Eosinophils are found in the lungs of humans with allergic asthma, as well as in the lungs of animals in models of this disease. Increasing evidence suggests that these cells are integral to the development of allergic asthma in C57BL/6 mice. However, the specific function of eosinophils that is required for this event is not known. In this study, we experimentally validate a dynamic computational model and perform follow-up experimental observations to determine the mechanism of eosinophil modulation of T cell recruitment to the lung during development of allergic asthma. We find that eosinophils deficient in IL-13 were unable to rescue airway hyperresponsiveness, T cell recruitment to the lungs, and Th2 cytokine/chemokine production in ΔdblGATA eosinophil-deficient mice, even if Th2 cells were present. However, eosinophil-derived IL-13 alone was unable to rescue allergic asthma responses in the absence of competence of other IL-13–producing cells. We further computationally investigate the role of other cell types in the production of IL-13, which led to the various predictions including early and late pulses of IL-13 during airway hyperresponsiveness. These experiments suggest that eosinophils and T cells have an interdependent relationship, centered on IL-13, which regulates T cell recruitment to the lung and development of allergic asthma.

Glucocorticoids are considered first-line therapy in a variety of eosinophilic disorders. They lead to a transient, profound decrease in circulating human eosinophils within hours of administration. The phenomenon of glucocorticoid-induced eosinopenia has been the basis for the use of glucocorticoids in eosinophilic disorders, and it has intrigued clinicians for 7 decades, yet its mechanism remains unexplained. To investigate, we first studied the response of circulating eosinophils to in vivo glucocorticoid administration in 3 species and found that the response in rhesus macaques, but not in mice, closely resembled that in humans. We then developed an isolation technique to purify rhesus macaque eosinophils from peripheral blood and performed live tracking of zirconium-89-oxine–labeled eosinophils by serial positron emission tomography/computed tomography imaging, before and after administration of glucocorticoids. Glucocorticoids induced rapid bone marrow homing of eosinophils. The kinetics of glucocorticoid-induced eosinopenia and bone marrow migration were consistent with those of the induction of the glucocorticoid-responsive chemokine receptor CXCR4, and selective blockade of CXCR4 reduced or eliminated the early glucocorticoid-induced reduction in blood eosinophils. Our results indicate that glucocorticoid-induced eosinopenia results from CXCR4-dependent migration of eosinophils to the bone marrow. These findings provide insight into the mechanism of action of glucocorticoids in eosinophilic disorders, with implications for the study of glucocorticoid resistance and the development of more targeted therapies. The human study was registered at ClinicalTrials.gov as #NCT02798523.

To the Editor, Glucocorticoids (GC) are the mainstay of therapy for a variety of eosinophil-associated disorders, including hypereosinophilic syndromes (HES), 1 although responses are not universal and GC are

Eosinophils are critical cellular mediators in allergic asthma and inflammation, however signals that regulate their functions are unclear. The transcription factor STAT6 regulates Th2 cytokine responses, acting downstream of IL-4 and -13. We have previously shown that eosinophil derived IL-13 plays an important role in the recruitment of T cells to the lung, and subsequent development of allergic asthma. However whether eosinophils respond to Th2 signals to control allergic airway inflammation is unclear. Here, we show that STAT6−/− eosinophils are unable to induce the development of allergic lung inflammation, including recruitment of CD4+ T cells, mucous production and development of airways hyperresponsiveness. This is likely due to the reduced migration of STAT6−/− eosinophils to the lung and in response to eotaxin. These data indicate that like Th cells, eosinophils need to respond to Th2 cytokines via STAT6 during the development of allergic airway inflammation.

