Eosinophil Knockout Humans: Uncovering the Role of Eosinophils Through Eosinophil-Directed Biological Therapies.

The enigmatic eosinophil has emerged as an exciting component of the immune system, involved in a plethora of homeostatic and inflammatory responses. Substantial progress has been achieved through experimental systems manipulating eosinophils in vivo, initially in mice and more recently in humans. Researchers using eosinophil knockout mice have identified a contributory role for eosinophils in basal and inflammatory processes and protective immunity. Primarily fueled by the purported proinflammatory role of eosinophils in eosinophil-associated diseases, a series of anti-eosinophil therapeutics have emerged as a new class of drugs. These agents, which dramatically deplete eosinophils, provide a valuable opportunity to characterize the consequences of eosinophil knockout humans. Herein, we comparatively describe mouse and human eosinophil knockouts. We put forth the view that human eosinophils negatively contribute to a variety of diseases and, unlike mouse eosinophils, do not yet have an identified role in physiological health; thus, clarifying all roles of eosinophils remains an ongoing pursuit.

Leukotriene receptor antagonists as add-on therapy to antihistamines for urticaria: a systematic review and meta-analysis of randomized clinical trials.

BACKGROUND: The benefits and harms of adding antileukotrienes to H1-antihistamines for the management of urticaria (hives, itch, and/or angioedema) remain unclear. OBJECTIVE: We sought to systematically synthesize the treatment outcomes of antileukotrienes in combination with H1-antihistamines versus H1-antihistamines alone for acute and chronic urticaria. METHODS: As part of updating American Academy of Allergy, Asthma & Immunology and American College of Allergy, Asthma, and Immunology Joint Task Force on Practice Parameters urticaria guidelines, we searched MEDLINE, Embase, CENTRAL, LILACS, WPRIM, IBECS, ICTRP, CBM, CNKI, VIP, Wanfang, FDA, and EMA databases from inception to December 18th, 2023 for randomized controlled trials (RCTs) evaluating antileukotrienes and H1-antihistamines versus H1-antihistamines alone in patients with urticaria. Paired reviewers independently screened citations, extracted data, and assessed risk of bias. Random effects models pooled effect estimates for urticaria activity, itch, wheal, sleep, quality of life, and harms. The GRADE approach informed certainty of evidence ratings. Open Science Framework registration: https://osf.io/h2bfx/. RESULTS: Thirty-four RCTs enrolled 3,324 children and adults. Compared to H1-antihistamines alone, the combination of a leukotriene receptor antagonist (LTRA) with H1-antihistamines probably modestly reduces urticaria activity (mean difference: -5.04, 95%CI -6.36 to -3.71; 7-day Urticaria Activity Score) with moderate certainty. We made similar findings for itch and wheal severity, and quality of life. Adverse events were probably not different between groups (moderate certainty), however, no RCT reported on neuropsychiatric adverse events. CONCLUSION: Among patients with urticaria, adding LTRAs to H1-antihistamines probably modestly improves urticaria activity with little to no increase in overall adverse events. The added risk of neuropsychiatric adverse events in this population with LTRAs is small and uncertain.

Topical corticosteroids for hives and itch (urticaria): systematic review and Bayesian meta-analysis of randomized trials.

