Benralizumab for allergic asthma: a randomised, double-blind, placebo-controlled, trial.

RATIONALE AND OBJECTIVE: Benralizumab induces rapid and near-complete depletion of eosinophils from blood and lung tissue. We investigated whether benralizumab could attenuate allergen-induced late asthmatic responses (LAR) in participants with allergic asthma. METHODS AND MEASUREMENTS: Participants with allergic asthma who demonstrated increased sputum eosinophils and LAR at screening were randomised to benralizumab 30 mg or matched placebo given every 4 weeks for 8 weeks (3 doses). Allergen challenges were performed at weeks 9 and 12 when blood, sputum, bone marrow and bronchial tissue eosinophils and LAR were assessed. MAIN RESULTS: Forty-six participants (mean age, 30.9 years) were randomised to benralizumab (n = 23) or placebo (n = 23). Eosinophils were significantly reduced in the benralizumab group compared with placebo in blood at 4 weeks and sputum and bone marrow at 9 weeks after treatment initiation. At 7 h after an allergen challenge at week 9, sputum eosinophilia was significantly attenuated in the benralizumab group compared to placebo (least squares mean difference -5.81% [95% CI, -10.69, -0.94]; P = 0.021); however, the LAR was not significantly different (least squares mean difference 2.54% [95% CI, 3.05, 8.12]; P = 0.363). Adverse events were reported for 7 (30.4%) and 14 (60.9%) participants in the benralizumab and placebo groups, respectively. CONCLUSION: Benralizumab administration over 8 weeks resulted in a significant attenuation of blood, bone marrow and sputum eosinophilia in participants with mild allergic asthma; however, there was no change in the LAR, suggesting that eosinophils alone are not a key component of allergen-induced bronchoconstriction.

Autoantibody-mediated Macrophage Dysfunction in Patients with Severe Asthma with Airway Infections.

Rationale: Localized autoimmune responses have been reported in patients with severe eosinophilic asthma, characterized by eosinophil degranulation and airway infections. Objective: To determine the presence of autoantibodies against macrophage scavenger receptors within the airways and their effects on macrophage function and susceptibility to infection. Methods: Anti-EPX (eosinophil peroxidase), anti-MARCO (macrophage receptor with collagenous structure) IgG titers, and T1 and T2 (type 1/2) cytokines were measured in 221 sputa from 143 well-characterized patients with severe asthma. Peripheral monocytes and MDMs (monocyte-derived macrophages) isolated from healthy control subjects were treated with immunoprecipitated immunoglobulins from sputa with high anti-MARCO titers or nonspecific IgG to assess uptake of Streptococcus pneumoniae or response to the bacterial product LPS. Measurements and Main Results: Anti-MARCO IgG was detected in 36% of patients, with significantly higher titers (up to 1:16) in patients with mixed granulocytic sputa, indicative of airway infections. Multivariate regression analysis confirmed increased frequency of degranulation (free eosinophil granules), increased blood eosinophils (indicative of high T2 burden), increased sputum total cell count, peripheral blood leukocytes (indicative of infection), and lymphopenia were associated with increased anti-MARCO IgG titers; IL-15 (odds ratio [OR], 1.79; confidence interval [CI], 1.19-2.70), IL-13 (OR, 1.06; CI, 1.02-1.12), and IL-12p70 (OR, 3.34; CI, 1.32-8.40) were the associated cytokines. Patients with anti-MARCO antibodies had higher chances of subsequent infective versus eosinophilic exacerbations (P = 0.01). MDMs treated with immunoprecipitated immunoglobulins (anti-MARCO+ sputa) had reduced bacterial uptake by 39% ± 15% and significantly reduced release of IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05) in response to an LPS stimulus. Conclusions: Autoantibodies against macrophage scavenger receptors in eosinophilic asthma airways may impede effective host defenses and lead to recurrent infective bronchitis.

Circulating anti-nuclear autoantibodies in COVID-19 survivors predict long COVID symptoms.

