Mast cells control lung type 2 inflammation via prostaglandin E2-driven soluble ST2.

Severe asthma and sinus disease are consequences of type 2 inflammation (T2I), mediated by interleukin (IL)-33 signaling through its membrane-bound receptor, ST2. Soluble (s)ST2 reduces available IL-33 and limits T2I, but little is known about its regulation. We demonstrate that prostaglandin E2 (PGE2) drives production of sST2 to limit features of lung T2I. PGE2-deficient mice display diminished sST2. In humans with severe respiratory T2I, urinary PGE2 metabolites correlate with serum sST2. In mice, PGE2 enhanced sST2 secretion by mast cells (MCs). Mice lacking MCs, ST2 expression by MCs, or E prostanoid (EP)2 receptors by MCs showed reduced sST2 lung concentrations and strong T2I. Recombinant sST2 reduced T2I in mice lacking PGE2 or ST2 expression by MCs back to control levels. PGE2 deficiency also reversed the hyperinflammatory phenotype in mice lacking ST2 expression by MCs. PGE2 thus suppresses T2I through MC-derived sST2, explaining the severe T2I observed in low PGE2 states.

Glucagon-like Peptide-1 Receptor Pathway Attenuates Platelet Activation in Aspirin-Exacerbated Respiratory Disease.

Platelets are key contributors to allergic asthma and aspirin-exacerbated respiratory disease (AERD), an asthma phenotype involving platelet activation and IL-33-dependent mast cell activation. Human platelets express the glucagon-like peptide-1 receptor (GLP-1R). GLP-1R agonists decrease lung IL-33 release and airway hyperresponsiveness in mouse asthma models. We hypothesized that GLP-1R agonists reduce platelet activation and downstream platelet-mediated airway inflammation in AERD. GLP-1R expression on murine platelets was assessed using flow cytometry. We tested the effect of the GLP-1R agonist liraglutide on lysine-aspirin (Lys-ASA)-induced changes in airway resistance, and platelet-derived mediator release in a murine AERD model. We conducted a prospective cohort study comparing the effect of pretreatment with liraglutide or vehicle on thromboxane receptor agonist-induced in vitro activation of platelets from patients with AERD and nonasthmatic controls. GLP-1R expression was higher on murine platelets than on leukocytes. A single dose of liraglutide inhibited Lys-ASA-induced increases in airway resistance and decreased markers of platelet activation and recruitment to the lung in AERD-like mice. Liraglutide attenuated thromboxane receptor agonist-induced activation as measured by CXCL7 release in plasma from patients with AERD and CD62P expression in platelets from both patients with AERD (n = 31) and nonasthmatic, healthy controls (n = 11). Liraglutide, a Food and Drug Administration-approved GLP-1R agonist for treatment of type 2 diabetes and obesity, attenuates in vivo platelet activation in an AERD murine model and in vitro activation in human platelets in patients with and without AERD. These data advance the GLP-1R axis as a new target for platelet-mediated inflammation warranting further study in asthma.

IL-4Rα signaling promotes barrier-altering oncostatin M and IL-6 production in aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is a severe disease involving dysregulated type 2 inflammation. However, the role other inflammatory pathways play in AERD is poorly understood. OBJECTIVE: We sought to broadly define the inflammatory milieu of the upper respiratory tract in AERD and to determine the effects of IL-4Rα inhibition on mediators of nasal inflammation. METHODS: Twenty-two AERD patients treated with dupilumab for 3 months were followed over 3 visits and compared to 10 healthy controls. Nasal fluid was assessed for 45 cytokines and chemokines using Olink Target 48. Blood neutrophils and cultured human mast cells, monocytes/macrophages, and nasal fibroblasts were assessed for response to IL-4/13 stimulation in vitro. RESULTS: Of the nasal fluid cytokines measured, nearly one third were higher in AERD patients compared to healthy controls, including IL-6 and the IL-6 family-related cytokine oncostatin M (OSM), both of which correlated with nasal albumin levels, a marker of epithelial barrier dysregulation. Dupilumab significantly decreased many nasal mediators, including OSM and IL-6. IL-4 stimulation induced OSM production from mast cells and macrophages but not from neutrophils, and OSM and IL-13 stimulation induced IL-6 production from nasal fibroblasts. CONCLUSION: In addition to type 2 inflammation, innate and IL-6-related cytokines are also elevated in the respiratory tract in AERD. Both OSM and IL-6 are locally produced in nasal polyps and likely promote pathology by negatively affecting epithelial barrier function. IL-4Rα blockade, although seemingly directed at type 2 inflammation, also decreases mediators of innate inflammation and epithelial dysregulation, which may contribute to dupilumab's therapeutic efficacy in AERD.

