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Whether children and people with asthma and allergic diseases are at increased risk for severe acute respiratory syndrome virus 2 (SARS-CoV-2) infection is unknown.Our aims were to determine the incidence of SARS-CoV-2 infection in households with children and to also determine whether self-reported asthma and/or other allergic diseases are associated with infection and household transmission.For 6 months, biweekly nasal swabs and weekly surveys were conducted within 1394 households (N = 4142 participants) to identify incident SARS-CoV-2 infections from May 2020 to February 2021, which was the pandemic period largely before a vaccine and before the emergence of SARS-CoV-2 variants. Participant and household infection and household transmission probabilities were calculated by using time-to-event analyses, and factors associated with infection and transmission risk were determined by using regression analyses.In all, 147 households (261 participants) tested positive for SARS-CoV-2. The household SARS-CoV-2 infection probability was 25.8%; the participant infection probability was similar for children (14.0% [95% CI = 8.0%-19.6%]), teenagers (12.1% [95% CI = 8.2%-15.9%]), and adults (14.0% [95% CI = 9.5%-18.4%]). Infections were symptomatic in 24.5% of children, 41.2% of teenagers, and 62.5% of adults. Self-reported doctor-diagnosed asthma was not a risk factor for infection (adjusted hazard ratio [aHR] = 1.04 [95% CI = 0.73-1.46]), nor was upper respiratory allergy or eczema. Self-reported doctor-diagnosed food allergy was associated with lower infection risk (aHR = 0.50 [95% CI = 0.32-0.81]); higher body mass index was associated with increased infection risk (aHR per 10-point increase = 1.09 [95% CI = 1.03-1.15]). The household secondary attack rate was 57.7%. Asthma was not associated with household transmission, but transmission was lower in households with food allergy (adjusted odds ratio = 0.43 [95% CI = 0.19-0.96]; P = .04).Asthma does not increase the risk of SARS-CoV-2 infection. Food allergy is associated with lower infection risk, whereas body mass index is associated with increased infection risk. Understanding how these factors modify infection risk may offer new avenues for preventing infection.

Hypereosinophilic syndrome (HES) is a diverse group of disorders characterized by peripheral blood eosinophilia of 1.5 × 10

PDF file - 488K, Insertion into the mouse TC21 gene causes a null allele. Supplemental Table 1. Loss of TC21 partially rescues Nf1 mutant embryonic lethality. Supplemental Figure 2: Loss of TC21 decreases the survival of NPCis mice by increasing brain tumors. Supplemental Figure 3. Cell death in spheres and TGFbeta1 expression in sciatic nerves and neurofibromas. Supplemental Figure 4: Nf1-/- DRGs and spheres express high levels of phospho-AKT but not phospho-SMAD2/3. Supplemental Figure 5: Expression of TGFbeta ligands and receptors in mouse MPNST and human MPNST cell lines

Eosinophils develop in the bone marrow from hematopoietic progenitors into mature cells capable of a plethora of immunomodulatory roles via the choreographed process of eosinophilopoiesis. However, the gene regulatory elements and transcription factors (TFs) orchestrating this process remain largely unknown. The potency and resulting diversity fundamental to an eosinophil’s complex immunomodulatory functions and tissue specialization likely result from dynamic epigenetic regulation of the eosinophil genome, a dynamic eosinophil regulome. In this study, we applied a global approach using broad-range, next-generation sequencing to identify a repertoire of eosinophil-specific enhancers. We identified over 8200 active enhancers located within 1–20 kB of expressed eosinophil genes. TF binding motif analysis revealed PU.1 (Spi1) motif enrichment in eosinophil enhancers, and chromatin immunoprecipitation coupled with massively parallel sequencing confirmed PU.1 binding in likely enhancers of genes highly expressed in eosinophils. A substantial proportion (>25%) of these PU.1-bound enhancers were unique to murine, culture-derived eosinophils when compared among enhancers of highly expressed genes of three closely related myeloid cell subsets (macrophages, neutrophils, and immature granulocytes). Gene ontology analysis of eosinophil-specific, PU.1-bound enhancers revealed enrichment for genes involved in migration, proliferation, degranulation, and survival. Furthermore, eosinophil-specific superenhancers were enriched in genes whose homologs are associated with risk loci for eosinophilia and allergic diseases. Our collective data identify eosinophil-specific enhancers regulating key eosinophil genes through epigenetic mechanisms (H3K27 acetylation) and TF binding (PU.1).

The Human Epidemiology and Response to SARS-CoV-2 (HEROS) is a prospective multi-city 6-month incidence study which was conducted from May 2020-February 2021. The objectives were to identify risk factors for SARS-CoV-2 infection and household transmission among children and people with asthma and allergic diseases, and to use the host nasal transcriptome sampled longitudinally to understand infection risk and sequelae at the molecular level. To overcome challenges of clinical study implementation due to the coronavirus pandemic, this surveillance study used direct-to-participant methods to remotely enroll and prospectively follow eligible children who are participants in other NIH-funded pediatric research studies and their household members. Households participated in weekly surveys and biweekly nasal sampling regardless of symptoms. The aim of this report is to widely share the methods and study instruments and to describe the rationale, design, execution, logistics and characteristics of a large, observational, household-based, remote cohort study of SARS-CoV-2 infection and transmission in households with children. The study enrolled a total of 5,598 individuals, including 1,913 principal participants (children), 1,913 primary caregivers, 729 secondary caregivers and 1,043 other household children. This study was successfully implemented without necessitating any in-person research visits and provides an approach for rapid execution of clinical research.

