Human Precision-cut Lung Slices Research Articles

Electronic Cigarette Exposure Increases the Severity of Influenza a Virus Infection via TRAIL Dysregulation in Human Precision-Cut Lung Slices.

The use of electronic nicotine dispensing systems (ENDS), also known as electronic cigarettes (ECs), is common among adolescents and young adults with limited knowledge about the detrimental effects on lung health such as respiratory viral infections and underlying mechanisms. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a protein of the TNF family involved in cell apoptosis, is upregulated in COPD patients and during influenza A virus (IAV) infections, but its role in viral infection during EC exposures remains unclear. This study was aimed to investigate the effect of ECs on viral infection and TRAIL release in a human lung precision-cut lung slices (PCLS) model, and the role of TRAIL in regulating IAV infection. PCLS prepared from lungs of nonsmoker healthy human donors were exposed to EC juice (E-juice) and IAV for up to 3 days during which viral load, TRAIL, lactate dehydrogenase (LDH), and TNF-α in the tissue and supernatants were determined. TRAIL neutralizing antibody and recombinant TRAIL were utilized to determine the contribution of TRAIL to viral infection during EC exposures. E-juice increased viral load, TRAIL, TNF-α release and cytotoxicity in IAV-infected PCLS. TRAIL neutralizing antibody increased tissue viral load but reduced viral release into supernatants. Conversely, recombinant TRAIL decreased tissue viral load but increased viral release into supernatants. Further, recombinant TRAIL enhanced the expression of interferon-β and interferon-λ induced by E-juice exposure in IAV-infected PCLS. Our results suggest that EC exposure in human distal lungs amplifies viral infection and TRAIL release, and that TRAIL may serve as a mechanism to regulate viral infection. Appropriate levels of TRAIL may be important to control IAV infection in EC users.

Cell Type-Specific Anti-Viral Effects of Novel SARS-CoV-2 Main Protease Inhibitors.

Recently, we have described novel pyridyl indole esters and peptidomimetics as potent inhibitors of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) main protease. Here, we analysed the impact of these compounds on viral replication. It has been shown that some antivirals against SARS-CoV-2 act in a cell line-specific way. Thus, the compounds were tested in Vero, Huh-7, and Calu-3 cells. We showed that the protease inhibitors at 30 µM suppress viral replication by up to 5 orders of magnitude in Huh-7 cells, while in Calu-3 cells, suppression by 2 orders of magnitude was achieved. Three pyridin-3-yl indole-carboxylates inhibited viral replication in all cell lines, indicating that they might repress viral replication in human tissue as well. Thus, we investigated three compounds in human precision-cut lung slices and observed donor-dependent antiviral activity in this patient-near system. Our results provide evidence that even direct-acting antivirals may act in a cell line-specific manner.

Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.

GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma.

A Par3/LIM Kinase/Cofilin Pathway Mediates Human Airway Smooth Muscle Relaxation by TAS2R14.

TAS2Rs (bitter taste receptors) are GPCRs (G protein-coupled receptors) expressed on human airway smooth muscle (HASM) cells; when activated by receptor agonists they evoke marked airway relaxation. In both taste and HASM cells, TAS2Rs activate a canonical Gβγ-mediated stimulation of Ca2+ release from intracellular stores by activation of PLCβ (phospholipase Cβ). Alone, this [Ca2+]i signaling does not readily account for relaxation, particularly since bronchoconstrictive agonists acting at Gq-coupled receptors also increase [Ca2+]i. We established that TAS2R14 activation in HASM promotes relaxation through F-actin (filamentous actin) severing. This destabilization of actin was from agonist-promoted activation (dephosphorylation) of cofilin, which was pertussis toxin sensitive. Cofilin dephosphorylation was due to TAS2R-mediated deactivation of LIM domain kinase. The link between early receptor action and the distal cofilin dephosphorylation was found to be the polarity protein partitioning defective 3 (Par3), a known binding partner with PLCβ that inhibits LIM kinase. The physiologic relevance of this pathway was assessed using knock-downs of cofilin and Par3 in HASM cells and in human precision-cut lung slices. Relaxation by TAS2R14 agonists was ablated with knock-down of either protein as assessed by magnetic twisting cytometry in isolated cells or intact airways in the slices. Blocking [Ca2+]i release by TAS2R14 inhibited agonist-promoted cofilin dephosphorylation, confirming a role for [Ca2+]i in actin-modifying pathways. These results further elucidate the mechanistic basis of TAS2R-mediated HASM relaxation and point toward nodal points that may act as asthma or chronic obstructive pulmonary disease response modifiers or additional targets for novel bronchodilators.

