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A key issue for research on COVID-19 pathogenesis is the lack of biopsies from patients and of samples at the onset of infection. To overcome these hurdles, hamsters were shown to be useful models for studying this disease. Here, we further leverage the model to molecularly survey the disease progression from time-resolved single-cell RNA sequencing data collected from healthy and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected Syrian and Roborovski hamster lungs. We compare our data to human COVID-19 studies, including bronchoalveolar lavage, nasal swab, and postmortem lung tissue, and identify a shared axis of inflammation dominated by macrophages, neutrophils, and endothelial cells, which we show to be transient in Syrian and terminal in Roborovski hamsters. Our data suggest that, following SARS-CoV-2 infection, commitment to a type 1- or type 3-biased immunity determines moderate versus severe COVID-19 outcomes, respectively., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Prospective and longitudinal data on pulmonary injury over one year after acute coronavirus disease 2019 (COVID-19) are sparse. We aim to determine reductions in pulmonary function and respiratory related quality of life up to 12 months after acute COVID-19., Methods: Patients with acute COVID-19 were enrolled into an ongoing single-centre, prospective observational study and prospectively examined 6 weeks, 3, 6 and 12 months after onset of COVID-19 symptoms. Chest CT-scans, pulmonary function and symptoms assessed by St. Georges Respiratory Questionnaire were used to evaluate respiratory limitations. Patients were stratified according to severity of acute COVID-19., Results: Median age of all patients was 57 years, 37.8% were female. Higher age, male sex and higher BMI were associated with acute-COVID-19 severity (p < 0.0001, 0.001 and 0.004 respectively). Also, pulmonary restriction and reduced carbon monoxide diffusion capacity was associated with disease severity. In patients with restriction and impaired diffusion capacity, FVC improved over 12 months from 61.32 to 71.82, TLC from 68.92 to 76.95, D LCO from 60.18 to 68.98 and K CO from 81.28 to 87.80 (percent predicted values; p = 0.002, 0.045, 0.0002 and 0.0005). The CT-score of lung involvement in the acute phase was associated with restriction and reduction in diffusion capacity in follow-up. Respiratory symptoms improved for patients in higher severity groups during follow-up, but not for patients with initially mild disease., Conclusion: Severity of respiratory failure during COVID-19 correlates with the degree of pulmonary function impairment and respiratory quality of life in the year after acute infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pneumonia is one of the leading causes of death worldwide. The course of the disease is often highly dynamic with unforeseen critical deterioration within hours in a relevant proportion of patients. Besides antibiotic treatment, novel adjunctive therapies are under development. Their additive value needs to be explored in preclinical and clinical studies and corresponding therapy schedules require optimization prior to introduction into clinical practice. Biomathematical modeling of the underlying disease and therapy processes might be a useful aid to support these processes. We here propose a biomathematical model of murine immune response during infection with Streptococcus pneumoniae aiming at predicting the outcome of different treatment schedules. The model consists of a number of non-linear ordinary differential equations describing the dynamics and interactions of the pulmonal pneumococcal population and relevant cells of the innate immune response, namely alveolar- and inflammatory macrophages and neutrophils. The cytokines IL-6 and IL-10 and the chemokines CCL2, CXCL1 and CXCL5 are considered as major mediators of the immune response. We also model the invasion of peripheral blood monocytes, their differentiation into macrophages and bacterial penetration through the epithelial barrier causing blood stream infections. We impose therapy effects on this system by modelling antibiotic therapy and treatment with the novel C5a-inactivator NOX-D19. All equations are derived by translating known biological mechanisms into equations and assuming appropriate response kinetics. Unknown model parameters were determined by fitting the predictions of the model to time series data derived from mice experiments with close-meshed time series of state parameters. Parameter fittings resulted in a good agreement of model and data for the experimental scenarios. The model can be used to predict the performance of alternative schedules of combined antibiotic and NOX-D19 treatment. We conclude that we established a comprehensive biomathematical model of pneumococcal lung infection, immune response and barrier function in mice allowing simulations of new treatment schedules. We aim to validate the model on the basis of further experimental data. We also plan the inclusion of further novel therapy principles and the translation of the model to the human situation in the near future., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: MS receives funding from Pfizer Inc. for epidemiologic modelling of pneumonia. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors have declared that no competing interests exist.

