Production Of CysLTs Research Articles

Endogenous PGD2 acting on DP2 receptor counter regulates Schistosoma mansoni infection-driven hepatic granulomatous fibrosis.

Identifying new molecular therapies targeted at the severe hepatic fibrosis associated with the granulomatous immune response to Schistosoma mansoni infection is essential to reduce fibrosis-related morbidity/mortality in schistosomiasis. In vitro cell activation studies suggested the lipid molecule prostaglandin D2 (PGD2) as a potential pro-fibrotic candidate in schistosomal context, although corroboratory in vivo evidence is still lacking. Here, to investigate the role of PGD2 and its cognate receptor DP2 in vivo, impairment of PGD2 synthesis by HQL-79 (an inhibitor of the H-PGD synthase) or DP2 receptor inhibition by CAY10471 (a selective DP2 antagonist) were used against the fibrotic response of hepatic eosinophilic granulomas of S. mansoni infection in mice. Although studies have postulated PGD2 as a fibrogenic molecule, HQL-79 and CAY10471 amplified, rather than attenuated, the fibrotic response within schistosome hepatic granulomas. Both pharmacological strategies increased hepatic deposition of collagen fibers - an unexpected outcome accompanied by further elevation of hepatic levels of the pro-fibrotic cytokines TGF-β and IL-13 in infected animals. In contrast, infection-induced enhanced LTC4 synthesis in the schistosomal liver was reduced after HQL-79 and CAY10471 treatments, and therefore, inversely correlated with collagen production in granulomatous livers. Like PGD2-directed maneuvers, antagonism of cysteinyl leukotriene receptors CysLT1 by MK571 also promoted enhancement of TGF-β and IL-13, indicating a key down-regulatory role for endogenous LTC4 in schistosomiasis-induced liver fibrosis. An ample body of data supports the role of S. mansoni-driven DP2-mediated activation of eosinophils as the source of LTC4 during infection, including: (i) HQL-79 and CAY10471 impaired systemic eosinophilia, drastically decreasing eosinophils within peritoneum and hepatic granulomas of infected animals in parallel to a reduction in cysteinyl leukotrienes levels; (ii) peritoneal eosinophils were identified as the only cells producing LTC4 in PGD2-mediated S. mansoni-induced infection; (iii) the magnitude of hepatic granulomatous eosinophilia positively correlates with S. mansoni-elicited hepatic content of cysteinyl leukotrienes, and (iv) isolated eosinophils from S. mansoni-induced hepatic granuloma synthesize LTC4 in vitro in a PGD2/DP2 dependent manner. So, our findings uncover that granulomatous stellate cells-derived PGD2 by activating DP2 receptors on eosinophils does stimulate production of anti-fibrogenic cysLTs, which endogenously down-regulates the hepatic fibrogenic process of S. mansoni granulomatous reaction - an in vivo protective function which demands caution in the future therapeutic attempts in targeting PGD2/DP2 in schistosomiasis.

Short-term high fat feeding induces inflammatory responses of tuft cells and mucosal barrier cells in the murine stomach.

Feeding mice with a high fat diet (HFD) induces inflammation and results in changes of gene expression and cellular composition in various tissues throughout the body, including the gastrointestinal tract. In the stomach, tuft cells expressing the receptor GPR120 are capable of sensing saturated long chain fatty acids (LCFAs) and thus may be involved in initiating mechanisms of mucosal inflammation. In this study, we assessed which cell types may additionally be affected by high fat feeding and which candidate molecular mediators might contribute to mucosa-protective immune responses. A high fat dietary intervention for 3 weeks caused an expansion of tuft cells that was accompanied by a higher frequency of mucosal mast cells and surface mucous cells which are a known source of the insult-associated cytokine interleukin 33 (IL-33). Our data demonstrate that both brush and mucosal mast cells comprise the enzyme ALOX5 and its activating protein FLAP and thus have the capacity for synthesizing leukotriene (LT). In HFD mice, several tuft cells showed a perinuclear colocalization of ALOX5 with FLAP which is indicative of an active LT synthesis. Monitoring changes in the expression of genes encoding elements of LT synthesis and signaling revealed that transcript levels of the leukotriene C4 synthase, LTC4S, catalyzing the first step in the biosynthesis of cysteinyl (cys) LTs, and the cysLT receptors, cysLTR2 and cysLTR3, were upregulated in mice on HFD. These mice also showed an increased expression level of IL-33 receptors, the membrane-bound ST2L and soluble isoform sST2, as well as the mast cell-specific protease MCPT1. Based on these findings it is conceivable that upon sensing saturated LCFAs tuft cells may elicit inflammatory responses which result in the production of cysLTs and activation of surface mucous cells as well as mucosal mast cells regulating gastric mucosal function and integrity.

