A novel quinoline with airway relaxant effects and anti-inflammatory properties.

BACKGROUND: In chronic pulmonary diseases characterized by inflammation and airway obstruction, such as asthma and COPD, there are unmet needs for improved treatment. Quinolines is a group of small heterocyclic compounds that have a broad range of pharmacological properties. Here, we investigated the airway relaxant and anti-inflammatory properties of a novel quinoline (RCD405). METHODS: The airway relaxant effect of RCD405 was examined in isolated airways from humans, dogs, rats and mice. Murine models of ovalbumin (OVA)-induced allergic asthma and LPS-induced airway inflammation were used to study the effects in vivo. RCD405 (10 mg/kg) or, for comparisons in selected studies, budesonide (3 mg/kg), were administered intratracheally 1 h prior to each challenge. Airway responsiveness was determined using methacholine provocation. Immune cell recruitment to bronchi was measured using flow cytometry and histological analyses were applied to investigate cell influx and goblet cell hyperplasia of the airways. Furthermore, production of cytokines and chemokines was measured using a multiplex immunoassay. The expression levels of asthma-related genes in murine lung tissue were determined by PCR. The involvement of NF-κB and metabolic activity was measured in the human monocytic cell line THP-1. RESULTS: RCD405 demonstrated a relaxant effect on carbachol precontracted airways in all four species investigated (potency ranking: human = rat > dog = mouse). The OVA-specific IgE and airway hyperresponsiveness (AHR) were significantly reduced by intratracheal treatment with RCD405, while no significant changes were observed for budesonide. In addition, administration of RCD405 to mice significantly decreased the expression of proinflammatory cytokines and chemokines as well as recruitment of immune cells to the lungs in both OVA- and LPS-induced airway inflammation, with a similar effect as for budesonide (in the OVA-model). However, the effect on gene expression of Il-4, IL-5 and Il-13 was more pronounced for RCD405 as compared to budesonide. Finally, in vitro, RCD405 reduced the LPS-induced NF-κB activation and by itself reduced cellular metabolism. CONCLUSIONS: RCD405 has airway relaxant effects, and it reduces AHR as well as airway inflammation in the models used, suggesting that it could be a clinically relevant compound to treat inflammatory airway diseases. Possible targets of this compound are complexes of mitochondrial oxidative phosphorylation, resulting in decreased metabolic activity of targeted cells as well as through pathways associated to NF-κB. However, further studies are needed to elucidate the mode of action.

Granzyme B is elevated in esophageal biopsies from children with eosinophilic esophagitis.

OBJECTIVES: Eosinophilic esophagitis (EoE) is an immune-mediated antigen-triggered inflammatory disease of the esophagus. Our aim was to investigate inflammatory responses by an ex vivo biopsy provocation-based method, stimulating biopsies with milk, wheat, and egg extracts. METHODS: An experimental study was conducted on esophageal biopsies from children who underwent esophagogastroduodenoscopy. Supernatants were collected before and after stimulation of the biopsies with food extracts and analyzed for 45 different inflammatory markers. Biopsies were also stained for histological analyzes. RESULTS: Study subjects included 13 controls, 9 active EoE, and 4 EoE in remission, median age 12 years. Of the 45 markers analyzed, three had significant differences between controls and patients with active EoE, Granzyme B, (GzmB), IL-1ra, and CXCL8 (p < .05). Levels of GzmB were higher, and levels of IL-1ra were lower in patients with active EoE compared with controls and EoE in remission both at baseline and after food extract stimulation. CXCL8 increased in active EoE compared with controls only after stimulation. The number of histologically detected GzmB-positive cells were significantly higher in patients with active EoE in contrast to control and EoE remission (p < .05). CONCLUSIONS: The levels of the barrier-damaging protease GzmB were higher in the supernatant both before and after stimulation with food extract ex vivo in patients with active EoE. GzmB was also observed histologically in biopsies from patients with active EoE. The presence of elevated serine protease GzmB in esophageal mucosa of children with active EoE suggests a role in the pathogenesis of this disorder.