Rationale: During pneumonia, pathogen-host interaction evokes inflammation and lung barrier dysfunction. Tie2 activation by angiopoietin-1 reduces, whereas Tie2 blockade by angiopoietin-2 increases, inflammation and permeability during sepsis. The role of angiopoietin-1/-2 in pneumonia remains unidentified., Objectives: To investigate the prognostic and pathogenic impact of angiopoietins in regulating pulmonary vascular barrier function and inflammation in bacterial pneumonia., Methods: Serum angiopoietin levels were quantified in pneumonia patients of two independent cohorts (n = 148, n = 395). Human postmortem lung tissue, pneumolysin- or angiopoietin-2-stimulated endothelial cells, isolated perfused and ventilated mouse lungs, and mice with pneumococcal pneumonia were investigated., Measurements and Main Results: In patients with pneumonia, decreased serum angiopoietin-1 and increased angiopoietin-2 levels were observed as compared with healthy subjects. Higher angiopoietin-2 serum levels were found in patients with community-acquired pneumonia who died within 28 days of diagnosis compared with survivors. Receiver operating characteristic analysis revealed improved prognostic accuracy of CURB-65 for 28-day survival, intensive care treatment, and length of hospital stay if combined with angiopoietin-2 serum levels. In vitro, pneumolysin enhanced endothelial angiopoietin-2 release, angiopoietin-2 increased endothelial permeability, and angiopoietin-1 reduced pneumolysin-evoked endothelial permeability. Ventilated and perfused lungs of mice with angiopoietin-2 knockdown showed reduced permeability on pneumolysin stimulation. Increased pulmonary angiopoietin-2 and reduced angiopoietin-1 mRNA expression were observed in Streptococcus pneumoniae-infected mice. Finally, angiopoietin-1 therapy reduced inflammation and permeability in murine pneumonia., Conclusions: These data suggest a central role of angiopoietin-1/-2 in pneumonia-evoked inflammation and permeability. Increased angiopoietin-2 serum levels predicted mortality and length of hospital stay, and angiopoietin-1 may provide a therapeutic target for severe pneumonia.

Descriptive histopathology of mouse models of pneumonia is essential in assessing the outcome of infections, molecular manipulations, or therapies in the context of whole lungs. Quantitative comparisons between experimental groups, however, have been limited to laborious stereology or ill-defined scoring systems that depend on the subjectivity of a more or less experienced observer. Here, we introduce self-learning digital image analyses that allow us to transform optical information from whole mouse lung sections into statistically testable data. A pattern-recognition-based software and a nuclear count algorithm were adopted to quantify user-defined pathologies from whole slide scans of lungs infected with Streptococcus pneumoniae or influenza A virus compared with PBS-challenged lungs. The readout parameters "relative area affected" and "nuclear counts per area" are proposed as relevant criteria for the quantification of lesions from hematoxylin and eosin-stained sections, also allowing for the generation of a heat map of, for example, immune cell infiltrates with anatomical assignments across entire lung sections. Moreover, when combined with immunohistochemical labeling of marker proteins, both approaches are useful for the identification and counting of, for example, immune cell populations, as validated here by direct comparisons with flow cytometry data. The solutions can easily and flexibly be adjusted to specificities of different models or pathogens. Automated digital analyses of whole mouse lung sections may set a new standard for the user-defined, high-throughput comparative quantification of histological and immunohistochemical images. Still, our algorithms established here are only a start, and need to be tested in additional studies and other applications in the future.

Pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. Murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. Despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. Here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by Streptococcus (S.) pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Legionella pneumophila, Escherichia coli, Middle East respiratory syndrome (MERS) coronavirus, influenza A virus (IAV) and superinfection of IAV-incuced pneumonia with S. pneumoniae. Systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. We therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogen- and model-specific patterns of pneumonia. Other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. The substantial differences between the model-specific pathologies underscore the necessity of pathogen- and model-adapted criteria for the comparative quantification of experimental outcomes. These criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models.