The IRE1α Inhibitor KIRA6 Blocks Leukotriene Biosynthesis in Human Phagocytes

The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) has been linked to inflammation and lipid mediator production. Here we report that the potent IRE1α inhibitor, KIRA6, blocks leukotriene biosynthesis in human phagocytes activated with lipopolysaccharide (LPS) plus N-formyl-methionyl-leucyl-phenylalanine (fMLP) or thapsigargin (Tg). The inhibition affects both leukotriene B4 (LTB4) and cysteinyl leukotriene (cys-LTs) production at submicromolar concentration. Macrophages made deficient of IRE1α were still sensitive to KIRA6 thus demonstrating that the compound’s effect on leukotriene production is IRE1α-independent. KIRA6 did not exhibit any direct inhibitory effect on key enzymes in the leukotriene pathway, as assessed by phospholipase A2 (PLA2), 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S) enzyme activity measurements in cell lysates. However, we find that KIRA6 dose-dependently blocks phosphorylation of p38 and ERK, mitogen-activated protein kinases (MAPKs) that have established roles in activating cytosolic PLA2α (cPLA2α) and 5-LOX. The reduction of p38 and ERK phosphorylation is associated with a decrease in cPLA2α phosphorylation and attenuated leukotriene production. Furthermore, KIRA6 inhibits p38 activity, and molecular modelling indicates that it can directly interact with the ATP-binding pocket of p38. This potent and unexpected, non-canonical effect of KIRA6 on p38 and ERK MAPKs and leukotriene biosynthesis may account for some of the immune-modulating properties of this widely used IRE1α inhibitor.

Leukotriene C4 synthase is a novel PPARγ target gene, and leukotriene C4 and D4 activate adipogenesis through cysteinyl LT1 receptors in adipocytes

Leukotriene (LT) C4 synthase (LTC4S) catalyzes the conversion from LTA4 to LTC4, which is a proinflammatory lipid mediator in asthma and other inflammatory diseases. LTC4 is metabolized to LTD4 and LTE4, all of which are known as cysteinyl (Cys) LTs and exert physiological functions through CysLT receptors. LTC4S is expressed in adipocytes. However, the function of CysLTs and the regulatory mechanism in adipocytes remain unclear. In this study, we investigated the expression of LTC4S and production of CysLTs in murine adipocyte 3T3-L1 cells and their underlying regulatory mechanisms. Expression of LTC4S and production of LTC4 and CysLTs increased during adipogenesis, whereas siRNA-mediated suppression of LTC4S expression repressed adipogenesis by reducing adipogenic gene expression. The CysLT1 receptor, one of the two LTC4 receptors, was expressed in adipocytes. LTC4 and LTD4 increased the intracellular triglyceride levels and adipogenic gene expression, and their enhancement was suppressed by co-treatment with pranlukast, a CysLT1 receptor antagonist. Moreover, the expression profiles of LTC4S gene/protein during adipogenesis resembled those of peroxisome proliferator-activated receptor (PPAR) γ. LTC4S expression was further upregulated by treatment with troglitazone, a PPARγ agonist. Promoter-luciferase and chromatin immunoprecipitation assays showed that PPARγ directly bound to the PPAR response element of the LTC4S gene promoter in adipocytes. These results indicate that the LTC4S gene expression was enhanced by PPARγ, and LTC4 and LTD4 activated adipogenesis through CysLT1 receptors in 3T3-L1 cells. Thus, LTC4S and CysLT1 receptors are novel potential targets for the treatment of obesity.

Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically initiate lung type 2 inflammation.