BACKGROUND: Autoimmunity has been reported in patients with severe coronavirus disease 2019 (COVID-19). We investigated whether anti-nuclear/extractable-nuclear antibodies (ANAs/ENAs) were present up to a year after infection, and if they were associated with the development of clinically relevant post-acute sequalae of COVID-19 (PASC) symptoms. METHODS: A rapid-assessment line immunoassay was used to measure circulating levels of ANAs/ENAs in 106 convalescent COVID-19 patients with varying acute phase severities at 3, 6 and 12 months post-recovery. Patient-reported fatigue, cough and dyspnoea were recorded at each time point. Multivariable logistic regression model and receiver operating curves were used to test the association of autoantibodies with patient-reported outcomes and pro-inflammatory cytokines. RESULTS: Compared to age- and sex-matched healthy controls (n=22) and those who had other respiratory infections (n=34), patients with COVID-19 had higher detectable ANAs at 3 months post-recovery (p<0.001). The mean number of ANA autoreactivities per individual decreased between 3 and 12 months (from 3.99 to 1.55) with persistent positive titres associated with fatigue, dyspnoea and cough severity. Antibodies to U1-snRNP and anti-SS-B/La were both positively associated with persistent symptoms of fatigue (p<0.028, area under the curve (AUC) 0.86) and dyspnoea (p<0.003, AUC=0.81). Pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α and C-reactive protein predicted the elevated ANAs at 12 months. TNF-α, D-dimer and interleukin-1ß had the strongest association with symptoms at 12 months. Regression analysis showed that TNF-α predicted fatigue (ß=4.65, p=0.004) and general symptomaticity (ß=2.40, p=0.03) at 12 months. INTERPRETATION: Persistently positive ANAs at 12 months post-COVID are associated with persisting symptoms and inflammation (TNF-α) in a subset of COVID-19 survivors. This finding indicates the need for further investigation into the role of autoimmunity in PASC.

Airway autoantibodies are determinants of asthma severity.

BACKGROUND: Local airway autoimmune responses may contribute to steroid dependence and persistent eosinophilia in severe asthma. Auto-IgG antibodies directed against granule proteins such as eosinophil peroxidase (EPX), macrophage scavenger receptor with collagenous structure (MARCO) and nuclear/extranuclear antigens (antinuclear antibodies (ANAs)) have been reported. Our objective was to describe the prevalence and clinical characteristics of asthmatic patients with airway autoreactivity, and to assess if this could be predicted from clinical history of autoreactivity. METHODS: We analysed anti-EPX, anti-MARCO and ANAs in 218 sputum samples collected prospectively from 148 asthmatic patients, and evaluated their association with lung function parameters, blood/airway inflammation, severity indices and exacerbations. Additionally, 107 of these patients consented to fill out an autoimmune checklist to determine personal/family history of systemic autoimmune disease and symptoms. RESULTS: Out of the 148 patients, 59 (40%) were anti-EPX IgG+, 53 (36%) were anti-MARCO IgG+ and 64 out of 129 (50%) had ≥2 nuclear/extranuclear autoreactivities. A composite airway autoreactivity score (CAAS) demonstrated that 82 patients (55%) had ≥2 airway autoreactivities (considered as CAAS+). Increased airway eosinophil degranulation (OR 15.1, 95% CI 1.1-199.4), increased blood leukocytes (OR 3.5, 95% CI 1.3-10.1) and reduced blood lymphocytes (OR 0.19, 95% CI 0.04-0.84) predicted CAAS+. A third of CAAS+ patients reported an exacerbation, associated with increased anti-EPX and/or anti-MARCO IgG (p<0.05). While no association was found between family history or personal diagnosis of autoimmune disease, 30% of CAAS+ asthmatic patients reported sicca symptoms (p=0.02). Current anti-inflammatory (inhaled/oral corticosteroids and/or adjunct anti-interleukin-5 biologics) treatment does not attenuate airway autoantibodies, irrespective of eosinophil suppression. CONCLUSION: We report 55% of moderate-severe asthmatic patients to have airway autoreactivity that persists despite anti-inflammatory treatment and is associated with exacerbations.

A Historical Perspective on the Reversal of Anticoagulants.

There has been a landmark shift in the last several decades in the management and prevention of thromboembolic events. From the discovery of parenteral and oral agents requiring frequent monitoring as early as 1914, to the development of direct oral anticoagulants (DOACs) that do not require monitoring or dose adjustment in the late 20th century, great advances have been achieved. Despite the advent of these newer agents, bleeding continues to be a key complication, affecting 2 to 4% of DOAC-treated patients per year. Bleeding is associated with substantial morbidity and mortality. Although specific reversal agents for DOACs have lagged the release of these agents, idarucizumab and andexanet alfa are now available as antagonists. However, the efficacy of these reversal agents is uncertain, and complications, including thrombosis, have not been adequately explored. As such, guidelines continue to advise the use of nonspecific prohemostatic agents for patients requiring reversal of the anticoagulant effect of these drugs. As the indications for DOACs and the overall prevalence of their use expand, there is an unmet need for further studies to determine the efficacy of specific compared with nonspecific pro-hemostatic reversal agents. In this review, we will discuss the evidence behind specific and nonspecific reversal agents for both parenteral and oral anticoagulants.