Background: Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide. Despite effective antimicrobial therapy, CAP can induce pulmonary endothelial hyperpermeability resulting in life-threatening lung failure due to an exaggerated host-pathogen interaction. Treatment of acute lung injury is mainly supportive because key elements of inflammation-induced barrier disruption remain undetermined. Angiopoietin-1 (Ang-1)-mediated Tie2 activation reduces, and the Ang-1 antagonist Ang-2 increases, inflammation and endothelial permeability in sepsis. Vasculotide (VT) is a polyethylene glycol-clustered Tie2-binding peptide that mimics the actions of Ang-1. The aim of our study was to experimentally test whether VT is capable of diminishing pneumonia-induced lung injury., Methods: VT binding and phosphorylation of Tie2 were analyzed using tryptophan fluorescence spectroscopy and phospho-Tie-2 enzyme-linked immunosorbent assay. Human and murine lung endothelial cells were investigated by immunofluorescence staining and electric cell-substrate impedance sensing. Pulmonary hyperpermeability was quantified in VT-pretreated, isolated, perfused, and ventilated mouse lungs stimulated with the pneumococcal exotoxin pneumolysin (PLY). Furthermore, Streptococcus pneumoniae-infected mice were therapeutically treated with VT., Results: VT showed dose-dependent binding and phosphorylation of Tie2. Pretreatment with VT protected lung endothelial cell monolayers from PLY-induced disruption. In isolated mouse lungs, VT decreased PLY-induced pulmonary permeability. Likewise, therapeutic treatment with VT of S. pneumoniae-infected mice significantly reduced pneumonia-induced hyperpermeability. However, effects by VT on the pulmonary or systemic inflammatory response were not observed., Conclusions: VT promoted pulmonary endothelial stability and reduced lung permeability in different models of pneumococcal pneumonia. Thus, VT may provide a novel therapeutic perspective for reduction of permeability in pneumococcal pneumonia-induced lung injury.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease in which the amount of emphysema and airway disease may be very different between individuals, even in end-stage disease. Emphysema formation may be linked to the involvement of the small pulmonary vessels. The NAPDH oxidase (Nox) family is emerging as a key disease-related factor in vascular diseases, but currently its role in hypoxia-induced pulmonary remodelling in COPD remains unclear.Here we investigate the role of p22phox, a regulatory subunit of Nox, in COPD lungs, hypoxic pulmonary vasoconstriction (HPV), hypoxia-induced pulmonary vascular remodelling and pulmonary hypertension.In COPD, compared to control lungs, p22phox expression was significantly reduced. The expression was correlated positively with mean pulmonary arterial pressure and oxygenation index and negatively with the diffusing capacity of the lung for carbon monoxide (p<0.02). This suggests a role of p22phox in ventilation/perfusion ratio matching, vascular remodelling and loss of perfused lung area. In p22phox -/- mice, HPV was significantly impaired. In the chronic hypoxic setting, lack of p22phox was associated with improved right ventricular function and decreased pulmonary vascular remodelling.p22phox-dependent Nox plays an important role in the COPD phenotype, by its action on phase II HPV and chronic vascular remodelling., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

The cation channel transient receptor potential vanilloid (TRPV) 4 is expressed in endothelial and immune cells; however, its role in acute lung injury (ALI) is unclear. The functional relevance of TRPV4 was assessed in vivo, in isolated murine lungs, and in isolated neutrophils. Genetic deficiency of TRPV4 attenuated the functional, histological, and inflammatory hallmarks of acid-induced ALI. Similar protection was obtained with prophylactic administration of the TRPV4 inhibitor, GSK2193874; however, therapeutic administration of the TRPV4 inhibitor, HC-067047, after ALI induction had no beneficial effect. In isolated lungs, platelet-activating factor (PAF) increased vascular permeability in lungs perfused with trpv4(+/+) more than with trpv4(-/-) blood, independent of lung genotype, suggesting a contribution of TRPV4 on blood cells to lung vascular barrier failure. In neutrophils, TRPV4 inhibition or deficiency attenuated the PAF-induced increase in intracellular calcium. PAF induced formation of epoxyeicosatrienoic acids by neutrophils, which, in turn, stimulated TRPV4-dependent Ca(2+) signaling, whereas inhibition of epoxyeicosatrienoic acid formation inhibited the Ca(2+) response to PAF. TRPV4 deficiency prevented neutrophil responses to proinflammatory stimuli, including the formation of reactive oxygen species, neutrophil adhesion, and chemotaxis, putatively due to reduced activation of Rac. In chimeric mice, however, the majority of protective effects in acid-induced ALI were attributable to genetic deficiency of TRPV4 in parenchymal tissue, whereas TRPV4 deficiency in circulating blood cells primarily reduced lung myeloperoxidase activity. Our findings identify TRPV4 as novel regulator of neutrophil activation and suggest contributions of both parenchymal and neutrophilic TRPV4 in the pathophysiology of ALI.

