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.

Defining the contractile prostanoid component in hyperosmolar-induced bronchoconstriction in human small airways.

Exercise-induced bronchoconstriction (EIB) is thought to be triggered by increased osmolarity at the airway epithelium. The aim of this study was to define the contractile prostanoid component of EIB, using an ex vivo model where intact segments of bronchi (inner diameter 0.5-2 mm) isolated from human lung tissue and subjected to mannitol. Exposure of bronchial segments to hyperosmolar mannitol evoked a contraction (64.3 ± 3.5 %) which could be prevented either by elimination of mast cells (15.8 ± 4.3 %) or a combination of cysteinyl leukotriene (cysLT1), histamine (H1) and thromboxane (TP) receptor antagonists (11.2 ± 2.3 %). Likewise, when antagonism of TP receptor was exchanged for inhibition of either cyclooxygenase-1 (8 ± 2.5 %), hematopoietic prostaglandin (PG)D synthase (20.7 ± 5.6 %), TXA synthase (14.8 ± 4.9 %), or the combination of the latter two (12.2 ± 4.6 %), the mannitol-induced contraction was prevented, suggesting that the TP-mediated component is induced by PGD2 and TXA2 generated by COX-1 and their respective synthases.

Activation of succinate receptor 1 boosts human mast cell reactivity and allergic bronchoconstriction.

BACKGROUND: SUCNR1 is a sensor of extracellular succinate, a Krebs cycle intermediate generated in excess during oxidative stress and has been linked to metabolic regulation and inflammation. While mast cells express SUCNR1, its role in mast cell reactivity and allergic conditions such as asthma remains to be elucidated. METHODS: Cord blood-derived mast cells and human mast cell line LAD-2 challenged by SUCNR1 ligands were analyzed for the activation and mediator release. Effects on mast cell-dependent bronchoconstriction were assessed in guinea pig trachea and isolated human small bronchi challenged with antigen and anti-IgE, respectively. RESULTS: SUCNR1 is abundantly expressed on human mast cells. Challenge with succinate, or the synthetic non-metabolite agonist cis-epoxysuccinate, renders mast cells hypersensitive to IgE-dependent activation, resulting in augmented degranulation and histamine release, de novo biosynthesis of eicosanoids and cytokine secretion. The succinate-potentiated mast cell reactivity was attenuated by SUCNR1 knockdown and selective SUCNR1 antagonists and could be tuned by pharmacologically targeting protein kinase C and extracellular signal-regulated kinase. Both succinate and cis-epoxysuccinate dose-dependently potentiated antigen-induced contraction in a mast cell-dependent guinea pig airway model, associated with increased generation of cysteinyl-leukotrienes and histamine in trachea. Similarly, cis-epoxysuccinate aggravated IgE-receptor-induced contraction of human bronchi, which was blocked by SUCNR1 antagonism. CONCLUSION: SUCNR1 amplifies IgE-receptor-induced mast cell activation and allergic bronchoconstriction, suggesting a role for this pathway in aggravation of allergic asthma, thus linking metabolic perturbations to mast cell-dependent inflammation.

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.

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.

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.

Dividing neutrophils in subsets reveals a significant role for activated neutrophils in the development of airway hyperreactivity.

BACKGROUND: Previous research has emphasized the importance of eosinophils in allergic asthma, while paying less attention to neutrophils. The known functionality of neutrophils in the inflammatory process has recently changed and knowledge about subsets of neutrophils, as characterized by their expression of CD16 and CD62L, has surfaced. Their specific roles in asthma are still unknown. OBJECTIVE: To study the functional differences between subsets of neutrophils by characterizing the impact of individual subsets on airway smooth muscle reactivity. METHODS: The direct effect of neutrophils on airway hyperresponsiveness was assessed by co-culturing different subsets of neutrophils (produced by LPS in vitro stimulation) with human isolated small airways or murine tracheae with subsequent evaluation of smooth muscle reactivity to bradykinin in myographs. Supernatants and tissue were saved for ELISA and immunohistochemistry. RESULTS: The CD16high CD62Ldim neutrophils were found to enhance the response to bradykinin in both human isolated small airways and murine tracheae. No such effects were obtained for the other subsets. The response is due to an upregulation of bradykinin receptor 2 through release of TNFα from the neutrophil. CONCLUSIONS AND CLINICAL RELEVANCE: The present study introduces a new concept regarding the role of neutrophils and defines a novel direct link between a specific activated neutrophil subset and airway smooth muscle, establishing neutrophils as important players in the development of asthmatic airway hyperactivity.