Tyrosine kinase inhibitors and tumor lysis syndrome in hematologic malignancies: A systemic review.

BACKGROUND: Effective treatments for hematologic malignancies include therapies that target tyrosine kinase (TK) signaling pathways. Tumor lysis syndrome (TLS) is an oncologic emergency that can occur due to rapid turnover following the initiation of treatments for hematologic malignancy. The incidence of TLS is under-reported and it is unclear as to whether TK inhibitors (TKIs) are associated with TLS. OBJECTIVE: To conduct a systematic review to determine the incidence of TLS with TKIs. METHODS: A search was performed using EMBASE, MEDLINE, and Web of Science electronic databases, as well as a manual search of the American Society of Hematology and American Society of Clinical Oncology abstract databases. Keywords included: "tumor lysis syndrome," "tyrosine kinase inhibitors," "lymphoma," and "leukemia." RESULTS: We identified a total of 57 publications that commented on the incidence of TLS with TKIs for hematologic malignancy. Thirty-nine of those publications reported TLS as an adverse event. TLS was described as an adverse event among essentially all the subclasses of TKIs that are used to manage hematologic malignancies. CONCLUSION: The overall number of articles commenting on TLS as an adverse event is sparse and there needs to be more transparency regarding the incidence of TLS when employing newer targeted therapies. Physicians should consider the risk of TLS on an individual basis and the added risk of TLS when using TKIs to treat hematologic malignancy.

The Role of the TL1A/DR3 Axis in the Activation of Group 2 Innate Lymphoid Cells in Subjects with Eosinophilic Asthma.

Rationale: Group 2 innate lymphoid cells (ILC2s) are critical for type 2 inflammation. In murine models of asthma, some ILC2s remain activated in the absence of epithelial cell-derived cytokine signaling, implicating alternate stimulatory pathways. DR3 (death receptor 3), a member of the tumor necrosis factor receptor superfamily, is expressed on ILC2s. Genome-wide association studies report an association between DR3 ligand, TL1A (tumor necrosis factor-like protein 1A), and chronic inflammatory conditions.Objectives: We investigated the TL1A/DR3 axis in airway ILC2 biology in eosinophilic asthma.Methods: Stable subjects with mild asthma were subject to allergen inhalation challenge, and DR3 expression on sputum cells was assessed. We investigated cytokine regulation of DR3 expression on ILC2s and steroid sensitivity. Airway TL1A was assessed in sputum from subjects with mild asthma and subjects with prednisone-dependent severe eosinophilic asthma.Measurements and Main Results: There was a significant increase in sputum DR3+ ILC2s 24 hours after allergen challenge, and DR3 expression on ILC2s was upregulated by IL-2, IL-33, or TSLP in vitro. Stimulation with TL1A significantly increased IL-5 expression by ILC2s and was attenuated by dexamethasone, an effect that was negated in the presence of TSLP. Airway TL1A levels were increased 24 hours after allergen challenge in subjects with mild asthma but were significantly greater in those with severe eosinophilic asthma. The highest levels were detected in subjects with severe asthma with airway autoimmune responses. C1q+ immune complexes from the sputa of subjects with severe asthma with high autoantibody levels stimulated TL1A production by monocytes.Conclusions: The TL1A/DR3 axis is a costimulator of ILC2s in asthma, particularly in the airways of patients with a predisposition to autoimmune responses.

Weight-adjusted Intravenous Reslizumab in Severe Asthma with Inadequate Response to Fixed-Dose Subcutaneous Mepolizumab.

