P2X7 Receptor-Induced Human Mast Cell Degranulation Is Enhanced by Interleukin 33.

MCs are tissue-resident immune cells that strategically reside in barrier organs and respond effectively to a wide range of stimuli, such as IL-33, a mediator released upon epithelial damage. Adenosine triphosphate (ATP) accumulates at sites of tissue injury and is known to modulate MC activities. This study investigated how an inflammatory tissue environment rich in IL-33 modulates the ATP-mediated activation of MCs. Human primary MCs primed with IL-33 displayed a strongly increased response to ATP but not ADP. This resulted in increased degranulation, IL-8 release, and pERK1/2 signalling. Such effects are unique to IL-33 stimulation and not shared by the epithelial alarmin, TSLP. MC exposure to IL-33 also increased membrane expression of purinergic and ATP-binding P2X receptors. The use of selective P2X receptor inhibitors identified P2X7 receptor as the key mediator of the enhanced ATP-induced ERK1/2 signalling and degranulation in IL-33-primed MCs. Whilst the inhibition of P2X1 and P2X4 receptors had no effect on MC degranulation, inhibiting these receptors together with P2X7 resulted in further decreased MC-mediated degranulation. These data therefore point toward the potential mechanisms by which IL-33 contributes to the modulation of ATP-mediated activation in human MCs.

Lack of a transcriptional response of primary bronchial epithelial cells from patients with asthma and controls to IL-33.

IL-33 and IL-1RL1 are well-replicated asthma genes that act in a single pathway toward type-2 immune responses. IL-33 is expressed by basal epithelial cells, and the release of IL-33 upon epithelial damage can activate innate lymphoid cells, T helper-2 cells, basophilic granulocytes, and mast cells through a receptor complex containing IL-1RL1. However, it is unknown how bronchial epithelial cells respond to IL-33, and whether this response is increased in the disease. We aimed to characterize the IL-33-driven transcriptomic changes in cultured primary bronchial epithelial cells from patients with asthma and healthy controls. Primary bronchial epithelial cells (PBECs) were obtained by bronchial brushing from six healthy control for air-liquid interface (ALI) cultures, whereas we selected eight healthy controls and seven patients with asthma for epithelial organoid cultures. We then stimulated the cultures for 24 h with recombinant IL-33 (rhIL33) at various concentrations with 1, 10, and 50 ng/mL for the ALI cultures and 20 ng/mL and 100 ng/mL for the organoid cultures, followed by RNA-sequencing and differential gene expression analysis. We did not detect any genome-wide significant differentially expressed genes after stimulation of PBECs with IL-33, irrespective of growth in three-dimensional (3-D) epithelial organoids or after differentiation in ALI cultures. These results were identical between PBECs obtained from patients with asthma or from healthy control subjects. We detected very low levels of IL-1RL1 gene expression in these airway epithelial cell cultures. We conclude that bronchial epithelial cells do not have a transcriptional response to IL-33, independent of their differentiation state. Hence, the airway epithelium acts as a source of IL-33 but does not seem to contribute to the response upon release of the alarmin after epithelial damage.NEW & NOTEWORTHY The IL-33/IL-1RL1 pathway stands as a formidable genetic predisposition for asthma, with ongoing clinical developments of various drugs designed to mitigate its influence in patients with asthma. The absence of a transcriptomic reaction to IL-33 within the bronchial epithelium holds significance in the pursuit of identifying biomarkers that can aid in pinpointing those individuals who would derive the greatest benefit from therapies targeting the IL-33 pathway.

Identification of asthma-associated microRNAs in bronchial biopsies.

BACKGROUND: Changes in microRNA (miRNA) expression can contribute to the pathogenesis of many diseases, including asthma. We aimed to identify miRNAs that are differentially expressed between asthma patients and healthy controls, and explore their association with clinical and inflammatory parameters of asthma. METHODS: Differentially expressed miRNAs were determined by small RNA sequencing on bronchial biopsies of 79 asthma patients and 82 healthy controls using linear regression models. Differentially expressed miRNAs were associated with clinical and inflammatory asthma features. Potential miRNA-mRNA interactions were analysed using mRNA data available from the same bronchial biopsies, and enrichment of pathways was identified with Enrichr and g:Profiler. RESULTS: In total, 78 differentially expressed miRNAs were identified in bronchial biopsies of asthma patients compared with controls, of which 60 remained differentially expressed after controlling for smoking and inhaled corticosteroid treatment. We identified several asthma-associated miRNAs, including miR-125b-5p and miR-223-3p, based on a significant association with multiple clinical and inflammatory asthma features and their negative correlation with genes associated with the presence of asthma. The most enriched biological pathway(s) affected by miR-125b-5p and miR-223-3p were inflammatory response and cilium assembly/organisation. Of interest, we identified that lower expression of miR-26a-5p was linked to more severe eosinophilic inflammation as measured in blood, sputum as well as bronchial biopsies. CONCLUSION: Collectively, we identified miR-125b-5p, miR-223-3p and miR-26a-5p as potential regulators that could contribute to the pathogenesis of asthma.