Trial of thromboxane receptor inhibition with ifetroban: TP receptors regulate eicosanoid homeostasis in aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is the triad of asthma, nasal polyposis, and respiratory reactions to COX-1 inhibitors. Overproduction of cysteinyl leukotrienes and underproduction of prostaglandin E2 (PGE2) are hallmarks of AERD. A mouse model predicted a key role for the thromboxane-prostanoid (TP) receptor in AERD. OBJECTIVE: Our aim was to determine whether ifetroban, a TP receptor antagonist, attenuates aspirin-induced respiratory symptoms in patients with AERD. METHODS: A total of 35 patients with AERD completed a 4-week double-blinded, placebo-controlled trial of ifetroban and underwent an oral aspirin challenge. The primary outcome was change in the provocative dose of aspirin that caused a 2-point increase in Total Nasal Symptom Score. Changes in lung function, eicosanoid levels, and platelet and mast cell activation were assessed. Cultured human nasal fibroblasts were stimulated with or without the TP agonist U46619 and assayed for prostanoid production. RESULTS: Ifetroban was well tolerated in AERD and did not change the mean 2-point increase in Total Nasal Symptom Score (P = .763). Participants taking ifetroban had greater aspirin-induced nasal symptoms and a greater decline in FEV1 value than did participants receiving placebo (-18.8% ± 3.6% with ifetroban vs -8.4% ± 2.1% with placebo [P = .017]). Four weeks of ifetroban significantly increased urinary leukotriene E4 levels and decreased nasal PGE2 levels compared with placebo. Peak aspirin-induced urinary thromboxane levels correlated with peak urinary leukotriene E4 and prostaglandin D2 metabolite levels in participants taking ifetroban. U46119 significantly potentiated the production of PGE2 by cultured nasal fibroblasts from subjects with AERD but not by cultured nasal fibroblasts from controls without polypoid sinusitis. CONCLUSION: Contrary to our hypothesis, TP receptor blockade worsened aspirin-induced reactions in AERD, possibly by exacerbating dysregulation of the eicosanoid system. TP signaling on stromal cells may be critical to maintaining PGE2 production when COX-2 function is low.

Developmental Process and Symmetry of Fetal Sulci in the Mesial Area Assessed by Ultrasound: A Cohort Study in Chinese Population.

OBJECTIVE: We aimed to investigate the progression of cortical development in Chinese population and to determine the rate of isolated asymmetric cortical development. We also explored the outcomes of these fetuses and determined whether cortical asymmetry represents normal individual physiological variation. METHODS: Our observational cohort study included 456 healthy singleton pregnant women who visited Peking University First Hospital between September 2020 and December 2021. We evaluated the progression and symmetry of the parieto-occipital sulcus, calcarine sulcus, and cingulate sulcus using a scoring system during routine fetal ultrasound examinations. The outcomes of the included fetuses after birth were assessed using the Ages and Stages Questionnaire, Third Edition (ASQ-3). RESULTS: The median gestational ages at which the parieto-occipital, calcarine, and cingulate sulci reached grade 1 were 22, 22, and 26 weeks, respectively. Among 456 included fetuses, 426 showed symmetric cortical development and 30 showed asymmetric cortical development during ultrasound examination. Fetuses with asymmetric cortical development underwent 'catch-up growth' and developed to the same grade in 2-6 weeks. All fetuses with symmetric or asymmetric cortical development had normal neurodevelopment after birth according to ASQ-3 assessment. CONCLUSION: The gestational age at which the parieto-occipital, calcarine, and cingulate sulci can be detected using ultrasound varies in different studies. Racial differences may be present in cortical development. Normal fetuses may physiologically have mildly asymmetric cortical development in the mesial area.

Leukotriene D4 paradoxically limits LTC4-driven platelet activation and lung immunopathology.

