Eosinophils in Health and Disease: A State-of-the-Art Review.

Eosinophils play a homeostatic role in the body's immune responses. These cells are involved in combating some parasitic, bacterial, and viral infections and certain cancers and have pathologic roles in diseases including asthma, chronic rhinosinusitis with nasal polyps, eosinophilic gastrointestinal disorders, and hypereosinophilic syndromes. Treatment of eosinophilic diseases has traditionally been through nonspecific eosinophil attenuation by use of glucocorticoids. However, several novel biologic therapies targeting eosinophil maturation factors, such as interleukin (IL)-5 and the IL-5 receptor or IL-4/IL-13, have recently been approved for clinical use. Despite the success of biologic therapies, some patients with eosinophilic inflammatory disease may not achieve adequate symptom control, underlining the need to further investigate the contribution of patient characteristics, such as comorbidities and other processes, in driving ongoing disease activity. New research has shown that eosinophils are also involved in several homeostatic processes, including metabolism, tissue remodeling and development, neuronal regulation, epithelial and microbiome regulation, and immunoregulation, indicating that these cells may play a crucial role in metabolic regulation and organ function in healthy humans. Consequently, further investigation is needed into the homeostatic roles of eosinophils and eosinophil-mediated processes across different tissues and their varied microenvironments. Such work may provide important insights into the role of eosinophils not only under disease conditions but also in health. This narrative review synthesizes relevant publications retrieved from PubMed informed by author expertise to provide new insights into the diverse roles of eosinophils in health and disease, with particular emphasis on the implications for current and future development of eosinophil-targeted therapies.

New Perspectives in the Diagnosis and Management of Allergic Fungal Airway Disease.

Allergy to airway-colonising, thermotolerant, filamentous fungi represents a distinct eosinophilic endotype of often severe lung disease. This endotype, which particularly affects adult asthma, but also complicates other airway diseases and sometimes occurs de novo, has a heterogeneous presentation ranging from severe eosinophilic asthma to lobar collapse. Its hallmark is lung damage, characterised by fixed airflow obstruction (FAO), bronchiectasis and lung fibrosis. It has a number of monikers including severe asthma with fungal sensitisation (SAFS) and allergic bronchopulmonary aspergillosis/mycosis (ABPA/M), but these exclusive terms constitute only sub-sets of the condition. In order to capture the full extent of the syndrome we prefer the inclusive term allergic fungal airway disease (AFAD), the criteria for which are IgE sensitisation to relevant fungi in association with airway disease. The primary fungus involved is Aspergillus fumigatus, but a number of other thermotolerant species from several genera have been implicated. The unifying mechanism involves germination of inhaled fungal spores in the lung in the context of IgE sensitisation, leading to a persistent and vigorous eosinophilic inflammatory response in association with release of fungal proteases. Most allergenic fungi, including Alternaria and Cladosporium species, are not thermotolerant and cannot germinate in the airways so only act as aeroallergens and do not cause AFAD. Studies of the airway mycobiome have shown that A. fumigatus colonises the normal as much as the asthmatic airway, suggesting it is the tendency to become IgE-sensitised that is the critical triggering factor for AFAD rather than colonisation per se. Treatment is aimed at preventing exacerbations with glucocorticoids and increasingly by the use of anti-T2 biological therapies. Anti-fungal therapy has a limited place in management, but is an effective treatment for fungal bronchitis which complicates AFAD in about 10% of cases.

Allergic fungal airways disease (AFAD): an under-recognised asthma endotype.

