Nasal polyp antibody-secreting cells display proliferation signature in aspirin-exacerbated respiratory disease.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) causes nasal obstruction and olfactory dysfunction. Aspirin-exacerbated respiratory disease (AERD) is the triad of CRSwNP, asthma, and respiratory reactions to COX-1 inhibitors. Patients with AERD have elevated nasal IL-5 levels and high numbers of antibody-secreting cells (ASCs), including plasma cells and plasmablasts, in their polyp tissue; in addition, their nasal polyp (NP) IgE levels are correlated with disease severity and recurrence of nasal polyposis. OBJECTIVE: We sought to explore differences in the transcriptomic profile, activation markers, and IL-5Rα expression and function of NP ASCs from patients with AERD and CRSwNP. METHODS: NP tissue was collected from patients with AERD and CRSwNP and digested into single-cell suspensions. NP cells were analyzed for protein expression by mass cytometry. For IL-5Rα functional studies, plasma cells were purified and cultured in vitro with or without IL-5 and analyzed by bulk RNA sequencing. RESULTS: Compared with polyp tissue from patients with CRSwNP, polyp tissue from patients with AERD contained significantly more ASCs and had increased ASC expression of IL-5Rα. ASCs from patients with AERD expressed higher protein levels of B-cell activation and regulatory markers (CD40, CD19, CD32, and CD38) and the proliferation marker Ki-67. ASCs from patients with AERD also expressed more IL5RA, IGHE, and cell cycle- and proliferation-related transcripts (CCND2, MKI67, CDC25A, and CDC25B) than did ASCs from patients with CRSwNP. Stimulation of plasma cells from patients with AERD with IL-5 induced key cell cycle genes (CCND2 and PTP4A3), whereas IL-5 stimulation of ASCs from patients with CRSwNP induced few transcriptomic changes. CONCLUSION: NP tissue ASCs from patients with AERD express higher levels of functional IL-5Rα and markers associated with cell cycling and proliferation than do ASCs from patients with aspirin-tolerant CRSwNP.

Inflammatory mediators in nasal secretions of patients with nasal polyposis with and without aspirin sensitivity.

BACKGROUND: The aim of this cross-sectional study was to compare the levels of inflammatory mediators in nasal secretions in patients with aspirin-exacerbated respiratory disease (AERD) and in those with nasal polyposis (NP) without aspirin-sensitivity and to correlate nasal fluid mediator concentrations with clinical parameters of the disease. METHODS: A total of 30 patients with AERD, 30 chronic rhinosinusitis (CRS) with NP patients without aspirin sensitivity (CRSwNP), and 30 control subjects without inflammation of the nasal mucosa (C), selected for surgical treatment entered the study. The total nasal symptom score (TNSS), endoscopic score (ES), and Lund-Mackay score (LMS), were evaluated. The concentrations of eosinophil cationic protein (ECP), tryptase, heat shock protein 70 (HSP70), substance P and Clara cell protein 16 (CC16) were determined in nasal secretions. RESULTS: Higher concentrations of ECP, tryptase, and HSP70 were measured in the AERD patients than in the CRSwNP patients and the C group (p < .001; p < .001, respectively for all mediators). However, levels of CC16 were higher in the C group than in the AERD and CRSwNP groups (p < .001; p < .001, respectively). A positive correlation between the TNSS and CC16 and a negative one between CC16 and tryptase levels were found in the C group. The CRSwNP group showed positive correlations between ECP, HSP70, and tryptase and negative correlations between substance P, ES, and LMS, as well as between CC16 and tryptase levels. In the AERD group, we found a positive correlation between HSP70 and ECP levels and a negative correlation between the TNSS and CC16 concentration. CONCLUSION: The obtained results indicate the increased production of mediators of eosinophil and mast cell function, and the decreased production of biomarker of respiratory epithelial function in AERD patients. Clinical and biochemical parameters correlate in different ways in the AERD and CRSwNP patients.

Mechanisms by which dupilumab normalizes eicosanoid metabolism and restores aspirin-tolerance in AERD: A hypothesis.