BACKGROUND: The 3 cysteinyl leukotrienes (cysLTs), leukotriene (LT) C4 (LTC4), LTD4, and LTE4, have different biologic half-lives, cellular targets, and receptor specificities. CysLT2R binds LTC4 and LTD4in vitro with similar affinities, but it displays a marked selectivity for LTC4in vivo. LTC4, but not LTD4, strongly potentiates allergen-induced pulmonary eosinophilia in mice through a CysLT2R-mediated, platelet- and IL-33-dependent pathway. OBJECTIVE: We sought to determine whether LTD4 functionally antagonizes LTC4 signaling at CysLT2R. METHODS: We used 2 different in vivo models of CysLT2R-dependent immunopathology, as well as ex vivo activation of mouse and human platelets. RESULTS: LTC4-induced CD62P expression; HMGB1 release; and secretions of thromboxane A2, CXCL7, and IL-33 by mouse platelets were all were blocked by a selective CysLT2R antagonist and inhibited by LTD4. These effects did not depend on CysLT1R. Inhaled LTD4 blocked LTC4-mediated potentiation of ovalbumin-induced eosinophilic inflammation; recruitment of platelet-adherent eosinophils; and increases in IL-33, IL-4, IL-5, and IL-13 levels in lung tissue. In contrast, the effect of administration of LTE4, the preferred ligand for CysLT3R, was additive with LTC4. The administration of LTD4 to Ptges-/- mice, which display enhanced LTC4 synthesis similar to that in aspirin-exacerbated respiratory disease, completely blocked the physiologic response to subsequent lysine-aspirin inhalation challenges, as well as increases in levels of IL-33, type 2 cytokines, and biochemical markers of mast cell and platelet activation. CONCLUSION: The conversion of LTC4 to LTD4 may limit the duration and extent of potentially deleterious signaling through CysLT2R, and it may contribute to the therapeutic properties of desensitization to aspirin in aspirin-exacerbated respiratory disease.

Type 2 Cysteinyl Leukotriene Receptors Drive IL-33-Dependent Type 2 Immunopathology and Aspirin Sensitivity.

Cysteinyl leukotrienes (cysLTs) facilitate mucosal type 2 immunopathology by incompletely understood mechanisms. Aspirin-exacerbated respiratory disease, a severe asthma subtype, is characterized by exaggerated eosinophilic respiratory inflammation and reactions to aspirin, each involving the marked overproduction of cysLTs. Here we demonstrate that the type 2 cysLT receptor (CysLT2R), which is not targeted by available drugs, is required in two different models to amplify eosinophilic airway inflammation via induced expression of IL-33 by lung epithelial cells. Endogenously generated cysLTs induced eosinophilia and expanded group 2 innate lymphoid cells (ILC2s) in aspirin-exacerbated respiratory disease-like Ptges-/- mice. These responses were mitigated by deletions of either Cysltr2 or leukotriene C4 synthase (Ltc4s). Administrations of either LTC4 (the parent cysLT) or the selective CysLT2R agonist N-methyl LTC4 to allergen sensitized wild-type mice markedly boosted ILC2 expansion and IL-5/IL-13 generation in a CysLT2R-dependent manner. Expansion of ILC2s and IL-5/IL-13 generation reflected CysLT2R-dependent production of IL-33 by alveolar type 2 cells, which engaged in a bilateral feed-forward loop with ILC2s. Deletion of Cysltr1 blunted LTC4-induced ILC2 expansion and eosinophilia but did not alter IL-33 induction. Pharmacological blockade of CysLT2R prior to inhalation challenge of Ptges-/- mice with aspirin blocked IL-33-dependent mast cell activation, mediator release, and changes in lung function. Thus, CysLT2R signaling, IL-33-dependent ILC2 expansion, and IL-33-driven mast cell activation are necessary for induction of type 2 immunopathology and aspirin sensitivity. CysLT2R-targeted drugs may interrupt these processes.

Platelet-driven leukotriene C4-mediated airway inflammation in mice is aspirin-sensitive and depends on T prostanoid receptors.