RATIONALE: Clinical benefits of fixed-dose 100-mg subcutaneous (SC) mepolizumab in prednisone-dependent patients are modest when sputum eosinophilia is not adequately controlled. OBJECTIVES: This study compared treatment response of weight-adjusted intravenous (IV) reslizumab in patients previously treated with 100-mg SC mepolizumab. METHODS: Ten prednisone-dependent patients with asthma (sputum eosinophils >3% and blood eosinophils >300 cells/µl), who had previously received mepolizumab (100 mg SC dosed every 4 wk [Q4W]) for at least 1 year, received two infusions of placebo (Q4W) followed by four infusions of 3.0 mg/kg reslizumab Q4W in a single-blind, placebo-controlled sequential trial. Primary outcomes were reduction of eosinophils in sputum and blood. Additional outcomes included FEV1, asthma control questionnaire, eosinophil peroxidase, IL-5, sputum and blood innate lymphoid cells group 2, eosinophil progenitor cells, and autoimmune responses. MEASUREMENTS AND MAIN RESULTS: IV reslizumab attenuated sputum eosinophils by 91.2% (P = 0.002), blood eosinophil counts by 87.4% (P = 0.004), and sputum eosinophil peroxidase levels by 65.5% (P = 0.03) compared with placebo. Attenuation of both local and systemic eosinophilia was associated with statistically significant improvements in FEV1 (P = 0.004) and asthma control questionnaire five-question instrument scores (P = 0.006). Decrease in percent sputum eosinophil was greater with reslizumab (by 42.7%) compared with mepolizumab (by 5.0%) and this was associated with greater improvement in asthma control questionnaire (P = 0.01; analysis of covariance of Δ between before and after treatment, mepolizumab vs. reslizumab, adjusted for baseline prednisone). Changes in sputum IL-5 and anti-eosinophil peroxidase IgG after anti-IL-5 therapy were predictors of response. CONCLUSIONS: Weight-adjusted IV reslizumab was superior to fixed-dose SC mepolizumab in attenuating airway eosinophilia in prednisone-dependent patients with asthma, with associated improvement in asthma control. Clinical trial registered with www.clinicaltrials.gov (NCT 02559791).

Human Bronchial Epithelial Cell-derived Factors from Severe Asthmatic Subjects Stimulate Eosinophil Differentiation.

Activated bronchial epithelial cells (BEC) release various alarmins, including thymic stromal lymphopoietin (TSLP), that drive type 2 inflammation. We hypothesize that BEC-derived factors promote in situ eosinophil differentiation and maturation, a process that is driven by an IL-5-rich microenvironment in asthmatic airways. To assess the eosinophilopoietic potential of epithelial-derived factors, eosinophil/basophil colony forming units (Eo/B-CFU) were enumerated in 14-day methylcellulose cultures of blood-derived nonadherent mononuclear cells incubated with BEC supernatants (BECSN) from healthy nonatopic controls (n = 8), mild atopic asthmatics (n = 9), and severe asthmatics (n = 5). Receptor-blocking antibodies were used to evaluate the contribution of alarmins. Modulation of the mRNA expression of transcription factors that are crucial for eosinophil differentiation was evaluated. BECSN stimulated the clonogenic expansion of eosinophil progenitors in vitro. In the presence of IL-5, Eo/B-CFU numbers were significantly greater in cocultures of BESCN from severe asthmatics compared with other groups. This was attenuated in the presence of a TSLP (but not an IL-33) receptor-blocking antibody. Recombinant human TSLP (optimal at 100 pg/ml) stimulated Eo/B-CFU growth, which was significantly enhanced in the presence of IL-5 (1 ng/ml). Overnight culture of CD34+ cells with IL-5 and TSLP synergistically increased GATA-binding factor 2 and CCAAT/enhancer-binding protein α mRNA expression. The eosinophilopoietic potential of factors derived from BEC is increased in severe asthma. Our data suggest that TSLP is a key alarmin that is produced by BECs and promotes in situ eosinophilopoiesis in a type 2-rich microenvironment.

IL-33 and Its Receptor ST2 after Inhaled Allergen Challenge in Allergic Asthmatics.