Aspirin-exacerbated respiratory disease (AERD) is associated with overproduction of proinflammatory cysteinyl leukotrienes (CysLTs), defective generation of anti-inflammatory prostaglandin E2 (PGE2), and reduced expression of the EP2 receptor for PGE2. Reduced PGE2 synthesis results from the downregulation of inducible COX-2. Because PGE2 signaling via EP2 inhibits the 5-lipoxygenase/leukotriene C4 synthase-dependent pathway, the deficient levels of both PGE2 and EP2 likely contribute to the excessive baseline production of cysteinyl leukotrienes in patients with AERD compared with in patients with aspirin-tolerant asthma. The COX-2 pathway is regulated by an autocrine metabolic loop involving IL-1ß, IL-1 receptor type I, EP2, COX-2, membrane-bound PGE2 prostaglandin E2 synthase-1, and PGE2. Previous studies reported that this metabolic loop is dysregulated in patients with AERD. When the downexpressed EP2 receptor is normalized, the entire loop returns to its normal function. Cotreatment of airway cells from healthy subjects with IL-4 and IFN-γ induces alterations in the metabolic loop similar to those seen in patients with AERD. In these patients, IL-4, which is produced in excess in airways of patients with AERD, likely contributes to the alteration of normal functioning of the autocrine metabolic loop involving IL-1ß, IL-1 receptor type I, EP2, COX-2, membrane-bound PGE2 prostaglandin E2 synthase-1, and PGE2. We hypothesized that by blocking IL-4 action, dupilumab normalizes EP2 expression and restores the normal functioning of the COX-2 pathway autocrine metabolic loop, thereby normalizing the synthesis of PGE2 and restoring aspirin tolerance.

Levels of eicosanoids in nasal secretions associated with nasal polyp severity in chronic rhinosinusitis.

Severe nasal polyposis and mucosal inflammation, in patients with chronic rhinosinusitis (CRS) may include a dysregulated eicosanoid profile, but a clinical role for eicosanoids in CRS with nasal polyps (NP; CRSwNP) remains to be elucidated. This study focused on assessing levels and clinical implications of inflammatory mediators in nasal secretions and urine from patients with different NP severity or Aspirin Exacerbated Respiratory Disease (AERD). Levels of leukotrienes E4 and B4, prostaglandins D2 and E2 as well as 15(S)-hydroxyeicosatetraenoic acid were measured with enzyme immunoassays and cytokines with magnetic bead immunoassays. Patients with CRSwNP were subdivided based on NP score; CRSwNP-low (NP score ≤ 4, n = 11) or CRSwNP-high (NP score ≥ 5, n = 32) and compared to CRS without polyps (CRSsNP, n = 12), CRSwNP-AERD (n = 11) and individuals without CRS (n = 25). Smell test score, fractional exhaled nitric oxide (FeNO), blood eosinophils and Sinonasal outcome test-22 were assessed as clinical markers. Leukotriene E4, prostaglandin D2 and 15(S)-hydroxyeicosatetraenoic acid in nasal secretions correlated with NP score. Nasal leukotriene E4 also correlated with FeNO and smell test score, with highest levels found in CRSwNP-AERD. Levels of prostaglandin D2 in nasal secretion as well as urinary levels of the prostaglandin D2 metabolite 11ß-prostaglandin F2α differed between CRSNP-high and CRSwNP-low. Urinary 11ß-prostaglandin F2α was associated with asthma comorbidity whereas a similar association with prostaglandin D2 in nasal secretions was not observed. In conclusion, subdividing patients based on NP severity in combination with analysis of eicosanoids in non-invasively collected nasal secretions, may have clinical implications when assessing CRS disease severity.

Transcriptome Analysis Identifies Doublesex and Mab-3 Related Transcription Factor (DMRT3) in Nasal Polyp Epithelial Cells of Patients Suffering from Non-Steroidal Anti-Inflammatory Drug-Exacerbated Respiratory Disease (AERD).