Aeroallergen sensing by airway epithelial cells triggers pathogenic immune responses leading to type 2 inflammation, the hallmark of chronic airway diseases such as asthma. Tuft cells are rare epithelial cells and the dominant source of interleukin-25 (IL-25), an epithelial cytokine, and cysteinyl leukotrienes (CysLTs), lipid mediators of vascular permeability and chemotaxis. How these two mediators derived from the same cell might cooperatively promote type 2 inflammation in the airways has not been clarified. Here, we showed that inhalation of the parent leukotriene C4 (LTC4) in combination with a subthreshold dose of IL-25 led to activation of two innate immune cells: inflammatory type 2 innate lymphoid cell (ILC2) for proliferation and cytokine production, and dendritic cells (DCs). This cooperative effect led to a much greater recruitment of eosinophils and CD4+ T cell expansion indicative of synergy. Whereas lung eosinophilia was dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells required signaling through CysLT1R and partially CysLT2R. Tuft cell–specific deletion of Ltc4s, the terminal enzyme required for CysLT production, reduced lung inflammation and the systemic immune response after inhalation of the mold aeroallergen Alternaria; this effect was further enhanced by concomitant blockade of IL-25. Our findings identified a potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs.

Cysteinyl leukotriene induces eosinophil extracellular trap formation via cysteinyl leukotriene 1 receptor in a murine model of asthma

Purpose Eosinophils are one of the main cells responsible to the inflammatory response in asthma by the release of inflammatory molecules such as cytokines, reactive oxygen species (ROS), cytotoxic granule, eosinophil extracellular trap (EET), and lipid mediators as cysteinyl leukotriene (cysLT). The interconnections between these molecules are not fully understood. Here, we attempted to investigate the cysLT participation in the mechanisms of EET formation in an asthma model of OVA challenge. Materials and methods Before intranasal challenge with OVA, BALB/cJ mice were treated with a 5-lipoxygenase-activating protein (FLAP) inhibitor (MK-886), or with a cysLT1 receptor antagonist (MK-571) and the lung and bronchoalveolar lavage fluid (BALF) were analyzed. Results We showed that OVA-challenged mice treated with MK-886 or MK-571 had a decrease in inflammatory cells, goblet cells hyperplasia, and eosinophil peroxidase (EPO) activity in the airway. However, only OVA-challenged mice treated with MK-571 had an improvement in lung function. Also, treatments with MK-886 or MK-571 decreased Th2 cytokines levels in the airway. Moreover, we observed that OVA-challenged mice treated with MK-886 or MK-571 had a decrease in EET formation in BALF. We also verified that EET release was not due to cell death because the cell viability remained the same among the groups. Conclusion We revealed that the decrease in cysLT production or cysLT1 receptor inhibition by MK-886 or/and MK-571 treatments, respectively reduced EET formation in BALF, showing that cysLT regulates the activation process of EET release in asthma.

Emerging role of cysteinyl LTs in cancer.

Cysteinyl leukotrienes (CysLTs) are inflammatory lipid mediators that play a central role in the pathophysiology of several inflammatory diseases. Recently, there has been an increased interest in determining how these lipid mediators orchestrate tumour development and metastasis through promoting a pro-tumour micro-environment. Up-regulation of CysLTs receptors and CysLTs production is found in a number of cancers and has been associated with increased tumorigenesis. Understanding the molecular mechanisms underlying the role of CysLTs and their receptors in cancer progression will help investigate the potential of targeting CysLTs signalling for anti-cancer therapy. This review gives an overview of the biological effects of CysLTs and their receptors, along with current knowledge of their regulation and expression. It also provides a recent update on the molecular mechanisms that have been postulated to explain their role in tumorigenesis and on the potential of anti-CysLTs in the treatment of cancer.