Recombinant multimeric dog allergen prevents airway hyperresponsiveness in a model of asthma marked by vigorous TH 2 and TH 17 cell responses.

BACKGROUND: Allergy to dogs affects around 10% of the population in developed countries. Immune therapy of allergic patients with dog allergen extracts has shown limited therapeutic benefit. METHODS: We established a mouse model of dog allergy by repeatedly administering dog dander and epithelium extracts via the intranasal route. We also assessed the efficacy of a recombinant multimeric protein containing Can f 1, f 2, f 4 and f 6 in preventing inflammatory responses to dog extracts. RESULTS: Repeated inhalation of dog extracts induced infiltration of the airways by TH 2 cells, eosinophils and goblet cells, reminiscent of the house dust mite (HDM) model of asthma. Dog extracts also induced robust airway hyperresponsiveness and promoted TH 17 cell responses, which was associated with a high neutrophilic infiltration of the airways. scRNA-Seq analysis of T helper cells in the airways pinpointed a unique gene signature for TH 17 cells. Analysis of T-cell receptors depicted a high frequency of clones that were shared between TH 17, TH 2 and suppressive Treg cells, indicative of a common differentiation trajectory for these subsets. Importantly, sublingual administration of multimeric Can f 1-2-4-6 protein prior to sensitization reduced airway hyperresponsiveness and type 2-mediated inflammation in this model. CONCLUSION: Dog allergen extracts induce robust TH 2 and TH 17 cell-mediated responses in mice. Recombinant Can f 1-2-4-6 can induce tolerance to complex dog allergen extracts.

Crystalline silica particles induce DNA damage in respiratory epithelium by ATX secretion and Rac1 activation.

Autotaxin (ATX) and its product lysophosphatidic acid (LPA) have been implicated in lung fibrosis and cancer. We have studied their roles in DNA damage induced by carcinogenic crystalline silica particles (CSi). In an earlier study on bronchial epithelia, we concluded that ATX, via paracrine signaling, amplifies DNA damage. This effect was seen at 6-16 h. A succeeding study showed that CSi induced NLRP3 phosphorylation, mitochondrial depolarization, double strand breaks (DSBs), and NHEJ repair enzymes within minutes. In the current study we hypothesized a role for the ATX-LPA axis also in this rapid DNA damage. Using 16HBE human bronchial epithelial cells, we show ATX secretion at 3 min, and that ATX inhibitors (HA130 and PF8380) prevented both CSi-induced mitochondrial depolarization and DNA damage (detected by γH2AX and Comet assay analysis). Experiments with added LPA gave similar rapid effects as CSi. Furthermore, Rac1 was activated at 3 min, and a Rac1 inhibitor (NSC23766) prevented mitochondrial depolarization and genotoxicity. In mice the bronchial epithelia exhibited histological signs of ATX activation and signs of DSBs (53BP1 positive nuclei) minutes after a single inhalation of CSi. Our data indicate that CSi rapidly activate the ATX-LPA axis and within minutes this leads to DNA damage in bronchial epithelial cells. Thus, ATX mediates very rapid DNA damaging effects of inhaled particles.

Mast cell-derived serotonin enhances methacholine-induced airway hyperresponsiveness in house dust mite-induced experimental asthma.

