Eosinophil extracellular DNA traps: molecular mechanisms and potential roles in disease

Eosinophil extracellular traps (EETs) are part of the innate immune response and are seen in multiple infectious, allergic, and autoimmune eosinophilic diseases. EETs are composed of a meshwork of DNA fibers and eosinophil granule proteins, such as major basic protein (MBP) and eosinophil cationic protein (ECP). Interestingly, the DNA within the EETs appears to have its origin in the mitochondria of eosinophils, which had released most their mitochondrial DNA, but were still viable, exhibiting no evidence of a reduced life span. Multiple eosinophil activation mechanisms are represented, whereby toll-like, cytokine, chemokine, and adhesion receptors can all initiate transmembrane signal transduction processes leading to the formation of EETs. One of the key signaling events required for DNA release is the activation of the NADPH oxidase. Here, we review recent progress made in the understanding the molecular mechanisms involved in DNA and granule protein release, discuss the presence of EETs in disease, speculate on their potential role(s) in pathogenesis, and compare available data on other DNA-releasing cells, particularly neutrophils.

Characterization of canine dendritic cells in healthy, atopic, and non-allergic inflamed skin

Atopic dermatitis in humans and dogs is a chronic relapsing allergic skin disease. Dogs show a spontaneous disease similar to the human counterpart and represent a model to improve our understanding of the immunological mechanisms, the pathogenesis of the disease, and new therapy development. The aim of the study was to determine the frequency and phenotype of dendritic cells (DC) in the epidermis and dermis of healthy, canine atopic dermatitis lesional, and non-allergic inflammatory skin to further validate the model and to obtain insights into the contribution of DC to the pathogenesis of skin diseases in dogs. We first characterized canine skin DC using flow-cytometric analysis of isolated skin DC combined with an immunohistochemical approach. A major population of canine skin dendritic cells was identified as CD1c(+)CD11c(+)CD14(-)CD80(+)MHCII(+)MAC387(-) cells, with dermal DC but not Langerhans cells expressing CD11b. In the epidermis of lesional canine atopic dermatitis and non-allergic inflammatory skin, we found significantly more dendritic cells compared with nonlesional and control skin. Only in canine atopic dermatitis skin did we find a subset of dendritic cells positive for IgE, in the epidermis and the dermis. Under all inflammatory conditions, dermal dendritic cells expressed more CD14 and CD206. MAC387(+) putative macrophages were absent in healthy but present in inflamed skin, in particular during non-allergic diseases. This study permits a phenotypic identification and differentiation of canine skin dendritic cells and has identified markers and changes in dendritic cells and macrophage populations related to allergic and non-allergic inflammatory conditions. Our data suggest the participation of dendritic cells in the pathogenesis of canine atopic dermatitis similar to human atopic dermatitis and further validate the only non-murine spontaneous animal model for this disease.

Immune dysregulation in flea allergy dermatitis--a model for the immunopathogenesis of allergic dermatitis

BACKGROUND: Flea allergy dermatitis (FAD) is a common skin disease in dogs and can be induced experimentally. It often coexists with other allergic conditions. So far no studies have investigated the quantitative production of cytokine mRNA in skin biopsies and peripheral blood mononuclear cells (PBMC) in flea allergic dogs. OBJECTIVE: The aim of our study was to improve the understanding of the immunopathogenesis of allergic dermatitis as a response to fleabites. MATERIAL AND METHODS: Allergic and non-allergic dogs were exposed to fleas. Before and after 4 days of flea exposure mRNA was isolated from biopsies and PBMC. Production of chymase, tryptase, IL-4, IL-5, IL-13, TNF-alpha and IFN-gamma mRNA was measured by real-time RT-PCR. The inflammatory infiltrate in the skin was scored semi-quantitatively. The number of eosinophils, mast cells (MC) and IgE+ cells/mm2 was evaluated to complete the picture. RESULTS: FAD was associated with a higher number of MC before flea exposure and with a significant increase of eosinophils after flea exposure as compared to non-allergic dogs. The number of IgE+ cells was higher in allergic dogs before and after flea exposure. In allergic dogs mRNA for most cytokines and proteases tested was higher before flea exposure than after flea exposure. After exposure to fleas an increased mRNA production was only observed in non-allergic dogs. In vitro stimulation with flea antigen resulted in a decreased expression of most cytokines in allergic dogs before flea exposure. In contrast, in PBMC, only increased levels of IL-4 and IL-5 mRNA were observed in allergic dogs before flea exposure. However, after flea exposure and additional stimulation with flea antigen the production of mRNA for all cytokines tested was significantly increased in allergic dogs. CONCLUSION: We demonstrated that the response in biopsies and PBMC is different and that FAD is associated with a TH2 response.