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is a syndrome characterised by chronic rhinosinusitis, nasal polyps, asthma and aspirin intolerance. An imbalance of eicosanoid metabolism with anover-production of cysteinyl leukotrienes (CysLTs) has been associated with AERD. However, the precise mechanisms underlying AERD are unknown. OBJECTIVE: To establish the transcriptome of the nasal polyp airway epithelial cells derived from AERD patients to discover gene expression patterns in this disease. METHODS: Nasal airway epithelial cells were isolated from 12 AERD polyps and 8 AERD non-polyp nasal mucosa samples as controls from the same subjects. Utilising the Illumina HiSeq 2500 platform, RNA samples were sequenced. Potential gene candidate DMRT3 was selected from the differentially-expressed genes for validation. RESULTS: Comparative transcriptome profiling of nasal epithelial cells was accomplished in AERD. A total of 20 genes had twofold mean regulation expression differences or greater. In addition, 8 genes were upregulated, including doublesex and mab-3 related transcription factor 3 (DMRT3), and 12 genes were downregulated. Differentially regulated genes comprised roles in inflammation, defence and immunity. Metabolic process and embryonic development pathways were significantly enriched. Enzyme-linked immune sorbent assay (ELISA) results of DMRT3 in AERD patients were significantly upregulated compared to controls (p = 0.03). Immunohistochemistry (IHC) of AERD nasal polyps localised DMRT3 and was predominantly released in the airway epithelia. CONCLUSION: Findings suggest that DMRT3 could be potentially involved in nasal polyp development in AERD patients. Furthermore, several genes are downregulated, hinting at the dedifferentiation phenomenon in AERD polyps. However, further studies are imperative to confirm the exact mechanism of polyp formation in AERD patients.

Aspirin Actions in Treatment of NSAID-Exacerbated Respiratory Disease.

Non-steroidal Anti-inflammatory drugs (NSAID)-exacerbated respiratory disease (N-ERD) is characterized by nasal polyposis, chronic rhinosinusitis, adult-onset asthma and hypersensitive reactions to cyclooxygenase-1 (COX-1) inhibitors. Among the available treatments for this disease, a combination of endoscopic sinus surgery followed by aspirin desensitization and aspirin maintenance therapy has been an effective approach. Studies have shown that long-term aspirin maintenance therapy can reduce the rate of nasal polyp recurrence in patients with N-ERD. However, the exact mechanism by which aspirin can both trigger and suppress airway disease in N-ERD remains poorly understood. In this review, we summarize current knowledge of aspirin effects in N-ERD, cardiovascular disease, and cancer, and consider potential mechanistic pathways accounting for the effects of aspirin in N-ERD.

Activation of the 15-lipoxygenase pathway in aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), and an intolerance of medications that inhibit cyclooxygenase-1. Patients with AERD have more severe upper and lower respiratory tract disease than do aspirin-tolerant patients with CRSwNP. A dysregulation in arachidonic acid metabolism is thought to contribute to the enhanced sinonasal inflammation in AERD. OBJECTIVE: Our aim was to utilize an unbiased approach investigating arachidonic acid metabolic pathways in AERD. METHODS: Single-cell RNA sequencing (10× Genomics, Pleasanton, Calif) was utilized to compare the transcriptional profile of nasal polyp (NP) cells from patients with AERD and patients with CRSwNP and map differences in the expression of select genes among identified cell types. Findings were confirmed by traditional real-time PCR. Lipid mediators in sinonasal tissue were measured by mass spectrometry. Localization of various proteins within NPs was assessed by immunofluorescence. RESULTS: The gene encoding for 15-lipooxygenase (15-LO), ALOX15, was significantly elevated in NPs of patients with AERD compared to NPs of patients with CRSwNP (P < .05) or controls (P < .001). ALOX15 was predominantly expressed by epithelial cells. Expression levels significantly correlated with radiographic sinus disease severity (r = 0.56; P < .001) and were associated with asthma. The level of 15-oxo-eicosatetraenoic acid (15-Oxo-ETE), a downstream product of 15-LO, was significantly elevated in NPs from patients with CRSwNP (27.93 pg/mg of tissue) and NPs from patients with AERD (61.03 pg/mg of tissue) compared to inferior turbinate tissue from controls (7.17 pg/mg of tissue [P < .001]). Hydroxyprostaglandin dehydrogenase, an enzyme required for 15-Oxo-ETE synthesis, was predominantly expressed in mast cells and localized near 15-LO+ epithelium in NPs from patients with AERD. CONCLUSIONS: Epithelial and mast cell interactions, leading to the synthesis of 15-Oxo-ETE, may contribute to the dysregulation of arachidonic acid metabolism via the 15-LO pathway and to the enhanced sinonasal disease severity observed in AERD.