BACKGROUND: Airway hyperresponsiveness (AHR) is a feature of asthma in which airways are hyperreactive to stimuli causing extensive airway narrowing. Methacholine provocations assess AHR in asthma patients mainly by direct stimulation of smooth muscle cells. Using in vivo mouse models, mast cells have been implicated in AHR, but the mechanism behind has remained unknown. METHODS: Cpa3Cre/+ mice, which lack mast cells, were used to assess the role of mast cells in house dust mite (HDM)-induced experimental asthma. Effects of methacholine in presence or absence of ketanserin were assessed on lung function and in lung mast cells in vitro. Airway inflammation, mast cell accumulation and activation, smooth muscle proliferation, and HDM-induced bronchoconstriction were evaluated. RESULTS: Repeated intranasal HDM sensitization induced allergic airway inflammation associated with accumulation and activation of lung mast cells. Lack of mast cells, absence of activating Fc-receptors, or antagonizing serotonin (5-HT)2A receptors abolished HDM-induced trachea contractions. HDM-sensitized mice lacking mast cells had diminished lung-associated 5-HT levels, reduced AHR and methacholine-induced airway contraction, while blocking 5-HT2A receptors in wild types eliminated AHR, implying that mast cells contribute to AHR by releasing 5-HT. Primary mouse and human lung mast cells express muscarinic M3 receptors. Mouse lung mast cells store 5-HT intracellularly, and methacholine induces release of 5-HT from lung-derived mouse mast cells and Ca2+ flux in human LAD-2 mast cells. CONCLUSIONS: Methacholine activates mast cells to release 5-HT, which by acting on 5-HT2A receptors enhances bronchoconstriction and AHR. Thus, M3-directed asthma treatments like tiotropium may also act by targeting mast cells.

IL-13 and IL-4, but not IL-5 nor IL-17A, induce hyperresponsiveness in isolated human small airways.

BACKGROUND: Specific inflammatory pathways are indicated to contribute to severe asthma, but their individual involvement in the development of airway hyperresponsiveness remains unexplored. OBJECTIVE: This experimental study in human small bronchi aimed to provide insight into which of the type 2 and type 17 cytokines cause hyperresponsiveness of airway smooth muscle. METHODS: Explanted small bronchi isolated from human lung tissue and human airway smooth muscle cells were treated for 2 and 1 day(s), respectively, with 100 ng/mL of IL-4, IL-5, IL-13, or IL-17A, and contractile responses, Ca2+ mobilization, and receptor expression were assessed. RESULTS: Treatment with IL-13 increased the potency of histamine, carbachol, and leukotriene D4 as contractile agonists. IL-4, but not IL-5 or IL-17A, also increased the potency of histamine. In human airway smooth muscle cells, IL-13 and IL-4, but not IL-5 and IL-17A, enhanced the histamine-induced Ca2+ mobilization that was accompanied with increased mRNA expression of histamine H1 and cysteinyl leukotriene CysLT1 receptors. RNA sequencing of isolated bronchi confirmed the IL-13-mediated upregulation of H1 and CysLT1 receptors, without showing an alteration of muscarinic M3 receptors. Dexamethasone had no effects on IL-13-induced hyperresponsiveness in human bronchi, the increased Ca2+ mobilization, or the enhanced receptor expression. In contrast, antagonism of the common receptor for IL-13 and IL-4 by the biologic dupilumab prevented the effects of both IL-13 and IL-4 in human bronchi and human airway smooth muscle cells. CONCLUSIONS: The glucocorticoid-insensitive hyperrresponsiveness in isolated human airways induced by IL-13 and IL-4 provides further evidence that the IL-4Rα pathway should be targeted as a new strategy for the treatment of airway hyperresponsiveness in asthma.

A new house dust mite-driven and mast cell-activated model of asthma in the guinea pig.