Pulmonary infection with influenza virus is frequently complicated by bacterial superinfection, with Streptococcus pneumoniae being the most prevalent causal pathogen and hence often associated with high morbidity and mortality rates. Local immunosuppression due to pulmonary influenza virus infection has been identified as a major cause of the pathogenesis of secondary bacterial lung infection. Thus, specific local stimulation of the pulmonary innate immune system in subjects with influenza virus infection might improve the host defense against secondary bacterial pathogens. In the present study, we examined the effect of pulmonary immunostimulation with Toll-like receptor 2 (TLR-2)-stimulating macrophage-activating lipopeptide 2 (MALP-2) in influenza A virus (IAV)-infected mice on the course of subsequent pneumococcal superinfection. Female C57BL/6N mice infected with IAV were treated with MALP-2 on day 5 and challenged with S. pneumoniae on day 6. Intratracheal MALP-2 application increased proinflammatory cytokine and chemokine release and enhanced the recruitment of leukocytes, mainly neutrophils, into the alveolar space of IAV-infected mice, without detectable systemic side effects. Local pulmonary instillation of MALP-2 in IAV-infected mice 24 h before transnasal pneumococcal infection considerably reduced the bacterial number in the lung tissue without inducing exaggerated inflammation. The pulmonary viral load was not altered by MALP-2. Clinically, MALP-2 treatment of IAV-infected mice increased survival rates and reduced hypothermia and body weight loss after pneumococcal superinfection compared to those of untreated coinfected mice. In conclusion, local immunostimulation with MALP-2 in influenza virus-infected mice improved pulmonary bacterial elimination and increased survival after subsequent pneumococcal superinfection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Dysregulation of the innate immune system drives lung injury and its systemic sequelae due to breakdown of vascular barrier function, harmful hyperinflammation and microcirculatory failure, which contribute to the unfavourable outcome of patients with severe pneumonia. A variety of promising therapeutic targets have been identified and numerous innovative therapeutic approaches demonstrated to improve lung injury in experimental preclinical studies. However, at present specific preventive or curative strategies for the treatment of lung failure in pneumonia in addition to antibiotics are still missing. The aim of this mini-review is to give a short overview of some, but not all, adjuvant therapeutic strategies for pneumonia and its most important complications, sepsis and acute respiratory distress syndrome, and briefly discuss future perspectives., (Copyright ©ERS 2015.)

In patients with chronic obstructive pulmonary disease (COPD), Moraxella catarrhalis infection of the lower airways is associated with chronic colonization and inflammation during stable disease and acute exacerbations. Chronic smoke exposure induces chronic inflammation and impairs mucociliary clearance, thus contributing to bacterial colonization of the lower airways in COPD patients. The human-specific carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 5, expressed in human airways, has been shown to contribute to epithelial colonization of CEACAM-binding pathogens. To investigate the impact of CEACAM5 expression on pulmonary M. catarrhalis colonization, we infected mice transgenic for human CEACAM5 (hCEACAM5) and wild type mice intratracheally with M. catarrhalis with or without preceding smoke exposure and analyzed bacterial colonization and local and systemic inflammation. Our results show that airway infection with M. catarrhalis accelerated acute local but not systemic inflammation, albeit independent of hCEACAM5 expression. Long-term smoke exposure alone or prior to M. catarrhalis infection did not contribute to increased local or systemic inflammation. No difference was found in pulmonary clearance of M. catarrhalis in hCEACAM5-transgenic mice compared with wild-type mice. Smoke exposure neither altered time nor extent of persistence of M. catarrhalis in the lungs of both genotypes. In conclusion, M. catarrhalis induced a local acute immune response in murine airways. Neither hCEACAM5 expression nor chronic smoke exposure nor a combination of both was sufficient as prerequisites for the establishment of chronic M. catarrhalis colonization. Our results demonstrate the difficulties in mirroring conditions of chronic airways colonization of M. catarrhalis in a murine model., (Copyright © 2015 the American Physiological Society.)

Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction.

Introduction: Lung-protective ventilation reduced acute respiratory distress syndrome (ARDS) mortality. To minimize ventilator-induced lung injury (VILI), tidal volume is limited, high plateau pressures are avoided, and positive end-expiratory pressure (PEEP) is applied. However, the impact of specific ventilatory patterns on VILI is not well defined. Increasing inspiratory time and thereby the inspiratory/expiratory ratio (I:E ratio) may improve oxygenation, but may also be harmful as the absolute stress and strain over time increase. We thus hypothesized that increasing inspiratory time and I:E ratio aggravates VILI., Methods: VILI was induced in mice by high tidal-volume ventilation (HVT 34 ml/kg). Low tidal-volume ventilation (LVT 9 ml/kg) was used in control groups. PEEP was set to 2 cm H2O, FiO2 was 0.5 in all groups. HVT and LVT mice were ventilated with either I:E of 1:2 (LVT 1:2, HVT 1:2) or 1:1 (LVT 1:1, HVT 1:1) for 4 hours or until an alternative end point, defined as mean arterial blood pressure below 40 mm Hg. Dynamic hyperinflation due to the increased I:E ratio was excluded in a separate group of animals. Survival, lung compliance, oxygenation, pulmonary permeability, markers of pulmonary and systemic inflammation (leukocyte differentiation in lung and blood, analyses of pulmonary interleukin-6, interleukin-1β, keratinocyte-derived chemokine, monocyte chemoattractant protein-1), and histopathologic pulmonary changes were analyzed., Results: LVT 1:2 or LVT 1:1 did not result in VILI, and all individuals survived the ventilation period. HVT 1:2 decreased lung compliance, increased pulmonary neutrophils and cytokine expression, and evoked marked histologic signs of lung injury. All animals survived. HVT 1:1 caused further significant worsening of oxygenation, compliance and increased pulmonary proinflammatory cytokine expression, and pulmonary and blood neutrophils. In the HVT 1:1 group, significant mortality during mechanical ventilation was observed., Conclusion: According to the "baby lung" concept, mechanical ventilation-associated stress and strain in overinflated regions of ARDS lungs was simulated by using high tidal-volume ventilation. Increase of inspiratory time and I:E ratio significantly aggravated VILI in mice, suggesting an impact of a "stress/strain × time product" for the pathogenesis of VILI. Thus increasing the inspiratory time and I:E ratio should be critically considered.