Cryopreserved human precision-cut lung slices provide an immune competent pulmonary test system for "on-demand" use and long-term cultures.

Human precision-cut lung slices (hPCLS), considered a highly relevant ex vivo model of the lung, offer native architecture and cells of the lung tissue including respiratory parenchyma, small airways, and immune competent cells. However, the irregular availability of donor lungs has limited the accessibility of this system. As described here, thousands of hPCLS can be created from 1 lung, cryopreserved, and used "on demand" by applying slicing and cryopreservation methodology improvements. Fresh and cryopreserved (∼7 and ∼34 weeks; F&C) hPCLS from 1 donor lung were cultured for up to 29 days and evaluated for biomass, viability, tissue integrity, and inflammatory markers in response to lipopolysaccharide (LPS; 5 µg/ml) and Triton X-100 (TX100; 0.1%) challenge (24 h) at days 1, 8, 15, 22, and 29 following culture initiation. The F&C hPCLS retained biomass, viability, and tissue integrity throughout the 29 days and demonstrated immune responsiveness with up to ∼30-fold LPS-induced cytokine increases. Histologically, more than 70% of normal cytomorphological features were preserved in all groups through day 29. Similar retention of tissue viability and immune responsiveness post cryopreservation (4-6 weeks) and culture (up to 14 days) was observed in hPCLS from additional 3 donor lungs. Banking cryopreserved hPCLS from various donors (and disease states) provides a critical element in researching human-derived pulmonary tissue. The retention of viability and functional responsiveness (≥4 weeks) allows evaluation of long-term, complex endpoints reflecting key events in Adverse Outcome Pathways and positions hPCLS as a valuable human-relevant model for use in regulatory applications.

Non-canonical integrin signaling activates EGFR and RAS-MAPK-ERK signaling in small cell lung cancer.

Background: Small cell lung cancer (SCLC) is an extremely aggressive cancer type with a patient median survival of 6-12 months. Epidermal growth factor (EGF) signaling plays an important role in triggering SCLC. In addition, growth factor-dependent signals and alpha-, beta-integrin (ITGA, ITGB) heterodimer receptors functionally cooperate and integrate their signaling pathways. However, the precise role of integrins in EGF receptor (EGFR) activation in SCLC remains elusive. Methods: We analyzed human precision-cut lung slices (hPCLS), retrospectively collected human lung tissue samples and cell lines by classical methods of molecular biology and biochemistry. In addition, we performed RNA-sequencing-based transcriptomic analysis in human lung cancer cells and human lung tissue samples, as well as high-resolution mass spectrometric analysis of the protein cargo from extracellular vesicles (EVs) that were isolated from human lung cancer cells. Results: Our results demonstrate that non-canonical ITGB2 signaling activates EGFR and RAS/MAPK/ERK signaling in SCLC. Further, we identified a novel SCLC gene expression signature consisting of 93 transcripts that were induced by ITGB2, which may be used for stratification of SCLC patients and prognosis prediction of LC patients. We also found a cell-cell communication mechanism based on EVs containing ITGB2, which were secreted by SCLC cells and induced in control human lung tissue RAS/MAPK/ERK signaling and SCLC markers. Conclusions: We uncovered a mechanism of ITGB2-mediated EGFR activation in SCLC that explains EGFR-inhibitor resistance independently of EGFR mutations, suggesting the development of therapies targeting ITGB2 for patients with this extremely aggressive lung cancer type.