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.

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.

Prostaglandin E2 inhibits mast cell-dependent bronchoconstriction in human small airways through the E prostanoid subtype 2 receptor.

BACKGROUND: Inhaled prostaglandin (PG) E2 might inhibit asthmatic responses, but the mechanisms involved remain undefined. OBJECTIVE: We sought to characterize the direct and indirect effects of PGE2 on human small airways with particular reference to the receptors mediating the responses. METHODS: Contraction and relaxation were studied in isolated human bronchi with an inner diameter of 1 mm or less. RESULTS: Low concentrations of PGE2 (0.01-1 µmol/L) relaxed the bronchi precontracted by histamine. The bronchodilator response was inhibited by the E prostanoid (EP) subtype 4 receptor antagonist ONO-AE3-208 but unaffected by the EP2 receptor antagonist PF-04418948. Higher concentrations of PGE2 (10-100 µmol/L) contracted the small airways. However, the TP receptor agonists U-46,619, PGF2α, and PGD2 were more potent than PGE2. Moreover, the bronchoconstrictor responses to PGE2 and all other tested prostanoids, including the EP1/EP3 receptor agonist 17-phenyl trinor PGE2 and the partial FP receptor agonist AL-8810, were uniformly abolished by the TP receptor antagonist SQ-29,548. In the presence of TP and EP4 antagonists, PGE2 inhibited the mast cell-mediated bronchoconstriction resulting from anti-IgE challenge. Measurement of the release of histamine and cysteinyl leukotrienes documented that this bronchoprotective action of PGE2 was mediated by the EP2 receptor, unrelated to bronchodilation, and increased with time of exposure. CONCLUSION: The pharmacology of PGE2 in isolated human small airways was different from its profile in animal models. This first demonstration of powerful EP2 receptor-mediated inhibition of IgE-dependent contractions in human airways introduces a new selective target for the treatment of asthma. This EP2 control of mast cell-mediated bronchoconstriction is presumably exaggerated in patients with aspirin-exacerbated respiratory disease.

An optimized method for IgE-mediated degranulation of human lung mast cells.

Background: Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Methods: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. Results: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. Conclusions: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.

Accumulation of tissue-resident natural killer cells, innate lymphoid cells, and CD8+ T cells towards the center of human lung tumors.

Lung cancer is a leading cause of cancer-related death worldwide. Despite recent advances in tissue immunology, little is known about the spatial distribution of tissue-resident lymphocyte subsets in lung tumors. Using high-parameter flow cytometry, we identified an accumulation of tissue-resident lymphocytes including tissue-resident NK (trNK) cells and CD8+ tissue-resident memory T (TRM) cells toward the center of human non-small cell lung carcinomas (NSCLC). Chemokine receptor expression patterns indicated different modes of tumor-infiltration and/or residency between trNK cells and CD8+ TRM cells. In contrast to CD8+ TRM cells, trNK cells and ILCs generally expressed low levels of immune checkpoint receptors independent of location in the tumor. Additionally, granzyme expression in trNK cells and CD8+ TRM cells was highest in the tumor center, and intratumoral CD49a+CD16- NK cells were functional and responded stronger to target cell stimulation than their CD49a- counterparts, indicating functional relevance of trNK cells in lung tumors. In summary, the present spatial mapping of lymphocyte subsets in human NSCLC provides novel insights into the composition and functionality of tissue-resident immune cells, suggesting a role for trNK cells and CD8+ TRM cells in lung tumors and their potential relevance for future therapeutic approaches.

Analysis of human lung mast cells by single cell RNA sequencing.

Mast cells are tissue-resident cells playing major roles in homeostasis and disease conditions. Lung mast cells are particularly important in airway inflammatory diseases such as asthma. Human mast cells are classically divided into the subsets MCT and MCTC, where MCT express the mast cell protease tryptase and MCTC in addition express chymase, carboxypeptidase A3 (CPA3) and cathepsin G. Apart from the disctintion of the MCT and MCTC subsets, little is known about the heterogeniety of human lung mast cells and a deep analysis of their heterogeniety has previously not been performed. We therefore performed single cell RNA sequencing on sorted human lung mast cells using SmartSeq2. The mast cells showed high expression of classical mast cell markers. The expression of several individual genes varied considerably among the cells, however, no subpopulations were detected by unbiased clustering. Variable genes included the protease-encoding transcripts CMA1 (chymase) and CTSG (cathepsin G). Human lung mast cells are predominantly of the MCT subset and consistent with this, the expression of CMA1 was only detectable in a small proportion of the cells, and correlated moderately to CTSG. However, in contrast to established data for the protein, CPA3 mRNA was high in all cells and the correlation of CPA3 to CMA1 was weak.