BACKGROUND: Animal models are extensively used to study underlying mechanisms in asthma. Guinea pigs share anatomical, pharmacological and physiological features with human airways and may enable the development of a pre-clinical in vivo model that closely resembles asthma. OBJECTIVES: To develop an asthma model in guinea pigs using the allergen house dust mite (HDM). METHODS: Guinea pigs were intranasally sensitized to HDM which was followed by HDM challenges once weekly for five weeks. Antigen-induced bronchoconstriction (AIB) was evaluated as alterations in Rn (Newtonian resistance), G (tissue damping) and H (tissue elastance) at the first challenge with forced oscillation technique (FOT), and changes in respiratory pattern upon each HDM challenge were assessed as enhanced pause (Penh) using whole-body plethysmography. Airway responsiveness to methacholine was measured one day after the last challenge by FOT. Inflammatory cells and cytokines were quantified in bronchoalveolar lavage fluid, and HDM-specific immunoglobulins were measured in serum by ELISA. Airway pathology was evaluated by conventional histology. RESULTS: The first HDM challenge after the sensitization generated a marked increase in Rn and G, which was abolished by pharmacological inhibition of histamine, leukotrienes and prostanoids. Repeated weekly challenges of HDM caused increase of Penh and a marked increase in airway hyperresponsiveness for all three lung parameters (Rn , G and H) and eosinophilia. Levels of IgE, IgG1 , IgG2 and IL-13 were elevated in HDM-treated guinea pigs. HDM exposure induced infiltration of inflammatory cells into the airways with a pronounced increase of mast cells. Subepithelial collagen deposition, airway wall thickness and goblet cell hyperplasia were induced by repeated HDM challenge. CONCLUSION AND CLINICAL RELEVANCE: Repeated intranasal HDM administration induces mast cell activation and hyperplasia together with an asthma-like pathophysiology in guinea pigs. This model may be suitable for mechanistic investigations of asthma, including evaluation of the role of mast cells.

Back to the future: re-establishing guinea pig in vivo asthma models.

Research using animal models of asthma is currently dominated by mouse models. This has been driven by the comprehensive knowledge on inflammatory and immune reactions in mice, as well as tools to produce genetically modified mice. Many of the identified therapeutic targets influencing airway hyper-responsiveness and inflammation in mouse models, have however been disappointing when tested clinically in asthma. It is therefore a great need for new animal models that more closely resemble human asthma. The guinea pig has for decades been used in asthma research and a comprehensive table of different protocols for asthma models is presented. The studies have primarily been focused on the pharmacological aspects of the disease, where the guinea pig undoubtedly is superior to mice. Further reasons are the anatomical and physiological similarities between human and guinea pig airways compared with that of the mouse, especially with respect to airway branching, neurophysiology, pulmonary circulation and smooth muscle distribution, as well as mast cell localization and mediator secretion. Lack of reagents and specific molecular tools to study inflammatory and immunological reactions in the guinea pig has however greatly diminished its use in asthma research. The aim in this position paper is to review and summarize what we know about different aspects of the use of guinea pig in vivo models for asthma research. The associated aim is to highlight the unmet needs that have to be addressed in the future.

Lipoxin A4 reduces house dust mite and TNFα-induced hyperreactivity in the mouse trachea.

Lipoxin A4 (LXA4) is considered a specialised pro-resolving mediator that decreases inflammation: however, pro-inflammatory effects have been described in the airways. Here, we investigated whether LXA4 could influence airway hyperreactivity induced in mouse trachea by house dust mite extract (HDM) or TNFα. Intranasal instillation of HDM caused a serotonin (5-HT) mediated airway hyperreactivity ex vivo (Emax: 78.1 ± 16.2 % versus control 12.8 ± 1.0 %) that was reduced by LXA4 installation one hour prior to HDM (Emax: 49.9 ± 11.4 %). Also, in isolated tracheal segments cultured for four days, HDM induced a hyperreactivity (Emax: 33.2 ± 3.1 % versus control 9.0 ± 0.7 %) that was decreased by LXA4 (Emax: 18.7 ± 1.5 %). One part of the HDM-induced hyperreactivity could be inhibited by the TNFα-inhibitor etanercept. TNFα-induced upregulation of 5-HT responses (Emax: 51.3 ± 1.2 % versus control 13.9 ± 0.5 %) was decreased by 10-1000 nM LXA4. In precontracted tracheal segments, LXA4 had no relaxing effect. Overall, LXA4 was able to decrease airway hyperreactivity induced by both HDM and TNFα, thus having a sub-acute anti-inflammatory effect in airway inflammation.

