Life-saving effect of pulmonary surfactant in premature babies.

The discovery and replacement of lung surfactant have helped increase survival rates for neonatal respiratory distress syndrome in extremely premature infants.

The pathogenesis of influenza in intact alveoli: virion endocytosis and its effects on the lung's air-blood barrier.

Lung infection by influenza A virus (IAV) is a major cause of global mortality from lung injury, a disease defined by widespread dysfunction of the lung's air-blood barrier. Endocytosis of IAV virions by the alveolar epithelium - the cells that determine barrier function - is central to barrier loss mechanisms. Here, we address the current understanding of the mechanistic steps that lead to endocytosis in the alveolar epithelium, with an eye to how the unique structure of lung alveoli shapes endocytic mechanisms. We highlight where future studies of alveolar interactions with IAV virions may lead to new therapeutic approaches for IAV-induced lung injury.

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.

Mitochondria of lung venular capillaries mediate lung-liver cross talk in pneumonia.

Failure of the lung's endothelial barrier underlies lung injury, which causes the high mortality acute respiratory distress syndrome (ARDS). Multiple organ failure predisposes to the mortality, but mechanisms are poorly understood. Here, we show that mitochondrial uncoupling protein 2 (UCP2), a component of the mitochondrial inner membrane, plays a role in the barrier failure. Subsequent lung-liver cross talk mediated by neutrophil activation causes liver congestion. We intranasally instilled lipopolysaccharide (LPS). Then, we viewed the lung endothelium by real-time confocal imaging of the isolated, blood-perfused mouse lung. LPS caused alveolar-capillary transfer of reactive oxygen species and mitochondrial depolarization in lung venular capillaries. The mitochondrial depolarization was inhibited by transfection of alveolar Catalase and vascular knockdown of UCP2. LPS instillation caused lung injury as indicated by increases in bronchoalveolar lavage (BAL) protein content and extravascular lung water. LPS or Pseudomonas aeruginosa instillation also caused liver congestion, quantified by liver hemoglobin and plasma aspartate aminotransferase (AST) increases. Genetic inhibition of vascular UCP2 prevented both lung injury and liver congestion. Antibody-mediated neutrophil depletion blocked the liver responses, but not lung injury. Knockdown of lung vascular UCP2 mitigated P. aeruginosa-induced mortality. Together, these data suggest a mechanism in which bacterial pneumonia induces oxidative signaling to lung venular capillaries, known sites of inflammatory signaling in the lung microvasculature, depolarizing venular mitochondria. Successive activation of neutrophils induces liver congestion. We conclude that oxidant-induced UCP2 expression in lung venular capillaries causes a mechanistic sequence leading to liver congestion and mortality. Lung vascular UCP2 may present a therapeutic target in ARDS.NEW & NOTEWORTHY We report that mitochondrial injury in lung venular capillaries underlies barrier failure in pneumonia, and venular capillary uncoupling protein 2 (UCP2) causes neutrophil-mediated liver congestion. Using in situ imaging, we found that epithelial-endothelial transfer of H2O2 activates UCP2, depolarizing mitochondria in venular capillaries. The conceptual advance from our findings is that mitochondrial depolarization in lung capillaries mediates liver cross talk through circulating neutrophils. Pharmacologic blockade of UCP2 could be a therapeutic strategy for lung injury.

Sessile alveolar macrophage connexin-43 determines mechano-immunity in the lung.

Although lung immunity is pathogen induced, the immunity can also be induced by mechanical distortion of the lung. The causal basis of the lung's mechanosensitive immunity remains unclear. Here, through live optical imaging of mouse lungs, we show that alveolar stretch due to hyperinflation induced prolonged cytosolic Ca2+ increases in sessile alveolar macrophages (AMs). Knockout studies revealed that the Ca2+ increases resulted from Ca2+ diffusion from the alveolar epithelium to sessile AMs through connexin 43 (Cx43)-containing gap junctions. Lung inflammation and injury in mice exposed to injurious mechanical ventilation were inhibited by AM-specific Cx43 knockout, or AM-specific delivery of a calcium inhibitor. We conclude, Cx43 gap junctions and calcium mobilization in sessile AMs determine the lung's mechanosensitive immunity, providing a therapeutic strategy against hyperinflation-induced lung injury.