Cysteinyl leukotrienes (cysLTs) are bronchoconstricting lipid mediators that amplify eosinophilic airway inflammation by incompletely understood mechanisms. We recently found that LTC4, the parent cysLT, potently activates platelets in vitro and induces airway eosinophilia in allergen-sensitized and -challenged mice by a platelet- and type 2 cysLT receptor-dependent pathway. We now demonstrate that this pathway requires production of thromboxane A2 and signaling through both hematopoietic and lung tissue-associated T prostanoid (TP) receptors. Intranasal administration of LTC4 to OVA-sensitized C57BL/6 mice markedly increased the numbers of eosinophils in the bronchoalveolar lavage fluid, while simultaneously decreasing the percentages of eosinophils in the blood by a TP receptor-dependent mechanism. LTC4 upregulated the expressions of ICAM-1 and VCAM-1 in an aspirin-sensitive and TP receptor-dependent manner. Both hematopoietic and nonhematopoietic TP receptors were essential for LTC4 to induce eosinophil recruitment. Thus, the autocrine and paracrine functions of thromboxane A2 act downstream of LTC4/type 2 cysLT receptor signaling on platelets to markedly amplify eosinophil recruitment through pulmonary vascular adhesion pathways. The findings suggest applications for TP receptor antagonists in cases of asthma with high levels of cysLT production.

Aspirin-Exacerbated Respiratory Disease Involves a Cysteinyl Leukotriene-Driven IL-33-Mediated Mast Cell Activation Pathway.

Aspirin-exacerbated respiratory disease (AERD), a severe eosinophilic inflammatory disorder of the airways, involves overproduction of cysteinyl leukotrienes (cysLTs), activation of airway mast cells (MCs), and bronchoconstriction in response to nonselective cyclooxygenase inhibitors that deplete homeostatic PGE2. The mechanistic basis for MC activation in this disorder is unknown. We now demonstrate that patients with AERD have markedly increased epithelial expression of the alarmin-like cytokine IL-33 in nasal polyps, as compared with polyps from aspirin-tolerant control subjects. The murine model of AERD, generated by dust mite priming of mice lacking microsomal PGE2 synthase (ptges(-/-) mice), shows a similar upregulation of IL-33 protein in the airway epithelium, along with marked eosinophilic bronchovascular inflammation. Deletion of leukotriene C4 synthase, the terminal enzyme needed to generate cysLTs, eliminates the increased IL-33 content of the ptges(-/-) lungs and sharply reduces pulmonary eosinophilia and basal secretion of MC products. Challenges of dust mite-primed ptges(-/-) mice with lysine aspirin induce IL-33-dependent MC activation and bronchoconstriction. Thus, IL-33 is a component of a cysLT-driven innate type 2 immune response that drives pathogenic MC activation and contributes substantially to AERD pathogenesis.

Thymic stromal lymphopoietin controls prostaglandin D2 generation in patients with aspirin-exacerbated respiratory disease.

BACKGROUND: Prostaglandin (PG) D2 is the dominant COX product of mast cells and is an effector of aspirin-induced respiratory reactions in patients with aspirin-exacerbated respiratory disease (AERD). OBJECTIVE: We evaluated the role of the innate cytokine thymic stromal lymphopoietin (TSLP) acting on mast cells to generate PGD2 and facilitate tissue eosinophilia and nasal polyposis in patients with AERD. METHODS: Urinary eicosanoid levels were measured in aspirin-tolerant control subjects and patients with AERD. Nasal polyp specimens from patients with AERD and chronic rhinosinusitis were analyzed by using quantitative PCR, Western blotting, and immunohistochemistry. Human cord blood-and peripheral blood-derived mast cells were stimulated with TSLP in vitro to assess PGD2 generation. RESULTS: Urinary levels of a stable PGD2 metabolite (uPGD-M) were 2-fold higher in patients with AERD relative to those in control subjects and increased further during aspirin-induced reactions. Peak uPGD-M levels during aspirin reactions correlated with reductions in blood eosinophil counts and lung function and increases in nasal congestion. Mast cells sorted from nasal polyps expressed PGD2 synthase (hematopoietic PGD2 synthase) mRNA at higher levels than did eosinophils from the same tissue. Whole nasal polyp TSLP mRNA expression correlated strongly with mRNA encoding hematopoietic PGD2 synthase (r = .75), the mast cell-specific marker carboxypeptidase A3 (r = .74), and uPGD-M (r = 0.74). Levels of the cleaved active form of TSLP were increased in nasal polyps from patients with AERD relative to those in aspirin-tolerant control subjects. Recombinant TSLP induced PGD2 generation by cultured human mast cells. CONCLUSIONS: Our study demonstrates that mast cell-derived PGD2 is a major effector of type 2 immune responses driven by TSLP and suggests that dysregulation of this innate system contributes significantly to the pathophysiology of AERD.

