the acute respiratory distress syndrome (ARDS) has not been demonstrated in situ in the lungs. Neither is it known how exogenous surfactant, which has failed to treat ARDS, affects in situ T. We aim to determine T in an acid-aspiration ARDS model before and after exogenous surfactant administration. In isolated rat lungs, we combine servo-nulling pressure measurement and confocal microscopy to determine alveolar liquid T according to the Laplace relation. Administering 0.01 N (pH 1.9) HCl solution by alveolar injection or tracheal instillation, to model gastric liquid aspiration, raises T. Subsequent surfactant administration fails to normalize T. Furthermore, in normal lungs, tracheal instillation of control saline or exogenous surfactant raises T. Lavaging the trachea with saline and injecting the lavage solution into the alveolus raises T, suggesting that tracheal instillation may wash T-raising airway contents to the alveolus. Adding 0.01 N HCl or 5 mM CaCl2-either of which aggregates mucins-to tracheal lavage solution reduces or eliminates the effect of lavage solution on alveolar T. Following tracheal saline instillation, liquid suctioned directly out of alveoli through a micropipette contains mucins. Additionally, alveolar injection of gastric mucin solution raises T. We conclude that 1) tracheal liquid instillation likely washes T-raising mucins to the alveolus and 2) even exogenous surfactant that could be delivered mucin-free to the alveolus might not normalize T in acid-aspiration ARDS. NEW & NOTEWORTHY We demonstrate in situ in isolated lungs that surface tension is elevated in an acid-aspiration acute respiratory distress syndrome (ARDS) model. Following tracheal liquid instillation, also in isolated lungs, we directly sample alveolar liquid. We find that liquid instillation into normal lungs washes mucins to the alveolus, thereby raising alveolar surface tension. Furthermore, even if exogenous surfactant could be delivered mucin-free to the alveolus, exogenous surfactant might fail to normalize alveolar surface tension in acid-aspiration ARDS.

Regulatory effects of miR-155 and miR-146a on repolarization and inflammatory cytokine secretion in human alveolar macrophages in vitro

Macrophages play an important role in inflammatory responses; however, miRNA-mediated repolarization of macrophages is essential for fulfilling this function. To clarify a series of changes at the RNA level in alveolar macrophages under normal and inflammatory conditions, bronchial alveolar lavage liquid (BALF) was collected from healthy volunteers or patients with pneumonia. This approach, which differs from that used in previously, provides more accurate information about the states of macrophages in different lung microenvironments. In this study, the density plots of macrophage subtypes (M1 and M2) in the BALF of healthy volunteers differed from that of the patients with pneumonia. The M2 subtype dominated in healthy volunteers and was rapidly repolarized to M1 in response to miRNA-mediated gene regulation. Differential miRNA expression in the two macrophage subtypes revealed lower expression of miR-155 and MIR-146a in patients with pneumonia compared with healthy volunteers; this may be related to inflammation and the use of anti-inflammatory drugs. We also found increased TNF-α and IL-6 expression at the RNA level, while macrophage galactose-type C-type lectin 1 (MGL-1) expression decreased with downregulation of miR-155 and miR-146a expression. These results indicate that the gene regulation mediated by miR-155 and miR-146a contributes to human alveolar macrophage phenotype repolarization, thus leading to an early switch from pro-inflammatory to anti-inflammatory cytokine production.

Glycosylation-dependent activation of epithelial sodium channel by solnatide

Dysfunction of the epithelial sodium channel (ENaC), which regulates salt and water homeostasis in epithelia, causes several human pathological conditions, including pulmonary oedema. This is a potentially lethal complication of acute lung injury at least partially caused by dysfunctional alveolar liquid clearance, which in turn impairs alveolar gas exchange. Solnatide (named TIP-peptide, AP301), a 17 residue peptide mimicking the lectin-like domain of TNF has been shown to activate ENaC in several experimental animal models of acute lung injury and is being evaluated as a potential therapy for pulmonary oedema. The peptide has recently completed phase 1 and 2a clinical trials. In this study, we identify a glycosylation-dependent mechanism that preserves ENaC function and expression. Since our previous data suggested that the pore-forming subunits of ENaC are essential for maximal current activation by solnatide, we performed single- and multi-N-glycosylation site mutations in αN232,293,312,397,511Q- and δN166,211,384Q-subunits, in order to identify crucial residues for interaction with solnatide within the extracellular loop of the channel. Additionally, we generated αL576X and αN232,293,312,397,511Q,L576X deletion mutants of ENaC-α, since we have previously demonstrated that the carboxy terminal domain of this subunit is also involved in its interaction with solnatide. In cells expressing αN232,293,312,397,511Q,L576Xβγ-hENaC or δN166,311,384Q,D552Xβγ-hENaC activation by solnatide, as measured in whole cell patch clamp mode, was completely abolished, whereas it was attenuated in αL576Xβγ-hENaC- and δD552Xβγ-hENaC-expressing cells. Taken together, our findings delineate an N-glycan dependent interaction between the TIP-peptide and ENaC leading to normalization of both sodium and fluid absorption in oedematous alveoli to non-oedematous levels.