A lung targeted miR-29 mimic as a therapy for pulmonary fibrosis.

SummaryBackgroundMicroRNAs are non-coding RNAs that negatively regulate gene networks. Previously, we reported that systemically delivered miR-29 mimic MRG-201 reduced fibrosis in animal models, supporting the consideration of miR-29-based therapies for idiopathic pulmonary fibrosis (IPF).MethodsWe generated MRG-229, a next-generation miR-29 mimic based on MRG-201 with improved chemical stability due to additional sugar modifications and conjugation with the internalization moiety BiPPB (PDGFbetaR-specific bicyclic peptide)1. We investigated the anti-fibrotic efficacy of MRG-229 on TGF-β1 treated human lung fibroblasts (NHLFs), human precision cut lung slices (hPCLS), and in vivo bleomycin studies; toxicology was assessed in two animal models, rats, and non-human primates. Finally, we examined miR-29b levels in a cohort of 46 and 213 patients with IPF diagnosis recruited from Yale and Nottingham Universities (Profile Cohort), respectively.FindingsThe peptide-conjugated MRG-229 mimic decreased expression of pro-fibrotic genes and reduced collagen production in each model. In bleomycin-treated mice, the peptide-conjugated MRG-229 mimic downregulated profibrotic gene programs at doses more than ten-fold lower than the original compound. In rats and non-human primates, the peptide-conjugated MRG-229 mimic was well tolerated at clinically relevant doses with no adverse findings observed. In human peripheral blood from IPF patients decreased miR-29 concentrations were associated with increased mortality in two cohorts potentially identified as a target population for treatment.InterpretationCollectively, our results provide support for the development of the peptide-conjugated MRG-229 mimic as a potential therapy in humans with IPF.FundingThis work was supported by NIH NHLBI grants UH3HL123886, R01HL127349, R01HL141852, U01HL145567.

Starving a Cell Promotes Airway Smooth Muscle Relaxation: Inhibition of Glycolysis Attenuates Excitation-Contraction Coupling.

Bronchomotor tone modulated by airway smooth muscle shortening represents a key mechanism that increases airway resistance in asthma. Altered glucose metabolism in inflammatory and airway structural cells is associated with asthma. Although these observations suggest a causal link between glucose metabolism and airway hyperresponsiveness, the mechanisms are unclear. We hypothesized that glycolysis modulates excitation-contraction coupling in human airway smooth muscle (HASM) cells. Cultured HASM cells from human lung donors were subject to metabolic screenings using Seahorse XF cell assay. HASM cell monolayers were treated with vehicle or PFK15 (1-(Pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one), an inhibitor of PFKFB3 (PFK-1,6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) that generates an allosteric activator for glycolysis rate-limiting enzyme PFK1 (phosphofructokinase 1), for 5-240 minutes, and baseline and agonist-induced phosphorylation of MLC (myosin light chain), MYPT1 (myosin phosphatase regulatory subunit 1), Akt, RhoA, and cytosolic Ca2+ were determined. PFK15 effects on metabolic activity and contractile agonist-induced bronchoconstriction were determined in human precision-cut lung slices. Inhibition of glycolysis attenuated carbachol-induced excitation-contraction coupling in HASM cells. ATP production and bronchodilator-induced cAMP concentrations were also attenuated by glycolysis inhibition in HASM cells. In human small airways, glycolysis inhibition decreased mitochondrial respiration and ATP production and attenuated carbachol-induced bronchoconstriction. The findings suggest that energy depletion resulting from glycolysis inhibition is a novel strategy for ameliorating HASM cell shortening and bronchoprotection of human small airways.

Acetylsalicylic Acid and Salicylic Acid Inhibit SARS-CoV-2 Replication in Precision-Cut Lung Slices.