Virus-induced IFN-α secretion by plasmacytoid dendritic cells (pDCs) is negatively impacted by IgE and has been linked to asthma exacerbations. Eosinophils, another contributor to type 2 inflammation, are also associated with asthma severity.We sought to investigate the impact of eosinophils on pDC antiviral interferon responses and determine whether anti-IL-5/5Rα therapy enhances pDC antiviral function.Blood pDCs purified from anonymous donors were stimulated in vitro with rhinovirus (RV)-16 in the presence or absence of eosinophils/eosinophil supernatants. IFN-α was measured in supernatants and RNA collected for bulk RNA-sequencing. Next, purified pDCs from 8 individuals with moderate to severe asthma, treated or not treated with anti-IL-5/5Rα therapy, were cultured ex vivo with or without RV; IFN-α secretion and differential gene expression analysis were compared between groups.Exposure to either eosinophils or eosinophil supernatants inhibited RV-induced pDC IFN-α secretion in a dose-dependent manner and did not impact pDC viability. Eosinophil-derived neurotoxin and TGF-β partially recapitulated pDC IFN-α inhibition. Transcriptome analysis revealed global repression of pDC interferon response patterns by eosinophils, most notably in basal expression of interferon-stimulated genes. Increased RV-induced IFN-α secretion and transcription as well as increased basal interferon-stimulated gene expression was detected in pDCs from participants treated with anti-IL-5/5Rα therapy.Our findings highlight a novel mechanism through which type 2 inflammation regulates pDC IFN-α responses relevant to RV respiratory infections in the context of eosinophilic airway disease, suggesting a potential mechanism through which eosinophil-depleting therapies may reduce severity of RV illnesses.

The eosinophil (Eos) surface phenotype and activation state is altered after recruitment into tissues and after exposure to pro-inflammatory cytokines. In addition, distinct Eos functional subsets have been described, suggesting that tissue-specific responses for Eos contribute to organ homeostasis. Understanding the mechanisms by which Eos subsets achieve their tissue-specific identity is currently an unmet goal for the eosinophil research community. Publicly archived expression data can be used to answer original questions, test and generate new hypotheses, and serve as a launching point for experimental design. With these goals in mind, we investigated the effect of genetic background, culture methods, and tissue residency on murine Eos gene expression using publicly available, genome-wide expression data. Eos differentiated from cultures have a gene expression profile that is distinct from that of native homeostatic Eos; thus, researchers can repurpose published expression data to aid in selecting the appropriate culture method to study their gene of interest. In addition, we identified Eos lung- and gastrointestinal-specific transcriptomes, highlighting the profound effect of local tissue environment on gene expression in a terminally differentiated granulocyte even at homeostasis. Expanding the “toolbox” of Eos researchers to include public-data reuse can reduce redundancy, increase research efficiency, and lead to new biological insights. Publicly archived eosinophil genome-wide expression data can be used to answer original questions, generate new hypotheses, and serve as a launching point for experimental design.

AIM To determine the vaccination rates in pediatric immunosuppression-dependent inflammatory bowel disease (IBD) and review the safety and efficacy of vaccinations in this population. METHODS The electronic medical records from October 2009 to December 2015 of patients diagnosed with IBD at 10 years of age or younger and prescribed anti-tumor necrosis factor alpha (anti-TNF-α) therapy were reviewed for clinical history, medication history, vaccination history, and hepatitis B and varicella titers. Literature discussing vaccination response in IBD patients were identified through search of the MEDLINE database and reviewed using the key words “inflammatory bowel disease”, “immunization”, “vaccination”, “pneumococcal”, “varicella”, and “hepatitis B”. Non-human and non-English language studies were excluded. Search results were reviewed by authors to select articles that addressed safety and efficacy of immunizations in inflammatory bowel disease. RESULTS A total of 51 patients diagnosed with IBD prior to the age of 10 and receiving anti-TNF-α therapy were identified. Thirty-three percent of patients (17/51) had incomplete or no documentation of vaccinations. Sixteen case reports, cohort studies, cross-sectional studies, and randomized trials were determined through review of the literature to describe the safety and efficacy of hepatitis B, pneumococcal, and varicella immunizations in adult and pediatric patients with IBD. These studies showed that patients safely tolerated the vaccines without significant adverse effects. Importantly, IBD patients receiving immunosuppressive medications, particularly anti-TNF-α treatment, have decreased vaccine response compared to controls. However, the majority of patients are still able to achieve protective levels of specific antibodies. CONCLUSION Immunizations have been shown to be well-tolerated and protective immunity can be achieved in patients with IBD requiring immunosuppressive therapy.

Eosinophils and their associated cytokines IL-4 and IL-5 are emerging as central orchestrators of the immune-metabolic axis. Herein, we demonstrate that cross-talk between the Ig-superfamily receptor CD300f and IL-5 is a key checkpoint that modifies the ability of eosinophils to regulate metabolic outcomes. Generation of Il5 Tg /Cd300f−/− mice revealed marked and distinct increases in eosinophil levels and their production of IL-4 in the white and brown adipose tissues. Consequently, Il5 Tg /Cd300f−/− mice had increased alternatively activated macrophage accumulation in the adipose tissue. Cd300f−/− mice displayed age-related accumulation of eosinophils and macrophages in the adipose tissue and decreased adipose tissue weight, which was associated with decreased diet-induced weight gain and insulin resistance. Notably, Il5 Tg /CD300f−/− were protected from diet-induced weight gain and glucose intolerance. These findings highlight the cross-talk between IL-5 receptor and CD300f as a novel pathway regulating adipose tissue eosinophils and offer new entry points for therapeutic intervention for obesity and its complications.