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.

Allergic bronchopulmonary aspergillosis: the hunt for a diagnostic serological marker in cystic fibrosis patients

The diagnosis of allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis patients remains challenging, mainly owing to overlapping symptoms of the underlying lung disease with clinical symptoms of ABPA. In addition, a varying mixture of diagnostic criteria, including clinical status, radiological findings and immunological measurements, has led to confusion and differing recommendations. In order to help simplify as well as standardize the diagnostic criteria for ABPA, different serological markers have been evaluated in the last 20 years and their usefulness has been assessed in many clinical studies. This review presents current diagnostic criteria of ABPA, with a special focus on serum markers supporting the diagnosis and explains why the hunt for a serological marker for ABPA is still ongoing.

A disease model for wheezing disorders in preschool children based on clinicians' perceptions

BACKGROUND: Wheezing disorders in childhood vary widely in clinical presentation and disease course. During the last years, several ways to classify wheezing children into different disease phenotypes have been proposed and are increasingly used for clinical guidance, but validation of these hypothetical entities is difficult. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to develop a testable disease model which reflects the full spectrum of wheezing illness in preschool children. We performed a qualitative study among a panel of 7 experienced clinicians from 4 European countries working in primary, secondary and tertiary paediatric care. In a series of questionnaire surveys and structured discussions, we found a general consensus that preschool wheezing disorders consist of several phenotypes, with a great heterogeneity of specific disease concepts between clinicians. Initially, 24 disease entities were described among the 7 physicians. In structured discussions, these could be narrowed down to three entities which were linked to proposed mechanisms: a) allergic wheeze, b) non-allergic wheeze due to structural airway narrowing and c) non-allergic wheeze due to increased immune response to viral infections. This disease model will serve to create an artificial dataset that allows the validation of data-driven multidimensional methods, such as cluster analysis, which have been proposed for identification of wheezing phenotypes in children. CONCLUSIONS/SIGNIFICANCE: While there appears to be wide agreement among clinicians that wheezing disorders consist of several diseases, there is less agreement regarding their number and nature. A great diversity of disease concepts exist but a unified phenotype classification reflecting underlying disease mechanisms is lacking. We propose a disease model which may help guide future research so that proposed mechanisms are measured at the right time and their role in disease heterogeneity can be studied.

Nicoletella semolina gen. nov., sp. nov., a new member of Pasteurellaceae isolated from horses with airway disease

Gram-negative, nonmotile bacteria that are catalase, oxidase, and urease positive are regularly isolated from the airways of horses with clinical signs of respiratory disease. On the basis of the findings by a polyphasic approach, we propose that these strains be classified as Nicoletella semolina gen. nov, sp. nov., a new member of the family Pasteurellaceae. N. semolina reduces nitrate to nitrite but is otherwise biochemically inert; this includes the lack of an ability to ferment glucose and other sugars. Growth is fastidious, and the isolates have a distinctive colony morphology, with the colonies being dry and waxy and looking like a semolina particle that can be moved around on an agar plate without losing their shape. DNA-DNA hybridization data and multilocus phylogenetic analysis, including 16S rRNA gene (rDNA), rpoB, and infB sequencing, clearly placed N. semolina as a new genus in the family Pasteurellaceae. In all the phylogenetic trees constructed, N. semolina is on a distinct branch displaying approximately 5% 16S rDNA, approximately 16% rpoB, and approximately 20% infB sequence divergence from its nearest relative within the family Pasteurellaceae. High degrees of conservation of the 16S rDNA (99.8%), rpoB (99.6%), and infB (99.7%) sequences exist within the species, indicating that N. semolina isolates not only are phenotypically homogeneous but also are genetically homogeneous. The type strain of N. semolina is CCUG43639(T) (DSM16380(T)).