A novel tumor necrosis factor-mediated mechanism of direct epithelial sodium channel activation.

Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

Lung ventilation injures areas with discrete alveolar flooding, in a surface tension-dependent fashion.

With proteinaceous-liquid flooding of discrete alveoli, a model of the edema pattern in the acute respiratory distress syndrome, lung inflation over expands aerated alveoli adjacent to flooded alveoli. Theoretical considerations suggest that the overexpansion may be proportional to surface tension, T. Yet recent evidence indicates proteinaceous edema liquid may not elevate T. Thus whether the overexpansion is injurious is not known. Here, working in the isolated, perfused rat lung, we quantify fluorescence movement from the vasculature to the alveolar liquid phase as a measure of overdistension injury to the alveolar-capillary barrier. We label the perfusate with fluorescence; micropuncture a surface alveolus and instill a controlled volume of nonfluorescent liquid to obtain a micropunctured-but-aerated region (control group) or a region with discrete alveolar flooding; image the region at a constant transpulmonary pressure of 5 cmH2O; apply five ventilation cycles with a positive end-expiratory pressure of 0-20 cmH2O and tidal volume of 6 or 12 ml/kg; return the lung to a constant transpulmonary pressure of 5 cmH2O; and image for an additional 10 min. In aerated areas, ventilation is not injurious. With discrete alveolar flooding, all ventilation protocols cause sustained injury. Greater positive end-expiratory pressure or tidal volume increases injury. Furthermore, we determine T and find injury increases with T. Inclusion of either plasma proteins or Survanta in the flooding liquid does not alter T or injury. Inclusion of 2.7-10% albumin and 1% Survanta together, however, lowers T and injury. Contrary to expectation, albumin inclusion in our model facilitates exogenous surfactant activity.

Surface tension in situ in flooded alveolus unaltered by albumin.

In the acute respiratory distress syndrome, plasma proteins in alveolar edema liquid are thought to inactivate lung surfactant and raise surface tension, T. However, plasma protein-surfactant interaction has been assessed only in vitro, during unphysiologically large surface area compression (%ΔA). Here, we investigate whether plasma proteins raise T in situ in the isolated rat lung under physiologic conditions. We flood alveoli with liquid that omits/includes plasma proteins. We ventilate the lung between transpulmonary pressures of 5 and 15 cmH2O to apply a near-maximal physiologic %ΔA, comparable to that of severe mechanical ventilation, or between 1 and 30 cmH2O, to apply a supraphysiologic %ΔA. We pause ventilation for 20 min and determine T at the meniscus that is present at the flooded alveolar mouth. We determine alveolar air pressure at the trachea, alveolar liquid phase pressure by servo-nulling pressure measurement, and meniscus radius by confocal microscopy, and we calculate T according to the Laplace relation. Over 60 ventilation cycles, application of maximal physiologic %ΔA to alveoli flooded with 4.6% albumin solution does not alter T; supraphysiologic %ΔA raise T, transiently, by 51 ± 4%. In separate experiments, we find that addition of exogenous surfactant to the alveolar liquid can, with two cycles of maximal physiologic %ΔA, reduce T by 29 ± 11% despite the presence of albumin. We interpret that supraphysiologic %ΔA likely collapses the interfacial surfactant monolayer, allowing albumin to raise T. With maximal physiologic %ΔA, the monolayer likely remains intact such that albumin, blocked from the interface, cannot interfere with native or exogenous surfactant activity.

Live imaging of the lung.

Live lung imaging has spanned the discovery of capillaries in the frog lung by Malpighi to the current use of single and multiphoton imaging of intravital and isolated perfused lung preparations incorporating fluorescent molecular probes and transgenic reporter mice. Along the way, much has been learned about the unique microcirculation of the lung, including immune cell migration and the mechanisms by which cells at the alveolar-capillary interface communicate with each other. In this review, we highlight live lung imaging techniques as applied to the role of mitochondria in lung immunity, mechanisms of signal transduction in lung compartments, studies on the composition of alveolar wall liquid, and neutrophil and platelet trafficking in the lung under homeostatic and inflammatory conditions. New applications of live lung imaging and the limitations of current techniques are discussed.