Lung diseases, including pneumonia and asthma, are among the most prevalent human disorders, and murine models have been established to investigate their pathobiology and develop novel treatment approaches. Whereas bronchoscopy is valuable for diagnostic and therapeutic procedures in patients, no equivalent for small rodents has been established. Here, we introduce a miniaturized video-bronchoscopy system offering new opportunities in experimental lung research. With an outer diameter of 0.75 mm, it is possible to advance the optics into the main bronchi of mice. An irrigation channel allows bronchoalveolar lavage and unilateral application of substances to one lung. Even a unilateral infection is possible, enabling researchers to use the contralateral lung as internal control.

Surfactant protein A has been shown to enhance opsonization and clearance of Staphylococcus aureus in vitro. Here, the phagocytosis of alveolar S. aureus was investigated in vivo using intravital microscopy. Fluorescence labelled S. aureus Newman cells were intratracheally administered to anesthetized mice and the alveolar surface was observed for fifteen minutes. Confirming previously reported in vitro data, surfactant protein A-deficient mice showed a significantly reduced uptake of bacteria compared to wild-type mice.

The lungs provide a large inner surface to guarantee respiration. In lung alveoli, a delicate membrane formed by endo- and epithelial cells with their fused basal lamina ensures rapid and effective gas exchange between alveolar and vascular compartments while concurrently forming a robust barrier against inhaled particles and microbes. However, upon infectious or sterile inflammatory stimulation, tightly regulated endothelial barrier leakiness is required for leukocyte transmigration. Further, endothelial barrier disruption may result in uncontrolled extravasation of protein-rich fluids. This brief review summarizes some important mechanisms of pulmonary endothelial barrier regulation and disruption, focusing on the role of specific cell populations, coagulation and complement cascades and mediators including angiopoietins, specific sphingolipids, adrenomedullin and reactive oxygen and nitrogen species for the regulation of pulmonary endothelial barrier function. Further, current therapeutic perspectives against development of lung injury are discussed.

Acute and sustained hypoxic pulmonary vasoconstriction (HPV), as well as chronic pulmonary hypertension (PH), is modulated by nitric oxide (NO). NO synthesis can be decreased by asymmetric dimethylarginine (ADMA), which is degraded by dimethylarginine dimethylaminohydrolase-1 (DDAH1). We investigated the effects of DDAH1 overexpression (DDAH1(tg)) on HPV and chronic hypoxia-induced PH. HPV was measured during acute (10 min) and sustained (3 h) hypoxia in isolated mouse lungs. Chronic PH was induced by the exposure of mice to 4 weeks of hypoxia. ADMA and cyclic 3',5'-guanosine monophosphate (cGMP) were determined by ELISA, and NO generation was determined by chemiluminescence. DDAH1 overexpression exerted no effects on acute HPV. However, DDAH1(tg) mice showed decreased sustained HPV compared with wild-type (WT) mice. Concomitantly, ADMA was decreased, and concentrations of NO and cGMP were significantly increased in DDAH1(tg). The administration of either Nω-nitro-l-arginine or 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one potentiated sustained HPV and partly abolished the differences in sustained HPV between WT and DDAH1(tg) mice. The overexpression of DDAH1 exerted no effect on the development of chronic hypoxia-induced PH. DDAH1 overexpression selectively decreased the sustained phase of HPV, partly via activation of the NO-cGMP pathway. Thus, increased ADMA concentrations modulate sustained HPV, but not acute HPV or chronic hypoxia-induced PH.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism by which pulmonary arteries constrict in hypoxic lung areas in order to redirect blood flow to areas with greater oxygen supply. Both oxygen sensing and the contractile response are thought to be intrinsic to pulmonary arterial smooth muscle cells. Here we speculated that the ideal site for oxygen sensing might instead be at the alveolocapillary level, with subsequent retrograde propagation to upstream arterioles via connexin 40 (Cx40) endothelial gap junctions. HPV was largely attenuated by Cx40-specific and nonspecific gap junction uncouplers in the lungs of wild-type mice and in lungs from mice lacking Cx40 (Cx40-/-). In vivo, hypoxemia was more severe in Cx40-/- mice than in wild-type mice. Real-time fluorescence imaging revealed that hypoxia caused endothelial membrane depolarization in alveolar capillaries that propagated to upstream arterioles in wild-type, but not Cx40-/-, mice. Transformation of endothelial depolarization into vasoconstriction involved endothelial voltage-dependent α1G subtype Ca2+ channels, cytosolic phospholipase A2, and epoxyeicosatrienoic acids. Based on these data, we propose that HPV originates at the alveolocapillary level, from which the hypoxic signal is propagated as endothelial membrane depolarization to upstream arterioles in a Cx40-dependent manner.