Retinoids stored locally in the lung are required to attenuate the severity of acute lung injury in male mice.

Retinoids are potent transcriptional regulators that act in regulating cell proliferation, differentiation, and other cellular processes. We carried out studies in male mice to establish the importance of local cellular retinoid stores within the lung alveolus for maintaining its health in the face of an acute inflammatory challenge induced by intranasal instillation of lipopolysaccharide. We also undertook single cell RNA sequencing and bioinformatic analyses to identify roles for different alveolar cell populations involved in mediating these retinoid-dependent responses. Here we show that local retinoid stores and uncompromised metabolism and signaling within the lung are required to lessen the severity of an acute inflammatory challenge. Unexpectedly, our data also establish that alveolar cells other than lipofibroblasts, specifically microvascular endothelial and alveolar epithelial cells, are able to take up lipoprotein-transported retinoid and to accumulate cellular retinoid stores that are directly used to respond to an acute inflammatory challenge.

The mitochondrial calcium uniporter of pulmonary type 2 cells determines severity of acute lung injury.

Acute Lung Injury (ALI) due to inhaled pathogens causes high mortality. Underlying mechanisms are inadequately understood. Here, by optical imaging of live mouse lungs we show that a key mechanism is the viability of cytosolic Ca2+ buffering by the mitochondrial Ca2+ uniporter (MCU) in the lung's surfactant-secreting, alveolar type 2 cells (AT2). The buffering increased mitochondrial Ca2+ and induced surfactant secretion in wild-type mice, but not in mice with AT2-specific MCU knockout. In the knockout mice, ALI due to intranasal LPS instillation caused severe pulmonary edema and mortality, which were mitigated by surfactant replenishment prior to LPS instillation, indicating surfactant's protective effect against alveolar edema. In wild-type mice, intranasal LPS, or Pseudomonas aeruginosa decreased AT2 MCU. Loss of MCU abrogated buffering. The resulting mortality was reduced by spontaneous recovery of MCU expression, or by MCU replenishment. Enhancement of AT2 mitochondrial buffering, hence endogenous surfactant secretion, through MCU replenishment might be a therapy against ALI.

Modulation of the NLRP3 inflammasome by Sars-CoV-2 Envelope protein.

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1ß and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1ß, levels of IL-1ß and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.

Molecular programs of fibrotic change in aging human lung.

Lung fibrosis is increasingly detected with aging and has been associated with poor outcomes in acute lung injury or infection. However, the molecular programs driving this pro-fibrotic evolution are unclear. Here we profile distal lung samples from healthy human donors across the lifespan. Gene expression profiling by bulk RNAseq reveals both increasing cellular senescence and pro-fibrotic pathway activation with age. Quantitation of telomere length shows progressive shortening with age, which is associated with DNA damage foci and cellular senescence. Cell type deconvolution analysis of the RNAseq data indicates a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts with age. Consistent with this pro-fibrotic profile, second harmonic imaging of aged lungs demonstrates increased density of interstitial collagen as well as decreased alveolar expansion and surfactant secretion. In this work, we reveal the transcriptional and structural features of fibrosis and associated functional impairment in normal lung aging.

Actin fence therapy with exogenous V12Rac1 protects against acute lung injury.

High mortality in acute lung injury (ALI) results from sustained proinflammatory signaling by alveolar receptors, such as TNF-α receptor type 1 (TNFR1). Factors that determine the sustained signaling are not known. Unexpectedly, optical imaging of live alveoli revealed a major TNF-α-induced surge of alveolar TNFR1 due to a Ca2+-dependent mechanism that decreased the cortical actin fence. Mouse mortality due to inhaled LPS was associated with cofilin activation, actin loss, and the TNFR1 surge. The constitutively active form of the GTPase, Rac1 (V12Rac1), given intranasally (i.n.) as a noncovalent construct with a cell-permeable peptide, enhanced alveolar filamentous actin (F-actin) and blocked the TNFR1 surge. V12Rac1 also protected against ALI-induced mortality resulting from i.n. instillation of LPS or of Pseudomonas aeruginosa. We propose a potentially new therapeutic paradigm in which actin enhancement by exogenous Rac1 strengthens the alveolar actin fence, protecting against proinflammatory receptor hyperexpression, and therefore blocking ALI.