OBJECTIVES A minimally invasive biomarker to monitor disease activity is one of the greatest unmet clinical needs of the pediatric eosinophilic esophagitis (EoE) population. We aimed to determine whether circulating eosinophil progenitors (EoPs) could be used as a biomarker to identify pediatric patients with active EoE. METHODS In a prospective observational study, peripheral blood samples, symptom history, and laboratory data were collected from pediatric patients undergoing endoscopy for evaluation of EoE on dietary therapy at Cincinnati Children's Hospital. Peripheral blood EoP level was determined by flow cytometry. RESULTS Thirty-four children with active (n = 16) and inactive (n = 18) EoE were included in the analysis. EoP levels in the peripheral blood were 3-fold higher in patients with active EoE than inactive EoE (P

BACKGROUND: Little is known about the endoscopic and histologic findings of non-esophageal eosinophilic gastrointestinal diseases (EGID). AIM: To characterize the presenting endoscopic and histologic findings in patients with eosinophilic gastritis (EG), eosinophilic gastroenteritis (EGE), and eosinophilic colitis (EC) at diagnosis and 6 months after initiating the treatment. METHODS: We conducted a retrospective cohort study at 6 US centers associated with the Consortium of Eosinophilic Gastrointestinal Researchers. Data abstracted included demographics, endoscopic findings, tissue eosinophil counts, and associated histologic findings at diagnosis and, when available, after initial treatment. RESULTS: Of 373 subjects (317 children and 56 adults), 142 had EG, 123 EGE, and 108 EC. Normal endoscopic appearance was the most common finding across all EGIDs (62% of subjects). Baseline tissue eosinophil counts were quantified in 105 (74%) EG, 36 (29%) EGE, and 80 (74%) EC subjects. The mean peak gastric eosinophil count across all sites was 87 eos/hpf for EG and 78 eos/hpf for EGE. The mean peak colonic eosinophil count for EC subjects was 76 eos/hpf (range 10–500). Of the 29% of subjects with post-treatment follow-up, most had an improvement in clinical, endoscopic, and histologic findings regardless of treatment utilized. Reductions in tissue eosinophilia correlated with improvements in clinical symptoms as well as endoscopic and histologic findings. CONCLUSIONS: In this large cohort, normal appearance was the most common endoscopic finding, emphasizing the importance of biopsy, regardless of endoscopic appearance. Decreased tissue eosinophilia was associated with improvement in symptoms, endoscopic, and histologic findings, showing that disease activity is reversible.

The literature related to eosinophilic gastritis (EG), gastroenteritis (EGE), and colitis (EC) is limited. We aimed to characterize rates of diagnosis, clinical features, and initial treatments for patients with EG, EGE, and EC.In this retrospective study, data were collected from 6 centers in the Consortium of Eosinophilic Gastrointestinal Researchers from 2005 to 2016. We analyzed demographics, time trends in diagnosis, medical history, presenting symptoms, disease overlap, and initial treatment patterns/responses.Of 373 subjects (317 children and 56 adults), 38% had EG, 33% EGE, and 29% EC. Rates of diagnosis of all diseases increased over time. There was no male predominance, and the majority of subjects had atopy. Presenting symptoms were similar between diseases with nausea/vomiting and abdominal pain, the most common. One hundred fifty-four subjects (41%) had eosinophilic inflammation outside of their primary disease location with the esophagus the second most common gastrointestinal (GI) segment involved. Multisite inflammation was more common in children than in adults (68% vs 37%; P0.001). Initial treatment patterns varied highly between centers. One hundred-nine subjects (29%) had follow-up within 6 months, and the majority had clinical, endoscopic, and histologic improvements.In this cohort, EG, EGE, and EC were diagnosed more frequently over time, and inflammation of GI segments outside the primary disease site co-occurrence of atopy was common with a lack of male predominance. Symptoms were similar between diseases, and initial treatment strategies were highly variable. Future investigation should assess the cause of the increased prevalence of eosinophilic GI disorders and prospectively assess outcomes to establish treatment algorithms.

Blood eosinophil progenitors (EoPs) correlate with tissue pathology during active eosinophilic esophagitis (EoE), providing additional evidence for blood EoP levels as a biomarker for disease activity and suggesting a role for EoPs in EoE pathogenesis.

Financial Support and AcknowledgementsSupport for this project was provided through a research training grant as part of the Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR) (U54 AI117804). CEGIR is part of the Rare Disease Clinical Research Network (RDCRN), an initiative of the Office of Rare Diseases Research (ORDR), NCATS, and is funded through collaboration between NIAID, NIDDK, and NCATS. CEGIR is also supported by patient advocacy groups including APFED CURED and EFC. This project also received support from NIH T32 DK007634 (CCR).Author DisclosersPatricia Fulkerson: Grant funding from the NIH; Consultant for Genentech, Inc; Research support from Knopp Biosciences, LLC.Gary Falk: Research support from Shire, Celgene, Adare, Regeneron. Consulting for ShireJonathan M. Spergel: Consultant for Regeneron, DBV Technology, Kaleo; Grant funding from DBV Technology, Aimmune Therapeutics, Food Allergy Research Education; Royalties from UpToDateNirmala Gonsalves: Royalties from UpToDate; Advisory board for AllakosSandeep K Gupta: Consultant for Alkalos, Abbott, QOL, Receptos; research support from ShireGlenn Furuta: Founder of EnteroTrack; Consultant for Shire; Royalties from UpToDateMarc E. Rothenberg: Consultant for Pulm One, Spoon Guru, ClostraBio, Celgene, Shire, Astra Zeneca, GlaxoSmithKline, Allakos, Adare, Regeneron and Novartis and has an equity interest in the first four listed and Immune Pharmaceuticals, and royalties from reslizumab (Teva Pharmaceuticals), PEESSv2 (Mapi Research Trust) and UpToDate. M.E.R. is an inventor of patents owned by Cincinnati Children’s.Evan Dellon: Consultant for Adare, Allakos, Alivio, Banner, Celgen/Receptos, Enumeral, GSK, Regeneron, Shire; Research funding from Adare, Celegene/Receptos, Miraca, Meritage, Nutricia, Regeneron, Shire, Educational grant from Banner, HoloclaraStudy HighlightsWhat is current knowledge?Eosinophilic gastrointestinal disorders (EGIDs) include eosinophilic esophagitis (EoE), eosinophilic gastritis (EG), gastroenteritis (EGE), and colitis (EC).Non-EoE EGIDs are rare with most studies limited to case reports or review of single center experiences.There are no widely established guidelines for the diagnosis of EG, EGE, or EC.What is new here?In this multicenter study, EG, EGE, and EC were all diagnosed with increasing frequency over the past decade.Presenting symptoms are non-specific and do not reliably distinguish between disorders.There was no male predominance and the majority of subjects had atopy.Co-occurrence of EG, EGE, and EC diagnoses is common, seen in 41% of patients.There is substantial variability between centers in initial treatment approaches.

