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Rodent models of lipopolysaccharide (LPS)–induced pulmonary inflammation are used for anti-inflammatory drug testing. We aimed to characterize mice responses to aerosolized LPS alone or with intraperitoneal (i.p.) delivery of alpha1-antitrypsin (AAT). Balb/c mice were exposed to clean air or aerosolized LPS (0.21 mg/mL) for 10 min per day, for 3 d. One hour after each challenge, animals were treated i.p. with saline or with (4 mg/kg body weight) one of the AAT preparations: native (AAT), oxidized (oxAAT), recombinant (recAAT), or peptide of AAT (C-36). Experiments were terminated 6 h after the last dose of AATs. Transcriptome data of mice lungs exposed to clean air versus LPS revealed 656 differentially expressed genes and 155 significant gene ontology terms, including neutrophil migration and toll-like receptor signaling pathways. Concordantly, mice inhaling LPS showed higher bronchoalveolar lavage fluid neutrophil counts and levels of myeloperoxidase, inducible nitric oxide synthase, IL-1β, TNFα, KC, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Plasma inflammatory markers did not increase. After i.p. application of AATs, about 1% to 2% of proteins reached the lungs but, except for GM-CSF, none of the proteins significantly influenced inflammatory markers. All AATs and C-36 significantly inhibited LPS-induced GM-CSF release. Surprisingly, only oxAAT decreased the expression of several LPS-induced inflammatory genes, such as Cxcl3, Cd14, Il1b, Nfkb1, and Nfkb2, in lung tissues. According to lung transcriptome data, oxAAT mostly affected genes related to transcriptional regulation while native AAT or recAAT affected genes of inflammatory pathways. Hence, we present a feasible mice model of local lung inflammation induced via aerosolized LPS that can be useful for systemic drug testing.

BackgroundThe coordinated transcriptional regulation of activated T-cells is based on the complex dynamic behavior of signaling networks. Given an external stimulus, T-cell gene expression is characterized by impulse and sustained patterns over the course. Here, we analyzed the temporal pattern of activation across different T-cell populations to develop consensus gene signatures for T-cell activation.MethodsWe applied a meta-analysis of anti-CD3/CD28 induced CD4+ T-cell activation kinetics of publicly available transcriptomewide time series using a random effects model. We used non-negative matrix factorization, an unsupervised deconvolution method, to infer changes in biological patterns over time. For verification and to further map a wider variety of the T-cell landscape, we performed a time series of transcriptome-wide RNA sequencing on activated blood T-cells. Lastly, we matched the identified consensus biomarker signatures to single-cell RNA sequencing (scRNA-Seq) data of autologous anti-CD19 chimeric antigen receptor (CAR) T-cells from 24 patients with large B cell lymphoma (LBCL) to characterize activation status of the cell product before infusion.ResultsWe identified time-resolved gene expression profiles comprising 521 genes of up to 10 disjunct time points during activation and different polarization conditions. The gene signatures include central transcriptional regulators of T-cell activation, representing successive waves as well as sustained patterns of induction. They cover early, intermediate, and late response expression rates across multiple T-cell populations, thus defining consensus biomarker signatures for T-cell activation. Intermediate and late response activation signatures in CAR T-cell infusion products were correlated to immune effector cell-associated neurotoxicity syndrome.ConclusionIn conclusion, we describe temporally resolved gene expression patterns across T-cell populations. These biomarker signatures are a valuable source for e.g., monitoring transcriptional changes during T-cell activation with a reasonable number of genes, annotating T-cell states in single-cell transcriptome studies or assessing dysregulated functions of human T-cell immunity.

We address the need for modelling and predicting adverse outcomes in immunotoxicology to improve non-clinical assessments of immunomodulatory therapy safety and efficacy. The integrated approach includes, first, the adverse outcome pathway concept established in the toxicology field, and, second, the systems medicine disease map approach for describing molecular mechanisms involved in a particular pathology. The proposed systems immunotoxicology workflow is demonstrated with CAR T cell treatment as a use case. To this end, the linear adverse outcome pathway (AOP) is expanded into a molecular interaction model in standard systems biology formats. Then it is shown how knowledge related to immunotoxic events can be integrated, encoded, managed and explored to benefit the research community. The map is accessible online via the MINERVA Platform for browsing, commenting and data visualisation (https://minerva.pages.uni.lu). Our work transforms a graphical illustration of an AOP into a digitally structured and standardised form, featuring precise and controlled vocabulary and supporting reproducible computational analyses. Because of annotations to source literature and databases, the map can be further expanded to match the evolving knowledge and research questions.

The American journal of pathology 192(2), 239-253 (2022). doi:10.1016/j.ajpath.2021.10.020, Published by Elsevier, New York [u.a.]

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.

Current in vitro and in vivo disease models have been reported to lack sufficient translation to human. Precision-Cut Lung Slices (PCLS) are viable sections of lung tissue and have been described to be a translational model for the ex vivo assessment of pharmacological and toxicological compounds. In most studies PCLS were cultured under static conditions. These lung sections, however, suffer from the limited viability. Here we present a novel modular microphysiological system (MPS) to prolong the cultivation of ex vivo lung tissue. A tailored MPS setup was designed using the PDMS free modular plug&play MPS construction kit. PCLS from mice were cultivated for up to one week under static versus perfused conditions. Using the MPS technology enabled a prolonged culture period with improved viability as shown by lowered lactate dehydrogenase release and improved membrane integrity. Using this technology might allow us to use PCLS for longer culture periods such as e.g. repeated dose toxicity or pharmacology studies.