Crystalline silica particles cause rapid NLRP3-dependent mitochondrial depolarization and DNA damage in airway epithelial cells.

BACKGROUND: Respirable crystalline silica causes lung carcinomas and many thousand future cancer cases are expected in e.g. Europe. Critical questions are how silica causes genotoxicity in the respiratory epithelium and if new cases can be avoided by lowered permissible exposure levels. In this study we investigate early DNA damaging effects of low doses of silica particles in respiratory epithelial cells in vitro and in vivo in an effort to understand low-dose carcinogenic effects of silica particles. RESULTS: We find DNA damage accumulation already after 5-10 min exposure to low doses (5 µg/cm2) of silica particles (Min-U-Sil 5) in vitro. DNA damage was documented as increased levels of γH2AX, pCHK2, by Comet assay, AIM2 induction, and by increased DNA repair (non-homologous end joining) signaling. The DNA damage response (DDR) was not related to increased ROS levels, but to a NLRP3-dependent mitochondrial depolarization. Particles in contact with the plasma membrane elicited a Ser198 phosphorylation of NLRP3, co-localization of NLRP3 to mitochondria and depolarization. FCCP, a mitochondrial uncoupler, as well as overexpressed NLRP3 mimicked the silica-induced depolarization and the DNA damage response. A single inhalation of 25 µg silica particles gave a similar rapid DDR in mouse lung. Biomarkers (CC10 and GPRC5A) indicated an involvement of respiratory epithelial cells. CONCLUSIONS: Our findings demonstrate a novel mode of action (MOA) for silica-induced DNA damage and mutagenic double strand breaks in airway epithelial cells. This MOA seems independent of particle uptake and of an involvement of macrophages. Our study might help defining models for estimating exposure levels without DNA damaging effects.

Mannitol triggers mast cell-dependent contractions of human small bronchi and prostacyclin bronchoprotection.

BACKGROUND: Clinical research supports that exercise-induced bronchoconstriction (EIB) is caused by hyperosmolar triggering of mast cells. The reaction can be mimicked by inhalation of mannitol, but it has paradoxically previously not been possible to replicate this mode of action of mannitol in isolated airways. OBJECTIVE: We sought to establish an ex vivo model of EIB in human small bronchi. METHODS: Small bronchi (inner diameter, 0.5-2 mm) from macroscopically healthy human lung tissue were obtained from 48 patients and mounted in organ baths. Contractions and mediator release were analyzed after challenge with hyperosmolar mannitol (850 mOsm). RESULTS: Ten minutes of exposure to mannitol caused a small initial contraction (12% ± 1% of maximum) that was followed by a second and much larger contraction (maximum effect [Emax], 47% ± 5%) when mannitol was washed out. The mast cell stabilizer cromolyn reduced the second contraction (Emax, 27% ± 3%). Furthermore, this main contraction was abolished by the combination of antagonists of histamine and cysteinyl leukotrienes in the presence of indomethacin. Mannitol increased the release of the mast cell mediators histamine (9.0-fold), cysteinyl leukotrienes (4.5-fold), and prostaglandin (PG) D2 (5.4-fold), as well as PGE2 (6.3-fold) and the prostacyclin metabolite 6-keto PGF1α (5.7-fold). In contrast, indomethacin alone enhanced the bronchoconstriction (Emax, 68% ± 6%). Likewise, receptor antagonists for PGE2 (EP2 and EP4) and prostacyclin (IP) also enhanced the mannitol-induced bronchoconstriction (Emax, 67% ± 5%, 66% ± 4%, and 68% ± 3%, respectively). In bronchi precontracted by carbachol, the IP receptor agonist cicaprost induced profound relaxation. CONCLUSION: This new protocol established an in vitro model for studies of EIB in isolated human bronchi. The IP receptor might be a new target for asthma treatment.

