Changes in targeted metabolomics in lung tissue of chronic obstructive pulmonary disease.

Background: Chronic obstructive pulmonary disease (COPD) is a common chronic lung disease and its incidence is steadily increasing. COPD patients and mouse models of COPD share some similarities in lung pathology and physiology. We performed this study to explore the potential metabolic pathways involved in the pathogenesis of COPD and to discover the COPD-associated biomarkers. Furthermore, we aimed to examine how much the mouse model of COPD was similar and different to human COPD in terms of the altered metabolites and pathways. Methods: Twenty human lung tissue samples (ten COPD and ten controls) and twelve mice lung tissue samples (six COPD and six controls) were analyzed by targeted HM350 metabolomics, and multivariate and pathway analysis were performed by Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Results: The counts of many metabolites such as amino acids, carbohydrates and carnitines were changed in both COPD patients and mice compared to controls, respectively. While lipid metabolism was changed only in COPD mice. After KEGG analysis, we found these altered metabolites involved in COPD through aging, apoptosis, oxidative stress and inflammation pathways. Conclusions: The expressions of metabolites changed in both COPD patients and cigarette smoke exposed (CS-exposed) mice. And there were also some differences between COPD patients and mouse models due to the differences between species. Our study suggested the dysregulation in amino acid metabolism, energy production pathway and perhaps lipid metabolism may be significantly related to the pathogenesis of COPD.

Application of Precision-Cut Lung Slices as an In Vitro Model for Research of Inflammatory Respiratory Diseases.

The leading cause of many respiratory diseases is an ongoing and progressive inflammatory response. Traditionally, inflammatory lung diseases were studied primarily through animal models, cell cultures, and organoids. These technologies have certain limitations, despite their great contributions to the study of respiratory diseases. Precision-cut lung slices (PCLS) are thin, uniform tissue slices made from human or animal lung tissue and are widely used extensively both nationally and internationally as an in vitro organotypic model. Human lung slices bridge the gap between in vivo and in vitro models, and they can replicate the living lung environment well while preserving the lungs' basic structures, such as their primitive cells and trachea. However, there is no perfect model that can completely replace the structure of the human lung, and there is still a long way to go in the research of lung slice technology. This review details and analyzes the strengths and weaknesses of precision lung slices as an in vitro model for exploring respiratory diseases associated with inflammation, as well as recent advances in this field.

Zinc Metalloprotease ProA from Legionella pneumophila Inhibits the Pro-Inflammatory Host Response by Degradation of Bacterial Flagellin.

The environmental bacterium Legionella pneumophila is an intracellular pathogen of various protozoan hosts and able to cause Legionnaires' disease, a severe pneumonia in humans. By encoding a wide selection of virulence factors, the infectious agent possesses several strategies to manipulate its host cells and evade immune detection. In the present study, we demonstrate that the L. pneumophila zinc metalloprotease ProA functions as a modulator of flagellin-mediated TLR5 stimulation and subsequent activation of the pro-inflammatory NF-κB pathway. We found ProA to be capable of directly degrading immunogenic FlaA monomers but not the polymeric form of bacterial flagella. These results indicate a role of the protease in antagonizing immune stimulation, which was further substantiated in HEK-BlueTM hTLR5 Detection assays. Addition of purified proteins, bacterial suspensions of L. pneumophila mutant strains as well as supernatants of human lung tissue explant infection to this reporter cell line demonstrated that ProA specifically decreases the TLR5 response via FlaA degradation. Conclusively, the zinc metalloprotease ProA serves as a powerful regulator of exogenous flagellin and presumably creates an important advantage for L. pneumophila proliferation in mammalian hosts by promoting immune evasion.