Fusion‐activated cation entry (FACE)viaP2X4couples surfactant secretion and alveolar fluid transport

Two fundamental mechanisms within alveoli are essential for lung function: regulated fluid transport and secretion of surfactant. Surfactant is secreted via exocytosis of lamellar bodies (LBs) in alveolar type II (ATII) cells. We recently reported that LB exocytosis results in fusion-activated cation entry (FACE) via P2X₄ receptors on LBs. We propose that FACE, in addition to facilitating surfactant secretion, modulates alveolar fluid transport. Correlative fluorescence and atomic force microscopy revealed that FACE-dependent water influx correlated with individual fusion events in rat primary ATII cells. Moreover, ATII cell monolayers grown at air-liquid interface exhibited increases in short-circuit current (Isc) on stimulation with ATP or UTP. Both are potent agonists for LB exocytosis, but only ATP activates FACE. ATP, not UTP, elicited additional fusion-dependent increases in Isc. Overexpressing dominant-negative P2X₄ abrogated this effect by ∼50%, whereas potentiating P2X4 lead to ∼80% increase in Isc. Finally, we monitored changes in alveolar surface liquid (ASL) on ATII monolayers by confocal microscopy. Only stimulation with ATP, not UTP, led to a significant, fusion-dependent, 20% decrease in ASL, indicating apical-to-basolateral fluid transport across ATII monolayers. Our data support the first direct link between LB exocytosis, regulation of surfactant secretion, and transalveolar fluid resorption via FACE.

The role of Cl− in the regulation of ion and liquid transport in the intact alveolus during β‐adrenergic stimulation

The epithelium of the developing lung displays an evolving liquid transport phenotype, in which Cl(-) secretion during fetal life is rapidly switched to Na(+) absorption perinatally. However, the mechanisms underlying the homeostasis of the thin layer of liquid lining the postnatal pulmonary epithelium remain elusive. In particular, it remains unclear whether the stimulated clearance of excess alveolar liquid is mediated via transepithelial Cl(-) transport. Our study is a pharmacological analysis with the aim of addressing this issue, which is of major physiological significance in cases of pulmonary oedema from any cause. We measured the rate of transepithelial liquid movement (J(v)) with (125)I-albumin, in the in situ perfused adult rat lung. Transepithelial Cl(-) transport was studied with the use of the Cl(-) channel inhibitor NPPB in the resting state and during stimulation with the β(2)-adrenergic agonist terbutaline. The study of J(v) in these conditions revealed the following findings: (1) there is net absorption of excess of alveolar liquid in the resting, unstimulated state, which is predominantly amiloride sensitive; (2) inhibition of Cl(-) transport with NPPB in the resting state results in a 1.6-fold increase in net absorption of alveolar liquid; and (3) the terbutaline-stimulated net absorption of the excess liquid is enhanced by 2.8-fold in the presence of NPPB. Our results are suggestive of the functional presence of secretory, but not absorptive, Cl(-) mechanisms and show that transepithelial Cl(-) transport is not part of the mechanism underlying lung liquid clearance in response to β-adrenergic stimulation.

CFTR is required for maximal transepithelial liquid transport in pig alveolar epithelia

A balance between alveolar liquid absorption and secretion is critical for maintaining optimal alveolar subphase liquid height and facilitating gas exchange in the alveolar space. However, the role of cystic fibrosis transmembrane regulator protein (CFTR) in this homeostatic process has remained elusive. Using a newly developed porcine model of cystic fibrosis, in which CFTR is absent, we investigated ion transport properties and alveolar liquid transport in isolated type II alveolar epithelial cells (T2AECs) cultured at the air-liquid interface. CFTR was distributed exclusively to the apical surface of cultured T2AECs. Alveolar epithelia from CFTR(-/-) pigs failed to increase liquid absorption in response to agents that increase cAMP, whereas cAMP-stimulated liquid absorption in CFTR(+/-) epithelia was similar to that in CFTR(+/+) epithelia. Expression of recombinant CFTR restored stimulated liquid absorption in CFTR(-/-) T2AECs but had no effect on CFTR(+/+) epithelia. In ex vivo studies of nonperfused lungs, stimulated liquid absorption was defective in CFTR(-/-) alveolar epithelia but similar between CFTR(+/+) and CFTR(+/-) epithelia. When epithelia were studied at the air-liquid interface, elevating cAMP levels increased subphase liquid height in CFTR(+/+) but not in CFTR(-/-) T2AECs. Our findings demonstrate that CFTR is required for maximal liquid absorption under cAMP stimulation, but it is not the rate-limiting factor. Furthermore, our data define a role for CFTR in liquid secretion by T2AECs. These insights may help to develop new treatment strategies for pulmonary edema and respiratory distress syndrome, diseases in which lung liquid transport is disrupted.