The Mitochondrial Calcium Uniporter of Pulmonary Type 2 Cells Determines Severity of ARDS.

Acute lung immunity to inhaled pathogens elicits defensive pneumonitis that may convert to the Acute Respiratory Distress Syndrome (ARDS), causing high mortality. Mechanisms underlying the conversion are not understood, but are of intense interest because of the ARDS-induced mortality in the ongoing Covid-19 pandemic. Here, by optical imaging of live lungs we show that key to the lethality is the functional status of mitochondrial Ca2+ buffering across the mitochondrial Ca2+ uniporter (MCU) in the alveolar type 2 cells (AT2), which protect alveolar stability. In mice subjected to ARDS by airway exposure to lipopolysaccharide (LPS), or to Pseudomonas aeruginosa, there was marked loss of MCU expression in AT2. The ability of mice to survive ARDS depended on the extent to which the MCU expression recovered, indicating that the viability of Ca2+ buffering by AT2 mitochondria critically determines ARDS severity. Mitochondrial transfer to enhance AT2 MCU expression might protect against ARDS.

Pulmonary surfactant and drug delivery: Vehiculization, release and targeting of surfactant/tacrolimus formulations.

This work explores the potential for strategizing pulmonary surfactant (PS) for drug delivery over the respiratory air-liquid interface: the interfacial delivery. The efficacy of PS- and interface-assisted drug vehiculization was determined both in vitro and in vivo using a native purified porcine PS combined with the hydrophobic anti-inflammatory drug Tacrolimus (TAC), a calcineurin inhibitor. In vitro assays were conducted in a novel double surface balance setup designed to emulate compression-expansion dynamics applied to interfacially connected drug donor and recipient compartments. In this setup, PS transported TAC efficiently over air-liquid interfaces, with compression/expansion breathing-like dynamics enhancing rapid interface-assisted diffusion and drug release. The efficacy of PS-assisted TAC vehiculization was also evaluated in vivo in a mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). In anesthetized mice, TAC combined with PS was intra-nasally (i.n) instilled prior administering i.n. LPS. PS/TAC pre-treatment caused greater TAC internalization into a higher number of lung cells obtained from bronchoalveolar lavages (BAL) than TAC pre-treatment alone. Additionally, the PS/TAC combination but not TAC or PS alone attenuated the LPS-induced pro-inflammatory effects reducing cells and proteins in BAL fluid. These findings indicated that PS-mediated increase in TAC uptake blunted the pro-injurious effects of LPS, suggesting a synergistic anti-inflammatory effect of PS/drug formulations. These in vitro and in vivo results establish the potential utility of PS to open novel effective delivery strategies for inhaled drugs.

Endothelial mitochondria determine rapid barrier failure in chemical lung injury.

Acid aspiration, which can result from several etiologies, including postoperative complications, leads to direct contact of concentrated hydrochloric acid (HCl) with the alveolar epithelium. As a result, rapid endothelial activation induces alveolar inflammation, leading to life-threatening pulmonary edema. Because mechanisms underlying the rapid endothelial activation are not understood, here we determined responses in real time through optical imaging of alveoli of live mouse lungs. By alveolar micropuncture, we microinfused concentrated HCl in the alveolar lumen. As expected, acid contact with the epithelium caused rapid, but transient, apical injury. However, there was no concomitant membrane injury to the endothelium. Nevertheless, H2O2-mediated epithelial-endothelial paracrine signaling induced endothelial barrier failure, as detected by microvascular dextran leakage and lung water quantification. Remarkably, endothelial mitochondria regulated the barrier failure by activating uncoupling protein 2 (UCP2), thereby inducing transient mitochondrial depolarization that led to cofilin-induced actin depolymerization. Knockdown, or endothelium-targeted deletion of UCP2 expression, blocked these responses, including pulmonary edema. To our knowledge, these findings are the first to mechanistically implicate endothelial mitochondria in acid-induced barrier deterioration and pulmonary edema. We suggest endothelial UCP2 may be a therapeutic target for acid-induced acute lung injury.

Regulatory T Cells Promote Macrophage Efferocytosis during Inflammation Resolution.