Background: Even protective ventilation may aggravate or induce lung failure, particularly in preinjured lungs. Thus, new adjuvant pharmacologic strategies are needed to minimize ventilator-induced lung injury (VILI). Intermedin/Adrenomedullin-2 (IMD) stabilized pulmonary endothelial barrier function in vitro. We hypothesized that IMD may attenuate VILI-associated lung permeability in vivo., Methodology/principal Findings: Human pulmonary microvascular endothelial cell (HPMVEC) monolayers were incubated with IMD, and transcellular electrical resistance was measured to quantify endothelial barrier function. Expression and localization of endogenous pulmonary IMD, and its receptor complexes composed of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) 1-3 were analyzed by qRT-PCR and immunofluorescence in non ventilated mouse lungs and in lungs ventilated for 6 h. In untreated and IMD treated mice, lung permeability, pulmonary leukocyte recruitment and cytokine levels were assessed after mechanical ventilation. Further, the impact of IMD on pulmonary vasoconstriction was investigated in precision cut lung slices (PCLS) and in isolated perfused and ventilated mouse lungs. IMD stabilized endothelial barrier function in HPMVECs. Mechanical ventilation reduced the expression of RAMP3, but not of IMD, CRLR, and RAMP1 and 2. Mechanical ventilation induced lung hyperpermeability, which was ameliorated by IMD treatment. Oxygenation was not improved by IMD, which may be attributed to impaired hypoxic vasoconstriction due to IMD treatment. IMD had minor impact on pulmonary leukocyte recruitment and did not reduce cytokine levels in VILI., Conclusions/significance: IMD may possibly provide a new approach to attenuate VILI.

Lung barrier protection by Sphingosine-1 Phosphate (S1P) has been demonstrated experimentally, but recent evidence suggests barrier disruptive properties of high systemic S1P concentrations. The S1P analog FTY720 recently gained an FDA approval for treatment of multiple sclerosis. In case of FTY720 treated patients experiencing multiple organ dysfunction syndrome the drug may accumulate due to liver failure, and the patients may receive ventilator therapy. Whereas low doses of FTY720 enhanced endothelial barrier function, data on effects of increased FTY720 concentrations are lacking. We measured transcellular electrical resistance (TER) of human umbilical vein endothelial cell (HUVEC) monolayers, performed morphologic analysis and measured apoptosis by TUNEL staining and procaspase-3 degradation in HUVECs stimulated with FTY720 (0.01-100 μM). Healthy C57BL/6 mice and mice ventilated with 17 ml/kg tidal volume and 100% oxygen for 2 h were treated with 0.1 or 2 mg/kg FTY720 or solvent, and lung permeability, oxygenation and leukocyte counts in BAL and blood were quantified. Further, electron microscopic analysis of lung tissue was performed. We observed barrier protective effects of FTY720 on HUVEC cell layers at concentrations up to 1 μM while higher concentrations induced irreversible barrier breakdown accompanied by induction of apoptosis. Low FTY720 concentrations (0.1 mg/kg) reduced lung permeability in mechanically ventilated mice, but 2 mg/kg FTY720 increased pulmonary vascular permeability in ventilated mice accompanied by endothelial apoptosis, while not affecting permeability in non-ventilated mice. Moreover, hyperoxic mechanical ventilation sensitized the pulmonary vasculature to a barrier disrupting effect of FTY720, resulting in worsening of ventilator induced lung injury. In conclusion, the current data suggest FTY720 induced endothelial barrier dysfunction, which was probably caused by proapoptotic effects and enhanced by mechanical ventilation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Airway hyperresponsiveness (AHR) is a hallmark of bronchial asthma. Important features of this exaggerated response to bronchoconstrictive stimuli have mostly been investigated in vivo in intact animals or in vitro in isolated tracheal or bronchial tissues. Both approaches have important advantages but also certain limitations. Therefore, the aim of our study was to develop an ex vivo model of isolated lungs from sensitized mice for the investigation of airway responsiveness (AR). BALB/c mice were sensitized by intraperitoneal ovalbumin (Ova) and subsequently challenged by Ova inhalation. In vivo AR was measured in unrestrained animals by whole body plethysmography after stimulation with aerosolized methacholine (MCh) with determination of enhanced pause (P(enh)). Twenty-four hours after each P(enh) measurement, airway resistance was continuously registered in isolated, perfused, and ventilated lungs on stimulation with inhaled or intravascular MCh or nebulized Ova. In a subset of experiments, in vivo AR was additionally measured in orotracheally intubated, spontaneously breathing mice 24 h after P(enh) measurement, and lungs were isolated further 24 h later. Isolated lungs of allergen-sensitized and -challenged mice showed increased AR after MCh inhalation or infusion as well as after specific provocation with aerosolized allergen. AR was increased on days 2 and 5 after Ova challenge and had returned to baseline on day 9. AHR in isolated lungs after aerosolized or intravascular MCh strongly correlated with in vivo AR. Pretreatment of isolated lungs with the beta(2)-agonist fenoterol diminished AR. In conclusion, this model provides new opportunities to investigate mechanisms of AHR as well as pharmacological interventions on an intact organ level.