Background: Food allergy affects up to 8% of the pediatric population. Despite ongoing efforts, treatment options remain limited. Novel models of food allergy are needed to study response patterns downstream of IgE-crosslinking and evaluate drugs modifying acute events. Here, we report a novel human ex vivo model that displays acute, allergen-specific, IgE-mediated smooth muscle contractions using precision cut intestinal slices (PCIS). Methods: PCIS were generated using gut tissue samples from children who underwent clinically indicated surgery. Viability and metabolic activity were assessed from 0-24h. Distribution of relevant cell subsets was confirmed using single cell nuclear sequencing. PCIS were passively sensitized using plasma from peanut allergic donors or peanut-sensitized non-allergic donors, and exposed to various stimuli including serotonin, histamine, FcɛRI-crosslinker and food allergens. Smooth muscle contractions and mediator release functioned as readouts. A novel program designed to measure contractions was developed to quantify responses. The ability to demonstrate the impact of antihistamines and immunomodulation from peanut oral immunotherapy (OIT) was assessed. Results: PCIS viability was maintained for 24h. Cellular distribution confirmed the presence of key cell subsets including mast cells. The video analysis tool reliably quantified responses to different stimulatory conditions. Smooth muscle contractions were allergen-specific and reflected the clinical phenotype of the plasma donor. Tryptase measurement confirmed IgE-dependent mast cell-derived mediator release. Antihistamines suppressed histamine-induced contraction and plasma from successful peanut OIT suppressed peanut-specific PCIS contraction. Conclusion: PCIS represent a novel human tissue-based model to study acute, IgE-mediated food allergy and pharmaceutical impacts on allergic responses in the gut.

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.

Metals and metal compounds are essential in daily life. Besides particulate materials nanofibrous material (nanowires) gain importance in innovative processes like optoelectronics. Toxicological data to assess the hazard is often available only for granular metals while data on nanowires is sparse. The project MetalSafety funded by the German BMBF aims at the development of an in vitro model to allow the grouping of different metal compounds with different bioavailability according to their toxicological hazard. Ex vivo and in vivo investigations are used to analyse the predictivity of the in vitro results and the underlying mode-of-action and dose-response relationship. Within this contribution the study concept is introduced. Silver and copper, both in particulate and nanofibrous form, are used across all examinations in vitro, ex vivo and in vivo. To allow an interspecies comparison cell lines derived from human and rat lung are used. Exposure to the materials can be investigated either submerse or with air-liquid-interface (ALI) conditions. The correlation between both species can also be examined ex vivo using precision-cut lung slices (PCLS) derived from human and rat lung tissue. Application in vivo by intratracheal instillation in rats is conducted to enable a validation of the in vitro and ex vivo screening models in a best case scenario under physiological conditions. Comparable endpoints in vitro, ex vivo as well as in vivo are measured during the study. This includes cytotoxicity endpoints and cytokine measurements as well as gene expression analyses. The lung tissue is also subjected to a histopathological examination.

In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. ‘host shutoff factor’) as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.

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.

Cell & tissue research 387(2), 249-260 (2022). doi:10.1007/s00441-021-03553-1, Published by Springer, Berlin ; Heidelberg

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.

Respiratory research 23, 201 (2022). doi:10.1186/s12931-022-02116-4, Published by BioMed Central, London

Human rhinovirus (RV) is a major risk factor for chronic obstructive pulmonary disease (COPD) and asthma exacerbations. The exploration of RV pathogenesis has been hampered by a lack of disease-relevant model systems. We performed a detailed characterization of host responses to RV infection in human lung tissue ex vivo and investigated whether these responses are disease relevant for patients with COPD and asthma. In addition, impact of the viral replication inhibitor rupintrivir was evaluated. Human precision-cut lung slices (PCLS) were infected with RV1B with or without rupintrivir. At Days 1 and 3 after infection, RV tissue localization, tissue viability, and viral load were determined. To characterize host responses to infection, mediator and whole genome analyses were performed. RV successfully replicated in PCLS airway epithelial cells and induced both antiviral and proinflammatory cytokines such as IFNa2a, CXCL10, CXCL11, IFN-g, TNFa, and CCL5. Genomic analyses revealed that RV not only induced antiviral immune responses but also triggered changes in epithelial cell-associated pathways. Strikingly, the RV response in PCLS was reflective of gene expression changes described in patients with COPD and asthma. Although RV-induced host immune responses were abrogated by rupintrivir, RV-triggered epithelial processes were largely refractory to antiviral treatment. Detailed analysis of RV-infected human PCLS and comparison with gene signatures of patients with COPD and asthma revealed that the human RV PCLS model represents disease-relevant biological mechanisms that can be partially inhibited by a well-known antiviral compound and provide an outstanding opportunity to evaluate novel therapeutics.

ObjectiveHuman precision cut lung slices (PCLS) are widely used as an ex vivo model system for drug discovery and development of new therapies. PCLS reflect the functional heterogeneity of lung tissue and possess relevant lung cell types. We thus determined the use of PCLS in studying non-coding RNAs notably miRNAs, which are important gene regulatory molecules. Since miRNAs play key role as mediators of respiratory diseases, they can serve as valuable prognostic or diagnostic biomarkers, and in therapeutic interventions, of lung diseases. A technical limitation though is the vast amount of agarose in PCLS which impedes (mi)RNA extraction by standard procedures. Here we modified our recently published protocol for RNA 29 isolation from PCLS to enable miRNA readouts. Results The modified method relies on the separation of lysis and precipitation steps, and a clean-up procedure with specific magnetic beads. We obtained successfully quality miRNA amenable for downstream applications such as RTqPCR and whole transcriptome miRNA analysis. Comparison of miRNA profiles in PCLS with published data from human lung, identified all important miRNAs regulated in IPF, COPD, asthma or lung cancer. Therefore, this shows suitability of the method for analyzing miRNA targets and biomarkers in the valuable human 38 PCLS model.