Hypereosinophilia (HE) is currently defined by a peripheral blood absolute eosinophil count (AEC) of ≥1,500 cells/microL. Although mild blood eosinophilia (AEC 500-1,500 cells/microL) is observed relatively frequently within the pediatric population, persistent HE is uncommon and should prompt additional clinical evaluation. While the clinical manifestations and underlying etiologies of HE in adults have been well-characterized, there is a paucity of data on HE in children. Limited evidence suggests that many similarities between adult and pediatric HE likely exist, but some important differences remain between these populations. The evaluation of HE in children can be challenging given the broad differential diagnosis, which includes primary hematologic disorders and secondary eosinophilia in which the increased eosinophil levels are propagated by disease states that promote eosinophil production and survival. On the basis of the underlying etiology, clinical manifestations can range from benign, self-resolving elevations in the AEC to life-threatening disorders with the potential for significant end-organ damage. Given the broad differential diagnosis of HE, it remains essential to systematically approach the evaluation of unexplained HE in children. This review will discuss the differential diagnosis for pediatric HE, highlighting etiologies that are more prevalent within the pediatric population. Additionally, a summary of the epidemiology of pediatric HE will be presented, with focus on some of the differences that exist between pediatric and adult HE. Finally, a directed approach to the diagnostic evaluation of children with HE will be discussed.

BACKGROUND: Recent data associate eosinophilic esophagitis (EoE) with IgG4 rather than IgE, but its significance and function have not been determined. Our aims were to measure esophageal IgG4 levels and to determine functional correlations as assessed by histologic and transcriptome analyses. METHODS: This case-control study included pediatric subjects with EoE (≥ 15 eosinophils/HPF) and non-EoE controls. Protein lysates were analyzed for IgA, IgM, and IgG1-IgG4 using the Luminex 100 system; IgE was quantified by ELISA. Esophageal biopsies were scored using the EoE histology scoring system. Transcripts were probed by the EoE diagnostic panel, designed to examine the expression of 96 esophageal transcripts. RESULTS: Esophageal IgG subclasses, IgA, and IgM, but not IgE, were increased in subjects with EoE relative to controls. The greatest change between groups was seen in IgG4 (4.2 mg/g protein [interquartile range: 1.0-13.1 mg/g protein] vs. 0.2 mg/g protein [0.1-0.9]; P < 0.0001). Tissue IgG4 levels correlated with esophageal eosinophil counts (P = 0.0006); histologic grade (P = 0.0011) and stage (P = 0.0112) scores; IL4, IL10, IL13, but not TGFB1, expression; and had strong associations with a subset of the EoE transcriptome. Esophageal IgG4 transcript expression was increased and correlated with IgG4 protein levels and IL10 expression. CONCLUSIONS: These findings extend prior studies on IgG4 in adult EoE to the pediatric population and provide deeper understanding of the potential significance and regulation of IgG4, demonstrating that IgG4 is a relevant feature of the disease, is closely related to esophageal eosinophil levels, type 2 immunity and T regulatory cytokines, and is likely produced locally.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a human cell surface protein expressed exclusively on eosinophils, mast cells, and basophils that, when engaged, induces eosinophil apoptosis and inhibits mast cell mediator release. This makes Siglec-8 a promising therapeutic target to treat diseases involving these cell types. However, pre-clinical studies of Siglec-8 targeting in vivo are lacking because this protein is only found in humans, apes, and some monkeys. Therefore, we have developed a mouse strain in which SIGLEC8 transcription is activated by Cre recombinase and have crossed this mouse with the eoCre mouse to achieve eosinophil-specific expression. We confirmed that Siglec-8 is expressed exclusively on the surface of mature eosinophils in multiple tissues at levels comparable to those on human blood eosinophils. Following ovalbumin sensitization and airway challenge, Siglec-8 knock-in mice generated a pattern of allergic lung inflammation indistinguishable from that of littermate controls, suggesting that Siglec-8 expression within the eosinophil compartment does not alter allergic eosinophilic inflammation. Using bone marrow from these mice, we demonstrated that, during maturation, Siglec-8 expression occurs well before the late eosinophil developmental marker CCR3, consistent with eoCre expression. Antibody ligation of the receptor induces Siglec-8 endocytosis and alters the phosphotyrosine profile of these cells, indicative of productive signaling. Finally, we demonstrated that mouse eosinophils expressing Siglec-8 undergo cell death when the receptor is engaged, further evidence that Siglec-8 is functional on these cells. These mice should prove useful to investigate Siglec-8 biology and targeting in vivo in a variety of eosinophilic disease models.

Human eosinophils have characteristic morphologic features, including a bilobed nucleus and cytoplasmic granules filled with cytotoxic and immunoregulatory proteins that are packaged in a specific manner. Eosinophil production in the bone marrow is exquisitely regulated by timely expression of a repertoire of transcription factors that work together via collaborative and hierarchical interactions to direct eosinophil development. In addition, proper granule formation, which occurs in a spatially organized manner, is an intrinsic checkpoint that must be passed for proper eosinophil production to occur. In eosinophil-associated disorders, eosinophils and their progenitors can be recruited in large numbers into tissues where they can induce proinflammatory organ damage in response to local signals. Eosinophils are terminally differentiated and do not proliferate once they leave the bone marrow. The cytokine IL-5 specifically enhances eosinophil production and, along with other mediators, promotes eosinophil activation. Indeed, eosinophil depletion with anti-IL-5 or anti-IL-5Rα is now proven to be clinically beneficial for several eosinophilic disorders, most notably severe asthma, and several therapeutics targeting eosinophil viability and production are now in development. Significant progress has been made in our understanding of eosinophil development and the consequences of tissue eosinophilia. Future research efforts focused on basic eosinophil immunobiology and translational efforts to assist in the diagnosis, treatment selection, and resolution of eosinophil-associated disorders will likely be informative and clinically helpful.