Steroids Inhibit Eosinophil Accumulation and Downregulate Hematopoietic Chemotaxic Prostaglandin D2 Receptor in Aspirin-Exacerbated Respiratory Disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by eosinophilic rhinosinusitis, nasal polyposis, aspirin sensitivity, and asthma. Aims/Objectives: This study aims to identify a mechanism to target for the future treatment of AERD via the elucidation of the effect of systemic steroids on the expression of hematopoietic prostaglandin D2 synthase (HPGDS) and chemotaxic prostaglandin D2 (DP2) receptor relative to eosinophil activation in the nasal polyps of patients with AERD. MATERIALS AND METHODS: Among 37 patients undergoing endoscopic sinus surgery, 28 received systemic steroids preoperatively. Nasal polyps were harvested from all 37 patients. After routine processing of paraffin sections, immunohistochemistry was performed using specific antibodies for HPGDS, eosinophil peroxidase (EPX), and DP2. RESULTS: Expression of HPGDS, DP2, and EPX by eosinophils was higher and more frequent in patients with non-preoperative steroid therapy. Likewise, HPGDS and DP2 were highly expressed in activated eosinophils in the nasal polyps, but not in normal eosinophils. CONCLUSION AND SIGNIFICANCE: This study provides clear evidence that systemic steroid therapy inhibits eosinophil activation and decreases HPGDS and DP2 expression in patients with AERD, indicating a reduction in prostaglandin D2 production and hence control hyperplasia of nasal polyps.

Roles of cysteinyl leukotrienes and their receptors in immune cell-related functions.

The cysteinyl leukotrienes (cys-LTs), leukotriene C4, (LTC4), LTD4, and LTE4, are lipid mediators of inflammation. LTC4 is the only intracellularly synthesized cys-LT through the 5-lipoxygenase and LTC4 synthase pathway and after transport is metabolized to LTD4 and LTE4 by specific extracellular peptidases. Each cys-LT has a preferred functional receptor in vivo; LTD4 to the type 1 cys-LT receptor (CysLT1R), LTC4 to CysLT2R, and LTE4 to CysLT3R (OXGR1 or GPR99). Recent studies in mouse models revealed that there are multiple regulatory mechanisms for these receptor functions and each receptor plays a distinct role as observed in different mouse models of inflammation and immune responses. This review focuses on the integrated host responses to the cys-LT/CysLTR pathway composed of sequential ligands with preferred receptors as seen from mouse models. It also discusses potential therapeutic targets for LTC4 synthase, CysLT2R, and CysLT3R.

IL-4/IFN-γ inflammatory cytokine profile induces a deficient regulation of the IL-1ß/IL-1RI/EP2/COX-2 pathway in nasal mucosa.

BACKGROUND: We hypothesized that the peculiar mixed interleukin-4 (IL-4/Th2) and interferon gamma INF-γ (INF-γ/Th1) inflammatory milieu found in the airways of patients with aspirin-exacerbated respiratory disease (AERD) is responsible for the altered regulation of the IL-1ß/IL-1RI-/EP2/COX-2 autocrine loop also found in these patients. The objective of the study is to demonstrate that IL-4 and INF-γ cytokines, are capable of inducing in healthy nasal mucosa (NM) the dysregulation of the autocrine loop of COX reported in AERD. SUBJECTS AND METHODS: Fibroblasts were obtained from NM (n = 8). To evaluate the role of IL-4 and IFN-γ on the autocrine loop, fibroblasts were incubated with or without IL-1ß, in the presence or absence of IL-4 and/or IFN-γ for 48 h. After this period, the expression of EP2, EP3, EP4, IL-1RI, COX-2 and mPGES-1 was measured by Western blot. RESULTS: Stimulation of fibroblasts with IL-1ß significantly increased the expression of EP2, but had no effects on EP3 and EP4 expression Incubation with IL-4 or IFN-γ alone was not able to modify the expression of any of the components of the autocrine loop. In contrast, co-treatment with IL-4 and IFN-γ was able to significantly inhibit IL-1ß-induced EP2, IL-1RI, COX-2 and mPGES-1. CONCLUSION: These results suggest that the mixed Th1/Th2 inflammatory pattern found in the airways of AERD patients might be responsible for the altered regulation of the COX pathway also reported in these asthma patients.