BACKGROUND: Topical corticosteroids are widely used as a treatment for itch and wheals (urticaria), but their benefits and harms are unclear. OBJECTIVE: To systematically synthesize the benefits and harms of topical corticosteroids for the treatment of urticaria. METHODS: We searched MEDLINE, EMBASE, and CENTRAL from database inception to March 23, 2024, for randomized trials addressing comparing topical corticosteroid to placebo for patients with urticaria (either chronic spontaneous or inducible urticaria or acute urticaria elicited from skin/intradermal allergy testing). Paired reviewers independently screened records, extracted data, and assessed risk of bias. Random-effects meta-analyses addressed urticaria severity, itch severity (numeric rating scale; range 0-10; higher is worse), and adverse events. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach informed certainty of evidence ratings. PROSPERO registration: CRD42023455182. RESULTS: Nineteen RCTs enrolled 379 participants with a median of mean age of 30.1 years (range 21.1 to 44.0). Compared to placebo, topical corticosteroids may reduce wheal size (ratio of means 0.47, 95%CI 0.38 to 0.59; low certainty) and itch severity (mean difference -1.30, 95%CI -5.07 to 2.46; very low certainty). Topical corticosteroids result in little to no difference in overall adverse events (94 fewer patients per 1000, 95%CrI 172 fewer to 12 more; high certainty). CONCLUSION: Compared to placebo, topical corticosteroids may result in a reduction of wheal size, and result in little to no difference in overall adverse events. Topical corticosteroids may reduce itch severity, but the evidence is very uncertain. Future large, randomized trials addressing the use of topical corticosteroids would further support optimal urticaria management.

Omalizumab for treatment of idiopathic angioedema.

BACKGROUND: Omalizumab has been found to improve outcomes in patients with chronic spontaneous urticaria (CSU). Idiopathic angioedema (IAE) is increasingly being recognized as a condition with similar underlying mechanisms as CSU and a form of CSU. We hypothesized that add-on therapy with omalizumab would benefit patients with uncontrolled IAE. OBJECTIVE: To study the safety and efficacy of omalizumab for the treatment of IAE in adults. METHODS: We conducted a randomized, placebo-controlled trial to study the efficacy of omalizumab in adults with 2 or more episodes of angioedema (AE) in the past 6 months for which no clinical or laboratory cause of AE could be found. A total of 10 patients were randomized on a 1:1 basis to receive omalizumab 300 mg subcutaneously or placebo every 4 weeks for 24 weeks with a 12-week follow-up period. The primary endpoint was the change in the Angioedema Activity Score. Secondary endpoints included the Angioedema Quality of Life Questionnaire, the Visual Analogue Scale, and the number of angioedema episodes per month. RESULTS: We observed improvement in the Angioedema Activity Score (-2.93 ln odds; 95% confidence interval [CI], -4.84 to -1.02; P = .003), Visual Analogue Scale (-3.49 ln odds; 95% CI, -6.58 to -0.40; P = .03), Angioedema Quality of Life Questionnaire (-9.43 score; 95% CI, -17.63 to -1.24; P = .03), and number of angioedema episodes per month (-1.93 ln count; 95% CI, -3.23 to -0.63; P = .005) in patients who received omalizumab vs placebo. CONCLUSION: This study provides preliminary prospective evidence that omalizumab improves outcomes in patients with IAE. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02966314. CLINICALTRIALS: gov URL:https://clinicaltrials.gov/ct2/show/NCT02966314?term=omalizumab&cond=Angioedema&draw=2&rank=1.

Orosomucoid-like 3 Supports Rhinovirus Replication in Human Epithelial Cells.

Polymorphism at the 17q21 gene locus and wheezing responses to rhinovirus (RV) early in childhood conspire to increase the risk of developing asthma. However, the mechanisms mediating this gene-environment interaction remain unclear. In this study, we investigated the impact of one of the 17q21-encoded genes, ORMDL3 (orosomucoid-like 3), on RV replication in human epithelial cells. ORMDL3 knockdown inhibited RV-A16 replication in HeLa, BEAS-2B, A549, and NCI-H358 epithelial cell lines and primary nasal and bronchial epithelial cells. Inhibition varied by RV species, as both minor and major group RV-A subtypes RV-B52 and RV-C2 were inhibited but not RV-C15 or RV-C41. ORMDL3 siRNA did not affect expression of the major group RV-A receptor ICAM-1 or initial internalization of RV-A16. The two major outcomes of ORMDL3 activity, SPT (serine palmitoyl-CoA transferase) inhibition and endoplasmic reticulum (ER) stress induction, were further examined: silencing ORMDL3 decreased RV-induced ER stress and IFN-ß mRNA expression. However, pharmacologic induction of ER stress and concomitant increased IFN-ß inhibited RV-A16 replication. Conversely, blockade of ER stress with tauroursodeoxycholic acid augmented replication, pointing to an alternative mechanism for the effect of ORMDL3 knockdown on RV replication. In comparison, the SPT inhibitor myriocin increased RV-A16 but not RV-C15 replication and negated the inhibitory effect of ORMDL3 knockdown. Furthermore, lipidomics analysis revealed opposing regulation of specific sphingolipid species (downstream of SPT) by myriocin and ORMDL3 siRNA, correlating with the effect of these treatments on RV replication. Together, these data revealed a requirement for ORMDL3 in supporting RV replication in epithelial cells via SPT inhibition.