Introduction: COL4A3 degradation is significantly increased in severe, exacerbating type 2 asthmatics and correlates with increased chymase positive mast cells (mouse model of asthma). However, whether chymase is sufficient/necessary for COL4A3 degradation and if this mode of action is responsive to therapeutic intervention, is unknown. We hypothesized, that mast cell chymase is necessary for COL4A3 degradation and that treatment with anti-IgE reduces COL4A3 degradation. Methods: The COL4A3 fibrolysis marker C4Ma3 (Nordic Bioscience, Denmark) was measured in female 6-8 week old Balb/C mice, which were sensitized (OVA i.p., day 1, 14, 21) and challenged with aerosol (OVA i.n., day 26, 27, 28) to induce acute allergic airway inflammation. Acute exacerbations were provoked by instillation of poly(cytidylic-inosinic, i.n. day 28) and Chymase was inhibited with Fulacimstat (i.t.). Allergic, uncontrolled asthmatics (n=19, Omalizumab treatment), were monitored over six months. Treatment response was set as Asthma Control Test (ACT) improvement of g3 points. Serum levels of C4Ma3 were determined at baseline, 3 and 6 months. Results: Chymase inhibition prevented OVA-challenge induced C4Ma3 increase. Recombinant chymase alone did not induce elevated C4Ma3 level. Ten out of 19 patients had ACT score increases of g3 points after treatment. Responders mean level of circulating C4Ma3 was 8.87 ng/mL (confidence interval (CI): 7.02-10.72ng/mL), which decreased to 6.57 ng/mL (CI: 4.95-8.1ng/mL, pl0.01) after six months treatment. Non-responders did not show any change in serum levels of after treatment. Conclusion: COL4A3 degradation depends on mast cell chymase in vivo and monitoring serum C4Ma3, may afford a novel avenue to stratify and monitor anti-IgE therapy in severe, allergic asthma.

Introduction: Remodeling of the airway wall is a hallmark feature of asthma. Under physiological conditions, a finely tuned balance maintains a functional state of the extracellular matrix. This balance is disrupted in asthma. COL4A3 is reduced 18-fold in lung tissue from asthmatics, however, the mechanism leading to the diminished levels of COL4A3 has remained elusive. Methods: The COL4A3 fibrolysis marker C4Ma3 (Nordic Bioscience, Denmark) was measured in the ALLIANCE cohort study participants (Fuchs et al. 2018). Pediatric controls/cases: 34/134; Adult control/cases 31/149. C4Ma3 data was correlated with clinical data, serum cytokine profiles (27-Bioplex, Biorad). Female 6-8 week old Balb/C mice were sensitized (OVA i.p., day 1, 14, 21) and challenged with aerosol (OVA i.n., day 26, 27, 28) to induce acute allergic airway inflammation. Acute exacerbations were provoked by instillation of poly(cytidylic-inosinic, i.n. day 28). Murine precision cut lung slices (PCLS) were used to elucidate the role of sensitisation for COL4A3 degradation. Results: Increased C4Ma3 correlated with asthma exacerbation (pl0.01) and elevated levels of Th2/Th9 cytokines in the ALLIANCE cohort. Serum levels of C4Ma3 were elevated in OVA challenged mice (pl0.0001) and correlated with lung tissue levels of mast cell chymase (pl0.01), but not with neutrophils or matrix-metallo-proteinase 9. C4MA3 was elevated in PCLS of allergic, HDM challenged mice (pl0.05) with no further increase after PolyIC. Conclusion: COL4A3 degradation (circulating C4Ma3), is elevated in severe, exacerbating type 2 asthmatics. C4Ma3 levels correlate with chymase positive mast cells in a mouse model of asthma exacerbation. These data highlight that the degradation of COL4A3 is a central part of the pathology of asthma.

Objective Human precision cut lung slices (PCLS) are widely used as an ex vivo model system for drug discovery and development of new therapies. PCLS reflect the functional heterogeneity of lung tissue and possess relevant lung cell types. We thus determined the use of PCLS in studying non-coding RNAs notably miRNAs, which are important gene regulatory molecules. Since miRNAs play key role as mediators of respiratory diseases, they can serve as valuable prognostic or diagnostic biomarkers, and in therapeutic interventions, of lung diseases. A technical limitation though is the vast amount of agarose in PCLS which impedes (mi)RNA extraction by standard procedures. Here we modified our recently published protocol for RNA isolation from PCLS to enable miRNA readouts. Results The modified method relies on the separation of lysis and precipitation steps, and a clean-up procedure with specific magnetic beads. We obtained successfully quality miRNA amenable for downstream applications such as RTqPCR and whole transcriptome miRNA analysis. Comparison of miRNA profiles in PCLS with published data from human lung, identified all important miRNAs regulated in IPF, COPD, asthma or lung cancer. Therefore, this shows suitability of the method for analyzing miRNA targets and biomarkers in the valuable human PCLS model.