Prostaglandin E2 decrease in induced sputum of hypersensitive asthmatics during oral challenge with aspirin.

BACKGROUND: A special regulatory role for prostaglandin E2 (PGE2 ) has been postulated in nonsteroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD). OBJECTIVE: To investigate the effect of systemic aspirin (acetylsalicylic acid) administration on airway PGE2 biosynthesis in induced sputum supernatant (ISS) among subjects with NERD or aspirin-tolerant asthma with chronic rhinosinusitis with nasal polyposis (ATA-CRSwNP), as well as healthy controls (HC). METHODS: Induced sputum (IS) was collected from patients with NERD (n = 26), ATA-CRSwNP (n = 17), and HC (n = 21) at baseline and after aspirin challenge. Sputum differential cell count and IS supernatant (ISS) levels of prostanoids, PGE2 , 8-iso-PGE2 , tetranor-PGE-M, 8-iso-PGF2 α, and leukotriene C4 , D4 , and E4 , were determined using mass spectrometry. Urinary excretion of LTE4 was measured by ELISA. RESULTS: NERD subjects had elevated sputum eosinophilic count as compared to ATA-CRSwNP and HC (median NERD 9.1%, ATA-CRSwNP 2.1%, and HC 0.4%; P < 0.01). Baseline ISS levels of PGE2 were higher in asthmatics as compared to HC at baseline (NERD vs HC P = 0.04, ATA-CRSwNP vs HC P < 0.05). Post-challenge ISS levels of PGE2 compared to baseline significantly decreased in NERD and HC (P < 0.01 and P = 0.01), but not in ATA-CRSwNP. In NERD, a similar decrease in PGE2 as in HC resulted from 2.8 times lower dose of aspirin. CONCLUSION: Aspirin-precipitated bronchoconstriction is associated with a decrease in airway PGE2 biosynthesis. These results support the mechanism of PGE2 biosynthesis inhibition as a trigger for bronchoconstriction in NERD.

Sinus Surgery Is Associated with a Decrease in Aspirin-Induced Reaction Severity in Patients with Aspirin Exacerbated Respiratory Disease.

BACKGROUND: Nasal polyps influence the burden of aspirin-exacerbated respiratory disease (AERD) by contributing to eicosanoid production. AERD is diagnosed through graded aspirin challenges. It is not known how sinus surgery affects aspirin challenge outcomes. OBJECTIVE: To investigate the effects of endoscopic sinus surgery (ESS) on aspirin-induced reaction severity and on the levels of eicosanoids associated with these reactions. METHODS: Twenty-eight patients with AERD were challenged with aspirin before and 3 to 4 weeks after ESS. Respiratory parameters and plasma and urine levels of eicosanoids were compared before and after challenges. RESULTS: Before ESS, AERD diagnosis was confirmed in all study patients by aspirin challenges that resulted in hypersensitivity reactions. After ESS, reactions to aspirin were less severe in all patients and 12 of 28 patients (43%, P < .001) had no detectable reaction. A lack of clinical reaction to aspirin was associated with lower peripheral blood eosinophilia (0.1 K/µL [interquartile range (IQR) 0.1-0.3] vs 0.4 K/µL [IQR 0.2-0.8]; P = .006), lower urinary leukotriene E4 levels after aspirin challenge (98 pg/mg creatinine [IQR 61-239] vs 459 pg/mg creatinine [IQR 141-1344]; P = .02), and lower plasma prostaglandin D2 to prostaglandin E2 ratio (0 [±0] vs 0.43 [±0.2]; P = .03), compared with those who reacted. CONCLUSIONS: Sinus surgery results in decreased aspirin sensitivity and a decrease in several plasma and urine eicosanoid levels in patients with AERD. Diagnostic aspirin challenges should be offered to patients with suspected AERD before ESS to increase diagnostic accuracy. Patients with established AERD could undergo aspirin desensitizations after ESS as the severity of their aspirin-induced hypersensitivity reactions lessens.