The term allergic fungal airways disease has a liberal definition based on IgE sensitisation to thermotolerant fungi and evidence of fungal-related lung damage. It arose from a body of work looking into the role of fungi in asthma. Historically fungi were considered a rare complication of asthma, exemplified by allergic bronchopulmonary aspergillosis; however, there is a significant proportion of individuals with Aspergillus fumigatus sensitisation who do not meet these criteria, who are at high risk for the development of lung damage. The fungi that play a role in asthma can be divided into two groups; those that can grow at body temperature referred to as thermotolerant, which are capable of both infection and allergy, and those that cannot but can still act as allergens in IgE sensitised individuals. Sensitisation to thermotolerant filamentous fungi (Aspergillus and Penicillium), and not non-thermotolerant fungi (Alternaria and Cladosporium) is associated with lower lung function and radiological abnormalities (bronchiectasis, tree-in-bud, fleeting shadows, collapse/consolidation and fibrosis). For antifungals to play a role in treatment, the focus should be on fungi capable of growing in the airways thereby causing a persistent chronic allergenic stimulus and releasing tissue damaging proteases and other enzymes which may disrupt the airway epithelial barrier and cause mucosal damage and airway remodelling. All patients with IgE sensitisation to thermotolerant fungi in the context of asthma and other airway disease are at risk of progressive lung damage, and as such should be monitored closely.

Fungal sensitization and positive fungal culture from sputum in children with asthma are associated with reduced lung function and acute asthma attacks respectively.

BACKGROUND: Sensitization to thermotolerant fungi, including filamentous fungi and Candida albicans, is associated with poor lung function in adults with severe asthma. Data in children are lacking. Environmental exposure to fungi is linked with acute severe asthma attacks, but there are few studies reporting the presence of fungi in the airways during asthma attacks. METHODS: We investigated the association between fungal sensitization and/or positive fungal sputum culture and markers of asthma severity in children with chronic and acute asthma. Sensitization was determined using serum-specific IgE and skin prick testing against a panel of five fungi. Fungal culture was focused towards detection of filamentous fungi from sputum samples. RESULTS: We obtained sensitization data and/or sputum from 175 children: 99 with chronic asthma, 39 with acute asthma and 37 controls. 34.1% of children with chronic asthma were sensitized to thermotolerant fungi compared with no children without asthma (p =< 0.001). These children had worse pre-bronchodilator lung function compared with asthmatics without sensitization including a lower FEV1 /FVC ratio (p < .05). The isolation rate of filamentous fungi from sputum was higher in children with acute compared with chronic asthma. CONCLUSIONS: Fungal sensitization is a feature of children with chronic asthma. Children sensitized to thermotolerant fungi have worse lung function, require more courses of systemic corticosteroids and have greater limitation of activities due to asthma. Asthma attacks in children were associated with the presence of filamentous fungi positive sputum culture. Mechanistic studies are required to establish whether fungi contribute directly to the development of acute asthma.

Efficacy and safety of mepolizumab in hypereosinophilic syndrome: A phase III, randomized, placebo-controlled trial.

BACKGROUND: Anti-IL-5 therapy is a potential treatment for patients with hypereosinophilic syndrome (HES), although its clinical efficacy is unclear. OBJECTIVE: We sought to investigate the clinical efficacy and safety of mepolizumab versus placebo in patients with HES. METHODS: This randomized, multicenter, double-blind, placebo-controlled, phase III trial was conducted across 39 centers in 13 countries. Eligible patients had FIP1L1-PDGFRA-negative HES, experienced 2 or more flares (worsening of HES-related symptoms or blood eosinophil count requiring therapeutic escalation) in the previous 12 months, and had a screening blood eosinophil count greater than or equal to 1000 cells/µL. Patients were randomized (1:1) to subcutaneous mepolizumab (300 mg) or placebo every 4 weeks for 32 weeks, plus existing HES therapy. The primary outcome was the proportion of patients with 1 or more flares (worsening of HES-related symptoms necessitating therapy escalation or ≥2 courses of blinded rescue oral corticosteroids) during the study; in addition, patients who withdrew early from the study were counted as having a flare. Safety end points were also assessed. RESULTS: The proportion of patients experiencing 1 or more flares/withdrawing from the study was 50% lower with mepolizumab versus placebo (15 of 54 [28%] vs 30 of 54 [56%]; P = .002). Logistic regression analysis was consistent with the primary analysis (odds ratio, 0.28; 95% CI, 0.12-0.64; P = .003). Similar proportions of patients in the mepolizumab and placebo groups experienced on-treatment adverse events (48 of 54 [89%] vs 47 of 54 [87%]). CONCLUSIONS: Compared with placebo, mepolizumab significantly reduced the occurrence of flares in patients with HES, with no new safety signals identified.