BACKGROUND: Previous murine models have demonstrated interleukin (IL)-33 to be an important mediator of type-2 inflammation and to promote airway hyperresponsiveness in allergic asthma. A number of inflammatory cells produce IL-33 and eosinophils express ST2 mRNA. The relationship between IL-33 and eosinophils in allergic asthma, however, remains unclear. OBJECTIVE: The aim of this work was to evaluate in vitro the effect of allergen inhalation on IL-33 levels and expression of its receptor (ST2L) on eosinophils in allergic asthmatics, and the effect of IL-33 stimulation on eosinophil activity. METHODS: Plasma and sputum IL-33, soluble ST2 (sST2) levels, and ST2L expression on eosinophils were measured in 10 healthy controls and 10 allergic asthmatics. Asthmatics underwent allergen and diluent inhalation challenges. Blood and sputum samples were collected to measure IL-33, sST2, and ST2L eosinophil expression before and 24 h after allergen inhalation. Purified blood eosinophils from allergic asthmatics were incubated overnight with IL-33 to assess ST2 and intracellular IL-5 expression. RESULTS: Baseline levels of IL-33 in sputum and sST2 in plasma and sputum were similar in allergic asthmatics compared to healthy controls. In addition, there was no difference in blood or sputum eosinophil ST2L expression in healthy controls versus allergic asthmatics. Eosinophil ST2L expression was significantly increased 24 h postallergen inhalation in allergic asthmatics. In vitro stimulation of human eosinophils with IL-33 and LPS significantly increased eosinophil ST2L expression and IL-33 stimulation increased intracellular IL-5 expression, which was attenuated by treatment with sST2 and ST2 blockade. CONCLUSION AND CLINICAL RELEVANCE: In mild asthmatics, there was a significant upregulation of ST2 surface expression on eosinophils from blood and sputum following allergen inhalation challenge. In vitro, IL-33 stimulation of eosinophils increases both ST2 membrane expression and IL-5 production. These results support a role for IL-33 in causing allergen-induced eosinophilia. Blockade of IL-33 and ST2 signaling may present a novel therapeutic avenue for asthma treatment.

Allergen-induced Increases in Sputum Levels of Group 2 Innate Lymphoid Cells in Subjects with Asthma.

RATIONALE: Group 2 innate lymphoid cells (ILC2), a major source of type 2 cytokines, initiate eosinophilic inflammatory responses in murine models of asthma. OBJECTIVES: To investigate the role of ILC2 in allergen-induced airway eosinophilic responses in subjects with atopy and asthma. METHODS: Using a diluent-controlled allergen challenge crossover study, where all subjects (n = 10) developed allergen-induced early and late responses, airway eosinophilia, and increased methacholine airway responsiveness, bone marrow, blood, and sputum samples were collected before and after inhalation challenge. MEASUREMENTS AND MAIN RESULTS: ILC2 (lin-FcεRI-CD45+CD127+ST2+) and CD4+T lymphocytes were enumerated by flow cytometry, as well as intracellular IL-5 and IL-13 expression. Steroid sensitivity of ILC2 and CD4+ T cells was investigated in vitro. A significant increase in total, IL-5+, IL-13+, and CRTH2+ ILC2 was found in sputum, 24 hours after allergen, coincident with a significant decrease in blood ILC2. Total, IL-5+, and IL-13+, but not CRTH2+, CD4+ T cells significantly increased at 24 and 48 hours after allergen in sputum. In blood and bone marrow, only CD4+ cells demonstrated increased activation after allergen. Airway eosinophilia correlated with IL-5+ ILC2 at all time points and allergen-induced changes in IL-5+ CD4+ cells at 48 hours after allergen. Dexamethasone significantly attenuated IL-2- and IL-33-stimulated IL-5 and IL-13 production by both cell types. CONCLUSIONS: Innate and adaptive immune cells are increased in the airways associated with allergic asthmatic responses. Total and type 2 cytokine-positive ILC2 are increased only within the airways, whereas CD4+ T lymphocytes demonstrated local and systemic increases. Steroid sensitivity of both cells may explain effectiveness of this therapy in those with mild asthma.

Regulation of human airway smooth muscle cell migration and relevance to asthma.

Airway remodelling is an important feature of asthma pathogenesis. A key structural change inherent in airway remodelling is increased airway smooth muscle mass. There is emerging evidence to suggest that the migration of airway smooth muscle cells may contribute to cellular hyperplasia, and thus increased airway smooth muscle mass. The precise source of these cells remains unknown. Increased airway smooth muscle mass may be collectively due to airway infiltration of myofibroblasts, neighbouring airway smooth muscle cells in the bundle, or circulating hemopoietic progenitor cells. However, the relative contribution of each cell type is not well understood. In addition, although many studies have identified pro and anti-migratory agents of airway smooth muscle cells, whether these agents can impact airway remodelling in the context of human asthma, remains to be elucidated. As such, further research is required to determine the exact mechanism behind airway smooth muscle cell migration within the airways, how much this contributes to airway smooth muscle mass in asthma, and whether attenuating this migration may provide a therapeutic avenue for asthma. In this review article, we will discuss the current evidence with respect to the regulation of airway smooth muscle cell migration in asthma.