Cutting edge: Leukotriene C4 activates mouse platelets in plasma exclusively through the type 2 cysteinyl leukotriene receptor.

Leukotriene C4 (LTC4) and its extracellular metabolites, LTD4 and LTE4, mediate airway inflammation. They signal through three specific receptors (type 1 cys-LT receptor [CysLT1R], CysLT2R, and GPR99) with overlapping ligand preferences. In this article, we demonstrate that LTC4, but not LTD4 or LTE4, activates mouse platelets exclusively through CysLT2R. Platelets expressed CysLT1R and CysLT2R proteins. LTC4 induced surface expression of CD62P by wild-type mouse platelets in platelet-rich plasma (PRP) and caused their secretion of thromboxane A2 and CXCL4. LTC4 was fully active on PRP from mice lacking either CysLT1R or GPR99, but completely inactive on PRP from CysLT2R-null (Cysltr2(-/-)) mice. LTC4/CysLT2R signaling required an autocrine ADP-mediated response through P2Y12 receptors. LTC4 potentiated airway inflammation in a platelet- and CysLT2R-dependent manner. Thus, CysLT2R on platelets recognizes LTC4 with unexpected selectivity. Nascent LTC4 may activate platelets at a synapse with granulocytes before it is converted to LTD4, promoting mediator generation and the formation of leukocyte-platelet complexes that facilitate inflammation.

Prostaglandin E2 deficiency uncovers a dominant role for thromboxane A2 in house dust mite-induced allergic pulmonary inflammation.

Prostaglandin E(2) (PGE(2)) is an abundant lipid inflammatory mediator with potent but incompletely understood anti-inflammatory actions in the lung. Deficient PGE(2) generation in the lung predisposes to airway hyperresponsiveness and aspirin intolerance in asthmatic individuals. PGE(2)-deficient ptges(-/-) mice develop exaggerated pulmonary eosinophilia and pulmonary arteriolar smooth-muscle hyperplasia compared with PGE(2)-sufficient controls when challenged intranasally with a house dust mite extract. We now demonstrate that both pulmonary eosinophilia and vascular remodeling in the setting of PGE(2) deficiency depend on thromboxane A(2) and signaling through the T prostanoid (TP) receptor. Deletion of TP receptors from ptges(-/-) mice reduces inflammation, vascular remodeling, cytokine generation, and airway reactivity to wild-type levels, with contributions from TP receptors localized to both hematopoietic cells and tissue. TP receptor signaling ex vivo is controlled heterologously by E prostanoid (EP)(1) and EP(2) receptor-dependent signaling pathways coupling to protein kinases C and A, respectively. TP-dependent up-regulation of intracellular adhesion molecule-1 expression is essential for the effects of PGE(2) deficiency. Thus, PGE(2) controls the strength of TP receptor signaling as a major bronchoprotective mechanism, carrying implications for the pathobiology and therapy of asthma.

Prostaglandin E2 resistance in granulocytes from patients with aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is an inflammatory condition of the respiratory tract and is characterized by overproduction of leukotrienes (LT) and large numbers of circulating granulocyte-platelet complexes. LT production can be suppressed by prostaglandin E(2) (PGE(2)) and the cyclic AMP-dependent protein kinase A (PKA). OBJECTIVE: To determine if PGE(2)-dependent control of LT production by granulocytes is dysregulated in AERD. METHODS: Granulocytes from well-characterized patients with and without AERD were activated ex vivo and subjected to a range of functional and biochemical analyses. RESULTS: Granulocytes from subjects with AERD generated more LTB4 and cysteinyl LTs than did granulocytes from controls with aspirin-tolerant asthma and controls without asthma. When compared with controls, granulocytes from subjects with AERD had comparable levels of EP(2) protein expression and PGE(2)-mediated cAMP accumulation, yet were resistant to PGE(2)-mediated suppression of LT generation. Percentages of platelet-adherent neutrophils correlated positively with LTB4 generation and inversely with responsiveness to PGE(2)-mediated suppression of LTB(4). The PKA inhibitor H89 potentiated LTB4 generation by control granulocytes but was inactive in granulocytes from individuals with AERD and had no effect on platelet P-selectin induction. Both tonic PKA activity and levels of PKA catalytic gamma subunit protein were significantly lower in granulocytes from individuals with AERD relative to those from controls. CONCLUSIONS: Impaired granulocyte PKA function in AERD may lead to dysregulated control of 5-lipoxygenase activity by PGE(2), whereas adherent platelets lead to increased production of LTs, which contributes to the features of persistent respiratory tract inflammation and LT overproduction.