Aspirin, with its active compound acetylsalicylic acid (ASA), shows antiviral activity against rhino- and influenza viruses at high concentrations. We sought to investigate whether ASA and its metabolite salicylic acid (SA) inhibit SARS-CoV-2 since it might use similar pathways to influenza viruses. The compound-treated cells were infected with SARS-CoV-2. Viral replication was analysed by RTqPCR. The compounds suppressed SARS-CoV-2 replication in cell culture cells and a patient-near replication system using human precision-cut lung slices by two orders of magnitude. While the compounds did not interfere with viral entry, it led to lower viral RNA expression after 24 h, indicating that post-entry pathways were inhibited by the compounds.

Spray drying siRNA-lipid nanoparticles for dry powder pulmonary delivery.

While all the siRNA drugs on the market target the liver, the lungs offer a variety of currently undruggable targets which could potentially be treated with RNA therapeutics. Hence, local, pulmonary delivery of RNA nanoparticles could finally enable delivery beyond the liver. The administration of RNA drugs via dry powder inhalers offers many advantages related to physical, chemical and microbial stability of RNA and nanosuspensions. The present study was therefore designed to test the feasibility of engineering spray dried lipid nanoparticle (LNP) powders. Spray drying was performed using 5% lactose solution (m/V), and the targets were set to obtain nanoparticle sizes after redispersion of spray-dried powders around 150 nm, a residual moisture level below 5%, and RNA loss below 15% at maintained RNA bioactivity. The LNPs consisted of an ionizable cationic lipid which is a sulfur-containing analog of DLin-MC3-DMA, a helper lipid, cholesterol, and PEG-DMG encapsulating siRNA. Prior to the spray drying, the latter process was simulated with a novel dual emission fluorescence spectroscopy method to preselect the highest possible drying temperature and excipient solution maintaining LNP integrity and stability. Through characterization of physicochemical and aerodynamic properties of the spray dried powders, administration criteria for delivery to the lower respiratory tract were fulfilled. Spray dried LNPs penetrated the lung mucus layer and maintained bioactivity for >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Additionally, the spray dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices at without increasing cytokine levels. This study verifies the successful spray drying procedure of LNP-siRNA systems maintaining their integrity and mediating strong gene silencing efficiency on mRNA and protein levels both in vitro and ex vivo. The successful spray drying procedure of LNP-siRNA formulations in 5% lactose solution creates a novel siRNA-based therapy option to target respiratory diseases such as lung cancer, asthma, COPD, cystic fibrosis and viral infections.

FDA-Approved Inhibitors of RTK/Raf Signaling Potently Impair Multiple Steps of In Vitro and Ex Vivo Influenza A Virus Infections.

Influenza virus (IV) infections pose a burden on global public health with significant morbidity and mortality. The limited range of currently licensed IV antiviral drugs is susceptible to the rapid rise of resistant viruses. In contrast, FDA-approved kinase inhibitors can be repurposed as fast-tracked host-targeted antivirals with a higher barrier of resistance. Extending our recent studies, we screened 21 FDA-approved small-molecule kinase inhibitors (SMKIs) and identified seven candidates as potent inhibitors of pandemic and seasonal IV infections. These SMKIs were further validated in a biologically and clinically relevant ex vivo model of human precision-cut lung slices. We identified steps of the virus infection cycle affected by these inhibitors (entry, replication, egress) and found that most SMKIs affected both entry and egress. Based on defined and overlapping targets of these inhibitors, the candidate SMKIs target receptor tyrosine kinase (RTK)-mediated activation of Raf/MEK/ERK pathways to limit influenza A virus infection. Our data and the established safety profiles of these SMKIs support further clinical investigations and repurposing of these SMKIs as host-targeted influenza therapeutics.

Notch1 Induces Defective Epithelial Surfactant Processing and Pulmonary Fibrosis.