IL-25 Receptor Expression on Airway Dendritic Cells after Allergen Challenge in Subjects with Asthma.

RATIONALE: IL-25 is an epithelial-derived cytokine, whose effects are mediated by the IL-25 receptor (IL-17RB), and that has been implicated in the pathogenesis of allergic disease and airway viral responses. Airway myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) are professional antigen-presenting cells. pDCs may play a protective role in asthma and are key players in the innate immune response through recognition of microbial products via Toll-like receptors (TLRs). The effects of inhaled allergens on the expression of IL-17RB by mDCs and pDCs, and the effects of IL-25 on pDCs, are unknown. OBJECTIVES: To evaluate allergen-induced changes in IL-17RB expression by mDCs and pDCs and to investigate the effects of IL-25 on pDCs. METHODS: Patients with mild atopic asthma (n = 13) were challenged with inhaled allergen. Blood and sputum DCs were enumerated and IL-17RB expression was determined by flow cytometry before and 7 and 24 hours after allergen challenge. The effects of IL-25 on pDCs in vitro were also assessed. MEASUREMENTS AND MAIN RESULTS: Inhaled allergen significantly increased mDC and pDC numbers in sputum but not in blood. The percentage of IL-17RB(+) mDCs and pDCs was significantly increased in blood and sputum 24 hours after challenge. IL-25 up-regulated TLR9 expression by pDCs and orchestrated the responses to TLR9 ligation. CONCLUSIONS: IL-17RB is up-regulated on blood and sputum mDCs and pDCs after allergen inhalation. IL-25 modulates pDC function through an effect on TLR9 expression.

IL-25 and IL-33 induce Type 2 inflammation in basophils from subjects with allergic asthma.

BACKGROUND: The alarmin cytokines IL-25 and IL-33 are key promoters of type 2 inflammation. Basophils respond to alarmin cytokines, however the relationship of these cytokines with basophil activation and recruitment in human studies of allergic asthma has not been well characterized. This study investigated the effect of IL-25 and IL-33 on basophils in a model of allergic asthma. METHODS: 10 mild allergic asthmatics underwent allergen and diluent inhalation challenges. Bone marrow aspirates were collected at pre-challenge and 24 h (h) post challenge. Peripheral blood and sputum samples were collected at pre-challenge, 7 h, and 24 h post-challenge to measure basophil expression of IL-17RB, ST2, and intracellular IL-25. Freshly isolated peripheral blood basophils from allergic donors were incubated overnight with IL-25 and IL-33, or sputum supernatant collected post-allergen to assess pro-inflammatory effects of mediators released in the airways. RESULTS: There were increased percentage of basophils expressing IL-17RB, ST2, and intracellular IL-25 collected from bone marrow, peripheral blood, and sputum after allergen inhalation challenge. In vitro stimulation with IL-25 and IL-33 increased the percentage of basophils expressing intracellular type 2 cytokines and surface activation markers, and primed eotaxin-induced migratory potential of basophils, which was mediated directly through IL-17RB and ST2, respectively. Stimulation of basophils with sputum supernatants collected post-allergen challenge up-regulated the percentage of basophils expressing markers of activation and intracellular type 2 cytokines, which was reversed following blockade of the common ß chain (ßc). CONCLUSIONS: Our findings indicate that the alarmin cytokines IL-33 and IL-25 increase basophil activation and migratory potential, and may pose as a novel therapeutic targets for the treatment of allergic asthma.

Allergen-Induced Increases in Interleukin-25 and Interleukin-25 Receptor Expression in Mature Eosinophils from Atopic Asthmatics.