Copy number variation of the Beta-defensin genes in europeans: no supporting evidence for association with lung function, chronic obstructive pulmonary disease or asthma

Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02-1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72-1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed.

Microbial communities in the respiratory tract of patients with interstitial lung disease

Background Molecular methods based on phylogenetic differences in the 16S rRNA gene are able to characterise the microbiota of the respiratory tract in health and disease. Objectives Our goals were (1) to characterise bacterial communities in lower and upper airways of patients with interstitial lung disease (ILD) and (2) to compare the results with the microbiota of patients with Pneumocystis pneumonia (PCP) and normal controls. Methods We examined the upper and lower respiratory tract of 18 patients with ILD of whom 5, 6, and 7 had idiopathic interstitial pneumonia (IIP), non-IIP and sarcoidosis, respectively. In addition, six immune-compromised patients with PCP and nine healthy subjects were included as controls. Exclusion criteria were recent bacterial/viral respiratory tract infection, HIV-positivity and subjects receiving antibiotic therapy. Bronchoalveolar lavage fluid and oropharyngeal swabs were simultaneously collected, and microbiota was characterised by ultra-deep 16S rRNA gene sequencing. Results The microbiota in lower airways of the majority of patients (30; 90%) primarily consisted of Prevotellaceae, Streptococcaceae and Acidaminococcaceae. α and β diversity measurements revealed no significant differences in airway microbiota composition between the five different groups of patients. Comparison of bacterial populations in upper and lower respiratory tract showed significant topographical discontinuities for 7 (23%) individuals. Conclusions IIP, non-IIP and sarcoidosis are not associated with disordered airway microbiota and a pathogenic role of commensals in the disease process is therefore unlikely. Nevertheless, molecular analysis of the topographical microbiota continuity along the respiratory tract may provide additional information to assist management of individual patients.

Abnormal small airways function in children with mild asthma

BACKGROUND Small airways disease is a hallmark in adults with persistent asthma, but little is known about small airways function in children with mild asthma and normal spirometry. We assessed ventilation heterogeneity, a marker of small airways function, with an easy tidal breath single-breath washout (SBW) technique in school-aged children with mild asthma and normal FEV1 and healthy age-matched control subjects. METHODS The primary outcome was the double-tracer gas phase III slope (SDTG), an index of ventilation heterogeneity in acinar airways derived from the tidal double-tracer gas SBW test. The second outcome was the nitrogen phase III slope (SN2), an index of global ventilation heterogeneity derived from the tidal nitrogen SBW test using pure oxygen. Triplicate SBW and spirometry tests were performed in healthy children (n = 35) and children with asthma (n = 31) at baseline and in children with asthma after bronchodilation. RESULTS Acinar (SDTG) but not global (SN2) ventilation heterogeneity was significantly increased in asthma despite normal FEV1. Of the 31 children with asthma, abnormal results were found for SDTG (≤ -2 z scores) in 11; forced expiratory flow, midexpiratory phase (FEF25%-75%) in three; and FEV1 in zero. After bronchodilation, SDTG, SN2, FEF25%-75%, and FEV1 significantly changed (mean [95% CI] change from baseline, 36% [15%-56%], 38% [18%-58%], 17% [9-25%], and 6% [3%-9%], respectively). CONCLUSIONS Abnormal acinar ventilation heterogeneity in one-third of the children suggests that small airways disease may be present despite rare and mild asthma symptoms and normal spirometry. The easy tidal SBW technique has considerable potential as a clinical and research outcome in children with asthma.

