IL-2-mediated hepatotoxicity: knowledge gap identification based on the irAOP concept.

Drug-induced hepatotoxicity constitutes a major reason for non-approval and post-marketing withdrawal of pharmaceuticals. In many cases, preclinical models lack predictive capacity for hepatic damage in humans. A vital concern is the integration of immune system effects in preclinical safety assessment. The immune-related Adverse Outcome Pathway (irAOP) approach, which is applied within the Immune Safety Avatar (imSAVAR) consortium, presents a novel method to understand and predict immune-mediated adverse events elicited by pharmaceuticals and thus targets this issue. It aims to dissect the molecular mechanisms involved and identify key players in drug-induced side effects. As irAOPs are still in their infancy, there is a need for a model irAOP to validate the suitability of this tool. For this purpose, we developed a hepatotoxicity-based model irAOP for recombinant human IL-2 (aldesleukin). Besides producing durable therapeutic responses against renal cell carcinoma and metastatic melanoma, the boosted immune activation upon IL-2 treatment elicits liver damage. The availability of extensive data regarding IL-2 allows both the generation of a comprehensive putative irAOP and to validate the predictability of the irAOP with clinical data. Moreover, IL-2, as one of the first cancer immunotherapeutics on the market, is a blueprint for various biological and novel treatment regimens that are under investigation today. This review provides a guideline for further irAOP-directed research in immune-mediated hepatotoxicity.

Interleukin-2-induced skin inflammation.

Recombinant human IL-2 has been used to treat inflammatory diseases and cancer; however, side effects like skin rashes limit the use of this therapeutic. To identify key molecules and cells inducing this side effect, we characterized IL-2-induced cutaneous immune reactions and investigated the relevance of CD25 (IL-2 receptor α) in the process. We injected IL-2 intradermally into WT mice and observed increases in immune cell subsets in the skin with preferential increases in frequencies of IL-4- and IL-13-producing group 2 innate lymphoid cells and IL-17-producing dermal γδ T cells. This overall led to a shift toward type 2/type 17 immune responses. In addition, using a novel topical genetic deletion approach, we reduced CD25 on skin, specifically on all cutaneous cells, and found that IL-2-dependent effects were reduced, hinting that CD25 - at least partly - induces this skin inflammation. Reduction of CD25 specifically on skin Tregs further augmented IL-2-induced immune cell infiltration, hinting that CD25 on skin Tregs is crucial to restrain IL-2-induced inflammation. Overall, our data support that innate lymphoid immune cells are key cells inducing side effects during IL-2 therapy and underline the significance of CD25 in this process.

Bacterial vesicles block viral replication in macrophages via TLR4-TRIF-axis.

Gram-negative bacteria naturally secrete nano-sized outer membrane vesicles (OMVs), which are important mediators of communication and pathogenesis. OMV uptake by host cells activates TLR signalling via transported PAMPs. As important resident immune cells, alveolar macrophages are located at the air-tissue interface where they comprise the first line of defence against inhaled microorganisms and particles. To date, little is known about the interplay between alveolar macrophages and OMVs from pathogenic bacteria. The immune response to OMVs and underlying mechanisms are still elusive. Here, we investigated the response of primary human macrophages to bacterial vesicles (Legionella pneumophila, Klebsiella pneumoniae, Escherichia coli, Salmonella enterica, Streptococcus pneumoniae) and observed comparable NF-κB activation across all tested vesicles. In contrast, we describe differential type I IFN signalling with prolonged STAT1 phosphorylation and strong Mx1 induction, blocking influenza A virus replication only for Klebsiella, E.coli and Salmonella OMVs. OMV-induced antiviral effects were less pronounced for endotoxin-free Clear coli OMVs and Polymyxin-treated OMVs. LPS stimulation could not mimic this antiviral status, while TRIF knockout abrogated it. Importantly, supernatant from OMV-treated macrophages induced an antiviral response in alveolar epithelial cells (AEC), suggesting OMV-induced intercellular communication. Finally, results were validated in an ex vivo infection model with primary human lung tissue. In conclusion, Klebsiella, E.coli and Salmonella OMVs induce antiviral immunity in macrophages via TLR4-TRIF-signaling to reduce viral replication in macrophages, AECs and lung tissue. These gram-negative bacteria induce antiviral immunity in the lung through OMVs, with a potential decisive and tremendous impact on bacterial and viral coinfection outcome. Video Abstract.

Mice inflammatory responses to inhaled aerosolized LPS: effects of various forms of human alpha1-antitrypsin.

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-1ß, 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.

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.

Precision cut intestinal slices, a novel model of acute food allergic reactions.

BACKGROUND: Food allergy affects up to 10% 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 to 24 h. Distribution of relevant cell subsets was confirmed using single nucleus RNA 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 24 h. 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.

Nintedanib modulates type III collagen turnover in viable precision-cut lung slices from bleomycin-treated rats and patients with pulmonary fibrosis.

BACKGROUND: Aberrant extracellular matrix (ECM) deposition and remodelling is important in the disease pathogenesis of pulmonary fibrosis (PF). We characterised neoepitope biomarkers released by ECM turnover in lung tissue from bleomycin-treated rats and patients with PF and analysed the effects of two antifibrotic drugs: nintedanib and pirfenidone. METHODS: Precision-cut lung slices (PCLS) were prepared from bleomycin-treated rats or patients with PF. PCLS were incubated with nintedanib or pirfenidone for 48 h, and levels of neoepitope biomarkers of type I, III and VI collagen formation or degradation (PRO-C1, PRO-C3, PRO-C6 and C3M) as well as fibronectin (FBN-C) were assessed in the culture supernatants. RESULTS: In rat PCLS, incubation with nintedanib led to a reduction in C3M, reflecting type III collagen degradation. In patient PCLS, incubation with nintedanib reduced the levels of PRO-C3 and C3M, thus showing effects on both formation and degradation of type III collagen. Incubation with pirfenidone had a marginal effect on PRO-C3. There were no other notable effects of either nintedanib or pirfenidone on the other neoepitope biomarkers studied. CONCLUSIONS: This study demonstrated that nintedanib modulates neoepitope biomarkers of type III collagen turnover and indicated that C3M is a promising translational neoepitope biomarker of PF in terms of therapy assessment.

