Jesper Säfholm

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.

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.

PGE2 maintains the tone of the guinea pig trachea through a balance between activation of contractile EP1 receptors and relaxant EP2 receptors

The guinea pig trachea (GPT) is commonly used in airway pharmacology. The aim of this study was to define the expression and function of EP receptors for PGE2 in GPT as there has been ambiguity concerning their role.

Bitter taste receptor agonists mediate relaxation of human and rodent vascular smooth muscle.

Taste-sensing type 2 receptors (TAS2Rs) have been implicated in extraoral functions. Airway smooth muscle expresses TAS2Rs and is strongly relaxed by TAS2R agonists. We hypothesised that TAS2R agonists might affect vascular smooth muscle as well. Moreover, the general pharmacological profile of TAS2R agonists, which are used to investigate the functions of TAS2R׳s, are undefined. The aim of this study was to pharmacologically characterise the effects of five prototype TAS2R agonists in vascular smooth muscle. Responses to the TAS2R agonists were investigated in guinea-pig aorta and taenia coli, mouse aorta (wild-type and caveolin-1-/- mice) and human pulmonary arteries. Chloroquine, denatonium, dextromethorphan, noscapine and quinine, agonists for TAS2R3, TAS2R4, TAS2R10 and TAS2R14, induced strong endothelium-independent relaxations (responses between 82-96% of maximal relaxations) in phenylephrine pre-contracted guinea-pig aorta that persisted in the presence of L-type Ca2+ and KCa1.1-channel blockers. Experiments in guinea-pig taenia coli revealed that denatonium and quinine also inhibited relaxations to phenylephrine, indicating antagonism of α-adrenoceptors. Only chloroquine and noscapine mediated relaxations when the guinea pig aorta was pre-contracted by U-46619 or PGF2α. Relaxations to chloroquine and noscapine after U-46619 pre-contractions were however markedly impaired in aortae from caveolin-1-/- mice. Chloroquine and noscapine mediated relaxations of human pulmonary arteries that expressed also mRNA for TAS2R3, TAS2R4, TAS2R10 and TAS2R14, at levels similar to that of the α1A adrenoceptor. Notwithstanding whether TAS2Rs are involved or not, TAS2R agonists have profound effects on vascular smooth muscle. Chloroquine and noscapine are of special interest as their effects cannot be accounted for by conventional pathways.

Budesonide prevents cytokine-induced decrease of the relaxant responses to formoterol and terbutaline, but not to salmeterol, in mouse trachea

During asthma exacerbations, increased airway inflammation may impair the effects of β2-adrenoceptor (β2AR) agonists. It is unclear whether this impairment is prevented by inhaled glucocorticoids (GCs). We have investigated the relaxation of carbachol-contracted mouse tracheal segments to the β2AR agonists formoterol, terbutaline, and salmeterol. The segments were pre-exposed for 4 days to the proinflammatory cytokines tumor necrosis factor α (100 ng/ml) and interleukin-1β (10 ng/ml) with or without the GC, budesonide (1 μM). Formoterol and terbutaline induced greater maximal relaxation (Rmax) than salmeterol. The cytokines decreased Rmax of all β2AR agonists, whereas budesonide had no effect. However, after concomitant treatment with cytokines and budesonide, the Rmax values of formoterol and terbutaline were not impaired, whereas budesonide did not prevent the decrease in the Rmax of salmeterol. A similar pattern was observed for cAMP production by the agonists. In tracheal smooth muscle, β2AR mRNA was not affected by the cytokines but increased with budesonide. However, the cytokines markedly increased cyclooxygenase (COX)-2 mRNA expression, which may lead to heterologous desensitization of β2AR. It is noteworthy that the cytokine-induced increase of COX-2 was blocked by concomitant budesonide suggesting that heterologous desensitization of β2AR by the cytokines may be prevented by budesonide treatment. Budesonide prevented cytokine-induced impairment of the tracheal relaxation and β2AR/cAMP signaling for formoterol but not for salmeterol. This suggests that differences exist between formoterol and salmeterol in β2AR coupling/activation and/or signal transduction upstream of cAMP. These results imply that maximal bronchodilator effects of formoterol, but not of salmeterol, are maintained by budesonide treatment during periods with increased inflammation, such as asthma exacerbations.