Effects of ex vivo γ-tocopherol on airway macrophage function in healthy and mild allergic asthmatics

Elevated inflammation and altered immune responses are features found in atopic asthmatic airways. Recent studies indicate γ-tocopherol (GT) supplementation can suppress airway inflammation in allergic asthma. We studied the effects of in vitro GT supplementation on receptor-mediated phagocytosis and expression of cell surface molecules associated with innate and adaptive immunity on sputum-derived macrophages. Cells from nonsmoking healthy (n = 6) and mild house dust mite-sensitive allergic asthmatics (n = 6) were treated ex vivo with GT (300 µM) or saline (control). Phagocytosis of opsonized zymosan A bioparticles (Saccharomyces cerevisiae) and expression of surface molecules associated with innate and adaptive immunity were assessed using flow cytometry. GT caused significantly decreased (p < 0.05) internalization of attached zymosan bioparticles and decreased (p < 0.05) macrophage expression of CD206, CD36 and CD86 in allergic asthmatics but not in controls. Overall, GT caused downregulation of both innate and adaptive immune response elements, and atopic status appears to be an important factor.

Genetically determined heterogeneity of lung disease in a mouse model of airway mucus obstruction

Mucus clearance is an important airway innate defense mechanism. Airway-targeted overexpression of the epithelial Na(+) channel β-subunit [encoded by sodium channel nonvoltage gated 1, beta subunit (Scnn1b)] in mice [Scnn1b-transgenic (Tg) mice] increases transepithelial Na(+) absorption and dehydrates the airway surface, which produces key features of human obstructive lung diseases, including mucus obstruction, inflammation, and air-space enlargement. Because the first Scnn1b-Tg mice were generated on a mixed background, the impact of genetic background on disease phenotype in Scnn1b-Tg mice is unknown. To explore this issue, congenic Scnn1b-Tg mice strains were generated on C57BL/6N, C3H/HeN, BALB/cJ, and FVB/NJ backgrounds. All strains exhibited a two- to threefold increase in tracheal epithelial Na(+) absorption, and all developed airway mucus obstruction, inflammation, and air-space enlargement. However, there were striking differences in neonatal survival, ranging from 5 to 80% (FVB/NJairways and a variable incidence of pulmonary hemorrhage and lung atelectasis. The spontaneous occurrence of a high surviving BALB/cJ line, which exhibited delayed onset of Na(+) hyperabsorption, provided evidence that: 1) air-space enlargement and postnatal death were only present when Na(+) hyperabsorption occurred early, and 2) inflammation and mucus obstruction developed whenever Na(+) hyperabsorption was expressed. In summary, the genetic context and timing of airway innate immune dysfunction critically determines lung disease phenotype. These mouse strains may be useful to identify key modifier genes and pathways.

Biomarkers of the involvement of mast cells, basophils and eosinophils in asthma and allergic diseases.

Biomarkers of disease activity have come into wide use in the study of mechanisms of human disease and in clinical medicine to both diagnose and predict disease course; as well as to monitor response to therapeutic intervention. Here we review biomarkers of the involvement of mast cells, basophils, and eosinophils in human allergic inflammation. Included are surface markers of cell activation as well as specific products of these inflammatory cells that implicate specific cell types in the inflammatory process and are of possible value in clinical research as well as within decisions made in the practice of allergy-immunology.

Precision medicine in patients with allergic diseases: Airway diseases and atopic dermatitis-PRACTALL document of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma & Immunology

In this consensus document we summarize the current knowledge on major asthma, rhinitis, and atopic dermatitis endotypes under the auspices of the PRACTALL collaboration platform. PRACTALL is an initiative of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma & Immunology aiming to harmonize the European and American approaches to best allergy practice and science. Precision medicine is of broad relevance for the management of asthma, rhinitis, and atopic dermatitis in the context of a better selection of treatment responders, risk prediction, and design of disease-modifying strategies. Progress has been made in profiling the type 2 immune response-driven asthma. The endotype driven approach for non-type 2 immune response asthma, rhinitis, and atopic dermatitis is lagging behind. Validation and qualification of biomarkers are needed to facilitate their translation into pathway-specific diagnostic tests. Wide consensus between academia, governmental regulators, and industry for further development and application of precision medicine in management of allergic diseases is of utmost importance. Improved knowledge of disease pathogenesis together with defining validated and qualified biomarkers are key approaches to precision medicine.