Brittany M. Salter

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.

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−Fc&egr;RI−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.

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/&mgr;l), 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 &Dgr; 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).

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.

Hematopoietic Processes in Eosinophilic Asthma

&NA; Airway eosinophilia is a hallmark of allergic asthma, and understanding mechanisms that promote increases in lung eosinophil numbers is important for effective pharmacotherapeutic development. It has become evident that expansion of hematopoietic compartments in the bone marrow (BM) promotes differentiation and trafficking of mature eosinophils to the airways. Hematopoietic progenitor cells egress the BM and home to the lungs, where in situ differentiation within the tissue provides an ongoing source of proinflammatory cells. In addition, hematopoietic progenitor cells in the airways can respond to locally derived alarmins to produce a panoply of cytokines, thereby themselves acting as effector proinflammatory cells that potentiate type 2 responses in eosinophilic asthma. In this review, we provide evidence for these findings and discuss novel targets for modulating eosinophilopoietic processes, migration, and effector function of precursor cells.

Glucagon‐like peptide‐1 receptor expression on human eosinophils and its regulation of eosinophil activation

Glucagon‐like peptide‐1 (GLP‐1) and its receptor are part of the incretin family of hormones that regulate glucose metabolism. GLP‐1 also has immune modulatory roles.

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.

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.

Dysregulation of Vascular Endothelial Progenitor Cells Lung-Homing in Subjects with COPD

Chronic obstructive pulmonary disease (COPD) is characterized by fixed airflow limitation and progressive decline of lung function and punctuated by occasional exacerbations. The disease pathogenesis may involve activation of the bone marrow stimulating mobilization and lung-homing of progenitor cells. We investigated the hypothesis that lower circulating numbers of vascular endothelial progenitor cells (VEPCs) are a consequence of increased lung-sequestration in COPD. Nonatopic, current or ex-smokers with diagnosed COPD and nonatopic, nonsmoking normal controls were enrolled. Blood and induced sputum extracted primitive hemopoietic progenitors (HPCs) and VEPC were enumerated by flow cytometry. Migration and adhesive responses to fibronectin were assessed. In sputum, VEPC numbers were significantly greater in COPD compared to normal controls. In blood, VEPCs were significantly lower in COPD versus normal controls. There were no differences in HPC levels between the two groups in either compartment. Functionally, there was a greater migrational responsiveness of progenitors from COPD subjects to stromal cell-derived factor-1alpha (SDF-1α) compared to normal controls. This was associated with greater numbers of CXCR4+ progenitors in sputum from COPD. Increased migrational responsiveness of progenitor cells may promote lung-homing of VEPC in COPD which may disrupt maintenance and repair of the airways and contribute to COPD disease pathogenesis.

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

&NA; 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 &agr; 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.