Rationale: Although type II alveolar epithelial cells (AEC2s) are chronically injured in idiopathic pulmonary fibrosis (IPF), they contribute to epithelial regeneration in IPF. Objectives: We hypothesized that Notch signaling may contribute to AEC2 proliferation, dedifferentiation characterized by loss of surfactant processing machinery, and lung fibrosis in IPF. Methods: We applied microarray analysis, kinome profiling, flow cytometry, immunofluorescence analysis, western blotting, quantitative PCR, and proliferation and surface activity analysis to study epithelial differentiation, proliferation, and matrix deposition invitro (AEC2 lines, primary murine/human AEC2s), ex vivo (human IPF-derived precision-cut lung slices), and invivo (bleomycin and pepstatin application, Notch1 [Notch receptor 1] intracellular domain overexpression). Measurements and Main Results: We document here extensive SP-B and -C (surfactant protein-B and -C) processing defects in IPF AEC2s, due to loss of Napsin A, resulting in increased intra-alveolar surface tension and alveolar collapse and induction of endoplasmic reticulum stress in AEC2s. In vivo pharmacological inhibition of Napsin A results in the development of AEC2 injury and overt lung fibrosis. We also demonstrate that Notch1 signaling is already activated early in IPF and determines AEC2 fate by inhibiting differentiation (reduced lamellar body compartment, reduced capacity to process hydrophobic SP) and by causing increased epithelial proliferation and development of lung fibrosis, putatively via altered JAK (Janus kinase)/Stat (signal transducer and activator of transcription) signaling in AEC2s. Conversely, inhibition of Notch signaling in IPF-derived precision-cut lung slices improved the surfactant processing capacity of AEC2s and reversed fibrosis. Conclusions: Notch1 is a central regulator of AEC2 fate in IPF. It induces alveolar epithelial proliferation and loss of Napsin A and of surfactant proprotein processing, and it contributes to fibroproliferation.

Competitive fitness of Pseudomonas aeruginosa isolates in human and murine precision-cut lung slices.

Chronic respiratory infections with the gram-negative bacterium Pseudomonas aeruginosa are an important co-morbidity for the quality of life and prognosis of people with cystic fibrosis (CF). Such long-term colonization, sometimes lasting up to several decades, represents a unique opportunity to investigate pathogen adaptation processes to the host. Our studies aimed to resolve if and to what extent the bacterial adaptation to the CF airways influences the fitness of the pathogen to grow and to persist in the lungs. Marker-free competitive fitness experiments of serial P. aeruginosa isolates differentiated by strain-specific SNPs, were performed with murine and human precision cut lung slices (PCLS). Serial P. aeruginosa isolates were selected from six mild and six severe CF patient courses, respectively. MPCLS or hPCLS were inoculated with a mixture of equal numbers of the serial isolates of one course. The temporal change of the composition of the bacterial community during competitive growth was quantified by multi-marker amplicon sequencing. Both ex vivo models displayed a strong separation of fitness traits between mild and severe courses. Whereas the earlier isolates dominated the competition in the severe courses, intermediate and late isolates commonly won the competition in the mild courses. The status of the CF lung disease rather than the bacterial genotype drives the adaptation of P. aeruginosa during chronic CF lung infection. This implies that the disease status of the lung habitat governed the adaptation of P. aeruginosa more strongly than the underlying bacterial clone-type and its genetic repertoire.

Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in Preclinical Models of Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two U.S. Food and Drug Administration-approved antifibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Objectives: Using an in silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. Methods: We investigated the antifibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: 1) invitro in normal human lung fibroblasts; 2) invivo in bleomycin and recombinant Ad-TGF-β (adenovirus transforming growth factor-β) murine models of pulmonary fibrosis; and 3) ex vivo in mice and human precision-cut lung slices from these two murine models as well as patients with IPF and healthy donors. Measurements and Main Results: In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone. Transcriptomic analyses of TGF-β-stimulated normal human lung fibroblasts identified specific gene sets associated with fibrosis, including epithelial-mesenchymal transition, TGF-β, and WNT signaling that was uniquely altered by saracatinib. Transcriptomic analysis of whole-lung extracts from the two animal models of pulmonary fibrosis revealed that saracatinib reverted many fibrogenic pathways, including epithelial-mesenchymal transition, immune responses, and extracellular matrix organization. Amelioration of fibrosis and inflammatory cascades in human precision-cut lung slices confirmed the potential therapeutic efficacy of saracatinib in human lung fibrosis. Conclusions: These studies identify novel Src-dependent fibrogenic pathways and support the study of the therapeutic effectiveness of saracatinib in IPF treatment.