The airway fungal microbiome in asthma.

BACKGROUND: Fungal involvement in asthma is associated with severe disease. The full spectrum of fungal species in asthma is not well described and is derived largely from insensitive culture techniques. OBJECTIVES: To use high-throughput sequencing to describe the airway mycobiota in asthmatics with and without fungal sensitization and healthy controls; to compare samples representing different airway compartments; to determine whether the mycobiota was influenced by the fungal composition of outdoor air; and to compare findings with clinically relevant outcomes. METHODS: We amplified the internal transcribed spacer region 2 of the nuclear ribosomal operon to identify the fungal species present. Ninety-seven subjects were recruited and provided sputum (83 asthmatics; 14 healthy subjects), with 29 also undergoing a bronchoscopy. A subset of airway samples were compared with matched outdoor air and mouthwash samples. RESULTS: Two hundred and six taxa at the species level were identified in sputum, most at low relative abundance. Aspergillus fumigatus, Candida albicans and Mycosphaerella tassiana had the highest relative abundances and were the most prevalent species across all subjects. The airway mycobiota consisted of a complex community with high diversity between individuals. Notable shifts in the balance of fungi detected in the lung were associated with asthma status, asthma duration and biomarkers of inflammation. Aspergillus tubingensis, a member of the Aspergillus niger species complex, was most prevalent from bronchoscopic protected brush samples and significantly associated with a low sputum neutrophilia. Cryptococcus pseudolongus, from the Cryptococcus humicola species complex, was more abundant from bronchoscopy samples than sputum, and differentially more abundant in asthma than health. CONCLUSIONS AND CLINICAL RELEVANCE: The airway mycobiota was dominated by a relatively small number of species, but was distinct from the oropharyngeal mycobiota and air samples. Members of the A. niger and C. humicola species complexes may play unexpected roles in the pathogenesis of asthma.

DP2 antagonism reduces airway smooth muscle mass in asthma by decreasing eosinophilia and myofibroblast recruitment.

Increased airway smooth muscle mass, a feature of airway remodeling in asthma, is the strongest predictor of airflow limitation and contributes to asthma-associated morbidity and mortality. No current drug therapy for asthma is known to affect airway smooth muscle mass. Although there is increasing evidence that prostaglandin D2 type 2 receptor (DP2) is expressed in airway structural and inflammatory cells, few studies have addressed the expression and function of DP2 in airway smooth muscle cells. We report that the DP2 antagonist fevipiprant reduced airway smooth muscle mass in bronchial biopsies from patients with asthma who had participated in a previous randomized placebo-controlled trial. We developed a computational model to capture airway remodeling. Our model predicted that a reduction in airway eosinophilia alone was insufficient to explain the clinically observed decrease in airway smooth muscle mass without a concomitant reduction in the recruitment of airway smooth muscle cells or their precursors to airway smooth muscle bundles that comprise the airway smooth muscle layer. We experimentally confirmed that airway smooth muscle migration could be inhibited in vitro using DP2-specific antagonists in an airway smooth muscle cell culture model. Our analyses suggest that fevipiprant, through antagonism of DP2, reduced airway smooth muscle mass in patients with asthma by decreasing airway eosinophilia in concert with reduced recruitment of myofibroblasts and fibrocytes to the airway smooth muscle bundle. Fevipiprant may thus represent a potential therapy to ameliorate airway remodeling in asthma.

Revisiting the NIH Taskforce on the Research needs of Eosinophil-Associated Diseases (RE-TREAD).

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.

Sputum Inflammatory Mediators Are Increased in Aspergillus fumigatus Culture-Positive Asthmatics.