Human eosinophils have characteristic morphologic features, including a bilobed nucleus and cytoplasmic granules filled with cytotoxic and immunoregulatory proteins that are packaged in a specific manner. Eosinophil production in the bone marrow is exquisitely regulated by timely expression of a repertoire of transcription factors that work together via collaborative and hierarchical interactions to direct eosinophil development. In addition, proper granule formation, which occurs in a spatially organized manner, is an intrinsic checkpoint that must be passed for proper eosinophil production to occur. In eosinophil-associated disorders, eosinophils and their progenitors can be recruited in large numbers into tissues where they can induce proinflammatory organ damage in response to local signals. Eosinophils are terminally differentiated and do not proliferate once they leave the bone marrow. The cytokine IL-5 specifically enhances eosinophil production and, along with other mediators, promotes eosinophil activation. Indeed, eosinophil depletion with anti-IL-5 or anti-IL-5Rα is now proven to be clinically beneficial for several eosinophilic disorders, most notably severe asthma, and several therapeutics targeting eosinophil viability and production are now in development. Significant progress has been made in our understanding of eosinophil development and the consequences of tissue eosinophilia. Future research efforts focused on basic eosinophil immunobiology and translational efforts to assist in the diagnosis, treatment selection, and resolution of eosinophil-associated disorders will likely be informative and clinically helpful.

Dynamic gene expression is a major mechanism that directs hematopoietic lineage commitment and differentiation. Recent advances have revealed an association between chromatin signatures and functional genetic sequences such that these chromatin signatures can be used to predict regulatory elements such as enhancers that may direct lineage differentiation. Our understanding of the genetic elements that regulate eosinophil development is very limited, likely due to the technical challenges in working with a rare complex cell. Herein, we describe protocols to sort mature eosinophils from the bone marrow of mice and to prepare chromatin that can be used for ChIP studies for genome-wide mapping of histone marks and transcription factors in mature eosinophils. Comprehensive epigenomic profiling during critical stages in eosinophil development will ultimately aid in defining the gene regulatory networks necessary to regulate eosinophil production.

Eosinophil-associated diseases (EADs) are rare, heterogeneous disorders characterized by the presence of eosinophils in tissues and/or peripheral blood resulting in immunopathology. The heterogeneity of tissue involvement, lack of sufficient animal models, technical challenges in working with eosinophils, and lack of standardized histopathologic approaches have hampered progress in basic research. Additionally, clinical trials and drug development for rare EADs are limited by the lack of primary and surrogate endpoints, biomarkers, and validated patient-reported outcomes. Researchers with expertise in eosinophil biology and eosinophil-related diseases reviewed the state of current eosinophil research, resources, progress, and unmet needs in the field since the 2012 meeting of the NIH Taskforce on the Research of Eosinophil-Associated Diseases (TREAD). RE-TREAD focused on gaps in basic science, translational, and clinical research on eosinophils and eosinophil-related pathogenesis. Improved recapitulation of human eosinophil biology and pathogenesis in murine models was felt to be of importance. Characterization of eosinophil phenotypes, the role of eosinophil subsets in tissues, identification of biomarkers of eosinophil activation and tissue load, and a better understanding of the role of eosinophils in human disease were prioritized. Finally, an unmet need for tools for use in clinical trials was emphasized. Histopathologic scoring, patient- and clinician-reported outcomes, and appropriate coding were deemed of paramount importance for research collaborations, drug development, and approval by regulatory agencies. Further exploration of the eosinophil genome, epigenome, and proteome was also encouraged. Although progress has been made since 2012, unmet needs in eosinophil research remain a priority. Review on eosinophil biology and eosinophil-related disorders (2012–2017) with a focus on continued unmet needs in eosinophil-associated diseases.

The production of mature eosinophils is a tightly orchestrated process with the aim to sustain normal eosinophil levels in tissues while also maintaining low numbers of these complex and sensitive cells in the blood. To identify regulators of homeostatic eosinophilopoiesis in mice, we took a global approach to identify genome-wide transcriptome and epigenome changes that occur during homeostasis at critical developmental stages, including eosinophil-lineage commitment and lineage maturation. Our analyses revealed a markedly greater number of transcriptome alterations associated with eosinophil maturation (1199 genes) than with eosinophil-lineage commitment (490 genes), highlighting the greater transcriptional investment necessary for differentiation. Eosinophil progenitors (EoPs) were noted to express high levels of granule proteins and contain granules with an ultrastructure distinct from that of mature resting eosinophils. Our analyses also delineated a 976-gene eosinophil-lineage transcriptome that included a repertoire of 56 transcription factors, many of which have never previously been associated with eosinophils. EoPs and eosinophils, but not granulocyte-monocyte progenitors (GMPs) or neutrophils, expressed Helios and Aiolos, members of the Ikaros family of transcription factors, which regulate gene expression via modulation of chromatin structure and DNA accessibility. Epigenetic studies revealed a distinct distribution of active chromatin marks between genes induced with lineage commitment and genes induced with cell maturation during eosinophil development. In addition, Aiolos and Helios binding sites were significantly enriched in genes expressed by EoPs and eosinophils with active chromatin, highlighting a potential novel role for Helios and Aiolos in regulating gene expression during eosinophil development.