BackgroundAsthma is a heterogeneous syndrome substantiating the urgent requirement for endotype-specific biomarkers. Dysbalance of fibrosis and fibrolysis in asthmatic lung tissue leads to reduced levels of the inflammation-protective collagen 4 (COL4A3).ObjectiveTo delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response.MethodsThe serological COL4A3 degradation marker C4Ma3 (Nordic Bioscience, Denmark) and serum cytokines were measured in the ALLIANCE cohort (paediatric cases/controls: n=134/n=35; adult cases/controls: n=149/n=31). Exacerbation of allergic airway disease in mice was induced by sensitising to ovalbumin (OVA), challenge with OVA aerosol and instillation of poly(cytidylic-inosinic). Fulacimstat (chymase inhibitor; Bayer) was used to determine the role of mast cell chymase in COL4A3 degradation. Patients with cystic fibrosis (n=14) and cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA; n=9) as well as patients with severe allergic uncontrolled asthma (n=19) were tested for COL4A3 degradation. Omalizumab (anti-IgE) treatment was assessed using the Asthma Control Test.ResultsSerum levels of C4Ma3 were increased in asthma in adults and children alike and linked to a more severe, exacerbating allergic asthma phenotype. In an experimental asthma mouse model, C4Ma3 was dependent on mast cell chymase. Serum C4Ma3 was significantly elevated in cystic fibrosis plus ABPA and at baseline predicted the success of the anti-IgE therapy in allergic, uncontrolled asthmatics (diagnostic OR 31.5).ConclusionC4Ma3 levels depend on lung mast cell chymase and are increased in a severe, exacerbating allergic asthma phenotype. C4Ma3 may serve as a novel biomarker to predict anti-IgE therapy response.

Smokers with apparently "healthy" lungs suffer from more severe and frequent viral respiratory infections, but the mechanisms underlying this observation are still unclear. Epithelial cells and dendritic cells (DC) form the first line of defense against inhaled noxes such as smoke or viruses. We therefore aimed to obtain insight into how cigarette smoke affects DCs and epithelial cells and how this influences the response to viral infection. Female C57BL/6J mice were exposed to cigarette smoke (CS) for 1 h daily for 24 days and then challenged i.n. with the viral mimic and Toll-like receptor 3 (TLR3) ligand poly (I:C) after the last exposure. DC subpopulations were analyzed 24 h later in whole lung homogenates by flow cytometry. Calu-3 cells or human precision-cut lung slices (PCLS) cultured at air-liquid interface were exposed to CS or air and subsequently inoculated with influenza H1N1. At 48 h post infection cytokines were analyzed by multiplex technology. Cytotoxic effects were measured by release of lactate dehydrogenase (LDH) and confocal imaging. In Calu-3 cells the trans-epithelial electrical resistance (TEER) was assessed. Smoke exposure of mice increased numbers of inflammatory and plasmacytoid DCs in lung tissue. Additional poly (I:C) challenge further increased the population of inflammatory DCs and conventional DCs, especially CD11b(+) cDCs. Smoke exposure led to a loss of the barrier function in Calu-3 cells, which was further exaggerated by additional influenza H1N1 infection. Influenza H1N1-induced secretion of antiviral cytokines (IFN-alpha 2a, IFN-lambda, interferon-gamma-induced protein 10 [IP-10]), pro-inflammatory cytokine IL-6, as well as T cell-associated cytokines (e.g., I-TAC) were completely suppressed in both Calu-3 cells and human PCLS after smoke exposure. In summary, cigarette smoke exposure increased the number of inflammatory DCs in the lung and disrupted epithelial barrier functions, both of which was further enhanced by viral stimulation. Additionally, the antiviral immune response to influenza H1N1 was strongly suppressed by smoke. These data suggest that smoke impairs protective innate mechanisms in the lung, which could be responsible for the increased susceptibility to viral infections in "healthy" smokers.

Background: Spleen tyrosine kinase (SYK) inhibitors have therapeutic potential for severe asthma. In rats, activity of SYK inhibitor BI 1002494 measured with a CD63 assay correlated well with bronchoconstriction and airway resistance (Lamb DJ, et al. JPET 2016; 357:554–61). Aim: To evaluate a CD63 assay as a biomarker for SYK inhibitor activity in humans. Methods: Healthy volunteer whole blood was sensitized with anti-dinitroprusside (DNP) immunoglobulin E (IgE) and challenged with DNP. SYK inhibitor activity was measured with surface expression of CD63 as an index of basophil degranulation. Results: Strong correlation between CD63 IC50 and histamine release IC50 (R2=0.998) across 14 SYK inhibitor compounds confirmed CD63 as a good surrogate for functional basophil degranulation. BI 1002494 had a median CD63 IC50 of 113 nM (95% CI 103, 135) across 153 consecutive assays, with inter-sample variability of 17–2530 nM. Individual concentration curve gradients for basophil activation by the SYK inhibitor did not correlate with IC50 (R2=0.15). The proportion of basophils activated by anti-DNP/DNP (14–100%) correlated with IC50 (R2=0.44), suggesting that the more IgE receptors (i.e. the more SYK molecules) engaged, the greater the amount of inhibitor required. Similar assay performance and IC50 values from clinical biomarker labs indicate that the assay is robust and translatable. For 1000 SYK inhibitors, poor correlation was found between enzymatic kinase activity and CD63 potency (R2=0.22), possibly due to differences in cellular permeability and plasma protein binding. Conclusion: Employing a CD63 assay in ex vivo stimulated human whole blood could be used for screening compounds for human SYK inhibitor activity.