Aspergillus fumigatus sensitization and culture in asthma are associated with disease severity and lung function impairment, but their relationship with airway inflammation is poorly understood. We investigated the profile of 24 sputum inflammatory mediators in A. fumigatus culture-positive or-negative moderate-to-severe asthmatics. Fifty-two subjects were recruited from a single center. A. fumigatus was cultured from 19 asthmatics. Asthma control, symptom score, lung function, and sputum cell count were not significantly different between the asthmatics with and without a positive A. fumigatus culture. All of the sputum mediators were numerically increased in subjects with a positive versus negative sputum A. fumigatus culture. Sputum TNF-R2 was significantly elevated (P=0.03) and the mediator that best distinguished A. fumigatus culture-positive from culture-negative subjects (receiver-operator characteristic area under the curve 0.66 [95% CI: 0.51 to 0.82, P=0.045]). A. fumigates-positive culture in moderate-to-severe asthma is associated with increased inflammatory sputum mediators.

The Ser82 RAGE Variant Affects Lung Function and Serum RAGE in Smokers and sRAGE Production In Vitro.

INTRODUCTION: Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model. METHODS: Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified. RESULTS: Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production. CONCLUSIONS: This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism.

Fevipiprant, a prostaglandin D2 receptor 2 antagonist, in patients with persistent eosinophilic asthma: a single-centre, randomised, double-blind, parallel-group, placebo-controlled trial.

BACKGROUND: Eosinophilic airway inflammation is often present in asthma, and reduction of such inflammation results in improved clinical outcomes. We hypothesised that fevipiprant (QAW039), an antagonist of prostaglandin D2 receptor 2, might reduce eosinophilic airway inflammation in patients with moderate-to-severe eosinophilic asthma. METHODS: We performed a single-centre, randomised, double-blind, parallel-group, placebo-controlled trial at Glenfield Hospital (Leicester, UK). We recruited patients with persistent, moderate-to-severe asthma and an elevated sputum eosinophil count (≥2%). After a 2-week single-blind placebo run-in period, patients were randomly assigned (1:1) by the trial pharmacist, using previously generated treatment allocation cards, to receive fevipiprant (225 mg twice per day orally) or placebo, stratified by the use of oral corticosteroid treatment and bronchoscopy. The 12-week treatment period was followed by a 6-week single-blind placebo washout period. The primary outcome was the change in sputum eosinophil percentage from baseline to 12 weeks after treatment, analysed in the intention-to-treat population. All patients who received at least one dose of study drug were included in the safety analyses. This trial is registered with ClinicalTrials.gov, number NCT01545726, and with EudraCT, number 2011-004966-13. FINDINGS: Between Feb 10, 2012, and Jan 30, 2013, 61 patients were randomly assigned to receive fevipiprant (n=30) or placebo (n=31). Three patients in the fevipiprant group and four patients in the placebo group withdrew because of asthma exacerbations. Two patients in the fevipiprant group were incorrectly given placebo (one at the mid-treatment visit and one throughout the course of the study). They were both included in the fevipiprant group for the primary analysis, but the patient who was incorrectly given placebo throughout was included in the placebo group for the safety analyses. Between baseline and 12 weeks after treatment, sputum eosinophil percentage decreased from a geometric mean of 5·4% (95% CI 3·1-9·6) to 1·1% (0·7-1·9) in the fevipiprant group and from 4·6% (2·5-8·7) to 3·9% (CI 2·3-6·7) in the placebo group. Compared with baseline, mean sputum eosinophil percentage was reduced by 4·5 times in the fevipiprant group and by 1·3 times in the placebo group (difference between groups 3·5 times, 95% CI 1·7-7·0; p=0·0014). Fevipiprant had a favourable safety profile, with no deaths or serious adverse events reported. No patient withdrawals were judged by the investigator to be related to the study drug. INTERPRETATION: Fevipiprant reduces eosinophilic airway inflammation and is well tolerated in patients with persistent moderate-to-severe asthma and raised sputum eosinophil counts despite inhaled corticosteroid treatment. FUNDING: Novartis Pharmaceuticals, AirPROM project, and the UK National Institute for Health Research.