Endocannabinoid receptor CB2R is significantly expressed in aspirin-exacerbated respiratory disease: a pilot study.

BACKGROUND: The endocannabinoid system represents a highly conserved, innate signaling network with direct and indirect control of eicosanoid-mediated inflammation. Activation of the type 2 cannabinoid receptor (CB2R) leads to decreased type 2 inflammation and reduced production of arachidonic acid (AA). Given that altered AA metabolism is associated with aspirin-exacerbated respiratory disease (AERD), we hypothesized that expression of the CB2R gene CNR2 is increased in AERD. METHODS: Nasal polyps from consecutive patients undergoing endoscopic sinus surgery for AERD or allergic fungal rhinosinusitis (AFRS) were prospectively evaluated. Control sphenoid mucosa was collected from patients undergoing endoscopic skull base procedures. Expression and localization of endocannabinoid receptors were evaluated by quantitative reverse transcript-polymerase chain reaction (qRT-PCR) and immunohistochemistry. A 2-group unpaired t test with unequal variances was used to evaluate group differences. RESULTS: Thirteen subjects were included in this pilot study, including 5 controls, 5 AFRS patients, and 3 AERD patients. Upregulated expression of CNR2 was detected in subjects with AERD vs both AFRS (p = 0.049) and controls (p = 0.047), with a mean increase of 5.2-fold. No significant differences in expression of the CB1R gene CNR1 were detected between control and AFRS groups. Immunohistochemistry predominantly localized CB1R and CB2R expression to the surface epithelium in all subjects. CONCLUSION: The endocannabinoid system is an emerging immunomodulatory network that may be involved in AERD. This is the first study of CB2R in sinonasal disease, showing significantly increased transcription in nasal polyps from subjects with AERD. Additional study is warranted to further evaluate the contribution and therapeutic potential of this novel finding in chronic rhinosinusitis.

Aspirin-Exacerbated Respiratory Disease.

Aspirin-exacerbated respiratory disease (AERD) is characterized by the triad of asthma, sinonasal polyposis, and aspirin intolerance. The hallmark of the disease is baseline overproduction of cysteinyl leukotrienes via the 5-lipoxygenase pathway, exacerbated by ingestion of aspirin. Patients with AERD have high rates of recidivistic polyposis following sinus surgery, although the improvement in quality of life following surgery is similar to aspirin-tolerant patients. The diagnosis is secured by a positive aspirin provocation test, usually administered by a medical allergist. Aspirin therapy is a unique treatment consideration for patients with AERD.

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-tobench 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.

[Induced sputum supernatant prostaglandin E2 during oral aspirin challenge of asthmatic patients with and without aspirin hypersensitivity and healthy controls--pilot study]. / Wplyw dawki prowokacyjnej aspiryny na poziom prostaglandyny E2 w plwocinie indukowanej u chorych na astme z i bez nadwrazliwosci na aspiryne oraz u osób zdrowych--badanie pilotazowe.

The aim of this pilot study was to evaluate changes in the concentration of prostaglandin E2 (PGE2) in induced sputum supernatant in 3 groups: sub- jects with NSAID-exacerbated respira- tory disease (NERD), aspirin tolerant asthma (ATA) and healthy controls (HC), before and after oral aspirin chal- lenge test. The study was conducted in the years 2014-2015 at the Clinical Department of the Pulmonology Clinic at the University Hospital in Cracow. 43 patients were enrolled in the study (NERD - n = 15, ATA - n = 15 and HC - n = 13). All of them underwent a placebo-controlled oral aspirin challenge. Sputum was induced 24 hours before the challenge and immediately after the test. Induced sputum was processed in order to obtain cystospin slides to depict inflammatory cell patterns and supernatants, in which PGE2 was measured. The concentration of PGE2 was determined using mass spectrometry coupled with gas chromatography (gas chromatography/mass spectrometry - GC/MS). After aspirin challenge, the concentration of PGE2 in induced sputum supernatant decreased in both asthmatics hypersensitive to aspirin (p = 0.01) and those who tolerated aspirin well (p = 0.17). The change in the healthy control group was not statistically significant. These results support the cyclooxygenase theory of PGE2 inhibition by aspirin. However, the mechanism of bronchoconstriction after aspirin administration alone in patients with NSAID-exacerbated respiratory disease remains unclear.