Interleukin-6-dependent epithelial fluidization initiates fibrotic lung remodeling.

Chronic disease results from the failure of tissues to maintain homeostasis. In the lung, coordinated repair of the epithelium is essential for preserving homeostasis. In animal models and human lung disease, airway epithelial cells mobilize in response to lung injury, resulting in the formation of airway-like cysts with persistent loss of functional cell types and parenchymal architecture. Using live-cell imaging of human lung epithelial cultures and mouse precision-cut lung slices, we demonstrated that distal airway epithelia are aberrantly fluidized both after injury and in fibrotic lung disease. Through transcriptomic profiling and pharmacologic stimulation of epithelial cultures, we identified interleukin-6 (IL-6) signaling as a driver of tissue fluidization. This signaling cascade occurred independently of canonical Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling but instead was dependent on a downstream SRC family kinase (SFK)-yes-associated protein (YAP) axis. Airway epithelial-fibroblast cocultures revealed that the fibrotic mesenchyme acts as a source of IL-6 family cytokines, which drive airway fluidization. Inhibition of the IL-6-SFK-YAP cascade was sufficient to prevent fluidization in both in vitro and ex vivo models. Last, we demonstrated a reduction in fibrotic lung remodeling in mice through genetic or pharmacologic targeting of IL-6-related signaling. Together, our findings illustrate the critical role of airway epithelial fluidization in coordinating the balance between homeostatic lung repair and fibrotic airspace remodeling.

Pulmonary Endothelial Activation with COVID‐19: Possible Role of Reactive Oxygen Species

IntroductionRecent research suggests that endothelial activation plays a role in COVID‐19 pathogenesis by promoting a pro‐coagulative and pro‐inflammatory state. However, the mechanism by which the endothelium is activated in COVID‐19 is unclear.ObjectiveTo investigate the mechanism by which COVID‐19 activates the pulmonary endothelium.HypothesisThe pulmonary endothelium generates reactive oxygen species (ROS) upon exposure to the “inflammatory load” of the systemic circulation.MethodsCOVID‐19 was recreated in vitro and ex vivo, by exposing human lung endothelial cells (EC) or donor human lung slices (human precision‐cut lung slices or huPCLS) to medium supplemented with serum from COVID‐19 affected subjects. Sera were acquired from patients with COVID‐19 infection admitted to the Intensive Care Unit of the Hospital at the University of Pennsylvania. ROS (fluorescent dye, CellROX) and intercellular adhesion molecule (ICAM‐1) levels were assessed by fluorescence labeling and imaging.ResultsBoth EC activation (as monitored by ROS production) and pro‐inflammatory phenotype (as assessed by ICAM‐1), were significantly higher with COVID‐19 as compared to normal subjects.ConclusionsThe endothelium is activated with COVID‐19 via ROS production; thus, the ROS produced drive a pro‐inflammatory phenotype by inducing the expression of ICAM‐1, a pivotal marker of endothelium inflammation. As ROS mediates EC activation and inflammation during COVID‐19, ROS blockade could be a therapeutic target in maintaining vascular health.

Nrf2 Activation Protects Against Organic Dust and Hydrogen Sulfide Exposure Induced Epithelial Barrier Loss and K. pneumoniae Invasion.