Chromosomal abnormalities and structural defects in fetuses with increased nuchal translucency at a Chinese tertiary medical center.

Objectives: To explore the pregnancy outcomes of fetuses with increased NT thickness. Methods: This was a retrospective study of fetuses with increased NT (≥95th centile) at 11-14 weeks of gestation between January 2020 and November 2020. Results: Among 264 fetuses with increased NT, the median of CRL and NT was 61.2 mm and 2.41 mm. Among them, 132 pregnancy women chose invasive prenatal diagnosis (43 cases of chorionic villus sampling (CVS), 89 cases of amniocentesis). Eventually, 16 cases of chromosomal abnormalities were discovered, including 6 cases (6.4%) of trisomy 21, 4 cases (3%) of trisomy 18, 1 case (0.8%) of 45, XO, 1 case (0.8%) of 47, XXY and 4 cases (3.03%) of CNV abnormalities. The major structural defects included hydrops (6.4%), cardiac defects (3%), and urinary anomalies (2.7%). The incidences of chromosomal abnormalities and structural defects in the NT < 2.5 mm group were 1.3 and 6%, while the incidences were 8.8 and 28.9% in the NT≥2.5 group. Conclusion: Increased NT was associated with high risk of chromosomal abnormalities and structural anomalies. Chromosomal abnormalities and structural defects could be detected when NT thickness was between 95th centile and 2.5 mm.

Study on the degradation and pyrolysis of 2-fluoromethcathinone.

Gas chromatography-mass spectrometry (GC-MS) is the first choice for law enforcement agencies in various countries to analyze new psychoactive substances (NPS) because of its advantages and complete databases. For synthetic cathinone-type NPS (SCat), alkalization and extraction processes before GC-MS analysis are essential. However, the base form of SCat is unstable, causing it to quickly degrade in solution and cause pyrolysis at the GC-MS injection inlet. In this study, we investigated the degradation of ethyl acetate and pyrolysis at the GC-MS injection inlet of 2-fluoromethcathinone (2-FMC), the most unstable SCat. Using gas chromatography-quadruple/time-of-flight mass spectrometry (GC-Q/TOF-MS) combined with the predicted data from theoretical calculations and the analysis of mass spectrometry (MS) fragmentation, the structures of 15 2-FMC degradation and pyrolysis products were identified. Among them, 11 products were produced during degradation, and six products were obtained from pyrolysis (two of which were the same as the degradation products). At the same time, the degradation and pyrolysis pathways of 2-FMC were provided. The balance between keto-enol and enamine-imine tautomerism triggered the primary degradation pathway of 2-FMC. The subsequent degradation started from the tautomer with a hydroxyimine structure, including imine hydrolysis, oxidation, imine-enamine tautomerism, intramolecular ammonolysis of halobenzene, and hydration to generate a series of degradation products. The secondary degradation reaction was the ammonolysis of ethyl acetate to yield N-[1-(2'-fluorophenyl)-1-oxopropan-2-yl]-N-methylacetamide and the byproduct, N-[1-(2'-fluorophenyl)-1-oxopropan-2-yl]-N-methylformamide. In the pyrolysis of 2-FMC, the major reactions were dehydrogenation, intramolecular ammonolysis of halobenzene, and defluoromethane. The achievements of this manuscript not only study 2-FMC degradation and pyrolysis but also lay the foundation for the study of SCat stability and their accurate analysis by GC-MS.

Prostaglandin E(2) exerts homeostatic regulation of pulmonary vascular remodeling in allergic airway inflammation.