Lung Regulatory T Cells Express Adiponectin Receptor 1: Modulation by Obesity and Airway Allergic Inflammation.

Regulatory T cells (Tregs) decrease in the adipose tissue upon weight gain, contributing to persistent low-grade inflammation in obesity. We previously showed that adipose tissue Tregs express the adiponectin receptor 1 (AdipoR1); however, the expression in lung Tregs is still unknown. Here, we aimed to determine whether Helios+ and Helios- Treg subsets expressed AdipoR1 in the lungs of obese mice and whether different obesity grades affected the expression upon allergic lung inflammation. For diet-induced obesity (DIO), mice were fed a high-fat diet (HFD) for up to 15 weeks (overweight), 21 weeks (obesity), and 26 weeks (morbid obesity). Overweight and morbidly obese mice were sensitized and challenged with ovalbumin (OVA) to induce allergic lung inflammation. The AdipoR1 expression was reduced significantly in the lung Helios+ and Helios- Tregs of obese mice compared with lean mice. Airway allergic inflammation showed reduced AdipoR1 expression in lung Foxp3+ Tregs. Obesity significantly exacerbated the eosinophilic airway inflammation and reduced the number of Helios+ Tregs in lung and adipose tissue in the obesity-associated asthma model. Upon further weight gain, AdipoR1-expressing Tregs in the lungs of allergic mice were increased, whereas AdipoR1-expressing Tregs in adipose tissue were reduced. These data suggest that obesity-associated adipose tissue inflammation may exacerbate allergic inflammation by downregulating the AdipoR1+ Tregs in the lungs.

Organic dust, causing both oxidative stress and Nrf2 activation, is phagocytized by bronchial epithelial cells.

Inhalation of organic dust (OD) from swine confinement facilities leads to pulmonary inflammation, airway hyperresponsiveness, and oxidative stress. In mice, pretreatment with a hydroxyl radical scavenger prevents airway inflammation and airway hyperresponsiveness (AHR) induced by OD exposure. We sought to determine a mechanism by which OD could induce oxidative stress in bronchial epithelial cells. Human bronchial epithelial cells (BEAS-2B or NHBE) were treated with various concentrations of OD, followed by evaluation of intracellular oxidative stress using 2',7'-dichlorofluorescein diacetate (DCFDA). After stimulation with OD, gene expression of antioxidant genes was assessed by real-time quantitative PCR followed by quantification of Nrf2 nuclear translocation using a luciferase reporter assay. Phagocytic markers (CD36 and CD68) were analyzed by FACS. Cells were treated with an actin inhibitor, cytochalasin D, before OD exposure and evaluated for Nrf2 nuclear translocation and DCFDA. Mice were pretreated with sulforaphane, the Nrf2 activator, before OD exposure and evaluated for pulmonary inflammation and airway reactivity. OD induced a time- and concentration-dependent increase in DCFDA. mRNA expression levels of Nrf2-dependent genes and Nrf2 nuclear translocation were increased after OD exposure. OD exposure increased the expression of CD68 and CD36. Cytochalasin D prevented oxidative stress and Nrf2 nuclear translocation after OD. Pretreatment with sulforaphane prevented OD-induced inflammation and AHR while increasing the uptake of OD in bronchial epithelial cells. Bronchial epithelial cells can phagocytose OD, resulting in an increase in endogenous oxidative stress. Nrf2-dependent mechanisms mediate the antioxidant response to OD.

Lipid Mediator Quantification in Isolated Human and Guinea Pig Airways: An Expanded Approach for Respiratory Research.