First report on BaltCRP, a cysteine-rich secretory protein (CRISP) from Bothrops alternatus venom: Effects on potassium channels and inflammatory processes

CRISPs represent a family of cysteine-rich secretory proteins with molecular mass between 20 and 30 kDa and a highly conserved specific pattern of 16 cysteine residues. In this work, we isolated and characterized a novel CRISP from Bothrops alternatus venom, named BaltCRP, also evaluating its effects on different isoforms of potassium channels (Kv1.1; Kv1.2; Kv1.3; Kv1.4; Kv1.5; Kv2.1; Kv10.1 and Shaker) and on inflammatory processes in vivo. This toxin has a molecular mass of 24.4 kDa and pI around 7.8. Electrophysiological experiments using voltage clamp techniques showed that BaltCRP can affect the currents of Kv1.1; Kv1.3; Kv2.1 and Shaker channels. In addition, BaltCRP induced inflammatory responses characterized by an increase of leukocytes in the peritoneal cavity of mice, also stimulating the production of mediators such IL-6, IL-1β, IL-10, PGE2, PGD2, LTB4 and CysLTs. Altogether, these results demonstrated that BaltCRP can help understand the biological effects evoked by snake venom CRISPs, which could eventually lead to the development of new molecules with therapeutic potential.

Exosomes and cells from lung cancer pleural exudates transform LTC4 to LTD4, promoting cell migration and survival via CysLT1

Tumor-derived exosomes can modulate the cancer microenvironment and induce metastatic spread. Exosomes may carry enzymes for leukotriene (LT) biosynthesis, but the role of exosomal LTs has not been studied in cancer. We isolated exosomes and malignant cells from pleura exudates from 14 patients with non-small cell lung cancer. Lipidomic profiles, migration and apoptosis were determined. Both exosomes and primary cancer cells contained γ-glutamyl transpeptidase 1 (GGT-1) and avidly transformed exogenous LTC4 to pro-tumorigenic LTD4, for the cells to levels 100-fold above their endogenous CysLT production. This suggests that cancer cells promote their own survival via LTD4 if supplied with LTC4, which in the exudates was produced by monocytic cells. Furthermore, exosomes promoted migration of cancer cells, which was counteracted by the CysLT1 antagonist montelukast. Montelukast also induced apoptosis of cancer cells, and this was partially inhibited by exosomes. Our results demonstrate how cancer cells and exosomes, together with monocytic cells in lung cancer tissue, can produce high amounts of LTD4, to stimulate cancer cell migration and survival. This suggests that part of the pro-metastatic effect of exosomes is mediated by the leukotriene machinery, further supporting the use of CysLT1 antagonists for lung cancer therapy.

DMSO-induced drastic changes in cellular processes and epigenetic landscape in vitro

Classical FcεRI-induced mast cell (MC) activation causes synthesis of arachidonic acid (AA)–derived eicosanoids (leukotriene [LT] C4, prostaglandin [PG] D2, and thromboxane A2), which mediate vascular leak, bronchoconstriction, and effector cell chemotaxis. Little is known about the significance and regulation of eicosanoid generation in response to nonclassical MC activation mechanisms.We sought to determine the regulation and significance of MC-derived eicosanoids synthesized in response to IL-33, a cytokine critical to innate type 2 immunity.We used an ex vivo model of mouse bone marrow–derived mast cells and an IL-33–dependent in vivo model of aspirin-exacerbated respiratory disease (AERD).IL-33 potently liberates AA and elicits LTC4, PGD2, and thromboxane A2 production by bone marrow–derived mast cells. Unexpectedly, the constitutive function of COX-1 is required for IL-33 to activate group IVa cytosolic phospholipase A2 with consequent AA release for synthesis of all eicosanoids, including CysLTs. In contrast, COX-1 was dispensable for FcεRI-driven CysLT production. Inhibition of COX-1 prevented IL-33–induced phosphorylation of extracellular signal-related kinase, an upstream effector of cytosolic phospholipase A2, which was restored by exogenous PGH2, implying that the effects of COX-1 required its catalytic function. Administration of a COX-1–selective antagonist to mice completely prevented the generation of both PGD2 and LTC4 in a model of AERD in which MC activation is IL-33 driven.MC-intrinsic COX-1 amplifies IL-33–induced activation in the setting of innate type 2 immunity and might help explain the phenomenon of therapeutic desensitization to aspirin by nonselective COX inhibitors in patients with AERD.