Nonselective inhibition of PG synthesis augments inflammation in mouse models of airway disease, but the roles of individual PGs are not completely clarified. To investigate the role of PGE(2) in a mouse model of airway inflammation induced by a natural allergen, we used mice lacking the critical terminal synthetic enzyme, microsomal PGE(2) synthase (mPGES)-1. Mice lacking mPGES-1 (ptges(-/-) mice) and wild-type C57BL/6 controls were challenged intranasally with low doses of an extract derived from the house dust mite Dermatophagoides farinae (Der f). The levels of PGE(2) in the bronchoalveolar lavage fluids of Der f-treated ptges(-/-) mice were approximately 80% lower than the levels in wild-type controls. Der f-induced bronchovascular eosinophilia was modestly enhanced in the ptges(-/-) mice. Both Der f-treated strains showed similar increases in serum IgE and IgG1, as well as comparable levels of Th1, Th2, and Th17 cytokine production by Der f-stimulated spleen cells. These findings indicated that mPGES-1-derived PGE(2) was not required for allergen sensitization or development of effector T cell responses. Unexpectedly, the numbers of vascular smooth muscle cells and the thickness of intrapulmonary vessels were both markedly increased in the Der f-treated ptges(-/-) mice. These vascular changes were suppressed by the administration of the stable PGE(2) analog 16, 16-dimethyl PGE(2), or of selective agonists of the E-prostanoid (EP) 1, EP2, and EP3 receptors, respectively, for PGE(2). Thus, mPGES-1 and its product, PGE(2), protect the pulmonary vasculature from remodeling during allergen-induced pulmonary inflammation, and these effects may be mediated by more than one EP receptor.

Characterization of a novel human mast cell line that responds to stem cell factor and expresses functional FcεRI.

BACKGROUND: Studies of human mast cells (MCs) are constrained by the paucity of functional cell lines, the expense of maintaining MCs in culture, and technical complexities. OBJECTIVE: We derived and characterized a human MC line that arose spontaneously from a culture of nontransformed hematopoietic progenitor cells. METHODS: CD34(+) enriched mononuclear cells derived from a donor with aspirin-exacerbated respiratory disease were cultured for 8 weeks with stem cell factor and IL-6 and with IL-3 for the first week only. The cells (termed LUVA cells) survived and proliferated without further addition of any growth factors and have been maintained in culture for approximately 2 years. RESULTS: LUVA cells possess metachromatic cytoplasmic granules that are immunoreactive for tryptase, cathepsin G, and carboxypeptidase A3. They express transcripts encoding FcεRI, c-kit, chymase, tryptase, histidine decarboxylase, carboxypeptidase A3, and the type 1 receptor for cysteinyl leukotrienes. Flow cytometry confirmed uniform expression of FcεRI, c-kit, and FcγRII. FcεRI cross-linkage induced the release of ß-hexosaminidase, prostaglandin D(2), thromboxane A(2), and macrophage inflammatory protein 1ß. Immortalization was not associated with either a known genomic mutation of c-kit in the donor or a somatic mutation of c-kit within the cells, and it was not associated with c-kit autophosphorylation. CONCLUSIONS: LUVA cells are an immortalized human MC line that can be maintained without stem cell factor and display high levels of normally signaling c-kit and FcεRI. These cells will prove valuable for functional human MC studies.

YY1 affects the levels and function of fibulin-5 in ox-LDL-treated vascular smooth muscle cells.

Fibulin-5 is reportedly involved in the pathological process of atherosclerosis (AS) where low expression has been frequently observed in ruptured atherosclerotic plaques. The aim of the present study was to determine the effects of fibulin-5 on the responses of vascular smooth muscle cells (VSMC) to oxidized low-density lipoprotein (ox-LDL). The expression of fibulin-5 was studied in human aortic-VSMCs (HA-VSMCs) treated with ox-LDL. Fibulin-5 was first overexpressed by the transfection of Ov-Fibulin-5 plasmids in HA-VSMCs challenged with ox-LDL to investigate its influence on cell proliferation, migration and invasion using Cell Counting Kit-8, wound healing and Transwell assays. Yin Yang-1 (YY1) was bioinformatically predicted to bind to the promoter sites of fibulin-5, which was subsequently confirmed by dual-luciferase reporter gene assay. Fibulin-5 overexpression was able to suppress cell proliferation, invasion and migration, which was effectively reversed by YY1 silencing by the transfection of siRNA-Fibulin-5 plasmids which could induced fibulin-5 silencing. YY1 binding sites in the promoter region of fibulin-5 were identified and confirmed in vitro by chromatin immunoprecipitation assay and dual-luciferase reporter gene assay. The present results suggested that as a modulator of fibulin-5, YY1 alleviated ox-LDL-induced proliferation, invasion, migration and phenotypic transition from differentiated contractile phenotype to dedifferentiated phenotype in VSMCs. However, the mechanism underlying the YY1-mediated regulation of fibulin-5 expression needs to be confirmed further in vivo. Nevertheless, targeting fibulin-5 and YY1 could be further developed for AS therapy.

