Elizabeth A. Schwantes

Characterizing asthma from a drop of blood using neutrophil chemotaxis

Significance Asthma is a chronic inflammatory disorder that is notoriously difficult to diagnose, characterize, and properly treat with tests that are available to clinicians today. Therefore, clinicians would benefit from additional tools that objectively characterize asthma in patients. In this work, we describe a handheld microfluidic device that discriminates asthma from allergic rhinitis patients based on neutrophil function—an inflammatory cell that has been implicated in the pathogenesis of asthma. The device can sort neutrophils from a drop of whole blood within minutes, and was used in a clinical setting to characterize asthmatic and nonasthmatic patients. This technology provides a new tool for clinicians to characterize asthma based on cellular function. Asthma is a chronic inflammatory disorder that affects more than 300 million people worldwide. Asthma management would benefit from additional tools that establish biomarkers to identify phenotypes of asthma. We present a microfluidic solution that discriminates asthma from allergic rhinitis based on a patient’s neutrophil chemotactic function. The handheld diagnostic device sorts neutrophils from whole blood within 5 min, and generates a gradient of chemoattractant in the microchannels by placing a lid with chemoattractant onto the base of the device. This technology was used in a clinical setting to assay 34 asthmatic (n = 23) and nonasthmatic, allergic rhinitis (n = 11) patients to establish domains for asthma diagnosis based on neutrophil chemotaxis. We determined that neutrophils from asthmatic patients migrate significantly more slowly toward the chemoattractant compared with nonasthmatic patients (P = 0.002). Analysis of the receiver operator characteristics of the patient data revealed that using a chemotaxis velocity of 1.55 μm/min for asthma yields a diagnostic sensitivity and specificity of 96% and 73%, respectively. This study identifies neutrophil chemotaxis velocity as a potential biomarker for asthma, and we demonstrate a microfluidic technology that was used in a clinical setting to perform these measurements.

Airway neutrophil inflammatory phenotype in older subjects with asthma

Asthma in the elderly has been inadequately studied. The age demographic >65 years old is the fastest growing in the United States, and asthma in the elderly population is frequently underdiagnosed and undertreated(1). Furthermore, elderly asthmatics also have a higher rate of severe exacerbations, emergency department visits, and hospitalizations than younger asthmatics which represents significant disease impairment and risk(2). The airway inflammation in older asthma subjects differs from younger patients, which suggests that the phenotype of asthma in older asthmatics may be different. An understanding of this phenotype of asthma will be important for the diagnosis of the disease, determining optimal therapies, and improvement in the morbidity and mortality in the growing elderly asthmatic population.

Identification of Genes Expressed by Human Airway Eosinophils after an In Vivo Allergen Challenge

Background The mechanism for the contribution of eosinophils (EOS) to asthma pathophysiology is not fully understood. Genome-wide expression analysis of airway EOS by microarrays has been limited by the ability to generate high quality RNA from sufficient numbers of airway EOS. Objective To identify, by genome-wide expression analyses, a compendium of expressed genes characteristic of airway EOS following an in vivo allergen challenge. Methods Atopic, mild asthmatic subjects were recruited for these studies. Induced sputum was obtained before and 48h after a whole lung allergen challenge (WLAC). Individuals also received a segmental bronchoprovocation with allergen (SBP-Ag) 1 month before and after administering a single dose of mepolizumab (anti-IL-5 monoclonal antibody) to reduce airway EOS. Bronchoalveolar lavage (BAL) was performed before and 48 h after SBP-Ag. Gene expression of sputum and BAL cells was analyzed by microarrays. The results were validated by qPCR in BAL cells and purified BAL EOS. Results A total of 299 transcripts were up-regulated by more than 2-fold in total BAL cells following SBP-Ag. Mepolizumab treatment resulted in a reduction of airway EOS by 54.5% and decreased expression of 99 of the 299 transcripts. 3 of 6 post-WLAC sputum samples showed increased expression of EOS-specific genes, along with the expression of 361 other genes. Finally, the intersection of the 3 groups of transcripts (increased in BAL post SBP-Ag (299), decreased after mepolizumab (99), and increased in sputum after WLAC (365)) was composed of 57 genes characterizing airway EOS gene expression. Conclusion We identified 57 genes that were highly expressed by BAL EOS compared to unseparated BAL cells after in vivo allergen challenge. 41 of these genes had not been previously described in EOS and are thus potential new candidates to elucidate EOS contribution to airway biology.

Interferon gene expression in sputum cells correlates with the Asthma Index Score during virus‐induced exacerbations

The majority of asthma exacerbations are related to viral respiratory infections. Some, but not all, previous studies have reported that low interferon responses in patients with asthma increase the risk for virus‐induced exacerbations.

IL-3 and TNFα increase Thymic Stromal Lymphopoietin Receptor (TSLPR) expression on eosinophils and enhance TSLP-stimulated degranulation

BackgroundThymic stromal lymphopoietin (TSLP) and eosinophils are prominent components of allergic inflammation. Therefore, we sought to determine whether TSLP could activate eosinophils, focusing on measuring the regulation of TSLPR expression on eosinophils and degranulation in response to TSLP, as well as other eosinophil activation responses.MethodsEosinophil mRNA expression of TSLPR and IL-7Rα was examined by real-time quantitative PCR of human eosinophils treated with TNFα and IL-5 family cytokines, and TSLPR surface expression on eosinophils was analyzed by flow cytometry. Eosinophils were stimulated with TSLP (with and without pre-activation with TNFα and IL-3) and evaluated for release of eosinophil derived neurotoxin (EDN), phosphorylation of STAT5, and survival by trypan blue exclusion. A blocking antibody for TSLPR was used to confirm the specificity of TSLP mediated signaling on eosinophil degranulation.ResultsEosinophil expression of cell surface TSLPR and TSLPR mRNA was upregulated by stimulation with TNFα and IL-3. TSLP stimulation resulted in release of EDN, phosphorylation of STAT5 as well as promotion of viability and survival. TSLP-stimulated eosinophil degranulation was inhibited by a functional blocking antibody to TSLPR. Pre-activation of eosinophils with TNFα and IL-3 promoted eosinophil degranulation at lower concentrations of TSLP stimulation.ConclusionsThis study demonstrates that eosinophils are activated by TSLP and that eosinophil degranulation in response to TSLP may be enhanced on exposure to cytokines present in allergic inflammation, indicating that the eosinophil has the capacity to participate in TSLP-driven allergic responses.