BACKGROUND: Interleukin (IL)-25 plays a pivotal role in type 2 immune responses. In a baseline cross-sectional study, we previously showed that IL-25 plasma levels and IL-25 receptor (IL-25R: IL-17RA, IL-17RB, and IL-17RA/RB) expression on mature blood eosinophils are increased in atopic asthmatics compared to normal nonatopic controls. This study investigated allergen-induced changes in IL-25 and IL-25R expression in eosinophils from asthmatics. METHODS: Dual responder atopic asthmatics (n = 14) were enrolled in this randomized diluent-controlled crossover allergen challenge study. Blood was collected before and 24 h after the challenge. The surface expression of IL-25R was evaluated by flow cytometry on eosinophils and Th2 memory cells. In addition, plasma levels of IL-25 were measured by ELISA, and functional responses to IL-25 including type 2 cytokine expression, degranulation, and the migrational responsiveness of eosinophils were evaluated in vitro. RESULTS: Following the allergen but not the diluent inhalation challenge, significant increases in the expression of IL-17RB and IL-17RA/B were found on eosinophils but not on Th2 memory cells. IL-25 plasma levels and the number of eosinophils but not of Th2 memory cells expressing intracellular IL-25 increased significantly in response to the allergen but not the diluent challenge. Stimulation with physiologically relevant concentrations of IL-25 in vitro caused (i) degranulation of eosinophils (measured by eosinophil peroxidase release), (ii) enhanced intracellular expression of IL-5 and IL-13, and (iii) priming of eosinophil migration to eotaxin. IL-25 stimulated intracellular cytokine expression, and the migration of eosinophils was blocked in the presence of a neutralizing IL-25 antibody. CONCLUSIONS: Our findings suggest that the IL-25/IL-25R axis may play an important role in promoting the recruitment and proinflammatory function of eosinophils in allergic asthma.

The effects of a CXCR1/CXCR2 antagonist on neutrophil migration in mild atopic asthmatic subjects.

BACKGROUND: Neutrophils are effector cells recruited to airways in patients with asthma. Migration of neutrophils occurs predominantly through activation of the CXCR1 and CXCR2 receptors by CXC chemokines, including IL-8 and Gro-α. The dual CXCR1/CXCR2 antagonist SCH 527123 has been developed to target neutrophil migration to alleviate airway neutrophilia. This study investigated the effects of SCH 527123 on neutrophil levels within the bone marrow, peripheral blood and airways, and on isolated bone marrow and peripheral blood neutrophil migration from mild allergic asthmatics. METHODS: Thirteen subjects with mild allergic asthma completed a double blind, placebo-controlled, multi-center crossover study and were randomized to daily dosing of 30 mg SCH 527123 and placebo for 8 days. Subjects provided bone marrow, peripheral blood and sputum samples pre-dosing and on the last day of dosing. Neutrophil numbers were quantified in all samples and chemotaxis assays were performed on neutrophils purified from bone marrow and peripheral blood. RESULTS: Neutrophil numbers fell significantly in the peripheral blood and sputum following treatment with SCH 527123 compared to placebo treatment. No change in neutrophil numbers was observed in bone marrow. SCH 527123 reduced IL-8-induced migration of purified peripheral blood neutrophils (p < 0.05), but had limited effects on migration of neutrophils purified from bone marrow. CONCLUSIONS: The results from this study demonstrate that oral administration of the dual CXCR1/CXCR2 antagonist SCH 527123 reduces neutrophil levels in the circulation and airways through inhibition of migration. There were no toxic effects of SCH 527123 on granulocytic progenitor cells in the bone marrow.

Thymic stromal lymphopoietin activation of basophils in patients with allergic asthma is IL-3 dependent.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) released after antigenic stimulation of allergic asthmatic airways is a key initiator of type 2 inflammation. Basophils are important effectors of allergic inflammation in the airways. Murine basophils have been shown to respond to TSLP independently of IL-3 by increasing functional thymic stromal lymphopoietin receptor (TSLPR) expression. OBJECTIVE: The purpose of this study was to investigate the effect of TSLP stimulation on human basophil function. METHODS: Ten patients with mild allergic asthma underwent diluent and allergen inhalation challenges. Peripheral blood and sputum samples were collected at baseline and 7 and 24 hours after challenge, and bone marrow samples were collected at baseline and 24 hours after challenge to measure basophil TSLPR expression. In vitro experiments were conducted on purified human basophils to measure the effect of TSLP on degranulation, expression of activation markers and TH2 cytokines, and eotaxin-induced shape change. RESULTS: Allergen inhalation increased basophil numbers in the airways and significantly upregulated the expression of activation markers, TH2 intracellular cytokines, and receptors for TSLP, IL-3, and eotaxin in blood, bone marrow, and sputum basophils. In vitro stimulation with TSLP primed basophil migration to eotaxin and induced rapid and sustained basophil activation mediated directly through TSLPR and indirectly through an IL-3-mediated basophil autocrine loop. Basophils responded to TSLP at a similar magnitude and potency as the well-described basophil-activating stimuli IL-3 and anti-IgE. CONCLUSION: Our findings indicate that basophil activation during early- and late-phase responses to inhaled allergen might be driven at least in part by TSLP.