Eosinophil cytolysis on Immunoglobulin G is associated with microtubule formation and suppression of rho-associated protein kinase signalling.

BACKGROUND: The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Cell-free eosinophil granules are found in tissues in eosinophilic diseases, including asthma. This suggests that eosinophils have lysed and released cellular content, likely harming tissues. OBJECTIVE: The present study explores the mechanism of CD32- and αMß2 integrin-dependent eosinophil cytolysis of IL3-primed blood eosinophils seeded on heat-aggregated immunoglobulin G (HA-IgG). METHODS: Cytoskeletal events and signalling pathways potentially involved in cytolysis were assessed using inhibitors. The level of activation of the identified events and pathways involved in cytolysis was measured. In addition, the links between these identified pathways and changes in degranulation (exocytosis) and adhesion were analysed. RESULTS: Cytolysis of IL3-primed eosinophils was dependent on the production of reactive oxygen species (ROS) and downstream phosphorylation of p-38 MAPK. In addition, formation of microtubule (MT) arrays was necessary for cytolysis and was accompanied by changes in MT dynamics as measured by phosphorylation status of stathmin and microtubule-associated protein 4 (MAP4), the latter of which was regulated by ROS production. Reduced ROCK signalling preceded cytolysis, which was associated with eosinophil adhesion and reduced migration. CONCLUSION AND CLINICAL RELEVANCE: In this CD32- and αMß2 integrin-dependent adhesion model, lysing eosinophils exhibit reduced migration and ROCK signalling, as well as both MT dynamic changes and p-38 phosphorylation downstream of ROS production. We propose that interfering with these pathways would modulate eosinophil cytolysis and subsequent eosinophil-driven tissue damage.

Effect of clinically significant thresholds of eosinophil elevation on health care resource use in asthma.

BACKGROUND: Blood eosinophil counts correlate with exacerbations, but there is a lack of consensus on a clinically relevant definition of eosinophil count elevation. OBJECTIVE: To analyze health care resource use among patients with elevated blood eosinophil counts defined at 150 cells/µL or greater and 300 cells/µL or greater. METHODS: Data on patients who received a diagnosis of asthma between 2007 and 2016 were extracted from EMRClaims + database. Patients were defined as having elevated eosinophil counts if any test result during 3 months before follow-up found blood eosinophil count of 150 cells/µL or more or 300 cells/µL or more. Hospitalizations, emergency department visits, outpatient visits, and associated costs were compared. With logistic regression, likelihood of hospitalization was assessed in the presence of eosinophil elevation. RESULTS: Among 3687 patients who met the study criteria, 1152 received a test within 3 months before the follow-up period, of whom 644 (56%) had elevated eosinophil counts of 150 cells/µL or greater and 322 (29%) had eosinophil counts of 300 cells/µL or greater. Overall, the mean (SD) number of hospitalizations for patients with elevated eosinophil counts vs the comparator was significantly greater (0.29 [0.92] vs 0.17 [0.57], P < .001 at ≥150 cells/µL and 0.30 [0.95] vs 0.18 [0.61] at ≥300 cells/µL, P = .001). The total mean cost was significantly greater for patients with elevated eosinophil counts (at ≥150 cells/µL: $10,262 vs $7149, P < .001 and at ≥300 cells/µL: $9966 vs $7468, P = .003). CONCLUSION: Patients with asthma incurred greater health care resource use when their blood eosinophil counts were elevated at 150 cells/µL or greater and 300 cells/µL or greater as measured within 3 months of follow-up.