Eosinophilic esophagitis (EoE) is a chronic, food antigen–driven gastrointestinal disease that is characterized by esophageal eosinophilia. Currently, there are no Food and Drug Administration (FDA)-approved treatments for EoE, but the two most commonly prescribed therapies include topical corticosteroids and food elimination diets. Clinical trials have revealed a significant proportion of cases that are resistant to topical corticosteroids, and although we define EoE as a food-antigen driven disease, not all patients with EoE respond to elimination diets or even elemental diets. The varied response to treatments highlights the heterogeneity of EoE and the need for new treatment strategies. Despite the clinical differences in treatment response, predicting the outcome remains difficult since factors including age, histologic severity at diagnosis, atopic history, and anthropometrics are not predictive of treatment response. In our practice at an academic pediatric referral center, we observe distinct clinical EoE phenotypes, including cases with atopy, connective tissue disorders, or responsiveness to a proton pump inhibitor. Similar to the work in progress with asthma, stratification of patients with EoE by clinical phenotypes and/or molecular endotypes will likely assist with therapy selection and prediction of natural history. Molecular analysis with gene expression panels also show promise in helping us classify patients based on molecular endotypes. In additional to the clinical and molecular classifications, more accurate histological diagnostic criteria for EoE may help us tease out small differences between patient cohorts. Despite the leaps in knowledge over the past decade regarding EoE pathogenesis, it remains a challenge to predict the response to treatment. Future studies focused on molecular, genetic, and immunologic analysis of larger patient cohorts are needed to assist in identifying EoE phenotypes and endotypes as we attempt to improve patient outcomes in pediatric EoE.

We come together to mourn the untimely passing of Dr. James (Jamie) J. Lee, Professor of Biochemistry and Molecular Biology in the Division of Pulmonary Medicine at Mayo Clinic Arizona. A leading scientist, talented teacher, prominent role model, and Eosino-philosopher-in-Chief, Jamie's many enthusiasms and joy for science inspired us all. Together with his wife and research partner, Dr. Nancy Lee, Jamie's academic career was dedicated to improving our understanding of eosinophils and to exploring their contributions to chronic respiratory diseases and cancer. This article is protected by copyright. All rights reserved.

Eosinophils originate in the bone marrow from an eosinophil lineage–committed, IL-5Rα–positive, hematopoietic progenitor (eosinophil progenitor). Indeed, IL-5 is recognized as a critical regulator of eosinophilia and has effects on eosinophil progenitors, eosinophil precursors, and mature eosinophils. However, substantial levels of eosinophils remain after IL-5 neutralization or genetic deletion, suggesting that there are alternative pathways for promoting eosinophilia. In this study, we investigated the contributory role of IL-5 accessory cytokines on the final stages of eosinophil differentiation. IL-5 stimulation of low-density bone marrow cells resulted in expression of a panel of cytokines and cytokine receptors, including several ligand–receptor pairs. Notably, IL-4 and IL-4Rα were expressed by eosinophil precursors and mature eosinophils. Signaling through IL-4Rα promoted eosinophil maturation when IL-5 was present, but IL-4 stimulation in the absence of IL-5 resulted in impaired eosinophil survival, suggesting that IL-4 cooperates with IL-5 to promote eosinophil differentiation. In contrast, CCL3, an eosinophil precursor–produced chemokine that signals through CCR1, promotes terminal differentiation of CCR1-positive eosinophil precursors in the absence of IL-5, highlighting an autocrine loop capable of sustaining eosinophil differentiation. These findings suggest that brief exposure to IL-5 is sufficient to initiate a cytokine cooperative network that promotes eosinophil differentiation of low-density bone marrow cells independent of further IL-5 stimulation.

BACKGROUND: Allergen-specific IgG(4) (sIgG(4)) antibodies are often associated with tolerance, but sIgG(4) antibodies to causally relevant foods have been reported recently in adults with eosinophilic esophagitis (EoE). Prevalence and levels of food sIgG(4) are not well established in the general pediatric population. OBJECTIVE: We sought to investigate serum food sIgG(4) with component diagnostics in children with EoE and children from an unselected birth cohort and to explore the effects of sex, age, and milk consumption on sIgG(4) levels. METHODS: Sera from 71 pediatric patients with EoE and 210 early adolescent children from an unselected birth cohort (Project Viva) were assayed for sIgG(4) and specific IgE (sIgE) to major cow’s milk (CM) proteins (α-lactalbumin, β-lactoglobulin, and caseins) and to wheat, soy, egg, and peanut proteins. RESULTS: In the EoE cohort high-titer sIgG(4) (≥10 μg/mL) to CM proteins was more common than in control sera and achieved odds ratios for EoE ranging from 5.5 to 8.4. sIgE levels to CM proteins were mostly 4 IU/mL or less in patients with EoE, such that sIgG(4)/sIgE ratios were often 10,000 or greater. When adjusted for age and milk consumption, high-titer sIgG(4) to CM proteins was strongly associated with EoE, with an odds ratio of greater than 20 to all 3 CM proteins in boys. CONCLUSIONS: sIgG(4) to CM proteins are common and high titer in children with EoE. Although it is not clear that this response is pathogenic, sIgG(4) levels imply that these antibodies are an important feature of the local immune response that gives rise to EoE. (J Allergy Clin Immunol 2018;142:139–48.)

Background Eosinophilia is associated with various conditions, including allergic, infectious, and neoplastic disorders. The diagnostic differential is broad, and data on hypereosinophilia in pediatric patients are limited. Objective The objectives of this study were to identify cases of hypereosinophilia in a tertiary pediatric medical center, determine clinical characteristics and disease associations, and estimate the incidence of hypereosinophilia in the hospital and geographic populations. Methods A retrospective chart review included patients younger than 18 years presenting to a tertiary pediatric medical center (January 1, 2008, to May 31, 2017) with absolute eosinophil counts (AECs) greater than or equal to 1.50 thousand eosinophils/microliter (K/μL) recorded on at least 2 occasions at least 4 weeks apart (N = 176). Clinical characteristics, laboratory values, treatment course, and associated diagnoses were evaluated. Results The most common cause of hypereosinophilia in this cohort was secondary hypereosinophilia. Atopic dermatitis, graft-versus-host disease, sickle cell disease, and parasitic infections were the most common conditions associated with hypereosinophilia. Median age at diagnosis was 4.6 (interquartile range, 1.5-10.5) years. Median peak AEC was 3.16 (2.46-4.78) K/μL. Hypereosinophilia occurred most frequently in patients aged between 6 and 11 years (24.4%) and younger than 1 year (18.2%). Patients with neoplasms and immune deficiencies had significantly higher peak AECs than did patients with overlap hypereosinophilic syndrome and atopic diseases (P Conclusions Pediatric hypereosinophilia has an incidence of 54.4 per 100,000 persons per year, with children younger than 1 year and aged 6 to 11 years accounting for most affected patients. Pediatric hypereosinophilia is not uncommon and remains underrecognized, highlighting a need for clinicians to identify patients who meet criteria for hypereosinophilia and to pursue a thorough evaluation.