Di-Tyrosine Crosslinking and NOX4 Expression as Oxidative Pathological Markers in the Lungs of Patients with Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a noninflammatory progressive lung disease. Oxidative damage is a hallmark of IPF, but the sources and consequences of oxidant generation in the lungs are unclear. In this study, we addressed the link between the H2O2-generating enzyme NADPH oxidase 4 (NOX4) and di-tyrosine (DT), an oxidative post-translational modification in IPF lungs. We performed immunohistochemical staining for DT and NOX4 in pulmonary tissue from patients with IPF and controls using validated antibodies. In the healthy lung, DT showed little or no staining and NOX4 was mostly present in normal vascular endothelium. On the other hand, both markers were detected in several cell types in the IPF patients, including vascular smooth muscle cells and epithelium (bronchial cells and epithelial cells type II). The link between NOX4 and DT was addressed in human fibroblasts deficient for NOX4 activity (mutation in the CYBA gene). Induction of NOX4 by Transforming growth factor beta 1 (TGFß1) in fibroblasts led to moderate DT staining after the addition of a heme-containing peroxidase in control cells but not in the fibroblasts deficient for NOX4 activity. Our data indicate that DT is a histological marker of IPF and that NOX4 can generate a sufficient amount of H2O2 for DT formation in vitro.

Presence of airborne microplastics in human lung tissue.

Plastics are ubiquitously used by societies, but most of the plastic waste is deposited in landfills and in the natural environment. Their degradation into submillimetre fragments, called microplastics, is a growing concern due to potential adverse effects on the environment and human health. Microplastics are present in the air and may be inhaled by humans, but whether they have deleterious effects on the respiratory system remain unknown. In this study, we determined the presence of microplastics in human lung tissues obtained at autopsies. Polymeric particles (n = 33) and fibres (n = 4) were observed in 13 of 20 tissue samples. All polymeric particles were smaller than 5.5 µm in size, and fibres ranged from 8.12 to 16.8 µm. The most frequently determined polymers were polyethylene and polypropylene. Deleterious health outcomes may be related to the heterogeneous characteristics of these contaminants in the respiratory system following inhalation.

AGR3 Regulates Airway Epithelial Junctions in Patients with Frequent Exacerbations of COPD.

Background: The mechanisms underlying differences in the susceptibility to chronic obstructive pulmonary disease (COPD) exacerbations between patients are not well understood. Recent studies have shown that the patients with frequent COPD exacerbations is related to specific protein expression in lung tissue. Anterior gradient 3 (AGR3) is expressed in airway epithelial cells in the lung and proteomic analysis revealed that its expression is decreased in patients with frequent COPD exacerbations. Moreover, the loss of epithelial integrity might facilitate trans-epithelial permeability of pathogens in such patients. This study was performed to determine that AGR3 protein play a role in COPD frequency exacerbators. Methods: Human lung tissues were collected from current-smoking patients (Control; n = 15) as well as patients with infrequent COPD exacerbations (IFCOPD; n = 18) and frequent COPD exacerbations (FCOPD; n = 8). While AGR3 protein expression was measured by immunohistochemistry and western blotting, AGR mRNA expression was determined by real time quantitative polymerase chain reaction (RT-qPCR). Furthermore, adherent junctions (AJs) and tight junctions (TJs) protein expression in human lung tissues were measured by immunohistochemistry. The effects of cigarette smoke extract (CSE) on AJ and TJ protein and mRNA expression in BEAS-2B cells were assessed by western blotting and RT-qPCR. In addition, the effect of AGR3 overexpression and knockdown on AJ and TJ protein expression was determined. Results: AGR3 was mainly expressed in the airway epithelium and AGR3-positive products were localized in the cytoplasm. Western blotting and RT-qPCR results showed that AGR3 protein (p = 0.009) and mRNA (p = 0.04) expression in the FCOPD group was significantly lower than that in the IFCOPD group. Moreover, E-cadherin, occludin, and zonula occludens-1 (ZO-1) expression was lower in the FCOPD group than in the IFCOPD group. The protein and mRNA expression of E-cadherin, occludin, and ZO-1 was decreased within 24 h post-CSE exposure. AGR3 overexpression rescued CSE-induced downregulation of E-cadherin, occludin, and ZO-1. Conclusion: Difference in AGR3 expression in the lung tissue might be correlated with increased susceptibility to COPD exacerbation. AGR3 can prevent CSE-induced downregulation of E-cadherin, occludin, and ZO-1 in airway epithelial cells. Loss of AGR3 might promote viral and bacterial infection and induce immune inflammation to increase COPD exacerbation.

Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.

ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.

De novo synthesis of serine and glycine fuels purine nucleotide biosynthesis in human lung cancer tissues.

Nucleotide synthesis is essential to proliferating cells, but the preferred precursors for de novo biosynthesis are not defined in human cancer tissues. We have employed multiplexed stable isotope-resolved metabolomics to track the metabolism of [13C6]glucose, D2-glycine, [13C2]glycine, and D3-serine into purine nucleotides in freshly resected cancerous and matched noncancerous lung tissues from nonsmall cell lung cancer (NSCLC) patients, and we compared the metabolism with established NSCLC PC9 and A549 cell lines in vitro Surprisingly, [13C6]glucose was the best carbon source for purine synthesis in human NSCLC tissues, in contrast to the noncancerous lung tissues from the same patient, which showed lower mitotic indices and MYC expression. We also observed that D3-Ser was preferentially incorporated into purine rings over D2-glycine in both tissues and cell lines. MYC suppression attenuated [13C6]glucose, D3-serine, and [13C2]glycine incorporation into purines and reduced proliferation in PC9 but not in A549 cells. Using detailed kinetic modeling, we showed that the preferred use of glucose as a carbon source for purine ring synthesis in NSCLC tissues involves cytoplasmic activation/compartmentation of the glucose-to-serine pathway and enhanced reversed one-carbon fluxes that attenuate exogenous serine incorporation into purines. Our findings also indicate that the substrate for de novo nucleotide synthesis differs profoundly between cancer cell lines and fresh human lung cancer tissues; the latter preferred glucose to exogenous serine or glycine but not the former. This distinction in substrate utilization in purine synthesis in human cancer tissues should be considered when targeting one-carbon metabolism for cancer therapy.

Establishment of a biobank for human lung tissues of congenital diaphragmatic hernia and congenital pulmonary airway malformation.

BACKGROUND: Human tissue samples are an invaluable and little available source of information for translational studies of congenital lung diseases such as Congenital Diaphragmatic Hernia (CDH) or Congenital Pulmonary Airway Malformation (CPAM). PURPOSE: We aimed to establish a human lung tissue biobank of CDH and CPAM patients together with age-matched controls, coupled with a clinical database. METHODS: Pathology records from autopsies or surgical specimens for CDH and CPAM cases between 1980 and 2017 were reviewed. For surviving individuals, clinical patient data was obtained from corresponding pediatric surgery reports. Formalin-fixed, paraffin-embedded tissues of patients and age-matched controls were systematically stored for further translational studies. RNA integrity was determined on selected CDH blocks. RESULTS: A total of 16 CDH and 18 CPAM and age-matched control lung tissue blocks were included in our biobank. Ages ranged from 22 to 41 weeks of gestation (GA) in CDH (33.9 ±â€¯6.35 weeks) and 26 weeks (GA) and 12 years in CPAM (2.3 ±â€¯3.7 y). RNA isolation from CDH and control blocks yielded good RNA quality (OD 260/280 ratio: 2.01-2.09, OD 260/230 ratio: 2.04-2.09). CONCLUSION: We established a unique human biobank for CDH and CPAM tissues. The combination with clinical patient data will allow us to design future translational studies to improve our understanding of the disease pathogenesis of these congenital malformations.

Identification of proteomic signatures associated with COPD frequent exacerbators.