Add-on tiotropium versus step-up inhaled corticosteroid plus long-acting beta-2-agonist in real-world patients with asthma.

Background: A step-up approach (increasing inhaled corticosteroid [ICS] dose and/or add-on treatment) is recommended for asthma that is uncontrolled despite ICS plus long-acting beta-2-agonist (LABA) combination treatment. Understanding the impact of different treatment options on health outcomes can help guide treatment decision-making. Objective: To compare the effectiveness of add-on tiotropium 1.25 µg (two puffs once daily) versus an increased ICS plus LABA dose in a real-world cohort of patients with asthma initiated on ICS plus LABA. Methods: De-identified data from patients ages ≥12 years and with asthma who were initiated on ICS plus LABA, and then had tiotropium added (Tio group; index date) or an ICS plus LABA dose increased (inc-ICS group; index date) were collected from two medical and pharmacy claims data bases (2014-2018). To account for population/group differences, propensity score matching was performed. The primary end point was the exacerbation risk after the index date. Secondary end points included exacerbation rates 6 and 12 months postindex, health-care resource utilization, costs, and short-acting beta-2-agonist (SABA) refills 12 months postindex. Results: Overall, 7857 patients (Tio group, 2619; inc-ICS group, 5238) were included. The exacerbation risk was 35% lower in the Tio group than in the inc-ICS group (hazard ratio 0.65 [95% confidence interval, 0.43-0.99]; p = 0.044). Exacerbation rates in the Tio group also were significantly lower within 6 and 12 months postindex (64% and 73%, respectively). All-cause and asthma-related emergency department (ED) visits were 47% and 74% lower, respectively (p < 0.0001 for both), and all-cause and asthma-related hospitalizations were 48% (p < 0.01) and 76% (p < 0.001) lower, respectively, in the Tio group. Also, significantly fewer patients in the Tio group versus the inc-ICS group required SABA refills (56% versus 67%, p < 0.0001). Conclusion: Add-on tiotropium significantly decreased the risk and rate of exacerbations, decreased all-cause and asthma-related ED visits and hospitalizations, and reduced SABA refills compared with increasing the ICS plus LABA dose. The findings supported the use of add-on tiotropium for patients with uncontrolled asthma taking ICS plus LABA.

Characterizing asthma from a drop of blood using neutrophil chemotaxis.

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.

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.

Role of food and aeroallergen sensitization in eosinophilic esophagitis in adults.

BACKGROUND: Evaluation of IgE-mediated food sensitivity is frequently performed for patients with eosinophilic esophagitis (EoE). However, the clinical relevance of identifying IgE-mediated sensitivity to foods in adults is unclear. OBJECTIVE: To determine whether EoE associated with food or aeroallergen sensitivity represents a phenotype of EoE with distinct clinical or biological features. METHODS: A medical record review identified 257 patients with a diagnosis of EoE evaluated in the adult allergy clinic at the University of Wisconsin Hospital and Clinics from 2008 to 2013. Patient records were reviewed to capture measures of disease severity, endoscopy results, pathology reports, allergy testing, medical management and patient-reported outcomes. RESULTS: Evaluation of food sensitization with skin prick testing and/or serum IgE was performed for 93% of patients. Sensitization to at least 1 food was identified in 54% of patients who were more likely to report concomitant asthma, allergic rhinitis, eczema, and/or food allergy compared with nonfood sensitive patients. Aeroallergen sensitivity was identified in 87% of patients tested. Clinical characteristics, including EoE symptoms, disease severity, endoscopic findings, peripheral eosinophilia, and patient-reported outcomes, did not differ between food sensitive and non-food sensitive patients. However, on endoscopy, aeroallergen sensitive patients were more likely to have strictures and less likely to exhibit felinization compared with non-aeroallergen sensitized patients. CONCLUSION: Adults with EoE and IgE-mediated food sensitivity are not phenotypically different than non-food sensitive patients. There is no clear clinical utility in identifying food sensitivity in adults with EoE. Further studies are needed to determine whether aeroallergen sensitivity represents a distinct phenotype of EoE.