Most in vivo studies of granulocytes draw conclusions about their trafficking based on examination of their steady-state tissue/blood levels, which result from a combination of tissue homing, survival, and egress, rather than direct examination of cellular trafficking. Herein, we developed a unique cell transfer system involving the adoptive transfer of a genetically labeled, bone-marrow–derived unique granulocyte population (eosinophils) into an elicited inflammatory site, the allergic lung. A dual polychromatic FACS-based biomarker-labeling system based on the IL4-eGFP transgene (4get) or Cd45.1 allele was used to track i.v. transferred eosinophils into the airway following allergen or T H 2-associated stimuli in the lung in multiple mouse strains. The system was amenable to reverse tagging of recipients, thus allowing transfer of nonlabeled eosinophils and competitive tracking of multiple populations of eosinophils in vivo. The half-life of eosinophils in the blood was 3 h, and migration to the lung was dependent upon the dosage of transferred eosinophils, sensitive to pertussis toxin pretreatment, peaked at ∼24 h after adoptive transfer, and revealed a greater than 8-d eosinophil half-life in the lung. Eosinophil migration to the lung was dependent upon recipient IL-5 and IL-13 receptor α1 and donor eosinophil C-C chemokine receptor type 3 (CCR3) and interleukin 1 receptor-like 1 (ST2) in vivo. Taken together, this unique eosinophil transfer system provides an unprecedented opportunity to examine airway eosinophil migration without the need for extensive efforts to acquire donor source and time-consuming genetic crossing and has already been used to identify a long eosinophil half-life in the allergic lung and a definite role for ST2 in regulating eosinophil trafficking.

Inflammatory bowel diseases (IBDs, e.g., Crohn's disease and ulcerative colitis) are thought to be a consequence of an uncontrolled inflammatory response against luminal antigens, including commensal bacteria. The observed link between eosinophil levels and severity and remission rates in IBD has led to speculation that eosinophils may contribute to the antimicrobial inflammatory response in IBD.Eosinophils express the necessary cellular machinery (innate immune receptors, proinflammatory cytokines, antibacterial proteins, and DNA traps) to mount an efficient antibacterial response; however, the rapid decline in eosinophil numbers following acute systemic bacterial infection suggests a very limited role for eosinophils in bacterial responses.We describe the clinical evidence of eosinophil involvement in IBD, summarize the in-vitro and in-vivo evidence of eosinophil antibacterial activity and the biology of eosinophils focusing on eosinophil-mediated bactericidal mechanisms and the involvement of eosinophil-derived granule proteins in this response, and conceptualize the contribution of eosinophils to a response against commensal bacteria in IBD.

Hypereosinophilic syndromes (HESs) are a diverse group of conditions characterized by clinical manifestations attributable to eosinophilia and eosinophilic infiltration of tissues. HESs are chronic disorders with significant morbidity and mortality. Although the availability of targeted chemotherapeutic agents, including imatinib, has improved quality of life and survival in some patients with HESs, additional agents with increased efficacy and decreased toxicity are sorely needed. The purpose of this review is to provide an overview of eosinophil biology with an emphasis on potential targets of pharmacotherapy and to provide a summary of potential eosinophil-targeting agents, including those in development, in clinical trials, or approved for other disorders.

Oeosinophils are pleiotropic multi-functional leukocytes that are typically associated with the initiation and propagation of inflammatory responses, particularly helminth infection and allergic disease. However, expanding evidence supports a broader role for oeosinophils in homoeostatic function and organ development and modulation of local immune responses via interaction with other effector cells. In this review, the biology of oeosinophils in the healthy gut is summarised. In particular, the molecular steps involved in oeosinophil development and trafficking are described, with special attention to the important role of the transcription factor GATA-1, the oeosinophil-selective cytokine IL-5 and the eotaxin subfamily of chemokines. In addition, the regulation of oeosinophil survival by inhibitory and death receptors and the expanding role for oeosinophils in health and disease are reviewed.

Spi-C is an E26 transformation-specific family transcription factor that is highly related to PU.1 and Spi-B. Spi-C is expressed in developing B cells, but its function in B cell development and function is not well characterized. To determine whether Spi-C functions as a negative regulator of Spi-B (encoded by Spib), mice were generated that were germline knockout for Spib and heterozygous for Spic (Spib−/−Spic+/−). Interestingly, loss of one Spic allele substantially rescued B cell frequencies and absolute numbers in Spib−/− mouse spleens. Spib−/−Spic+/− B cells had restored proliferation compared with Spib−/− B cells in response to anti-IgM or LPS stimulation. Investigation of a potential mechanism for the Spib−/−Spic+/− phenotype revealed that steady-state levels of Nfkb1, encoding p50, were elevated in Spib−/−Spic+/− B cells compared with Spib−/− B cells. Spi-B was shown to directly activate the Nfkb1 gene, whereas Spi-C was shown to repress this gene. These results indicate a novel role for Spi-C as a negative regulator of B cell development and function.