The clinical importance of prostaglandins and leukotrienes in asthma is well recognized; however, the biochemical role of other lipid mediators (often termed oxylipins) in the regulation of airway tone and inflammation remains unclear. We therefore developed a workflow to investigate oxylipin physiology and pharmacology in two in vitro models, the intact human bronchus and the guinea pig trachea. Airways were isolated and smooth muscle contraction was measured in an organ bath following stimulation with either anti-IgE or ovalbumin. The associated release of oxylipins over time into the organ bath was quantified using three developed LC-MS/MS methods capable of collectively measuring 130 compounds. Oxylipin extraction recoveries were 71% on average, method accuracy was 90-98%, coefficient of variation was 4.3-9.4%, and matrix effects were on average 11%. At baseline, low levels of primarily prostaglandins and associated metabolites were observed in both tissue preparations. The mast cell-induced airway constriction caused release of leukotrienes and further elevations in prostaglandin levels. In total, 57 oxylipins from the human bronchus, and 42 from guinea pig trachea, were detected at 60 min post-stimulation in the organ bath. Chiral analysis demonstrated that 5-hydroxyeicosatetraenoic acid (5-HETE) in the human bronchus preparation was not produced by 5-LOX enzymatic activity (enantiomeric excess [ee] = 10%), as opposed to 12( S)-HETE, 14( S)-, and 17( S)-hydroxy docosahexaenoic acid (HDoHE; ee = 100%), highlighting that chiral chromatography is necessary for correct biological interpretation. Unexpectedly, prostaglandin D2 and its metabolites remained elevated 24 h after the challenges, suggesting a sustained activation of mast cells not previously described. The reported translational methodology provides a new platform for comprehensive studies to elucidate the origin and functions of individual oxylipins in various airway responses.

Effects of tiotropium bromide on airway hyperresponsiveness and inflammation in mice exposed to organic dust.

INTRODUCTION: Acute exposure to organic dust (OD) in pig barns induces intense airway inflammation with neutrophilia and hyperresponsiveness. This reaction is likely associated with increased cholinergic activity. Therefore, the involvement of cholinergic mechanisms in the reaction to acute exposure of OD was investigated in mice using the long-acting muscarinic antagonist tiotropium. METHODS: BALB/c mice received tiotropium (2-200 ng) intranasally on day 1 of the study. On days 2-4, mice received vehicle or OD (25 µg) intranasally. Airway hyperresponsiveness to methacholine was assessed 24 h following the last OD exposure. Bronchoalveolar lavage (BAL) fluid, lung tissue and blood were collected for analyses. RESULTS: Organic dust elevated airway responsiveness to methacholine compared with controls (PBS) assessed as Newtonian resistance (1.5 ±â€¯0.1 vs 0.9 ±â€¯0.1 cm H2O x s/mL), tissue damping (12.4 ±â€¯1.4 vs 8.9 ±â€¯0.9 cm H2O∙s/mL) and tissue elastance (41.1 ±â€¯5.3 vs 27.2 ±â€¯2.5 cm H2O∙s/mL). Tiotropium (200 ng) decreased the Newtonian resistance and tissue damping after exposure to PBS or OD. Organic dust exposure increased inflammatory cells in BAL fluid by almost 400%, mainly due to neutrophil influx, which was unaffected by tiotropium. Organic dust increased levels of mainly Th1 mediators. Tiotropium treatment attenuated OD-induced release of IL-2, IL-4 and IL-6. CONCLUSIONS: Tiotropium decreased the OD-induced increase of specific cytokines without influencing the OD-induced increase of airway responsiveness and neutrophil infiltration into the lungs. We conclude that the cholinergic pathway contributes to the pro-inflammatory effects caused by inhalation of OD from pig barns.

Prominent release of lipoxygenase generated mediators in a murine house dust mite-induced asthma model.