Low E-prostanoid 2 receptor levels and deficient induction of the IL-1ß/IL-1 type I receptor/COX-2 pathway: Vicious circle in patients with aspirin-exacerbated respiratory disease.

BACKGROUND: We hypothesized that the 2 reported alterations in aspirin-exacerbated respiratory disease (AERD), reduced expression/production of COX-2/prostaglandin (PG) E2 and diminished expression of E-prostanoid (EP) 2 receptor, are closely linked. OBJECTIVE: We sought to determine the mechanisms involved in the altered regulation of the COX pathway in patients with AERD. METHODS: Fibroblasts were obtained from nasal mucosa; samples of control subjects (NM-C, n = 8) and from nasal polyps from patients with aspirin-exacerbated respiratory disease (NP-AERD, n = 8). Expression of the autocrine loop components regulating PGE2 production and signaling, namely IL-1 type I receptor (IL-1RI), COX-2, microsomal prostaglandin E synthase 1 (mPGES-1), and EP receptors, was assessed at baseline and after stimulation with IL-1ß, PGE2, and specific EP receptor agonists. RESULTS: Compared with NM-C fibroblasts, basal expression levels of IL-1RI and EP2 receptor were lower in NP-AERD fibroblasts. IL-1ß-induced IL-1RI, COX-2, and mPGES-1 expression levels were also lower in these cells. Levels of IL-1RI positively correlated with COX-2 and mPGES-1 expression in both NM-C and NP-AERD fibroblasts. Incubation with either exogenous PGE2 or selective EP2 agonist significantly increased expression of IL-1RI in NM-C fibroblasts and had hardly any effect on NP-AERD fibroblasts. Alterations in IL-1RI, COX-2, and mPGES-1 expression that were found in NP-AERD fibroblasts were corrected when EP2 receptor expression was normalized by transfection of NP-AERD fibroblasts. CONCLUSION: Altered expression of EP2 in patients with AERD contributes to deficient induction of IL-1RI, reducing the capacity of IL-1ß to increase COX-2 and mPGES-1 expression, which results in low PGE2 production. This impairment in the generation of PGE2 subsequently reduces its ability to induce IL-1RI.

Systemic expression of inflammatory mediators in patients with chronic rhinosinusitis and nasal polyps with and without Aspirin Exacerbated Respiratory Disease.

BACKGROUND: Systemic reactions are related to the pathogenesis of Aspirin Exacerbated Respiratory Disease (AERD). With this work we wanted to study the changes in the systemic levels of inflammatory mediators in both baseline and after oral aspirin challenge in patients with and without AERD. METHODS: Patients with nasal polyposis and asthma with AERD (n=20) and without (n=18) were orally challenged with aspirin in a single-blind placebo controlled study. Serum samples and urine were collected before and 6h after placebo and aspirin oral challenges. Serum levels of inflammatory mediators were assayed by using the Luminex technology and ELISA. The concentrations of 9-alpha, 11-beta prostaglandin F2, and leukotriene E4 (uLTE4) were measured in urine samples by ELISA. The expression of T-cell surface markers was analyzed in peripheral blood mononuclear cells isolated before and after the challenges. RESULTS: AERD patients showed significantly higher baseline levels of s-IL-5R-alpha, uLTE4 and percentage of CD4(+)CD25(+)CD127(pos) and CD4(+)CD45RA(-)CD45RO(+) but decreased levels of TGF-ß1 and number of CD4(+)CD25(+)CD127(neg) cells. Aspirin challenge induced the release of uLTE4, IL-6 and increased the number of CD4(+)CD45RA(-)CD45RO(+) memory T-cells only in AERD patients but failed to reduce the levels of sCD40L as observed in non-AERD subjects. Further, IL-8 and sIL-5R-alpha levels directly correlated with the PD20ASA and the effects of aspirin on IL-6 and number of memory T-cells was more pronounced in subjects showing more strong reaction (bronchial and nasal). CONCLUSIONS: AERD patients have a differential baseline inflammatory pattern that supports the role inflammation as underlying mechanism of the disease. Systemic response to oral aspirin challenge was related to an increase in serum IL-6 and the number of circulating memory T-cells in AERD patients.