Generalized underrepresentation of IFN-gamma has been implicated in the development of allergic asthma. However, the role of local IFN-gamma in the lung during the development of this disease has not been completely elucidated. We studied the influence of local pulmonary IFN-gamma expression on the development of allergen-induced lung inflammation. To restrict our analysis to IFN-gamma expression in the lung and to exclude influences of systemic IFN-gamma production, we generated a transgenic mouse line with a targeted deletion of the IFN-gamma gene and constitutive, lung-specific IFN-gamma expression (Clara cell 10 [CC10]-IFN-gamma-tg-IFN-gamma-KO mice), and compared allergen-induced airway inflammation in these mice with that of wild-type and IFN-gamma- KO mice on the C57BL/6 background. Cytokine quantification in lungs of mice with allergic airway inflammation revealed that pulmonary IFN-gamma expression increased expression of IL-5 and IL-13. Consistent with this observation, eosinophilia in bronchoalveolar lavage of CC10-IFN-gamma-tg-IFN-gamma-KO mice was profoundly increased, indicating that this critical component of asthma is enhanced by local IFN-gamma expression. In contrast, airway hyperresponsiveness and anti-ovalbumin-IgE serum levels were reduced by local IFN-gamma expression. Together, our results demonstrate pleiotropic action of constitutive IFN-gamma expression in the lung, and question the therapeutic value of IFN-gamma in allergic asthma. Local expression of IFN-gamma in the lung increases markers of allergic airway inflammation, but decreases airway hyperresponsiveness in a murine model of allergic-asthma.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia and death from infectious diseases in industrialized countries. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX)-derived prostaglandins, such as PGE(2), are considered to be important regulators of lung function. Herein, we tested the hypothesis that pneumococci induced COX-2-dependent PGE(2) production in pulmonary epithelial cells. Pneumococci-infected human pulmonary epithelial BEAS-2B cells released PGE(2). Expression of COX-2 but not COX-1 was dose and time dependently increased in S. pneumoniae-infected BEAS-2B cells as well as in lungs of mice with pneumococcal pneumonia. S. pneumoniae induced degradation of IkappaBalpha and DNA binding of NF-kappaB. A specific peptide inhibitor of the IkappaBalpha kinase complex blocked pneumococci-induced PGE(2) release and COX-2 expression. In addition, we noted activation of p38 MAPK and JNK in pneumococci-infected BEAS-2B cells. PGE(2) release and COX-2 expression were reduced by p38 MAPK inhibitor SB-202190 but not by JNK inhibitor SP-600125. We analyzed interaction of kinase pathways and NF-kappaB activation: dominant-negative mutants of p38 MAPK isoforms alpha, beta(2), gamma, and delta blocked S. pneumoniae-induced NF-kappaB activation. In addition, recruitment of NF-kappaB subunit p65/RelA and RNA polymerase II to the cox2 promoter depended on p38 MAPK but not on JNK activity. In summary, p38 MAPK- and NF-kappaB-controlled COX-2 expression and subsequent PGE(2) release by lung epithelial cells may contribute significantly to the host response in pneumococcal pneumonia.

