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.

Development of a Wearable Ultrasonic Auricular Vagus Nerve Stimulator.

This paper discusses the design and development of a wearable and portable ultrasound auricular nerve stimulator. The device is in the form of a headset that presses against the Cymba Concha depression in the ear and stimulates the auricular vagus nerve with ultrasound energy. This paper reviews the development, design, and material structures for such a system, including the characterization of the working prototypes. The devices are being supplied to various organizations and universities for clinical trials in which effectiveness will be assessed. The device is light, compact, and an effective neurostimulator to induce calm. To the best of the author's knowledge the device is the first fully-built ultrasonic auricular nerve stimulator in the world.

Increased epithelial mTORC1 activity in chronic rhinosinusitis with nasal polyps.

Background: The airway epithelium plays a central role in the pathogenesis of chronic respiratory diseases such as asthma and chronic rhinosinusitis with nasal polyps (CRSwNP), but the mechanisms by which airway epithelial cells (EpCs) maintain inflammation are poorly understood. Objective: We hypothesized that transcriptomic assessment of sorted airway EpCs across the spectrum of differentiation would allow us to define mechanisms by which EpCs perpetuate airway inflammation. Methods: Ethmoid sinus EpCs from adult patients with CRS were sorted into 3 subsets, bulk RNA sequenced, and analyzed for differentially expressed genes and pathways. Single cell RNA-seq (scRNA-seq) datasets from eosinophilic and non-eosinophilic CRSwNP and bulk RNA-seq of EpCs from mild/moderate and severe asthma were assessed. Immunofluorescent staining and ex vivo functional analysis of sinus EpCs were used to validate our findings. Results: Analysis within and across purified EpC subsets revealed an enrichment in glycolytic programming in CRSwNP vs CRSsNP. Correlation analysis identified mammalian target of rapamycin complex 1 (mTORC1) as a potential regulator of the glycolytic program and identified EpC expression of cytokines and wound healing genes as potential sequelae. mTORC1 activity was upregulated in CRSwNP, and ex vivo inhibition demonstrated that mTOR is critical for EpC generation of CXCL8, IL-33, and CXCL2. Across patient samples, the degree of glycolytic activity was associated with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in severe asthma. Conclusions: Together, these findings highlight a metabolic axis required to support epithelial generation of cytokines critical to both chronic T2 and non-T2 inflammation in CRSwNP and asthma.

Optimizing cryopreservation of nasal polyp tissue for cellular functional studies and single-cell RNA sequencing.

KEY POINTS: Mast cell numbers were reduced in samples cryopreserved as whole tissue chunks. Thawed epithelial cells had reduced proliferation rates when preserved as dissociated cell suspensions. The right cryopreservation method to choose may depend on the goals and cell-type focus of the project.

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.

Rapid and sustained effect of dupilumab on clinical and mechanistic outcomes in aspirin-exacerbated respiratory disease.

BACKGROUND: Dupilumab, a mAb targeting IL-4Rα, improves upper and lower airway symptoms in patients with aspirin-exacerbated respiratory disease (AERD), but the mechanisms leading to clinical improvement are not fully elucidated. OBJECTIVE: Our aim was to identify the mechanistic basis of clinical improvement in patients with AERD treated with dupilumab. METHODS: A total of 22 patients with AERD were treated with dupilumab for 3 months for severe asthma and/or chronic rhinosinusitis with nasal polyps. Clinical outcomes were assessed at baseline and at 1 and 3 months after initiation of dupilumab. Nasal fluid, urine, blood, and inferior turbinate scrapings were collected at the 3 time points for determination of mediator levels, cellular assays, and RNA sequencing. RESULTS: Participants had rapid improvement in clinical measures, including sense of smell, sinonasal symptoms, and lung function after 1 month of treatment with dupilumab; the improvements were sustained after 3 months of dupilumab. Baseline severity of smell loss was correlated with lower nasal prostaglandin E2 levels. Dupilumab increased nasal prostaglandin E2 level and decreased levels of nasal albumin, nasal and urinary leukotriene E4, and serum and nasal IgE. Transcripts related to epithelial dysfunction and leukocyte activation and migration were downregulated in inferior turbinate tissue after treatment with dupilumab. There were no dupilumab-induced changes in nasal eosinophilia. CONCLUSION: Inhibition of IL-4Rα in AERD led to rapid improvement in respiratory symptoms and smell, with a concomitant improvement in epithelial barrier function, a decrease in inflammatory eicosanoid levels, and an increase in the anti-inflammatory eicosanoid prostaglandin E2 level. The therapeutic effects of dupilumab are likely due to decreased IL-4Rα signaling on respiratory tissue granulocytes, epithelial cells, and B cells.

Mepolizumab targets multiple immune cells in aspirin-exacerbated respiratory disease.

BACKGROUND: Eosinophilic asthma and nasal polyposis are hallmarks of aspirin-exacerbated respiratory disease (AERD), and IL-5 inhibition has been shown to provide therapeutic benefit. However, IL-5Rα is expressed on many cells in addition to eosinophils, and the mechanisms by which IL-5 inhibition leads to clinical benefit in eosinophilic asthma and nasal polyposis are unlikely to be due exclusively to antieosinophil effects. OBJECTIVE: We sought to identify the mechanisms by which anti-IL-5 treatment with mepolizumab improves respiratory inflammation in AERD. METHODS: The clinical characteristics, circulating granulocytes, nasal scraping transcripts, eosinophilic cationic protein, tryptase, and antibody levels, and urinary and nasal eicosanoid levels were measured for 18 subjects with AERD who were taking mepolizumab and compared with those of 18 matched subjects with AERD who were not taking mepolizumab. RESULTS: Subjects taking mepolizumab had significantly fewer peripheral blood eosinophils and basophils, and those cells that remained had higher surface CRTH2 expression than did the cells from subjects not taking mepolizumab. Nasal prostaglandin F2α, prostaglandin D2 metabolites, leukotriene B4, and thromboxane levels were lower in subjects taking mepolizumab, as were urinary levels of tetranor-prostaglandin D2 and leukotriene E4. The nasal epithelial cell transcripts that were overexpressed among subjects with AERD who were taking mepolizumab were enriched for genes involved in tight junction formation and cilium organization. Nasal and urinary prostaglandin E2, tryptase, and antibody levels were not different between the 2 groups. CONCLUSION: IL-5 inhibition in AERD decreases production of inflammatory eicosanoids and upregulates tight junction-associated nasal epithelial cell transcripts, likely due to decreased IL-5 signaling on tissue mast cells, eosinophils, and epithelial cells. These direct effects on multiple relevant immune cells contribute to the mechanism of benefit afforded by mepolizumab.