The bitter taste receptor (TAS2R) agonists denatonium and chloroquine display distinct patterns of relaxation of the guinea pig trachea.

Activation of taste receptors (TAS2Rs) by bitter taste agonists has been reported to cause bronchodilation. The aim of this study was to extend the information on the effects of bitter taste agonists on responses induced by different contractile mediators in a standard airway physiology preparation. Isometric responses were assessed in guinea pig trachea (GPT). TAS2R agonists were administered either to segments precontracted with different agonists for contraction or given before challenge with the different contractile stimuli, including antigen in tissues from ovalbumin-sensitized animals. TAS2R mRNA expression on GPT epithelium and smooth muscle was measured with real-time PCR. Denatonium, chloroquine, thiamine, and noscapine induced concentration-dependent relaxations (R(max): 98.3 ± 1.6, 100.0 ± 0.0, 100.0 ± 0.0, and 52.3 ± 1.1% of maximum, respectively, in the presence of indomethacin) in segments precontracted with carbachol. The receptors for denatonium (TAS2R4, TAS2R10) and chloroquine (TAS2R3, TAS2R10) were expressed in GPT. Whereas denatonium selectively inhibited contractions induced by carbachol, chloroquine uniformly inhibited contractions evoked by prostaglandin E(2), the thromboxane receptor agonist U-46619, leukotriene D(4), histamine, and antigen. The effects of denatonium, but not those of chloroquine, were partly inhibited by blockers of the large Ca(2+)-activated K(+) channels and decreased by an increase of the level of precontraction. In conclusion, TAS2R agonists mediated strong relaxations and substantial inhibition of contractions in GPT. Chloroquine and denatonium had distinct patterns of activity, indicating different signaling mechanisms. The findings reinforce the hypothesis that TAS2Rs are potential targets for the development of a new class of more efficacious agonists for bronchodilation.

Monensin inhibits mast cell mediated airway contractions in human and guinea pig asthma models.

Asthma is a common respiratory disease associated with airway hyperresponsiveness (AHR), airway inflammation and mast cell (MC) accumulation in the lung. Monensin, an ionophoric antibiotic, has been shown to induce apoptosis of human MCs. The aim of this study was to define the effect of monensin on MC responses, e.g., antigen induced bronchoconstriction, and on asthmatic features in models of allergic asthma. Tracheal segments from house dust mite (HDM) extract sensitized guinea pigs were isolated and exposed to monensin, followed by histological staining to quantify MCs. Both guinea pig tracheal and human bronchi were used for pharmacological studies in tissue bath systems to investigate the monensin effect on tissue viability and antigen induced bronchoconstriction. Further, an HDM-induced guinea pig asthma model was utilized to investigate the effect of monensin on AHR and airway inflammation. Monensin decreased MC number, caused MC death, and blocked the HDM or anti-IgE induced bronchoconstriction in guinea pig and human airways. In the guinea pig asthma model, HDM-induced AHR, airway inflammation and MC hyperplasia could be inhibited by repeated administration of monensin. This study indicates that monensin is an effective tool to reduce MC number and MCs are crucial for the development of asthma-like features.

Cysteinyl-maresin 3 inhibits IL-13 induced airway hyperresponsiveness through alternative activation of the CysLT1 receptor.