Exome-wide analysis of rare coding variation identifies novel associations with COPD and airflow limitation in MOCS3, IFIT3 and SERPINA12.

BACKGROUND: Several regions of the genome have shown to be associated with COPD in genome-wide association studies of common variants. OBJECTIVE: To determine rare and potentially functional single nucleotide polymorphisms (SNPs) associated with the risk of COPD and severity of airflow limitation. METHODS: 3226 current or former smokers of European ancestry with lung function measures indicative of Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2 COPD or worse were genotyped using an exome array. An analysis of risk of COPD was carried out using ever smoking controls (n=4784). Associations with %predicted FEV1 were tested in cases. We followed-up signals of interest (p<10(-5)) in independent samples from a subset of the UK Biobank population and also undertook a more powerful discovery study by meta-analysing the exome array data and UK Biobank data for variants represented on both arrays. RESULTS: Among the associated variants were two in regions previously unreported for COPD; a low frequency non-synonymous SNP in MOCS3 (rs7269297, pdiscovery=3.08×10(-6), preplication=0.019) and a rare SNP in IFIT3, which emerged in the meta-analysis (rs140549288, pmeta=8.56×10(-6)). In the meta-analysis of % predicted FEV1 in cases, the strongest association was shown for a splice variant in a previously unreported region, SERPINA12 (rs140198372, pmeta=5.72×10(-6)). We also confirmed previously reported associations with COPD risk at MMP12, HHIP, GPR126 and CHRNA5. No associations in novel regions reached a stringent exome-wide significance threshold (p<3.7×10(-7)). CONCLUSIONS: This study identified several associations with the risk of COPD and severity of airflow limitation, including novel regions MOCS3, IFIT3 and SERPINA12, which warrant further study.

Eosinophilia in Pulmonary Disorders.

Lung disease associated with marked peripheral blood eosinophilia is unusual and nearly always clinically significant. Once recognized, it is generally easy to manage, albeit with long-term systemic corticosteroids. A failure to respond to oral steroids in the context of good compliance suggests a malignant cause for the eosinophilia. An important development is the introduction of antieosinophil therapies, particularly those directed against the interleukin 5 pathway, which is hoped to provide benefit in the full spectrum of eosinophilic lung disease as well as asthma, reducing the burden of side effects and resultant comorbidities.

The role of phosphoinositide 3-kinases in neutrophil migration in 3D collagen gels.

The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.

Allergic fungal airway disease: pathophysiologic and diagnostic considerations.

PURPOSE OF REVIEW: Fungal spores are ubiquitously present in indoor and outdoor air. A number can act as aeroallergens in Immunoglobulin E (IgE)-sensitized individuals and some thermotolerant fungi germinate in the lung where they can cause a combined allergic and infective stimulus leading to a number of clinical presentations characterized by evidence of lung damage. We discuss which biomarkers are useful in helping to guide diagnosis, prognosis and treatment of allergic fungal airway disease (AFAD). RECENT FINDINGS: Diagnostic biomarkers, such as specific IgEs and fungal culture, for AFAD are limited by sensitivity, although this may be improved with novel agents such as specific IgEs to fungal components and quantitative PCR. Total IgE and hypereosinophilia are nonspecific and do not clearly relate to disease activity. High attenuation mucus and proximal bronchiectasis are specific, albeit insensitive markers of AFAD. Biomarkers that predict prognosis and treatment response are yet to be defined. SUMMARY: This review summarizes the fungi involved and the current debate regarding the diagnostic criteria to define fungal-associated lung disease. We advocate the phasing out of the term allergic bronchopulmonary aspergillosis and the use of a more inclusive term such as AFAD, together with a more liberal set of criteria based largely on IgE sensitization to thermotolerant fungi, which identifies those patients at risk of developing lung damage.