AIMS: Acute exacerbation is a major event that alters the natural course of chronic obstructive pulmonary disease (COPD), and recurrent exacerbation results in worse clinical outcomes and greater economic consequences. While some patients suffer frequent exacerbations, others experience no exacerbations; this study was designed to detect proteins that were differentially abundant in COPD frequent exacerbators and assess whether those expression profiles are unique among COPD patients. MAIN METHODS: Tandem mass tag labeled quantitative proteomics combined with two-dimensional liquid chromatography-tandem mass spectrometry was used to detect the changes in the lung proteome in COPD frequent exacerbators and infrequent exacerbators. A series of bioinformatics analyses were performed to screen potential signatures of COPD frequent exacerbations. The accuracy of proteomic results was further verified by western blot studies. KEY FINDINGS: Compared with infrequent exacerbators, 23 proteins in the lung tissues from frequent exacerbators showed significant degrees of differential expression; combined bioinformatics analyses of proteome indicated that the immune network for IgA production and the phenylalanine metabolism pathway were associated with frequent exacerbations. The Western blot analysis confirmed the expression pattern of three significantly regulated proteins (HLA-DQA1, pIgR and biglycan). SIGNIFICANCE: These findings indicate that immune response might play a key role in the pathophysiological mechanisms of COPD frequent exacerbations. Our results make a crucial contribution to the search for a comprehensive understanding of potential pathophysiological mechanisms associated with the frequent exacerbations of COPD, and might provide guidance for treating frequent exacerbations.

Peptidyl-Prolyl-cis/trans-Isomerases Mip and PpiB of Legionella pneumophila Contribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection.

The gammaproteobacterium Legionella pneumophila is the causative agent of Legionnaires' disease, an atypical pneumonia that manifests itself with severe lung damage. L. pneumophila, a common inhabitant of freshwater environments, replicates in free-living amoebae and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts, including protozoa and humans. Peptidyl-prolyl-cis/trans-isomerases (PPIases) are multifunctional proteins that are mainly involved in protein folding and secretion in bacteria. In L. pneumophila the surface-associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant-dependent sliding motility and adaptation to suboptimal temperatures, features that contribute to the environmental fitness of L. pneumophila Furthermore, they contribute to infection of the natural host Acanthamoeba castellanii as well as human macrophages and human explanted lung tissue. These effects were additive in the case of sliding motility or synergistic in the case of temperature tolerance and infection, as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators of L. pneumophila with compensatory action and pleiotropic effects.

Expression of MATE1, P-gp, OCTN1 and OCTN2, in epithelial and immune cells in the lung of COPD and healthy individuals.

BACKGROUND: Several inhaled drugs are dependent on organic cation transporters to cross cell membranes. To further evaluate their potential to impact on inhaled drug disposition, the localization of MATE1, P-gp, OCTN1 and OCTN2 were investigated in human lung. METHODS: Transporter proteins were analysed by immunohistochemistry in lung tissue from healthy subjects and COPD patients. Transporter mRNA was analysed by qPCR in lung tissue and in bronchoalveolar lavage (BAL) cells from smokers and non-smokers. RESULTS: We demonstrate for the first time MATE1 protein expression in the lung with localization to the apical side of bronchial and bronchiolar epithelial cells. Interestingly, MATE1 was strongly expressed in alveolar macrophages as demonstrated both in lung tissue and in BAL cells, and in inflammatory cells including CD3 positive T cells. P-gp, OCTN1 and OCTN2 were also expressed in the alveolar epithelial cells and in inflammatory cells including alveolar macrophages. In BAL cells from smokers, MATE1 and P-gp mRNA expression was significantly lower compared to cells from non-smokers whereas no difference was observed between COPD patients and healthy subjects. THP-1 cells were evaluated as a model for alveolar macrophages but did not reflect the transporter expression observed in BAL cells. CONCLUSIONS: We conclude that MATE1, P-gp, OCTN1 and OCTN2 are expressed in pulmonary lung epithelium, in alveolar macrophages and in other inflammatory cells. This is important to consider in the development of drugs treating pulmonary disease as the transporters may impact drug disposition in the lung and consequently affect pharmacological efficacy and toxicity.

Human Pulmonary 3D Models For Translational Research.

Lung diseases belong to the major causes of death worldwide. Recent innovative methodological developments now allow more and more for the use of primary human tissue and cells to model such diseases. In this regard, the review covers bronchial air-liquid interface cultures, precision cut lung slices as well as ex vivo cultures of explanted peripheral lung tissue and de-/re-cellularization models. Diseases such as asthma or infections are discussed and an outlook on further areas for development is given. Overall, the progress in ex vivo modeling by using primary human material could make translational research activities more efficient by simultaneously fostering the mechanistic understanding of human lung diseases while reducing animal usage in biomedical research.