In vitro lung research requires appropriate cell culture models that adequately mimic in vivo structure and function. Previously, researchers extensively used commercially available and easily expandable A549 and NCI-H441 cells, which replicate some but not all features of alveolar epithelial cells. Specifically, these cells are often restricted by terminally altered expression while lacking important alveolar epithelial characteristics. Of late, human primary alveolar epithelial cells (hPAEpCs) have become commercially available but are so far poorly specified. Here, we applied a comprehensive set of technologies to characterize their morphology, surface marker expression, transcriptomic profile, and functional properties. At optimized seeding numbers of 7,500 cells per square centimeter and growth at a gas–liquid interface, hPAEpCs formed regular monolayers with tight junctions and amiloride-sensitive transepithelial ion transport. Electron microscopy revealed lamellar body and microvilli formation characteristic for alveolar type II cells. Protein and single-cell transcriptomic analyses revealed expression of alveolar type I and type II cell markers; yet, transcriptomic data failed to detect NKX2-1, an important transcriptional regulator of alveolar cell differentiation. With increasing passage number, hPAEpCs transdifferentiated toward alveolar-basal intermediates characterized as SFTPC−, KRT8high, and KRT5− cells. In spite of marked changes in the transcriptome as a function of passaging, Uniform Manifold Approximation and Projection plots did not reveal major shifts in cell clusters, and epithelial permeability was unaffected. The present work delineates optimized culture conditions, cellular characteristics, and functional properties of commercially available hPAEpCs. hPAEpCs may provide a useful model system for studies on drug delivery, barrier function, and transepithelial ion transport in vitro. [ABSTRACT FROM AUTHOR]

Abstract: The ability of synthetic small interfering RNA (siRNA) to silence gene expression makes it a useful tool in biomedical research. However, effective and non-toxic functional siRNA delivery to mouse lungs in vivo is still a key challenge, and regulation of constitutively expressed genes is poorly characterized. Following in vitro validation of siRNA molecules, naked, stabilized siRNA (AtuRNAi) was applied intranasally (i.n.) by droplets or intratracheally (i.t.) by MicroSprayer™ in female C57BL/6 mice. Distribution of Cy3-tagged siRNAs was examined. Pulmonary expression of ubiquitously (lamin B1) or cell-specific (E-cadherin, VE-cadherin), constitutive genes was analysed by TaqMan-realtime-PCR. Further, formulated lipoplex-siRNA, which has enhanced transfection efficiency, was applied i.t. or intravenously (i.v.). Single i.t. as compared to i.n. application of unformulated siRNA resulted in higher delivery efficiency and homogenous pulmonary distribution. After inhalation of target-specific siRNA, reduction of epithelial E-cadherin by 21%, but no significant reduction of endothelial VE-cadherin or ubiquitously expressed lamin B1 was observed. Pharmacokinetic analysis revealed rapid transfer of intact siRNA molecules into the vascular system and accumulation in the kidneys, calling lung specificity into question. I.t. application of lipoplex-siRNA evoked inflammation. In contrast, i.v. application of lipoplex-siRNA specifically reduced expression of VE-cadherin mRNA by about 50% in lungs without evoking lung cellular influx. In conclusion, sufficient pulmonary distribution of aerosolized siRNA was attained in mice by MicroSprayer™, however development of appropriate siRNA carriers is highly desirable to improve lung-specific functional inhalative siRNA delivery. Pulmonary knockdown of constitutive endothelial targets by 50% was achieved by i.v. application of lipoplex-siRNA. [Copyright &y& Elsevier]

Stroke-induced immunosuppression contributes to the development of stroke-associated pneumonia (SAP). Experiments in mice demonstrated that apoptosis of IFN-γ producing cells and reduced IFN-γ secretion resulted in impaired immune responses and the development of pneumonia after middle cerebral artery occlusion (MCAo). In the present study, we investigated the efficacy of intratracheal IFN-γ treatment to prevent SAP and demonstrated that modest benefits on pulmonary cytokine response in IFN-γ treated stroke mice did not prevent spontaneously developing infections and even slightly reduced bacterial clearance of aspirated pneumococci. Our results suggest that pulmonary IFN-γ treatment is not an effective preventive measure for SAP. [Display omitted] • Apoptosis of IFN-γ producing cells and impaired IFN-γ secretion in lung and blood contribute to stroke-associated pneumonia • I.t. IFN-γ slightly improve pulmonary cytokine secretion after MCAo but finally cannot reconstitute the local immune competence • Intratracheal IFN-γ treatment does not prevent stroke-associated pneumonia (SAP) • Infarct size is not affected by i.t. IFN-γ application suggesting that treatment is safe and basically suitable for SAP [ABSTRACT FROM AUTHOR]