Background Glucocorticoids (GC) are considered first‐line therapy for most patients with hypereosinophilic syndrome (HES). Although response rates are generally high, many patients require moderate to high doses for control of eosinophilia and symptoms, and up to 15% of patients do not respond at all. Despite this, little is known about the mechanisms of GC resistance in patients with HES. Objective To explore the aetiology of GC resistance in HES. Methods Clinical data and samples from 26 patients with HES enrolled on a prospective study of GC responsiveness and 23 patients with HES enrolled on a natural history study of eosinophilia for whom response to GC was known were analysed retrospectively. Expression of GC receptor isoforms was assessed by quantitative RT‐PCR in purified eosinophils. Serum cytokine levels were quantified by suspension array assay in multiplex. Results Despite an impaired eosinophil response to GC after 7 days of treatment, the expected rise in absolute neutrophil count was seen in 7/7 GC‐resistant patients, suggesting that GC resistance in HES is not a global phenomenon. Eosinophil mRNA expression of glucocorticoid receptor (GR) isoforms (α, β, and P) was similar between GC‐sensitive (n = 20) and GC‐resistant (n = 9) patients with HES. Whereas geometric mean serum levels were also comparable between GC‐r (n = 11) and GC‐s (n = 19) for all cytokines tested, serum IL‐5 levels were >100 pg/mL only in GC‐r patients. Conclusions and Clinical Relevance These data suggest that the mechanism of GC resistance in HES is not due to a global phenomenon affecting all lineages, but may be due, at least in some patients, to impairment of eosinophil apoptosis by increased levels of IL‐5.

Glucocorticoids are first-line agents for the treatment of many eosinophil-associated disorders; however, their effects on human eosinophils remain poorly understood. To gain an unbiased, genome-wide view of the early transcriptional effects of glucocorticoids on human eosinophils in vivo, RNA sequencing was performed on purified blood eosinophils obtained before and 30, 60, and 120 minutes after administration of a single dose of oral prednisone (1 mg/kg) to three unrelated healthy subjects with hypereosinophilia of unknown significance. The resulting dataset is of high quality and suitable for differential expression analysis. Flow cytometry and qPCR were then performed on three additional cohorts of human subjects, to validate the key findings at the transcript and protein levels. The resulting datasets provide a resource for understanding the response of circulating human eosinophils to glucocorticoid administration.

Allergic asthma and inflammation is a disease of the airways. The prevalence of allergic responses and asthma are increasing worldwide, but it is still unclear why some predisposed individuals develop disease. Eosinophils and T helper 2 cells (TH2) and eosinophils are critical cellular mediators in asthma. The signal transducer and activator of transcription (STAT)-6 is a crucial mediator of TH2 cytokine responses, acting downstream of interleukin -4 and -13 (IL-4 and IL-13). We have previously shown that eosinophil derived IL-13 plays an important role in the recruitment of T cells to the lung, and subsequent development of allergic asthma. However whether eosinophils respond to TH2 signals to control allergic airway inflammation is unclear. The objective of this study is to understand the role of STAT6 in eosinophil-dependent regulation of allergic asthma and inflammation. Using ΔdblGATA mice lacking eosinophils and transfer of STAT6-/- eosinophils, we found that compared to ΔdblGATA reconstituted with WT eosinophils, eosinophils lacking STAT6 were unable to rescue recruitment of CD4+ T cells and eosinophils into the lungs compared to WT eosinophils. These data indicates that STAT6 is important in the recruitment of both T cells and eosinophils to the lung during the development of allergic airway inflammation. Ultimately, deciphering the mechanisms of intracellular and cytokine signals will afford us improved insights on developing therapeutic strategies in asthma.

RATIONALE:We have developed an oil-in-water based nanoemulsion (NE) that induces strong systemic and mucosal immunity against a variety of pathogens when used as a mucosal adjuvant. In an effort to enhance the mucosal immune responses of NE, we co-formulated it with retinoic acid (NERA). RA, a vitamin A derivative, has been shown to orchestrate T cell homing towards the gut as well as IgA production by retinaldehyde dehydrogenase (RALDH) in dendritic cells (DCs). METHODS: To examine the efficacy of NERA in-vitro, we simulated invivo intranasal immunization in a co-culture model. We treated the epithelial cells with different formulations of NE, followed by co-culturing with dendritic and T cells. Further, animals were intranasally immunized with ova-albumin (Ova) in combination to NE (with and without RA) to study RALDH production by mucosal DCs, T cell gut homing, IgA production and Th1/Th2/Th17 cytokine production. RESULTS: NE alone induces RALDH activity in co-cultured DCs and induces gut homing marker expression in-vitro. Also, immunization with NE-Ova showed increased RALDH activity in DCs, resulting in increased IgA production as well as T cell gut homing. Furthermore, antibody titers and cytokine data suggested increased Th1/Th17 response while suppressing Th2. In-vitro studies showed NE activates RALDH partially via Abstracts AB191 SUNDAY