PilY1 Promotes Legionella pneumophila Infection of Human Lung Tissue Explants and Contributes to Bacterial Adhesion, Host Cell Invasion, and Twitching Motility.

Legionnaires' disease is an acute fibrinopurulent pneumonia. During infection Legionella pneumophila adheres to the alveolar lining and replicates intracellularly within recruited macrophages. Here we provide a sequence and domain composition analysis of the L. pneumophila PilY1 protein, which has a high homology to PilY1 of Pseudomonas aeruginosa. PilY1 proteins of both pathogens contain a von Willebrand factor A (vWFa) and a C-terminal PilY domain. Using cellular fractionation, we assigned the L. pneumophila PilY1 as an outer membrane protein that is only expressed during the transmissive stationary growth phase. PilY1 contributes to infection of human lung tissue explants (HLTEs). A detailed analysis using THP-1 macrophages and A549 lung epithelial cells revealed that this contribution is due to multiple effects depending on host cell type. Deletion of PilY1 resulted in a lower replication rate in THP-1 macrophages but not in A549 cells. Further on, adhesion to THP-1 macrophages and A549 epithelial cells was decreased. Additionally, the invasion into non-phagocytic A549 epithelial cells was drastically reduced when PilY1 was absent. Complementation variants of a PilY1-negative mutant revealed that the C-terminal PilY domain is essential for restoring the wild type phenotype in adhesion, while the putatively mechanosensitive vWFa domain facilitates invasion into non-phagocytic cells. Since PilY1 also promotes twitching motility of L. pneumophila, we discuss the putative contribution of this newly described virulence factor for bacterial dissemination within infected lung tissue.

IL-17B is elevated in patients with pneumonia and mediates IL-8 production in bronchial epithelial cells.

The role of IL-17B in regulating pulmonary immunity and inflammation is unknown. In this study, we found that IL-17B concentrations were significantly elevated in adult and paediatric patients with community-acquired pneumonia relative to their corresponding healthy adult and paediatric controls. The increased concentrations of IL-17B significantly and positively correlated with chemokine IL-8 concentrations in clinical pneumonia. In vitro studies demonstrated that IL-17B could induce gene and protein expression of IL-8 in human bronchial epithelial cells, but not lung fibroblasts, which was regulated by the activation of Akt, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and nuclear factor-kappaB (NF-κB) signaling pathways. In vivo studies further showed that increased IL-17B levels significantly and positively correlated with IL-8 concentrations in experimental pneumonia. In conclusion, human pneumonia was associated with enhanced release of IL-17B, which might regulate pulmonary immunity and inflammation through the induction of IL-8 in bronchial epithelial cells.

Gene expression analysis of membrane transporters and drug-metabolizing enzymes in the lung of healthy and COPD subjects.

This study describes for the first time the expression levels of genes encoding membrane transporters and drug-metabolizing enzymes in the lungs of ex-smoking patients with chronic obstructive pulmonary disease (COPD). Membrane transporters and drug-metabolizing enzymes are key determinants of drug uptake, metabolism, and elimination for systemically administered as well as inhaled drugs, with consequent influence on clinical efficacy and patient safety. In this study, while no difference in gene expression was found between healthy and COPD subjects, we identified a significant regional difference in mRNA expression of both membrane transporters and drug-metabolizing enzymes between central and peripheral tissue in both healthy and COPD subjects. The majority of the differentially expressed genes were higher expressed in the central airways such as the transporters SLC2A1 (GLUT1), SLC28A3 (CNT3), and SLC22A4 (OCTN1) and the drug-metabolizing enzymes GSTZ1, GSTO2, and CYP2F1. Together, this increased knowledge of local pharmacokinetics in diseased and normal lung may improve modeling of clinical outcomes of new chemical entities intended for inhalation therapy delivered to COPD patients. In addition, based on the similarities between COPD and healthy subjects regarding gene expression of membrane transporters and drug-metabolizing enzymes, our results suggest that clinical pharmacological studies in healthy volunteers could be a valid model of COPD patients regarding drug disposition of inhaled drugs in terms of drug metabolism and drug transporters.