BACKGROUND: Cysteinyl-maresins, also known as maresin-conjugates in tissue regeneration (MCTRs), are recently discovered lipid mediators proposed to reduce airway inflammation. OBJECTIVE: To investigate the influence of MCTRs on IL-13-induced airway hyperresponsiveness in isolated human and mice airways. METHODS: Before responsiveness to contractile agonists were assessed in myographs, human small bronchi were cultured for 2 days and mouse tracheas were cultured for 1-4 days. During the culture procedure airways were exposed to interleukin (IL)-13 in the presence or absence of MCTRs. Signalling mechanisms were explored using pharmacologic agonists and antagonists, and genetically modified mice. RESULTS: IL-13 treatment increased contractions to histamine, carbachol and leukotriene D4 (LTD4) in human small bronchi, and to 5-hydroxytryptamine (5-HT) in mouse trachea. In both preparations, co-incubation of the explanted tissues with MCTR3 reduced the IL-13 induced enhancement of contractions. In mouse trachea, this inhibitory effect of MCTR3 was blocked by three different CysLT1 receptor antagonists (montelukast, zafirlukast and pobilukast) during IL-13 exposure. Likewise, MCTR3 failed to reduce the IL-13-induced 5-HT responsiveness in mice deficient of the CysLT1 receptor. However, co-incubation with the classical CysLT1 receptor agonist LTD4 did not alter the IL-13-induced 5-HT hyperreactivity. CONCLUSIONS: MCTR3, but not LTD4, decreased the IL-13-induced airway hyperresponsiveness by activation of the CysLT1 receptor. The distinct actions of the two lipid mediators on the CysLT1 receptor suggest an alternative signalling pathway appearing under inflammatory conditions, where this new action of MCTR3 implicates potential to inhibit airway hyperresponsiveness in asthma.

Inflammation-induced airway smooth muscle responsiveness is strain dependent in mice.

Different mouse strains display different degrees of inflammation-induced airway hyperresponsiveness in vivo. It is not known whether these variations are attributable to distinct properties of the airway smooth muscle. Therefore, tracheal ring segments from C57BL/6 and BALB/c mice were exposed to three different pro-inflammatory stimuli for 4 days while maintained under tissue-culture conditions: tumour necrosis factor α (100 ng/ml), the Toll-like receptor (TLR) 3 agonist polyI:C (10 µg/ml), and the TLR4 agonist LPS (10 µg/ml). The contractile responses to carbachol, 5-hydroxytryptamine (5-HT) and bradykinin were assessed after culture. In addition, gene expression of TLR1-TLR9, pivotal inflammatory signal transduction proteins (jun-kinase, p38 and p65) and critical negative regulators of inflammation (A20, Itch, Tax1bp1 and RNF11) were studied in tracheal smooth muscle strips, fresh and following treatment for 4 days with LPS, from both strains. No differences between the strains were detected regarding the response of freshly isolated preparations to carbachol, 5-HT and bradykinin. After stimulation with pro-inflammatory mediators, contractions in response to 5-HT and bradykinin, but not to carbachol, were up-regulated. This up-regulation was markedly larger in BALB/c than in C57BL/6 segments and depended on the type of inflammatory stimulus. Expression of the genes investigated did not differ between the two strains. These findings indicate that strain differences in airway hyperresponsiveness can be linked to differences in the responsiveness of airway smooth muscle to pro-inflammatory mediators per se. The differences do not appear to be due to differential expression of TLR or common inflammatory transduction and repressor proteins.

Prostaglandin D2 inhibits mediator release and antigen induced bronchoconstriction in the Guinea pig trachea by activation of DP1 receptors.

The mechanism by which cyclooxygenase (COX) inhibition increases antigen-induced responses in airways remains unknown. Male albino guinea pigs were sensitized to ovalbumin (OVA). Intact rings of the trachea were isolated and mounted in organ baths for either force measurements or lipid mediator release analysis by UPLC-MS/MS or EIA following relevant pharmacological interventions. First, challenge with OVA increased the release of all primary prostanoids (prostaglandin (PG) D2/E2/F2α/I2 and thromboxanes). This release was eliminated by unselective COX inhibition (indomethacin) whereas selective inhibition of COX-2 (lumiracoxib) did not inhibit release of PGD2 or thromboxanes. Additionally, the increased levels of leukotriene B4 and E4 after OVA were further amplified by unselective COX inhibition. Second, unselective inhibition of COX and selective inhibition of the prostaglandin D synthase (2-Phenyl-Pyrimidine-5-Carboxylic Acid (2,3-dihydro-indol-1-yl)-amide) amplified the antigen-induced bronchoconstriction which was reversed by exogenous PGD2. Third, a DP1 receptor agonist (BW 245c) concentration-dependently reduced the antigen-induced constriction as well as reducing released histamine and cysteinyl-leukotrienes, a response inhibited by the DP1 receptor antagonist (MK-524). In contrast, a DP2 receptor agonist (15(R)-15-methyl PGD2) failed to modulate the OVA-induced constriction. In the guinea pig trachea, endogenous PGD2 is generated via COX-1 and mediates an inhibitory effect of the antigen-induced bronchoconstriction via DP1 receptors inhibiting mast cell release of bronchoconstrictive mediators. Removal of this protective function by COX-inhibition results in increased release of mast cell mediators and enhanced bronchoconstriction.