Regulatory T (Treg) cell responses and apoptotic cell clearance (efferocytosis) represent critical arms of the inflammation resolution response. We sought to determine whether these processes might be linked through Treg-cell-mediated enhancement of efferocytosis. In zymosan-induced peritonitis and lipopolysaccharide-induced lung injury, Treg cells increased early in resolution, and Treg cell depletion decreased efferocytosis. In advanced atherosclerosis, where defective efferocytosis drives disease progression, Treg cell expansion improved efferocytosis. Mechanistic studies revealed the following sequence: (1) Treg cells secreted interleukin-13 (IL-13), which stimulated IL-10 production in macrophages; (2) autocrine-paracrine signaling by IL-10 induced Vav1 in macrophages; and (3) Vav1 activated Rac1 to promote apoptotic cell engulfment. In summary, Treg cells promote macrophage efferocytosis during inflammation resolution via a transcellular signaling pathway that enhances apoptotic cell internalization. These findings suggest an expanded role of Treg cells in inflammation resolution and provide a mechanistic basis for Treg-cell-enhancement strategies for non-resolving inflammatory diseases.

Crosstalk signaling between alveoli and capillaries.

Crosstalk signaling between the closely juxtaposed epithelial and endothelial membranes of pulmonary alveoli establishes the lung's immune defense against inhaled and blood-borne pathogens. The crosstalk can occur in a forward direction, as from alveolus to capillary, or in a reverse direction, as from capillary to alveolus. The crosstalk direction likely depends on the site at which pathogens first initiate signaling. Thus, forward crosstalk may occur when inhaled pathogens encounter the alveolar epithelium, while reverse crosstalk may result from interactions of blood-borne pathogens with the endothelium. Here, we review the factors that regulate these two directions of signaling.

Pulmonary vascular endothelium: the orchestra conductor in respiratory diseases: Highlights from basic research to therapy.

The European Respiratory Society (ERS) Research Seminar entitled "Pulmonary vascular endothelium: orchestra conductor in respiratory diseases - highlights from basic research to therapy" brought together international experts in dysfunctional pulmonary endothelium, from basic science to translational medicine, to discuss several important aspects in acute and chronic lung diseases. This review will briefly sum up the different topics of discussion from this meeting which was held in Paris, France on October 27-28, 2016. It is important to consider that this paper does not address all aspects of endothelial dysfunction but focuses on specific themes such as: 1) the complex role of the pulmonary endothelium in orchestrating the host response in both health and disease (acute lung injury, chronic obstructive pulmonary disease, high-altitude pulmonary oedema and pulmonary hypertension); and 2) the potential value of dysfunctional pulmonary endothelium as a target for innovative therapies.

Disruption of staphylococcal aggregation protects against lethal lung injury.

Infection by Staphylococcus aureus strain USA300 causes tissue injury, multiorgan failure, and high mortality. However, the mechanisms by which the bacteria adhere to, then stabilize on, mucosal surfaces before causing injury remain unclear. We addressed these issues through the first real-time determinations of USA300-alveolar interactions in live lungs. We found that within minutes, inhaled USA300 established stable, self-associated microaggregates in niches at curved, but not at flat, regions of the alveolar wall. The microaggregates released α-hemolysin toxin, causing localized alveolar injury, as indicated by epithelial dye loss, mitochondrial depolarization, and cytosolic Ca2+ increase. Spread of cytosolic Ca2+ through intercellular gap junctions to adjoining, uninfected alveoli caused pulmonary edema. Systemic pretreatment with vancomycin, a USA300-cidal antibiotic, failed to protect mice infected with inhaled WT USA300. However, vancomycin pretreatment markedly abrogated mortality in mice infected with mutant USA300 that lacked the aggregation-promoting factor PhnD. We interpret USA300-induced mortality as having resulted from rapid bacterial aggregation in alveolar niches. These findings indicate, for the first time to our knowledge, that alveolar microanatomy is critical in promoting the aggregation and, hence, in causing USA300-induced alveolar injury. We propose that in addition to antibiotics, strategies for bacterial disaggregation may constitute novel therapy against USA300-induced lung injury.

A three-dimensional model of human lung development and disease from pluripotent stem cells.

Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modelling, drug discovery and regenerative medicine. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.