BackgroundExtensive analysis of eosinophilic airway inflammation in human-relevant animal models is required to test novel, human-specific pharmaceuticals. This requires species, which show high genetic homology to humans such as non-human primates. Efficacy assessment of novel human-specific biologicals in eosinophilic airway inflammation is currently performed in the cost-intensive macaque asthma model.ObjectiveThe present study investigated whether marmoset monkeys (Callithrix jacchus), a small-bodied non-human primate species from the New World, develop eosinophilic airway inflammation in response to house dust mite allergen exposure (HDM, Dermatophagoides pteronyssinus).MethodsMarmoset monkeys were sensitized against HDM by subcutaneous (s.c.) injection and subsequent intratracheal (i.t.) HDM aerosol challenges. Airway and systemic immunologic reactions were monitored and sensitivity towards glucocorticoid therapy was assessed. The pulmonary immunologic response was analyzed by repetitive bronchoalveolar lavage (BAL).ResultsBronchoalveolar lavage fluid (BALF) exhibited increased levels of eosinophils, mast cells, and lymphocytes, as well as interleukin (IL)-13 after HDM challenges, compared to negative controls. The systemic immunologic response was assessed in peripheral blood mononuclear cells derived from sensitized animals, which secreted increased IL-13 and IFN-γ upon allergen stimulation in contrast to non-sensitized negative control animals. Although IgE was not detectable, HDM-specific serum IgG was elevated in sensitized animals. Both airway and systemic responses were reduced by treatment with glucocorticoids. However, lung function and pathological analyses did not reveal significant differences between groups.ConclusionIn conclusion, marmoset monkeys developed a mild HDM-induced eosinophilic airway inflammation useful for efficacy testing of novel human-specific biologicals.

Objectives In the context of cystic fibrosis, Pseudomonas aeruginosa biofilms often develop in the vicinity of airway mucus, which acts as a protective physical barrier to inhaled matter. However, mucus can also adsorb small drug molecules administered as aerosols, including antibiotics, thereby reducing their bioavailability. The efficacy of antibiotics is typically assessed by determining the MIC using in vitro assays. This widespread technique, however, does not consider either bacterial biofilm formation or the influence of mucus, both of which may act as diffusion barriers, potentially limiting antibiotic efficacy. Methods We grew P. aeruginosa biofilms in the presence or absence of human tracheal mucus and tested their susceptibility to tobramycin and colistin. Results A significant reduction of tobramycin efficacy was observed when P. aeruginosa biofilms were grown in the presence of mucus compared with those grown in the absence of mucus. Diffusion of tobramycin through mucus was reduced; however, this reduction was more pronounced in biofilm/mucus mixtures, suggesting that biofilms in the presence of mucus respond differently to antibiotic treatment. In contrast, the influence of mucus on colistin efficacy was almost negligible and no differences in mucus permeability were observed. Conclusions These findings underline the important role of mucus in the efficacy of anti-infective drugs.

Background Asthma is a widespread, multifactorial chronic airway disease. The influence of regulatory B cells on airway hyperreactivity (AHR) and remodeling in asthma is poorly understood. Objective Our aim was to analyze the role of B cells in a house dust mite (HDM)-based murine asthma model. Methods The influence of B cells on lung function, tissue remodeling, and the immune response were analyzed by using wild-type and B-cell–deficient (μMT) mice and transfer of IL-10–proficient and IL-10–deficient B cells to μMT mice. Results After HDM-sensitization, both wild-type and μMT mice developed AHR, but the AHR was significantly stronger in μMT mice, as confirmed by 2 independent techniques: invasive lung function measurement in vivo and examination of precision-cut lung slices ex vivo. Moreover, airway remodeling was significantly increased in allergic μMT mice, as shown by enhanced collagen deposition in the airways, whereas the numbers of FoxP3+ and FoxP3– IL-10–secreting regulatory T cells were reduced. Adoptive transfer of IL-10–proficient but not IL-10–deficient B cells into μMT mice before HDM-sensitization attenuated AHR and lung remodeling. In contrast, FoxP3+ regulatory T cells were equally upregulated by transfer of IL-10–proficient and IL-10–deficient B cells. Conclusion Our data in a murine asthma model illustrate a central role of regulatory B cells in the control of lung function and airway remodeling and may support future concepts for B-cell–targeted prevention and treatment strategies for allergic asthma.

Sensitization of the respiratory tract is an important occupational health challenge, and understanding the mechanistic basis of this effect is necessary to support the development of toxicological tools to detect chemicals that may cause it. Here we use the adverse outcome pathway (AOP) framework to organize information that may better inform our understanding of sensitization of the respiratory tract, building on a previously published skin sensitization AOP, relying on literature evidence linked to low-molecular-weight organic chemicals and excluding other known respiratory sensitizers acting via different molecular initiating events. The established key events (KEs) are as follows: (1) covalent binding of chemicals to proteins, (2) activation of cellular danger signals (inflammatory cytokines and chemokines and cytoprotective gene pathways), (3) dendritic cell activation and migration, (4) activation, proliferation, and polarization of T cells, and (5) sensitization of the respiratory tract. These events mirror the skin sensitization AOP but with specific differences. For example, there is some evidence that respiratory sensitizers bind preferentially to lysine moieties, whereas skin sensitizers bind to both cysteine and lysine. Furthermore, exposure to respiratory sensitizers seems to result in cell behavior for KEs 2 and 3, as well as the effector T cell response, in general skewing toward cytokine secretions predominantly associated with T helper 2 (Th2) response. Knowledge gaps include the lack of understanding of which KE(s) drive the Th2 polarization. The construction of this AOP may provide insight into predictive tests that would in combination support the discrimination of respiratory-sensitizing from non- and skin-sensitizing chemicals, a clear regulatory need.

In times of increasing costs for health insurances, obstructive lung diseases are a burden for both the patients and the economy. Pulmonary symptoms of asthma and chronic obstructive pulmonary disease (COPD) are similar; nevertheless, the diseases differ in pathophysiology and therapeutic approaches. Novel therapeutics are continuously developed, and nonhuman primates (NHPs) provide valuable models for investigating novel biologicals regarding efficacy and safety.This review discusses the role of nonhuman primate models for drug development in asthma and COPD and investigates whether alternative methods are able to prevent animal experiments.

Humanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed that humanized mice immunized with long-lived induced–dendritic cells loaded with the pp65 viral antigen (iDCpp65) exhibited a faster development and maturation of T cells. Herein, we evaluated these effects in a long-term (36 weeks) nonclinical model using two stem cell donors to assess efficacy and safety. Relative to baseline, iDCpp65 immunization boosted the output of effector memory CD4 + T cells in peripheral blood and lymph nodes. No weight loss, human malignancies, or systemic graft- versus -host (GVH) disease were observed. However, for one reconstitution cohort, some mice immunized with iDCpp65 showed GVH-like signs on the skin. Histopathology analyses of the inflamed skin revealed intrafollicular and perifollicular human CD4 + cells near F4/80 + mouse macrophages around hair follicles. In spleen, CD4 + cells formed large clusters surrounded by mouse macrophages. In plasma, high levels of human T helper 2–type inflammatory cytokines were detectable, which activated in vitro the STAT5 pathway of murine macrophages. Despite this inflammatory pattern, human CD8 + T cells from mice with GVH reacted against the pp65 antigen in vitro . These results uncover a dynamic cross-species interaction between human memory T cells and mouse macrophages in the skin and lymphatic tissues of humanized mice.

Asthma is a widespread chronic multifactorial disease of the airways which is often triggered by the perennial allergen house dust mite (HDM). Next to regulatory T cells (Tregs), IL10 producing regulatory B cells are crucial to substantially counteract inflammatory responses. However, their regulatory impact in asthma is inadequately known so far. To better understand the regulatory influence of B cells in allergic asthma, we employed a HDM-based experimental asthma model using wildtype (WT) and B cell deficient (µMT) mice. Compared to WT counterparts, HDM-sensitized µMT mice developed a significantly increased airway hyperreactivity (AHR), a cardinal feature of asthma, measured by invasive lung function. This observation was confirmed by independent precision cut lung slices (PCLS) technique, demonstrating stronger bronchoconstriction of explanted lung tissue slices of allergic µMT mice after methacholine provocation. Furthermore, allergic µMT mice displayed diminished levels of Tregs and Th2 cytokines, especially IL10, as determined by flow cytometry and immunoassays. Adoptive transfer of naive WT B cells followed by HDM-sensitization substantially attenuated AHR in µMT mice and boosted IL10 production and Treg expansion. To test the impact of IL10, B cells from IL10 deficient (IL10-/-) mice were transferred, but failed to improve lung function of allergic µMT mice. Furthermore, transfer of WT and IL10-/- B cells provoked comparable amounts of IL10 and Tregs. Taken together, our data indicate that B cells are critical in HDM-induced AHR, and that IL10 competent B cells may be central in regulating AHR in allergic airway inflammation.

Chronic P. aeruginosa infections of the respiratory tract pose a major health care problem. The bacterial flagellum represents a tightly regulated virulence factor involved in biofilm formation as well as immune recognition of P. aeruginosa. Aim of this study was to delineate the role of flagellin using precision-cut lung slices (PCLS) as an organotypic, immunocompetent model of the human lung. PCLS were prepared from tumour-free lung tissue and infected with wildtype (wt) or flagellin mutant (∆fliC) PA14 strains. Persistent infection of the tissue over 24h was achieved by treatment with subinhibitory concentrations of tobramycin. Bacterial load was determined and lung tissue viability as well as specific cytokine responses were measured to assess the immune response. While the initial attachment of P. aeruginosa to the lung tissue was enhanced by the presence of the flagellum, both wt as well as ∆fliC strains were able to colonize the tissue over 24h. Analysis of cytokine profiles revealed a strong upregulation of pro-inflammatory mediators such as IL-8 (6-fold), IL-6 (6-fold), TNF-α (20-fold), GM-CSF (25-fold), IL-1β (52-fold), and MIP-3α (19-fold) after 24h of infection with wt PA14 compared to non-infected control PCLS. Importantly, infection with ∆fliC PA14 induced 2- to 4-fold lower levels of the respective molecules than the wt strain. Taken together, we show here that wt as well as flagellar mutant P. aeruginosa strains can infect human lung tissue ex vivo whereby flagellin acts as a potent immune stimulus, which could have implications for both immune evasion and biofilm formation of P. aeruginosa. Future studies will look at the transformation of acute towards persistent infection.

Background Antiviral drugs such as rupintrivir may have an immune-modulatory effect in experimentally induced allergic asthma with subsequent RV infection. We infected lung slices of house-dust mite (HDM)-sensitized asthmatic mice ex vivo with human rhinovirus (RV) and investigated the effect of the antiviral drug rupintrivir on RV-induced cytokine response in lung tissue of HDM-sensitized mice ex vivo. Methods Mice were sensitized with HDM. Precision-cut lung slices (PCLS) were prepared from HDM-sensitized or non-sensitized mice. Lung slices were infected ex vivo with RV or RV together with rupintrivir. Modulation of immune responses was evaluated by cytokine secretion 48 h post infection. Results In vivo HDM sensitization resulted in a TH-2/TH-17-dominated cytokine response that persisted in PCLS ex vivo. RV infection of PCLS from non-sensitized mice resulted in the induction of an antiviral and pro-inflammatory immune response, as indicated by the secretion of IFN-α, IFN-β, IFN-γ, TNF-α, MCP-1, IP-10, IL-10, and IL-17A. In contrast, PCLS from HDM-sensitized mice showed an attenuated antiviral response, but exaggerated IL-4, IL-6, and IL-10 secretion upon infection. Rupintrivir inhibited exaggerated pro-inflammatory cytokine IL-6 and TH-2 cytokine IL-4 in HDM-sensitized mice. Conclusions In summary, this study demonstrates that treatment with rupintrivir influences virus-induced IL-4 and IL-6 cytokine release under experimental conditions ex vivo. Electronic supplementary material The online version of this article (10.1186/s12931-019-1175-y) contains supplementary material, which is available to authorized users.