A tiered approach to investigate the mechanism of anti-inflammatory activity of an herbal medicinal product containing a fixed combination of thyme herb and primula root extracts

BackgroundThe herbal medicinal product Bronchipret® TP film-coated tablets contains a fixed combination of thyme and primula dry extracts (BRO) and has long and successfully been used for the treatment of acute bronchitis. However, the underlying pharmacological mechanisms of action have not been determined so far. We report a tiered approach applying in vivo and in vitro studies to investigate the pharmacodynamic activity and underlying mechanisms of action, and to identify possible active ingredients contributing to the product’s pharmacodynamic activity.ResultsIn an LPS-induced rat model of bronchoalveolitis oral administration of BRO effectively ameliorated the influx of leukocytes into the lung. This was accompanied by reduced levels of leukotriene (LT) B4, cysteinyl-LTs (cysLT), and prostaglandin (PG) E2. We also found that BRO potently reduced the production of LTB4 in vitro in rat whole blood stimulated with the Ca2+-ionophore A23187 whereas the effect on PGE2 was much less pronounced. The transferability of these findings to human cells was assessed by measuring the effects of BRO on A23187-stimulated human monocytes and neutrophils. BRO and thyme extract were potent inhibitors of LTB4 and cysLT production. We further investigated the effects of BRO, thyme extract as well as single compounds of thyme extract on enzymatic activity of 5-lipoxygenase (5-LO). 5-LO activity was potently reduced by BRO and thyme extract. Of the single ingredients, thymoquinone and rosmarinic acid showed most potent inhibitory activity against 5-LO, with lower potency for thymol and carvacrol.ConclusionBRO attenuates inflammation-induced leukotriene formation in vivo and affects leukotriene biosynthesis in vitro via 5-LO inhibition. This inhibitory activity appears to be primarily related to the thyme extract component. However, none of the single ingredients tested seems to fully account for the activity of thyme extract alone. Rather, the interaction of different compounds seems to be required for its overall inhibitory effect on leukotriene production.

Prevalence of M75 Streptococcus pyogenes Strains Harboring slaA Gene in Patients Affected by Pediatric Obstructive Sleep Apnea Syndrome in Central Italy.

Recently we reported an association between pediatric obstructive sleep apnea syndrome (OSAS) and Group A streptococcus (GAS) sub-acute chronic tonsil colonization. We showed that GAS may contribute to tonsil hyperplasia via a streptolysin O (SLO)-dependent cysteinyl leukotrienes (CysLTs) production, which can trigger T and B cell proliferation. In the present study, we characterized the GAS strains isolated from pediatric OSAS patients in comparison with a panel of age and sex matched GAS strains unrelated to OSAS, but isolated in the same area and during the same period ranging from 2009 to 2013. We found that slaA gene, previously reported to be associated to CysLTs production pathway, was significantly associated to GAS OSAS strains. Moreover, the most numerous group (32%) of the GAS OSAS strains belonged to M75 type, and 6 out of 7 of these strains harbored the slaA gene. Multilocus Sequence Typing (MLST) experiments demonstrated that the clone emm75/ST49/ smeZ, slaA was associated to OSAS cases. In conclusion, we found an association between slaA gene and the GAS OSAS strains, and we showed that the clone emm75/ST49 harboring genes smeZ and slaA was exclusively isolated from patients affected by OSAS, thus suggesting that this genotype might be associated to the pathogenesis of OSAS, although further studies are needed to elucidate the possible role of SlaA in tonsil hypertrophy development.

Effects of leukotriene D4 nasal challenge on bronchial responsiveness and inflammation in asthmatic patients with allergic rhinitis.