Platelet activation markers overexpressed specifically in patients with aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by respiratory reactions on ingestion of COX-1 inhibitors and cysteinyl leukotriene overproduction. The hypersensitivity reaction is induced by low doses of aspirin that inhibit COX-1 in platelets. OBJECTIVE: We sought to explore the role of platelets in the pathogenesis of AERD in patients under stable conditions and during an aspirin challenge test. METHODS: Stable patients with AERD (n = 30), aspirin-tolerant asthma (ATA; n = 21), or idiopathic chronic eosinophilic pneumonia (n = 10) were enrolled. Platelet activation was estimated based on expression levels of P-selectin (CD62P), CD63, CD69, and GPIIb/IIIa (PAC-1) in peripheral platelets; percentages of circulating platelet-adherent leukocytes; and plasma levels of soluble P-selectin (sP-selectin) and soluble CD40 ligand (sCD40L). RESULTS: In the stable condition, expression of all surface markers on platelets, the percentage of platelet-adherent eosinophils, and the plasma levels of sP-selectin and sCD40L were significantly higher in patients with AERD compared with those in patients with ATA. P-selectin and CD63 expression on platelets and plasma sP-selectin and sCD40L levels were positively correlated with the percentage of platelet-adherent eosinophils. Among these markers, P-selectin expression and plasma sP-selectin levels positively correlated with urinary concentrations of leukotriene E4. Additionally, plasma sP-selectin and sCD40L levels were negatively correlated with lung function. In contrast, platelet activation markers in patients with AERD did not change during the aspirin challenge test. CONCLUSION: Peripheral platelets were activated more in patients with stable AERD compared with those in patients with stable ATA, patients with idiopathic chronic eosinophilic pneumonia, and control subjects. Platelet activation was involved in cysteinyl leukotriene overproduction and persistent airflow limitations in patients with AERD.

Impaired E Prostanoid2 Expression and Resistance to Prostaglandin E2 in Nasal Polyp Fibroblasts from Subjects with Aspirin-Exacerbated Respiratory Disease.

Recurrent, rapidly growing nasal polyps are hallmarks of aspirin-exacerbated respiratory disease (AERD), although the mechanisms of polyp growth have not been identified. Fibroblasts are intimately involved in tissue remodeling, and the growth of fibroblasts is suppressed by prostaglandin E2 (PGE2), which elicits antiproliferative effects mediated through the E prostanoid (EP)2 receptor. We now report that cultured fibroblasts from the nasal polyps of subjects with AERD resist this antiproliferative effect. Fibroblasts from polyps of subjects with AERD resisted the antiproliferative actions of PGE2 and a selective EP2 agonist (P < 0.0001 at 1 µM) compared with nasal fibroblasts from aspirin-tolerant control subjects undergoing polypectomy or from healthy control subjects undergoing concha bullosa resections. Cell surface expression of the EP2 receptor protein was lower in fibroblasts from subjects with AERD than in fibroblasts from healthy control subjects and aspirin-tolerant subjects (P < 0.01 for both). Treatment of the fibroblasts with trichostatin A, a histone deacetylase inhibitor, significantly increased EP2 receptor mRNA in fibroblasts from AERD and aspirin-tolerant subjects but had no effect on cyclooxygenase-2, EP4, and microsomal PGE synthase 1 (mPGES-1) mRNA levels. Histone acetylation (H3K27ac) at the EP2 promoter correlated strongly with baseline EP2 mRNA (r = 0.80; P < 0.01). These studies suggest that the EP2 promotor is under epigenetic control, and one explanation for PGE2 resistance in AERD is an epigenetically mediated reduction of EP2 receptor expression, which could contribute to the refractory nasal polyposis typically observed in this syndrome.