Food allergy is a growing global public health concern. As treatment strategies are currently limited to allergen avoidance and emergency interventions, there is an increasing demand for appropriate models of food allergy for the development of new therapeutics. Many models of food allergy rely heavily on the use of animals, and while useful, many are unable to accurately reflect the human system. In order to bridge the gap between in vivo animal models and clinical trials with human patients, human models of food allergy are of great importance. This review will summarize the commonly used human ex vivo and in vitro models of food allergy and highlight their advantages and limitations regarding how accurately they represent the human in vivo system. We will cover biopsy-based systems, precision cut organ slices, and coculture systems as well as organoids and organ-on-a-chip. The availability of appropriate experimental models will allow us to move forward in the field of food allergy research, to search for effective treatment options and to further explore the cause and progression of this disorder.

Food allergy is a growing global public health concern. As treatment strategies are currently limited to allergen avoidance and emergency interventions, there is an increasing demand for appropriate models of food allergy for the development of new therapeutics. Many models of food allergy rely heavily on the use of animals, and while useful, many are unable to accurately reflect the human system. In order to bridge the gap between in vivo animal models and clinical trials with human patients, human models of food allergy are of great importance. This review will summarize the commonly used human ex vivo and in vitro models of food allergy and highlight their advantages and limitations regarding how accurately they represent the human in vivo system. We will cover biopsy-based systems, precision cut organ slices, and coculture systems as well as organoids and organ-on-a-chip. The availability of appropriate experimental models will allow us to move forward in the field of food allergy research, to search for effective treatment options and to further explore the cause and progression of this disorder.

Millions of asthmatics suffer from virus induced exacerbation mainly caused by rhinovirus (RV). Insufficient virus elimination and inadequate immune response are assumed to be responsible for exacerbation. Therefore, in this study the impact of an asthmatic background on the anti-viral and pro-inflammatory immune response was investigated in precision-cut lung slices (PCLS) of HDM-sensitized mice. Balb/c mice were sensitized and challenged with HDM or saline i.n. for 28 days. Then PCLS were sliced and infected with RV1b (105 TCID50/mL) or UV-inactivated RV1b for 48 h. HDM sensitization resulted in a TH-2/TH-17 dominated allergic immune response with significantly higher secretion of IL-4, IL-5, IL-10, IL-17A and IP-10 which was maintained ex vivo. RV1b infection significantly upregulated anti-viral cytokines such as IFN-β (954±311 vs. 566±111 ng/mg protein) and IP-10 (61±17 vs. 40±6 pg/mg protein) in healthy vs. asthmatic tissue, respectively. Furthermore, RV1b exacerbated IL-6 (40±13 vs. 102±16 ng/mg protein) and IL-4 (823±201 vs. 1374±340 pg/mg protein) secretion in asthmatic tissue ex vivo in comparison to the corresponding UV-control but remained unchanged in healthy tissue. Rupintrivir treatment significantly reduced cytokine secretion of IFN-α, IP-10, IL-4, IL-6 and MCP-1 in asthmatic tissue but had only an impact on MCP-1 levels in healthy tissue. The in vivo established asthmatic disease background by HDM-challenge maintained in cell culture, leads to a significantly dysregulated immune response to virus infection ex vivo. Further studies are required to reveal pathways involved in the mechanisms of virus-induced exacerbation of asthma.

Pulmonary fibrosis comprises a multitude of rapidly progressing interstitial lung diseases assumed to develop from failure in the process of wound healing. Although TGFβ was reported to play a central role in the pathogenesis, it seems evident that also pro-inflammatory mediators contribute to the initiation and progression of lung fibrosis. In this study, the effect of TGFβ and TNFα on the induction of profibrotic mediators was investigated in human ex vivo lung tissue slices (Precision-Cut Lung Slices; PCLS). PCLS were prepared from tumor-free lung tissue sections and were cultured in the presence of TGFβ, TNFα or combination (TGFβ/TNFα) for up to 48h. Stimulation with TGFβ, TNFα or TGFβ/TNFα lead to specific up- or downregulation of distinct mediators in PCLS as compared to control samples, highlighting the necessity of combining key cytokines for the efficient induction of a pro-fibrotic profile. Upon others, TNFα as well as TGFβ/TNFα stimulation of PCLS lead to specific upregulation of IL-1β, while single stimulation with TGFβ showed no effect. IP10 and IFNγ levels remain unchanged in the presence of TNFα, while TGFβ/TNFα, or TGFβ alone lead to down-modulation of the expression. Stimulation of PCLS with either TNFα or TGFβ showed 2- to 3-fold up-regulation of PAI1. The combination of TNFα and TGFβ lead to further amplification of PAI1 mRNA levels to up to 7-fold. Importantly, ex vivo treatment with Pirfenidone efficiently reverts the TGFβ and TNFα-mediated modulation. We describe here the induction of a specific pro-fibrotic biomarker pattern in PCLS by TGFβ and TNFα stimulation, enabling the investigation of fibrotic signaling pathways with high translational relevance.