P2X Receptor-Dependent Modulation of Mast Cell and Glial Cell Activities in Neuroinflammation.

Localisation of mast cells (MCs) at the abluminal side of blood vessels in the brain favours their interaction with glial cells, neurons, and endothelial cells, resulting in the activation of these cells and the release of pro-inflammatory mediators. In turn, stimulation of glial cells, such as microglia, astrocytes, and oligodendrocytes may result in the modulation of MC activities. MCs, microglia, astrocytes, and oligodendrocytes all express P2X receptors (P2XRs) family members that are selectively engaged by ATP. As increased concentrations of extracellular adenosine 5'-triphosphate (ATP) are present in the brain in neuropathological conditions, P2XR activation in MCs and glial cells contributes to the control of their communication and amplification of the inflammatory response. In this review we discuss P2XR-mediated MC activation, its bi-directional effect on microglia, astrocytes and oligodendrocytes and role in neuroinflammation.

Differential DNA methylation in bronchial biopsies between persistent asthma and asthma in remission.

Approximately 40% of asthmatics experience remission of asthma symptoms. A better understanding of biological pathways leading to asthma remission may provide insight into new therapeutic targets for asthma. As an important mechanism of gene regulation, investigation of DNA methylation provides a promising approach. Our objective was to identify differences in epigenome wide DNA methylation levels in bronchial biopsies between subjects with asthma remission and subjects with persistent asthma or healthy controls.We analysed differential DNA methylation in bronchial biopsies from 26 subjects with persistent asthma, 39 remission subjects and 70 healthy controls, using the limma package. The comb-p tool was used to identify differentially methylated regions. DNA methylation of CpG-sites was associated to expression of nearby genes from the same biopsies to understand function.Four CpG-sites and 42 regions were differentially methylated between persistent asthma and remission. DNA methylation at two sites was correlated i n cis with gene expression at ACKR2 and DGKQ Between remission subjects and healthy controls 1163 CpG-sites and 328 regions were differentially methylated. DNA methylation was associated with expression of a set of genes expressed in ciliated epithelium.CpGs differentially methylated between remission and persistent asthma identify genetic loci associated with resolution of inflammation and airway responsiveness. Despite the absence of symptoms, remission subjects have a DNA methylation profile that is distinct from that of healthy controls, partly due to changes in cellular composition, with a higher gene expression signal related to ciliated epithelium in remission versus healthy controls.

A cellular census of human lungs identifies novel cell states in health and in asthma.

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.

The discovery of quinoline-3-carboxamides as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors.

With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50 = 220,000 nM, LE = 0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50 = 3,100 nM, LE = 0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50 = 9.9 nM, LE = 0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.

Syk-kinase inhibition prevents mast cell activation in nasal polyps.

BACKGROUND: Mast cells are crucial effector cells in the allergic cascade. The cross-linking of the high affinity IgE receptor (FcεRI) activates mast cells and basophils. Spleen tyrosine kinase (Syk) is positioned upstream of the IgE receptor signal transducing pathway and may represent an important target for the treatment of nasal inflammatory diseases. OBJECTIVE: We measured effects of a specific Syk inhibitor in the release of mast cell mediators in human cord blood-derived mast cells (CBDMCs) (in-vitro) and in human nasal tissue (ex-vivo). METHODS: Surgical samples were collected from patients with nasal polyposis who underwent sinus surgery. Tissue cubes of +- 0.9 mm3 were primed with myeloma IgE (1 microg/ml), preincubated with Syk inhibitor NVP-QAB205 in different concentrations and then stimulated with tissue culture medium, anti-IgE 10 microg/ml and anti-IgE 30 microg/ml. Supernatants were analysed for concentrations of histamine, LTC4/LTD4/LTE4 and PGD2. CBDMCs were likewise pre-incubated with compound, prior to stimulation with anti-IgE at 10 microg/ml. RESULTS: In CBDMCs, the Syk inhibitor prevented degranulation assessed by measurement of histamine release and the production of LTC4/LTD4/LTE4 and PGD2. Furthermore, the Syk inhibitor was similarly able to significantly inhibit the release of these granules and newly synthesized mediators by nasal polyp mast cells in a dose dependent manner. CONCLUSION: Although the critical role of Syk in the IgE receptor signal transduction pathway has been well documented in vitro, this study supports the importance of Syk in IgE receptor-mediated degranulation of mast cells ex-vivo within nasal tissue. Thus, inhibition of Syk may represent an important therapeutic strategy for the treatment of upper airway disease with mast cell involvement, such as allergic rhinitis.