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.

The Challenge of Long-Term Cultivation of Human Precision-Cut Lung Slices.

Human precision-cut lung slices (PCLS) have proven to be an invaluable tool for numerous toxicologic, pharmacologic, and immunologic studies. Although a cultivation period of <1 week is sufficient for most studies, modeling of complex disease mechanisms and investigating effects of long-term exposure to certain substances require cultivation periods that are much longer. So far, data regarding tissue integrity of long-term cultivated PCLS are incomplete. More than 1500 human PCLS from 16 different donors were cultivated under standardized, serum-free conditions for up to 28 days and the viability, tissue integrity, and the transcriptome was assessed in great detail. Even though viability of PCLS was well preserved during long-term cultivation, a continuous loss of cells was observed. Although the bronchial epithelium was well preserved throughout cultivation, the alveolar integrity was preserved for about 2 weeks, and the vasculatory system experienced significant loss of integrity within the first week. Furthermore, ciliary beat in the small airways gradually decreased after 1 week. Interestingly, keratinizing squamous metaplasia of the alveolar epithelium with significantly increasing manifestation were found over time. Transcriptome analysis revealed a significantly increased immune response and significantly decreased metabolic activity within the first 24 hours after PCLS generation. Overall, this study provides a comprehensive overview of histomorphologic and pathologic changes during long-term cultivation of PCLS.

Cigarette smoke alters inflammatory genes and the extracellular matrix - investigations on viable sections of peripheral human lungs.

Chronic obstructive pulmonary disease (COPD) is a complex chronic respiratory disorder often caused by cigarette smoke. Cigarette smoke contains hundreds of toxic substances. In our study, we wanted to identify initial mechanisms of cigarette smoke induced changes in the distal lung. Viable slices of human lungs were exposed 24 h to cigarette smoke condensate, and the dose-response profile was analyzed. Non-toxic condensate concentrations and lipopolysaccharide were used for further experiments. COPD-related protein and gene expression was measured. Cigarette smoke condensate did not induce pro-inflammatory cytokines and most inflammation-associated genes. In contrast, lipopolysaccharide significantly induced IL-1α, IL-1ß, TNF-α and IL-8 (proteins) and IL1B, IL6, and TNF (genes). Interestingly, cigarette smoke condensate induced metabolism- and extracellular matrix-associated proteins and genes, which were not influenced by lipopolysaccharide. Also, a significant regulation of CYP1A1 and CYP1B1, as well as MMP9 and MMP9/TIMP1 ratio, was observed which resembles typical findings in COPD. In conclusion, our data show that cigarette smoke and lipopolysaccharide induce significant responses in human lung tissue ex vivo, giving first hints that COPD starts early in smoking history.

COL4A3 is degraded in allergic asthma and degradation predicts response to anti-IgE therapy.

BACKGROUND: Asthma 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). OBJECTIVE: To delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response. METHODS: The 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. RESULTS: Serum 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). CONCLUSION: C4Ma3 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.

A modified protocol for successful miRNA profiling in human precision-cut lung slices (PCLS).

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.

Rhinovirus-induced Human Lung Tissue Responses Mimic Chronic Obstructive Pulmonary Disease and Asthma Gene Signatures.

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 IFNα2a, CXCL10, CXCL11, IFN-γ, TNFα, 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.

Cigarette Smoke Affects Dendritic Cell Populations, Epithelial Barrier Function, and the Immune Response to Viral Infection With H1N1.

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-α2a, IFN-λ, interferon-γ-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.

Human ex vivo and in vitro disease models to study food allergy.

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.

Bioreactor-based mass production of human iPSC-derived macrophages enables immunotherapies against bacterial airway infections.

The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45+CD11b+CD14+CD163+ cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa-mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies.

Human Teratoma-Derived Hematopoiesis Is a Highly Polyclonal Process Supported by Human Umbilical Vein Endothelial Cells.

Hematopoietic stem cells (HSCs) ensure a life-long regeneration of the blood system and are therefore an important source for transplantation and gene therapy. The teratoma environment supports the complex development of functional HSCs from human pluripotent stem cells, which is difficult to recapitulate in culture. This model mimics various aspects of early hematopoiesis, but is restricted by the low spontaneous hematopoiesis rate. In this study, a feasible protocol for robust hematopoiesis has been elaborated. We achieved a significant increase of the teratoma-derived hematopoietic population when teratomas were generated in the NSGS mouse, which provides human cytokines, together with co-injection of human umbilical vein endothelial cells. Since little is known about hematopoiesis in teratomas, we addressed localization and clonality of the hematopoietic lineage. Our results indicate that early human hematopoiesis is closely reflected in teratoma formation, and thus highlight the value of this model.

Human airway mucus alters susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, but not colistin.

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.

Alpha-1 Antitrypsin Inhibits ATP-Mediated Release of Interleukin-1ß via CD36 and Nicotinic Acetylcholine Receptors.

While interleukin (IL)-1ß 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-1ß secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1ß 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-1ß. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1ß regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1ß from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1ß release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1ß release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2ß, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1ß release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices.

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-α), and interleukin 1 alpha (IL-1α) 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.