Cystic fibrosis patients are highly susceptible to Pseudomonas aeruginosa which rapidly adapts to the lung microenvironment and causes chronic inflammation strongly contributing to disease pathogenesis. In the presented study, fresh human lung tissue was infected ex vivo with P. aeruginosa to investigate bacterial adaption to lung tissue and antibiotic treatment as well as the host immune response. Human lung tissue slices were infected with 105 PAO1, treated with 10, 20 or 40µg/ml tobramycin and incubated for 24h or 96h. Tissue viability was assessed by Calcein AM staining. Bacterial load was determined by dilution plating and enumeration of CFU. Bacterial colonization was imaged by confocal microscopy. Cytokine release was determined using ELISA.P. aeruginosa rapidly colonized the lung tissue and induced a strong and persistent host immune response as indicated by the release of IL-8, IL-6, IL-1ß and TNF-a. Tobramycin treatment reduced CFU and resulted in maintained tissue viability, but without full eradication of P. aeruginosa, thereby enabling the transition to persisting infection up to 96h. Interestingly, this adaption of P. aeruginosa was associated with a decreased sensitivity to repeated antibiotic treatment. In summary, P. aeruginosa infection and respective induction of the host immune response can be reflected in human lung tissue ex vivo. Furthermore, the data indicate that early stages of the transition of acute to persistent infection can be mimicked, thereby reflecting bacterial adaption to antibiotic treatment. This paves the way to future investigations on host-pathogen interactions as well as testing of novel anti-infective or immunomodulatory therapeutics.

Formation of metastases marks the main cause of death in cancer and is characterized by complex processes, which can only partly be reflected in most in-vitro models due to a lacking human microenvironment. Cisplatin, bevacizumab and vemurafenib were used to modulate different aspects of tumorigenesis in co-cultures of patient-derived, disseminated melanoma cells or MDA-MB-231 cells and fresh human lung tissue as well as fresh human tumor tissue slices. Neoangiogenetic biomarker VEGF was elevated 3.5fold in co-cultures and 5.4fold in lung tumor slices after 48h. Treatment with bevacizumab [200 mg/mL] suppressed VEGF-release 24fold in co-cultures and 25fold in lung tumor tissue slices after 48h, also leading to impaired endothelial cell migration of the supernatants (81% in co-cultures, 83% in tumor slices). Cisplatin treatment [50mM] led to a decline of viability in co-cultures by up to 37.5% and in tumor slices up to 48.7% after 72h. Co-cultures and tumor slices were less sensitive in regards to their respective anti-cancer drug efficacy than the 2D culture. To address patient-specific genetic disparities, co-cultures with disseminated melanoma cells were performed with both patient-derived cells carrying BRAF mutation V600E and non-mutated cells. Treatment with vemurafenib reduced V600E cancer cell number (IC50=6µM) after 48 hours while non-mutated cells showed no significant cancer cell decrease. Here we show that tumor cells in solid tumors and seeded into healthy lung tissue are sensitive to tumor treatments ex vivo. This work aims to translate cancer drug efficacy data from animals towards humans to optimize clinical trial outcome.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), and interleukin 1 alpha (IL-1alpha) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

Cigarette smoke (Cs) inhalation is one of the main reasons to develop chronic obstructive pulmonary disease (COPD), characterised by degradation of alveoli, emphysema development, inflammation and mucus hypersecretion. Mechanisms that underlie various components of COPD can be modelled in vitro, specifically using whole fresh cigarette smoke. We assessed the effects of Cs and Cs condensate (Csc) on fresh human lung tissue. Human Precision-Cut Lung Slices (PCLS) were exposed to Csc or whole Cs in an Air-Liquid Interface (ALI) using the in vitro exposure device P.R.I.T.® ExpoCube®. Tissue viability, release of cytokines and extracellular matrix (ECM) proteins were analysed after single and repeated exposure and cultivation of up to 96h. Inhibitors were applied to suppress inflammatory responses of tissue to Cs. Csc and Cs induced concentration-dependent loss of tissue viability in fresh lung tissue. Smoke exposure induced an increased release of pro-inflammatory cytokines IL-1a (~ two fold), IL-1v (~ three fold) and changes in ECM proteins, e.g. MMP-9 and pro-Collagen1a1 (~ two fold), as well as Receptor for Advanced Glycation Endproducts (RAGE) from human PCLS. Dexamethasone and Roflumilast inhibited Cs-induced increase of pro-inflammatory cytokines. Csc and Cs induced tissue injury, early biomarkers of inflammation and changes in ECM proteins in vital ex vivo human lung tissue. The exposure of the complex mixture of whole cigarette smoke closely reflects the in vivo situation in human lung tissue. Next we will assess the response of human PCLS exposed to Cs and infected with rhinovirus to study exacerbations.

While interleukin (IL)-1β is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1β secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1β is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1β. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1β regulate each other

Background: Pulmonary fibrosis (PF) is characterized by excessive extracellular matrix (ECM) remodeling. Clinically validated ECM neoepitopes markers, related to progressive PF, may be useful for the evaluation of potential anti-fibrotic effects. Aim: The aim was to evaluate ECM remodelling (ECMR) in a human ex vivo precision-cut lung slice (PCLS) model. Methods: Human PF tissue was collected from two donors during lung transplantation. Within 24hours, the lungs were processed into PCLSs. The slices were cultured 2 pr/well and in triplicates for 48hours in serum free medium with 1nM-10mM nintedanib or 100pM-1µM mTOR/PI3K inhibitor omipalisib (GSK2126458). Responsiveness was tested using Lipopolysaccaride (LPS) and cytotoxicity using lactate dehydrogenase (LDH). Markers of collagen type I, III and VI formation (P1NP, PRO-C3, PRO-C6) and type III collagen degradation (C3M) were assessed in the supernatants. The subpleural and central lung regions were used and evaluated by hematoxylin and eosin staining. Results: The tissue from both donors was responsive to LPS and no toxicity was seen with the selected compound doses using LDH. P1NP, PRO-C3, and C3M were significantly reduced by 1nM-1µM omipalisib (p