In asthmatic patients with allergic rhinitis (AR), increased cysteinyl leukotrienes (CysLTs) production in the secretion of nasal mucosa has been associated with greater bronchial hyperresponsiveness (BHR) after nasal allergen challenge. However, the role of CysLTs in eliciting BHR after nasal allergen challenge has not been evaluated. The aim of this study is to evaluate the effect of LTD4 nasal challenge on BHR and inflammation in asthmatic patients with AR. In this self-controlled study, fifteen eligible consecutively recruited subjects underwent methacholine (Mch) bronchial provocation test before and 30 minutes after LTD4 nasal provocation test. The cumulative concentration of LTD4 inducing a 60% increase in nasal airway resistance (PC60NAR) was calculated. The mean values of cumulative doses inducing a 20% decrease in forced expiratory flow in one second (PD20FEV1) for Mch before and after nasal challenge were compared. Fractional exhaled nitric oxide (FeNO), differential inflammatory cell counts in nasal lavage and induced sputum before and after nasal challenge were compared. House dust mites were the major allergens accounting for 10/15 (66.7%) of asthmatic patients with AR. The PC60NAR for LT was (8.39±3.48)×10-3 mg·mL-1. The PD20FEV1 before and after nasal challenge was 3.05±3.81 and 2.70±3.81 µmol, respectively (P=0.45). The percentages of eosinophils were (38.36±23.14)% and (45.70±24.86)% in nasal lavage, and (17.51±11.05)% and (24.29±16.52)% in induced sputum before and 24 hours after nasal challenge. The neutrophil counts were (60.64±23.14)% and (53.30±24.46)% in nasal lavage, and (53.83±23.27)% and (56.19±22.28)% in induced sputum before and 24 hours after nasal challenge. The values of FeNO were 40 [35] and 43 [30] ppb before and 24 hours after nasal challenge. No severe adverse effects were reported during the tests. Although most asthmatic patients with AR were sensitive to LTD4 nasal challenge, LTD4 nasal provocation tests do not confer any major effect on BHR. LTD4 might not play a vital role in eliciting bronchial responsiveness induced by nasal allergen challenge.

Exhaled breath condensate pH and cysteinyl leukotriens in patients with chronic cough secondary to acid gastroesophageal reflux

Chronic cough is one of the most common clinical problems and it may be secondary to different stimuli and diseases, including low-level physical and chemical stimulation of the esophageal–bronchial reflex, suggestive of cough-reflex hyperresponsiveness, in patients with gastroesophageal reflux; however, it is still debated whether gastroesophageal reflux could induce airway inflammation and acidification. The aim of this study was to investigate airway pH and cysteynil-leukotrienes (Cys-LTs) concentration (a marker of airway inflammation) in exhaled breath condensate (EBC). Patients with chronic cough and for which all known causes, excluding gastroesophageal reflux, had been investigated and ruled out, were enrolled in the study. All patients underwent 24 h pH monitoring, and EBC was collected to assess pH and Cys-LTs concentration. Forty-five patients were included in the study and those with gastroesophageal reflux had significantly lower EBC-pH and higher concentration of EBC-Cys-LTs. There was a linear inverse correlation between EBC-pH values and EBC-Cys-LTs logarithmically transformed, and a multivariate analysis confirmed that the only significant determinat variable of EBC-Cys-LTs was the presence of gastroesophageal reflux. This study adds knowledge on possible mechanisms related to chronic cough associated with gastroesophageal reflux, which seems to be strictly dependent on airway acidification and the production of Cys-LTs, therefore suggesting an underlying neurogenic inflammation with tachykinins involvement.

Aspirin Intolerance: Experimental Models for Bed-to-Bench.

Aspirin is the oldest non-steroidal anti-inflammatory drug (NSAID), and it sometimes causes asthma-like symptoms known as aspirin-exacerbated respiratory disease (AERD), which can be serious. Unwanted effects of aspirin (aspirin intolerance) are also observed in patients with food-dependent exercise-induced anaphylaxis, a type I allergy disease, and aspirin-induced urticaria (AIU). However the target and the mechanism of the aspirin intolerance are still unknown. There is no animal or cellular model of AERD, because its pathophysiological mechanism is still unknown, but it is thought that inhibition of cyclooxygenase by causative agents leads to an increase of free arachidonic acid, which is metabolized into cysteinyl leukotrienes (cysLTs) that provoke airway smooth muscle constriction and asthma symptoms. As the bed-to-bench approach, to confirm the clinical discussion in experimental cellular models, we have tried to develop a cellular model of AERD using activated RBL-2H3 cells, a rat mast cell like cell line. Indomethacin (another NSAID and also causes AERD), enhances in vitro cysLTs production by RBL-2H3 cells, while there is no induction of cysLTs production in the absence of inflammatory activation. Since this suggests that all inflammatory cells with activation of prostaglandin and cysLT metabolism should respond to NSAIDs, and then I have concluded that aspirin intolerance should be separated from subsequent bronchoconstriction. Evidence about the cellular mechanisms of NSAIDs may be employed for development of in vitro AERD models as the approach from bench-to-bed.