The profile of activation of lipid mediator (LM) pathways in asthmatic airway inflammation remains unclear. This experimental study quantified metabolite levels of ω3-, ω6- and ω9-derived polyunsaturated fatty acids in bronchoalveolar lavage fluid (BALF) after 4-weeks of repeated house dust mite (HDM) exposure in a murine (C57BL/6) asthma model. The challenge induced airway hyperresponsiveness, pulmonary eosinophil infiltration, but with low and unchanged mast cell numbers. Of the 112 screened LMs, 26 were increased between 2 to >25-fold in BALF with HDM treatment (p < 0.05, false discovery rate = 5%). While cysteinyl-leukotrienes were the most abundant metabolites at baseline, their levels did not increase after HDM treatment, whereas elevation of PGD2, LTB4 and multiple 12/15-lipoxygenase products, such as 5,15-DiHETE, 15-HEDE and 15-HEPE were observed. We conclude that this model has identified a global lipoxygenase activation signature, not linked to mast cells, but with aspects that mimic chronic allergic airway inflammation in asthma.

Human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional CD69-CD56dim cells.

BACKGROUND: In contrast to the extensive knowledge about human natural killer (NK) cells in peripheral blood, relatively little is known about NK cells in the human lung. Knowledge about the composition, differentiation, and function of human lung NK cells is critical to better understand their role in diseases affecting the lung, including asthma, chronic obstructive pulmonary disease, infections, and cancer. OBJECTIVE: We sought to analyze and compare the phenotypic and functional characteristics of NK cells in the human lung and peripheral blood at the single-cell level. METHODS: NK cells in human lung tissue and matched peripheral blood from 132 subjects were analyzed by using 16-color flow cytometry and confocal microscopy. RESULTS: CD56dimCD16+ NK cells made up the vast majority of NK cells in human lungs, had a more differentiated phenotype, and more frequently expressed educating killer cell immunoglobulin-like receptors compared with NK cells in peripheral blood. Despite this, human lung NK cells were hyporesponsive toward target cell stimulation, even after priming with IFN-α. Furthermore, we detected a small subset of NK cells expressing CD69, a marker of tissue residency. These CD69+ NK cells in the lung consisted predominantly of immature CD56brightCD16- NK cells and less differentiated CD56dimCD16+ NK cells. CONCLUSION: Here, we characterize the major NK cell populations in the human lung. Our data suggest a model in which the majority of NK cells in the human lung dynamically move between blood and the lung rather than residing in the lung as bona fide tissue-resident CD69+ NK cells.

Neutrophilic oxidative stress mediates organic dust-induced pulmonary inflammation and airway hyperresponsiveness.

Airway exposure to organic dust (OD) from swine confinement facilities induces airway inflammation dominated by neutrophils and airway hyperresponsiveness (AHR). One important neutrophilic innate defense mechanism is the induction of oxidative stress. Therefore, we hypothesized that neutrophils exacerbate airway dysfunction following OD exposure by increasing oxidant burden. BALB/C mice were given intranasal challenges with OD or PBS (1/day for 3 days). Mice were untreated or treated with a neutrophil-depleting antibody, anti-Ly6G, or the antioxidant dimethylthiourea (DMTU) prior to OD exposure. Twenty-four hours after the final exposure, we measured airway responsiveness in response to methacholine (MCh) and collected bronchoalveolar lavage fluid to assess pulmonary inflammation and total antioxidant capacity. Lung tissue was harvested to examine the effect of OD-induced antioxidant gene expression and the effect of anti-Ly6G or DMTU. OD exposure induced a dose-dependent increase of airway responsiveness, a neutrophilic pulmonary inflammation, and secretion of keratinocyte cytokine. Depletion of neutrophils reduced OD-induced AHR. DMTU prevented pulmonary inflammation involving macrophages and neutrophils. Neutrophil depletion and DMTU were highly effective in preventing OD-induced AHR affecting large, conducting airways and tissue elastance. OD induced an increase in total antioxidant capacity and mRNA levels of NRF-2-dependent antioxidant genes, effects that are prevented by administration of DMTU and neutrophil depletion. We conclude that an increase in oxidative stress and neutrophilia is critical in the induction of OD-induced AHR. Prevention of oxidative stress diminishes neutrophil influx and AHR, suggesting that mechanisms driving OD-induced AHR may be dependent on neutrophil-mediated oxidant pathways.