Agriculture workers report various respiratory symptoms owing to occupational exposure to organic dust (OD) and various gases. Previously, we demonstrated that pre-exposure to hydrogen sulfide (H2S) alters the host response to OD and induces oxidative stress. Nrf2 is a master-regulator of host antioxidant response and exposures to toxicants is known to reduce Nrf2 activity. The OD exposure-induced lung inflammation is known to increase susceptibility to a secondary microbial infection. We tested the hypothesis that repeated exposure to OD or H2S leads to loss of Nrf2, loss of epithelial cell integrity and that activation of Nrf2 rescues this epithelial barrier dysfunction. Primary normal human bronchial epithelial (NHBE) cells or mouse precision cut-lung slices (PCLS) were treated with media, swine confinement facility organic dust extract (ODE) or H2S or ODE+H2S for one or five days. Cells were also pretreated with vehicle control (DMSO) or RTA-408, a Nrf2 activator. Acute exposure to H2S and ODE+H2S altered the cell morphology, decreased the viability as per the MTT assay, and reduced the Nrf2 expression as well as increased the keap1 levels in NHBE cells. Repeated exposure to ODE or H2S or ODE+H2S induced oxidative stress and cytokine production, decreased tight junction protein occludin and cytoskeletal protein ezrin expression, disrupted epithelial integrity and resulted in increased Klebsiella pneumoniae invasion. RTA-408 (pharmacological activator of Nrf2) activated Nrf2 by decreasing keap1 levels and reduced ODE+H2S-induced changes including reversing loss of barrier integrity, inflammatory cytokine production and microbial invasion in PCLS but not in NHBE cell model. We conclude that Nrf2 activation has a partial protective function against ODE and H2S.

Human Precision-Cut Lung Slices: Generation of and Measurement of Contractility and Relaxation of Small Airways.

Lung slices have been used since the mid-1990's to study various aspects of lung biology that include, but are not limited to, mechanisms of airway contraction and relaxation; the pulmonary immune response in the context of inflammatory diseases of the lung like asthma and chronic obstructive pulmonary disease; mast cell-mediated airway contractility and inflammation; modulation of airway cells following pathogen exposure; and consequences of environmental toxicant exposure. Here we describe the generation of human precision-cut lung slices (hPCLS) and measurement of contraction and relaxation of small airways within the slices.

Infection of Human Precision-Cut Lung Slices with the Influenza Virus.

Viral infections are common causes of asthma exacerbations. To model these processes ex vivo, human precision-cut lung slices (PCLSs) can be used. Here we describe the infection of human PCLSs with the human influenza virus. We then provide methods to quantify the virus and reveal its localization within infected PCLSs and study consequences of infection, including cell death and production of cytokines, chemokine, and mucus. We also describe the stimulation of PCLSs with immune mediators such as pro-inflammatory tumor necrosis factor α (TNF-α). These models are useful to investigate mechanisms of virally induced asthma exacerbations and study modes of action and efficacy of antiviral and/or anti-inflammatory drugs.

Functional changes in long-term incubated rat precision-cut lung slices

BackgroundRespiratory diseases represent a global health burden. Because research on therapeutic strategies of airway diseases is essential, the technique of precision-cut lung slices (PCLS) has been developed and widely studied. PCLS are an alternative ex vivo model and have the potential to replace and reduce in vivo animal models. So far, the majority of studies was conducted with short-term cultivated PCLS (≤ 72 h). As there is large interest in research of chronic diseases and chronic toxicity, feasibility of cultivating human PCLS long-term over 2 weeks and recently over 4 weeks was investigated by another research group with successful results. Our aim was to establish a model of long-term cultivated rat PCLS over a period of 29 days.MethodsRat PCLS were cultured for 29 days and analysed regarding viability, histopathology, reactivity and gene expression at different time points during cultivation.ResultsCultivation of rat PCLS over a 29-day time period was successful with sustained viability. Furthermore, the ability of bronchoconstriction was maintained between 13 and 25 days, depending on the mediator. However, reduced relaxation, altered sensitivity and increased respiratory tone were observed. Regarding transcription, alteration in gene expression pattern of the investigated target genes was ascertained during long-term cultivation with mixed results. Furthermore, the preparation of PCLS seems to influence messenger ribonucleic acid (mRNA) expression of most target genes. Moreover, the addition of fetal bovine serum (FBS) to the culture medium did not improve viability of PCLS. In contrast to medium without FBS, FBS seems to affect measurements and resulted in marked cellular changes of metaplastic and/or regenerative origin.ConclusionsOverall, a model of long-term cultivated rat PCLS which stays viable for 29 days and reactive for at least 13 days could be established. Before long-term cultivated PCLS can be used for in-depth study of chronic diseases and chronic toxicity, further investigations have to be made.