CysLT1 Receptor Is Protective against Oxidative Stress in a Model of Irritant-Induced Asthma.

The bronchoconstrictive and proinflammatory properties of cysteinyl leukotrienes (cysLTs) in allergic asthma mediate their effects predominantly through the cysLT1 receptor (cysLT1R). However, the role of cysLTs and cysLT1R in innate immune-triggered asthma is largely unexplored. We explored the synthesis of cysLTs and cysLT1R as determinants of airway responses in an oxidative stress-induced model of irritant asthma. Wild-type (WT) mice exposed to 100 ppm Cl2 for 5 min had airway neutrophilia, increased cysLT production, and pulmonary expression of cysLT-related biosynthetic genes. CysLT1R-deficient (CysLTr1(-/-)) mice that were exposed to Cl2 demonstrated airway hyperresponsiveness to inhaled methacholine significantly greater than in WT BALB/c mice. Compared to WT mice, airway neutrophilia and keratinocyte chemoattractant production levels were higher in CysLTr1(-/-) mice and airway hyperresponsiveness was ameliorated using a granulocyte depletion Ab. CysLTr1(-/-) mice also demonstrated prolonged bronchial epithelial cell apoptosis following Cl2 WT mice showed increased antioxidant and NF erythroid 2-related factor 2 (Nrf2) gene expression, Nrf2 nuclear translocation in bronchial epithelial cells, and increased reduced glutathione/oxidized glutathione following Cl2 exposure whereas CysLTr1(-/-) mice did not. Furthermore, CysLTr1(-/-) mice demonstrated increased pulmonary E-cadherin expression and soluble E-cadherin shedding compared with WT mice. Loss of a functional cysLT1R results in aberrant antioxidant response and increased susceptibility to oxidative injury, apparently via a cysLT1R-dependent impairment of Nrf2 function.

Paediatric obstructive sleep apnoea syndrome (OSAS) is associated with tonsil colonisation by Streptococcus pyogenes

The involvement of pathogenic bacteria in obstructive sleep apnoea syndrome (OSAS) has yet to be elucidated. We investigated the possible role of group A streptococcus (GAS) in OSAS pathogenesis. In 40 tonsillectomized patients affected by OSAS and 80 healthy controls, significant (p < 0.0001) association of GAS with paediatric OSAS was found. Supernatant from streptolysin O (SLO)-producing GAS induced production of cysteinyl leukotrienes (CysLTs) in tonsil mononuclear cells (TMCs). CysLTs-treated TMCs showed significant (p < 0.05) proliferation of CD4+ T, CD19+ and CD19+CD27+CD38+ B lymphocytes. We discovered a SLO-dependent activation of CysLTs production through a pathway involving TOLL-like receptor 4 (TLR4), TIR-domain-containing adapter-inducing interferon-β (TRIF), Myeloid differentiation primary response gene 88 (MyD88), and p38 MAP Kinase. In conclusion, we hypothesise that GAS may contribute to paediatric tonsillar hyperplasia through CysLTs production induced by SLO, and this might explain its association with OSAS.

Adipokines and Cysteinyl Leukotrienes in the Pathogenesis of Asthma.

Background. The prevalence of obesity has increased dramatically over the last decades, and its association with asthma is being increasingly recognized. Aims. Our hypothesis is that increased leptin and decreased adiponectin levels in obese subjects play a direct role in regulating inflammation in asthmatics. We wanted to examine the hypothesis that cysteinyl leukotrienes (cys-LT), inflammatory mediators that are regulated by adipokines, are involved in the pathogenesis of asthma. Methods. We studied a population of asthmatics and nonasthmatics, who in turn were divided into obese and nonobese categories. We examined leptin and its ratio to adiponectin, in asthmatics and nonasthmatics, with and without obesity. In addition, we measured cys-LT levels in exhaled breath condensate (EBC) and in peripheral blood monocytes (PBM) in these groups. Results. Leptin levels were increased in obese asthmatics compared to obese nonasthmatics. The leptin/adiponectin (L/A) ratio was higher in obese asthmatics compared to obese nonasthmatics. EBC cys-LT levels were elevated in asthmatics compared to nonasthmatics. Discussion. Proinflammatory adipokines, released from adipose tissue, may promote an asthma phenotype through enhanced cys-LT production that may result in more prevalent and difficult to control airway disease.