Enhanced release of IgE-dependent early phase mediators from nasal polyp tissue.

BACKGROUND: The mast cell is a crucial effector cell in allergic rhinitis and other inflammatory diseases. During the acute allergic reaction preformed mediators such as histamine, but also de novo produced mediators such as leukotrienes (LTC4/D4/E4) and prostaglandins (PGD2) are released. Mast cells represent targets for therapeutic intervention, and thus a human ex-vivo model to stimulate mast cells taken from mucosal sites would be instrumental for drug intervention studies. We have aimed to activate mast cells within ex-vivo human nasal tissue by IgE/anti-IgE specific (epsilon chain specific) stimulations and in this respect to test the usability of nasal polyps versus inferior turbinates METHODS: Biopsy samples were collected from patients with nasal polyps and inferior turbinates from patients who underwent sinus or septal surgery. Tissue fragments were primed with IgE 1 mug/ml for 60 minutes and then stimulated for 30 minutes with tissue culture medium (negative control), anti-IgE 10 mug/ml, anti-IgE 30 mug/ml and ionomycin 10 muM (positive control). Histamine, leukotrienes and PGD2 were measured in supernatants. To help provide an understanding of the extent of the response, the number of tryptase and FcepsilonRIalpha positive cells was evaluated by means of immunohistochemistry and the FcepsilonRIalpha-chain was measured by means of quantitative PCR in the nasal polyp and inferior turbinate tissues. Finally, the correlation between IgE concentrations in the nasal tissue and the release of mediators was analysed. RESULTS: Stimulations with anti-IgE on IgE-primed nasal tissue fragments lead to a concentration-dependent release of histamine, leukotrienes and PGD2. The release of these early phase mediators was significantly higher in nasal polyps compared to inferior turbinates, although tryptase, FcepsilonRIalpha positive cells and FcepsilonRIalpha-chain transcripts were equally present in both groups. No correlation was found between baseline concentrations of IgE, and the release of histamine, LTC4/LTD4/LTE4 and PGD2 after stimulation. CONCLUSION: This human nasal challenge model mimics the allergic early phase reaction. The release of histamine, cys-leukotrienes and PGD2 was significantly higher in nasal polyps versus inferior turbinates, however, this observation could not be explained by differences in mast cell or FcepsilonRI+ cell numbers.

Human ClCa1 modulates anionic conduction of calcium-dependent chloride currents.

Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca(2+)-dependent Cl- currents. The present study examines the relationship between the hClCa1 protein and Ca(2+)-dependent Cl- currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl- currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca(2+)-dependent Cl- currents and show that hClCa1 increases the amplitude of Ca(2+)-dependent Cl- currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl- conductance while decreasing the G(SCN-)/G(Cl-) conductance ratio from approximately 2-3 to approximately 1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca(2+)-dependent Cl- channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca(2+)-dependent Cl- channels by lowering the energy barriers for ion translocation through the pore.

Discovery and biological evaluation of potent dual ErbB-2/EGFR tyrosine kinase inhibitors: 6-thiazolylquinazolines.

We have identified a novel class of 6-thiazolylquinazolines as potent and selective inhibitors of both ErbB-2 and EGFR tyrosine kinase activity, with IC(50) values in the nanomolar range. These compounds inhibited the growth of both EGFR (HN5) and ErbB-2 (BT474) over-expressing human tumor cell lines in vitro. Using xenograft models of the same cell lines, we found that the compounds given orally inhibited in vivo tumor growth significantly compared with control animals.