Eosinophilic esophagitis (EE) is an emerging disorder with a poorly understood pathogenesis. In order to define disease mechanisms, we took an empirical approach analyzing esophageal tissue by a genome-wide microarray expression analysis. EE patients had a striking transcript signature involving 1% of the human genome that was remarkably conserved across sex, age, and allergic status and was distinct from that associated with non-EE chronic esophagitis. Notably, the gene encoding the eosinophil-specific chemoattractant eotaxin-3 (also known as CCL26) was the most highly induced gene in EE patients compared with its expression level in healthy individuals. Esophageal eotaxin-3 mRNA and protein levels strongly correlated with tissue eosinophilia and mastocytosis. Furthermore, a single-nucleotide polymorphism in the human eotaxin-3 gene was associated with disease susceptibility. Finally, mice deficient in the eotaxin receptor (also known as CCR3) were protected from experimental EE. These results implicate eotaxin-3 as a critical effector molecule for EE and provide insight into disease pathogenesis.

Asthma is a complex inflammatory pulmonary disorder that is on the rise despite intense ongoing research. We aimed to elucidate novel pathways involved in the pathogenesis of asthma. Employing asthma models induced by different allergens (ovalbumin and Aspergillus fumigatus), we uncovered the involvement of two members of the small proline-rich protein (SPRR) family, SPRR2a and SPRR2b, known to be involved in epithelial differentiation but not allergic disease. In situ hybridization revealed induction of SPRR2 signal in a subset of bronchial epithelial cells and mononuclear cells associated with inflammation after allergen challenge. Allergen-induced SPRR2 mRNA accumulation in the lung occurred in a time-dependent manner, with peak expression 10-96 h after a second ovalbumin challenge. Transgenic overexpression of interleukin (IL)-13 in the lungs resulted in a marked increase of SPRR2 expression, and allergen-induced SPRR2 expression was significantly decreased in IL-13-deficient mice. Studies in gene-targeted mice revealed that allergen-induced SPRR2 was dependent upon STAT6. Finally, we aimed to determine if the induction of SPRR2 by allergen was tissue specific. Notably, SPRR2 was markedly increased in the small intestine after induction of allergic gastrointestinal inflammation. Thus, SPRR2 is an allergen- and IL-13-induced gene in experimental allergic responses that may be involved in disease pathophysiology.

Asthma, a complex chronic inflammatory pulmonary disorder, is on the rise despite intense ongoing research. To elucidate novel pathways involved in asthma pathogenesis, we used transcript expression profiling in a murine model of asthma. Employing asthma models induced by different allergens (ovalbumin and Aspergillus fumigatus) we uncovered the involvement of ADAM8, a member of a disintegrin and metalloproteinase (ADAM) family. In situ hybridization of mouse lungs revealed strong ADAM8 induction in peribronchial and perivascular inflammatory cells as well as in bronchiolar epithelial cells following allergen challenge. Sequence analysis of lung ADAM8 cDNA identified a novel splice variant of ADAM8 that contained an additional exon in juxtaposition to the transmembrane domain. Allergen-induced ADAM8 mRNA accumulation in the lung was dose- and time-dependent. Transgenic or pharmacologic delivery of interleukin (IL)-4 or IL-13 to the lungs resulted in a marked increase of ADAM8 expression. Gene-targeted mice studies revealed that ovalbumin-induced ADAM8 was largely dependent upon signal transducer and activator of transcription (STAT) 6 and the IL-4 receptor alpha-chain. Thus, ADAM8 is an allergen-, IL-4-, and IL-13-induced gene in the experimental asthmatic lung. Taken together with the role of ADAM33 in asthma, these results suggest that allergic lung responses involve the interplay of diverse members of the ADAM family.

The airways are lined by several distinct epithelial cells that play unique roles in pulmonary homeostasis; however, the mechanisms controlling their differentiation in health and disease are poorly understood. The winged helix transcription factor, FOXA2, is expressed in the foregut endoderm and in subsets of respiratory epithelial cells in the fetal and adult lung. Because targeted mutagenesis of the Foxa2 gene in mice is lethal before formation of the lung, its potential role in lung morphogenesis and homeostasis has not been determined. We selectively deleted Foxa2 in respiratory epithelial cells in the developing mouse lung. Airspace enlargement, goblet cell hyperplasia, increased mucin and neutrophilic infiltration were observed in lungs of the Foxa2-deleted mice. Experimental goblet cell hyperplasia caused by ovalbumin sensitization,interleukin 4 (IL4), IL13 and targeted deletion of the gene encoding surfactant protein C (SP-C), was associated with either absent or decreased expression of Foxa2 in airway epithelial cells. Analysis of lung tissue from patients with a variety of pulmonary diseases revealed a strong inverse correlation between FOXA2 and goblet cell hyperplasia. FOXA2 is required for alveolarization and regulates airway epithelial cell differentiation in the postnatal lung.

The analysis of polygenic diseases such as asthma poses a challenging problem. In an effort to provide unbiased insight into disease pathogenesis, we took an empirical approach involving transcript expression profiling of lung tissue from mice with experimental asthma. Asthmatic responses were found to involve sequential induction of 4.7% of the tested genome; notably, there was ectopic expression of a series of genes not previously implicated in allergic or pulmonary responses. Genes were widely distributed throughout all chromosomes, but preferentially included genes involved in immunity, development, and homeostasis. When asthma was induced by two independent experimental regimens, unique gene transcript profiles were found depending upon the mode of disease induction. However, the majority of genes were common to both models representing an asthma signature genome. Analysis of STAT6-deficient mice revealed that an unexpectedly large segment of the asthma genes were STAT6 independent; this correlated with sustained inflammatory events in these mice. Notably, induction of asthma in STAT6-deficient mice resulted in gene induction not seen in wild-type mice. These results raise concern that therapeutic blockade of STAT6 in the asthmatic setting may reprogram the genetic signature, resulting in alternative lung pathology, which we indeed observed in STAT6-deficient mice. These results provide unprecedented insight into the complex steps involved in the pathogenesis of allergic airway responses; as such, these results have significant therapeutic and clinical implications.