Structure identification and analysis of the suspected chemical precursor of 2-fluorodeschloroketamine and its decomposition products.

In this work, 1-[(2″-fluorophenyl)(methylimino)methyl]cyclopentan-1-ol (2-fluorodeschlorohydroxylimine) was identified as a suspected chemical precursor of 2-fluorodeschloroketamine (2-FDCK) using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-quadrupole/time-of-flight mass spectrometry (GC-Q/TOF-MS) and comparing the data with those of ketamine and its chemical precursor, hydroxylimine. Furthermore, the entire fragmentation pathway of 2-fluorodeschlorohydroxylimine was theorized from the GC-MS spectrum recorded using an electron ionization (EI) source, and the mechanisms and decomposition pathways of 2-fluorodeschlorohydroxylimine were elucidated. In protic solvents, the nitrogen atom in the C═N group of 2-fluorodeschlorohydroxylimine underwent a protonation reaction. Thereafter, the traces of water present in protic solvents promoted the hydrolysis of the protonated imine, and a carbon cation was obtained following the loss of methylamine. The carbon cation could follow the classical decomposition mechanism of imines and yield an α-hydroxyl ketone, which was the major decomposition product, (2'-fluorophenyl)(1″-hydroxycyclopentyl)methanone. The cation could also undergo a loop expansion rearrangement and yield another α-hydroxyl ketone, 2-(2'-fluorophenyl)-2-hydroxycyclohexan-1-one. The structures of the two aforementioned decomposition products were elucidated using several techniques including theoretical calculation, GC-MS, nuclear magnetic resonance (NMR), the prediction and assistance elucidation functions of ACDLabs-Structure Elucidator Suite, and the virtual separation technology of diffusion-ordered spectroscopy. The aforementioned study revealed important information about the chemical precursor of 2-FDCK and its decomposition. Furthermore, a set of methods for the qualitative analysis of 2-fluorodeschlorohydroxylimine were established, which facilitated accurate analysis of 2-fluorodeschlorohydroxylimine samples following decomposition or destruction.

Human airway mast cells proliferate and acquire distinct inflammation-driven phenotypes during type 2 inflammation.

Mast cells (MCs) play a pathobiologic role in type 2 (T2) allergic inflammatory diseases of the airway, including asthma and chronic rhinosinusitis with nasal polyposis (CRSwNP). Distinct MC subsets infiltrate the airway mucosa in T2 disease, including subepithelial MCs expressing the proteases tryptase and chymase (MCTC) and epithelial MCs expressing tryptase without chymase (MCT). However, mechanisms underlying MC expansion and the transcriptional programs underlying their heterogeneity are poorly understood. Here, we use flow cytometry and single-cell RNA-sequencing (scRNA-seq) to conduct a comprehensive analysis of human MC hyperplasia in CRSwNP, a T2 cytokine-mediated inflammatory disease. We link discrete cell surface phenotypes to the distinct transcriptomes of CRSwNP MCT and MCTC, which represent polarized ends of a transcriptional gradient of nasal polyp MCs. We find a subepithelial population of CD38highCD117high MCs that is markedly expanded during T2 inflammation. These CD38highCD117high MCs exhibit an intermediate phenotype relative to the expanded MCT and MCTC subsets. CD38highCD117high MCs are distinct from circulating MC progenitors and are enriched for proliferation, which is markedly increased in CRSwNP patients with aspirin-exacerbated respiratory disease, a severe disease subset characterized by increased MC burden and elevated MC activation. We observe that MCs expressing a polyp MCT-like effector program are also found within the lung during fibrotic diseases and asthma, and further identify marked differences between MCTC in nasal polyps and skin. These results indicate that MCs display distinct inflammation-associated effector programs and suggest that in situ MC proliferation is a major component of MC hyperplasia in human T2 inflammation.