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‐&agr;. 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. Graphical abstract Figure. No Caption available.

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.

Antagonising EP1 and EP2 receptors reveal that the TP receptor mediates a component of antigen-induced contraction of the guinea pig trachea

The role of different cyclooxygenase (COX) metabolites released after antigen exposure has been difficult to assess due to simultaneous release of dominant constrictors such as histamine and cysteinyl-leukotrienes (CysLT). In addition prostaglandin E2 (PGE2) also has a powerful effect on basal tone. The aim was to exclude PGE2, histamine and CysLTs from the antigen-induced contraction to define the possible involvement of remaining COX metabolites. Isometric force was measured in guinea pig trachea after exposure to cumulatively increasing concentrations of ovalbumin (OVA; 0.1 ng/ml to 0.1 mg/ml) in the absence or presence of biosynthesis inhibitors and receptor antagonists. Challenge with OVA induced a concentration-dependent contraction that reached 75% of maximal tissue response. COX-inhibition or a combination of EP1 and EP2 receptor antagonism (ONO-8130 and PF-04418948) completely abolished the tone, resulting in an augmented antigen response. COX inhibition in combination with either antihistamines or antileukotrienes (FLAP inhibitor or CysLT1-2 receptor antagonist) displayed no difference compared to COX inhibition alone. However, a combination of all three classes reduced the contraction to 30%, revealing an unknown contractile component. Exchanging COX inhibition with EP1-2 receptor antagonists together with antihistamines and antileukotrienes could not decrease the contraction more than to 50%. However, when a TP receptor antagonist (SQ-29,548) was further included, the maximal antigen contraction reached 30%, similar as previously, clearly revealing a TP-mediated contractile component. PGE2 primarily regulate the basal tone via EP1 and EP2, whereas prostanoids, such as TXA2 and PGD2, contribute as mediator of the antigen-response by activation of the TP receptor.

An optimized protocol for the isolation and functional analysis of human lung mast cells.

Background: Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, FcεRI, and CD117/KIT, the receptor for stem cell factor (SCF). There is a considerable heterogeneity among mast cells, both phenotypically and functionally. Human mast cells are generally divided into two main subtypes based on their protease content; the mucosa-associated MCT (tryptase positive and chymase negative mast cell) and the connective tissue associated-residing MCTC (tryptase and chymase positive mast cell). Human lung mast cells exhibit heterogeneity in terms of cellular size, expression of cell surface receptors, and secreted mediators. However, knowledge about human lung mast cell heterogeneity is restricted to studies using immunohistochemistry or purified mast cells. Whereas the former is limited by the number of cellular markers that can be analyzed simultaneously, the latter suffers from issues related to cell yield. Aim: To develop a protocol that enables isolation of human lung mast cells at high yields for analysis of functional properties and detailed analysis using single-cell based analyses of protein (flow cytometry) or RNA (RNA-sequencing) expression. Methods: Mast cells were isolated from human lung tissue by a sequential combination of washing, enzymatic digestion, mechanical disruption, and density centrifugation using Percoll (WEMP). As a comparison, we also isolated mast cells using a conventional enzyme-based protocol. The isolated cells were analyzed by flow cytometry. Results: We observed a significant increase in the yield of total human lung CD45+ immune cells and an even more pronounced increase in the yield of CD117+ mast cells with the WEMP protocol in comparison to the conventional protocols. In contrast, the frequency of the rare lymphocyte subset innate lymphoid cells group 2 (ILC2) did not differ between the two methods. Conclusion: The described WEMP protocol results in a significant increase in the yield of human lung mast cells compared to a conventional protocol. Additionally, the WEMP protocol enables simultaneous isolation of different immune cell populations such as lymphocytes, monocytes, and granulocytes while retaining their surface marker expression that can be used for advanced single-cell analyses including multi-color flow cytometry and RNA-sequencing.

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.