Semaphorin 7A is expressed on airway eosinophils and upregulated by IL-5 family cytokines.

Semaphorin 7A (sema7a) plays a major role in TGF-ß1-induced lung fibrosis. Based on the accumulating evidence that eosinophils contribute to fibrosis/remodeling in the airway, we hypothesized that airway eosinophils may be a significant source of sema7a. In vivo, sema7a was expressed on the surface of circulating eosinophils and upregulated on bronchoalveolar lavage eosinophils obtained after segmental bronchoprovocation with allergen. Based on mRNA levels in unfractionated and isolated bronchoalveolar cells, eosinophils are the predominant source of sema7a. In vitro, among the members of the IL-5-family cytokines, sema7a protein on the surface of blood eosinophils was increased more by IL-3 than by GM-CSF or IL-5. Cytokine-induced expression of cell surface sema7a required translation of newly synthesized protein. Finally, a recombinant sema7a induced alpha-smooth muscle actin production in human bronchial fibroblasts. semaphorin 7A is a potentially important modulator of eosinophil profibrotic functions in the airway remodeling of patients with chronic asthma.

Relevance of allergy in adult asthma.

Recent studies on asthma have demonstrated multiple phenotypes, based on the clinical characteristics of the disease. With the current interest in personalized medicine, the question arises whether the presence of allergic sensitization has any relevance for these phenotypes and the management of asthma. This review will examine the current knowledge of asthma phenotypes and the impact of atopy on asthma diagnosis and severity in adults. In addition, this review will address whether therapies targeted at the atopic axis help improve asthma outcomes, including lung function indices and exacerbations.

Biologic Therapy in Rare Eosinophil-Associated Disorders: Remaining Questions and Translational Research Opportunities.

Rare eosinophil-associated disorders (EADs), including hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis and eosinophilic gastrointestinal disorders, are a heterogeneous group of conditions characterized by blood and/or tissue hypereosinophilia and eosinophil-related clinical manifestations. Although the recent availability of biologic therapies that directly and indirectly target eosinophils has the potential to dramatically improve treatment options for all EADs, clinical trials addressing their safety and efficacy in rare EADs have been relatively few. Consequently, patient access to therapy is limited for many biologics, and the establishment of evidence-based treatment guidelines has been extremely difficult. In this regard, multicenter retrospective collaborative studies focusing on disease manifestations and treatment responses in rare EADs have provided invaluable data for physicians managing patients with these conditions and helped identify important questions for future translational research. During the Clinical Pre-Meeting Workshop held in association with the July 2023 biennial meeting of the International Eosinophil Society in Hamilton, Ontario, Canada, the successes and limitations of pivotal multicenter retrospective studies in EADs were summarized, and unmet needs regarding the establishment of guidelines for use of biologics in rare EADs were discussed. Key topics of interest included: 1) clinical outcome measures, 2) minimally invasive biomarkers of disease activity, 3) predictors of response to biologic agents, and 4) long-term safety of eosinophil depletion. Herein, we report a summary of these discussions, presenting a state-of-the-art overview of data currently available for each of these topics, the limitations of the data, and avenues for future data generation through implementation of multidisciplinary and multicenter studies.