The Peripheral Blood Eosinophil Proteome

A system-wide understanding of biological processes requires a comprehensive knowledge of the proteins in the biological system. The eosinophil is a type of granulocytic leukocyte specified early in hematopoietic differentiation that participates in barrier defense, innate immunity, and allergic disease. The proteome of the eosinophil is largely unannotated with under 500 proteins identified. We now report a map of the nonstimulated peripheral blood eosinophil proteome assembled using two-dimensional liquid chromatography coupled with high-resolution mass spectrometry. Our analysis yielded 100,892 unique peptides mapping to 7,086 protein groups representing 6,813 genes as well as 4,802 site-specific phosphorylation events. We account for the contribution of platelets that routinely contaminate purified eosinophils and report the variability in the eosinophil proteome among five individuals and proteomic changes accompanying acute activation of eosinophils by interleukin-5. Our deep coverage and quantitative analyses fill an important gap in the existing maps of the human proteome and will enable the strategic use of proteomics to study eosinophils in human diseases.

Characterization of leukotrienes in a pilot study of older asthma subjects

Leukotrienes are pro-inflammatory lipid mediators, which are important in asthma leading to its characteristic features of airway bronchoconstriction, wheezing, increased mucus secretion and decreased mucociliary clearance. Aging results in changes in immune function and we have previously demonstrated age-related differences in levels of neutrophil elastase, MMP-9, and IL-8 in asthma subjects. We sought to determine whether leukotriene levels differed between young and older asthmatics.

Nasal lavage VEGF and TNF-α levels during a natural cold predict asthma exacerbations

Asthma exacerbations contribute to significant morbidity, mortality and healthcare utilization. Furthermore, viral infections are associated with asthma exacerbations by mechanisms that are not fully understood.

Association of ORMDL3 with rhinovirus-induced endoplasmic reticulum stress and type I Interferon responses in human leucocytes.

Children with risk alleles at the 17q21 genetic locus who wheeze during rhinovirus illnesses have a greatly increased likelihood of developing childhood asthma. In mice, overexpression of the 17q21 gene ORMDL3 leads to airway remodelling and hyperresponsiveness. However, the mechanisms by which ORMDL3 predisposes to asthma are unclear. Previous studies have suggested that ORMDL3 induces endoplasmic reticulum (ER) stress and production of the type I interferon (IFN)‐regulated chemokine CXCL10.

Variability of blood eosinophil count as an asthma biomarker

In several recent clinical trials, the efficacy of novel targeted therapeutics, such as the monoclonal antibodies for interleukin (IL) 5 (eg, mepolizumab), IL-13 (eg, lebrikizumab), and IL-4 receptor (eg, dupilimab), was demonstratedmainly in a subgroup of patients with eosinophilic or TH2 endotype asthma.1e4 In these studies, sputum eosinophil counts, serum periostin level, and blood eosinophil counts identified the patients who were likely to respond. Furthermore, a post hoc analysis of patients with chronic obstructive pulmonary disease revealed that increased blood eosinophilia predicted response to the addition of inhaled corticosteroid.5 The blood eosinophil count is an attractive biomarker because of the ease of availability and its ability to identify which patients with asthma should receive novel therapeutics. However, is the blood eosinophil count a stable and reliable biomarker? Since 2008, we have recruited 82 individuals who have donated blood on at least 4 separate occasions (at least 8 weeks apart) for the purification of eosinophils and other blood cells for a total of 749 blood donations. Individuals were characterized initially with an extensive medical history, spirometry, exhaled nitric oxide, aeroallergen skin prick testing, and manual blood eosinophil count. At each donation visit, typically between 7 and 8 AM, an update to the medical history (including medication changes), symptom questionnaire, exhaled nitric oxide, and eosinophil count was obtained. If the individuals had an acute infection, were taking oral corticosteroids, or were receiving increased doses of inhaled steroids, they were not allowed to donate. Individuals were classified as having asthma only (13%), allergic rhinitis only (23%), or both allergic rhinitis and asthma (63%) based on self-report with confirmation based on medical history, prescribed medications, skin prick testing, and/or spirometry. Of these individuals, 55% were male, and the mean age at first donation was 34 years (range, 18e53 years). On the basis of the availability of repeated blood eosinophil counts, we sought to determine its variability and consider the suitability of eosinophil count as a biomarker using a linear mixed-effect model. Eosinophil counts were greater in individuals with asthma alone (geometric mean, 254 cells/mm3; 95% confidence interval [CI], 189e342 cells/mm3) and asthma with allergic rhinitis (244 cells/mm3; 95% CI, 213e280 cells/mm3) compared with those with allergic rhinitis alone (174 cells/mm3; 95% CI, 139e219 cells/mm3) (P 1⁄4 .05 and P 1⁄4 .01, respectively). The within-subject geometric coefficient of variation was 39.7%; in other words, a typical eosinophil count in any given individual is approximately 40% higher or lower than that individual’s mean. This variability