The role of 12/15-lipoxygenase in production of selected eicosanoids in allergic airway inflammation

PurposeTo evaluate the role of 12/15-lipoxygenase (LOX) in regulation of synthesis of selected eicosanoids in mice sensitized and challenged with Dermatophagoides pteronyssinus (Dp) allergen extract. Materials and methodsBoth C57Bl and 12/15-LOX knockout mice were sensitized by 2 intraperitoneal injections and subsequently challenged by inhalation with Dp allergen extract. Sham sensitized and challenged mice were used as controls. Samples of bronchoalveolar lavage (BAL) were used for assessment of prostaglandin E2 (PGE2), cysteinyl leukotreienes (cysLT), lipoxin A4 (LXA4) and 15-hydroxyeicosatetraenoic acid (15-HETE) concentration using ELISA method. Whole lung samples were used for isolation of RNA and evaluation of selected genes involved in eicosanoid metabolism, including cyclooxygenase-2 (COX-2), 12/15-LOX, 5-LOX and 5-LOX activated protein (FLAP). ResultsAllergen-induced airway inflammation was associated with significant (9-fold, 95% CI 8.068–9.932-fold; p<0.05) up-regulation of 12/15-LOX in wild type but not in the 12/15-LOX knockout mice in which 12/15-LOX mRNA remained undetectable. Lack of 12/15-LOX was associated with significant attenuation of production of 15-HETE in response to allergen challenge. On the contrary, the greatest up-regulation of COX-2 after allergen exposure was demonstrated in the 12/15-LOX knockout mice (4.3-fold vs sham group) and was significantly greater than in the wild type counterparts (5.185-fold, 95% CI 4.723–6.309-fold; p<0.05 vs wild type mice). Also, allergen challenged 12/15-LOX knockout mice were characterized by greater production of PGE2 and cysLT. ConclusionThe 12/15-LOX plays an important role in the metabolism of eicosanoids in response to allergen-induced airway inflammation.

The efficacy of single-high dose inhaled corticosteroid versus oral prednisone treatment on exhaled leukotriene and 8-isoprostane levels in mild to moderate asthmatic children with asthma exacerbation

BackgroundThe anti-inflammatory effect of high-dose inhaled corticosteroids (ICS) in children with asthma exacerbation is unknown. We aimed to investigate the efficacy of single-high dose ICS versus oral prednisone treatment followed by a course of six day high-dose ICS or oral prednisone (P) treatment on the concentrations of Cys-LTs and 8-isoprostane levels in the exhaled breath condensate (EBC) of children with asthma exacerbation. MethodsNinety-four children with moderate–severe asthma exacerbation were evaluated with asthma scores, peak expiratory flow rate (PEF), forced expiratory volume in first second (FEV1) and exhaled Cys-LT and 8-isoprostane levels before and after treatment. EBC was collected from 52 patients before and four hours after treatment with inhaled fluticasone propionate (FP) (4000μg) or P and after six days of treatment with FP-1000μg/day or P. Cys-LTs and 8-isoprostane concentrations were determined using a specific immunoassay kit. ResultsBoth single high-dose FP (n=59) and p (n=35) treatment resulted in a significant improvement in asthma score (p<0.0001), PEF (p<0.0001), and FEV1 (p<0.0001). Cys-LT concentration in the EBC decreased significantly both after the initial treatment (p=0.001), and at the end of the six-day period in the FP group (p<0.0001). 8-Isoprostane concentration was lower only after six days of treatment with FP-1000μg/day in the FP group (p=0.023). There was a significant decrease in exhaled Cys-LTs after four hours (p=0.012) and six days of P treatment (p=0.018) in children with asthma exacerbation. ConclusionsHigh-dose ICS treatment may be useful in the treatment of children with asthma exacerbation. The effects start as early as after four hours. The suppression of Cys-LTs production contributes to the early effects. Suppression of both Cys-LTs and oxidants may favourably contribute to the effects observed later.