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Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functions. In this study, we used ancestral sequence reconstruction to probe evolutionary and molecular determinants that distinguish biological activities within eosinophil members of the RNase 2/3 subfamily. Our investigation unveils functional, structural, and dynamical behaviors that differentiate the evolved ancestral ribonuclease (AncRNase) from its contemporary eosinophil RNase orthologs. Leveraging the potential of ancestral reconstruction for protein engineering, we used AncRNase predictions to design a minimal 4-residue variant that transforms human RNase 2 into a chimeric enzyme endowed with the antimicrobial and cytotoxic activities of RNase 3 members. This work provides unique insights into mutational and evolutionary pathways governing structure, function, and conformational states within the eosinophil RNase subfamily, offering potential for targeted modulation of RNase-associated functions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Eosinophil peroxidase (EPO) is the most abundant granule protein exocytosed by eosinophils, specialized human phagocytes. Released EPO catalyzes the formation of reactive oxidants from bromide, thiocyanate, and nitrite that kill tissue-invading parasites. However, EPO also plays a deleterious role in inflammatory diseases, making it a potential pharmacological target. A major hurdle is the high similarity to the homologous myeloperoxidase (MPO), which requires a detailed understanding of the small structural differences that can be used to increase the specificity of the inhibitors. Here, we present the first crystal structure of mature leukocyte EPO at 1.6 Å resolution together with analyses of its posttranslational modifications and biochemical properties. EPO has an exceptionally high number of positively charged surface patches but only two occupied glycosylation sites. The crystal structure further revealed the existence of a light (L) and heavy (H) chain as a result of proteolytic cleavage. Detailed comparison with the structure of human MPO allows us to identify differences that may contribute to the known divergent enzymatic properties. The crystal structure revealed fully established ester links between the prosthetic group and the protein, the comparably weak imidazolate character of the proximal histidine, and the conserved structure of the catalytic amino acids and Ca 2+ -binding site. Prediction of the structure of unprocessed proeosinophil peroxidase allows further structural analysis of the three protease cleavage sites and the potential pro-convertase recognition site in the propeptide. Finally, EPO biosynthesis and its biochemical and biophysical properties are discussed with respect to the available data from the well-studied MPO., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The first protein-binding allosteric RNA-cleaving DNAzyme (RCD) obtained by direct in vitro selection against eosinophil peroxidase (EPX), a validated marker for airway eosinophilia, is described. The RCD has nanomolar affinity for EPX, shows high selectivity against related peroxidases and other eosinophil proteins, and is resistant to degradation by mammalian nucleases. An optimized RCD was used to develop both fluorescence and lateral flow assays, which were evaluated using 38 minimally processed patient sputum samples (23 non-eosinophilic, 15 eosinophilic), producing a clinical sensitivity of 100 % and specificity of 96 %. This RCD-based lateral flow assay should allow for rapid evaluation of airway eosinophilia as an aid for guiding asthma therapy., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The endocannabinoids 2-arachidonoyl-glycerol and N -arachidonoyl-ethanolamine are lipids regulating many physiological processes, notably inflammation. Endocannabinoid hydrolysis inhibitors are now being investigated as potential anti-inflammatory agents. In addition to 2-arachidonoyl-glycerol and N -arachidonoyl-ethanolamine, the endocannabinoidome also includes other monoacylglycerols and N -acyl-ethanolamines such as 1-linoleoyl-glycerol (1-LG) and N -linoleoyl-ethanolamine (LEA). By increasing monoacylglycerols and/or N -acyl-ethanolamine levels, endocannabinoid hydrolysis inhibitors will likely increase the levels of their metabolites. Herein, we investigated whether 1-LG and LEA were substrates for the 15-lipoxygenase pathway, given that both possess a 1 Z ,4 Z -pentadiene motif, near their omega end. We thus assessed how human eosinophils and neutrophils biosynthesized the 15-lipoxygenase metabolites of 1-LG and LEA. Linoleic acid (LA), a well-documented substrate of 15-lipoxygenases, was used as positive control. N -13-hydroxy-octodecadienoyl-ethanolamine (13-HODE-EA) and 13-hydroxy-octodecadienoyl-glycerol (13-HODE-G) , the 15-lipoxygenase metabolites of LEA and 1-LG, were synthesized using Novozym 435 and soybean lipoxygenase. Eosinophils, which express the 15-lipoxygenase-1, metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was almost complete after five minutes. Substrate preference of eosinophils was LA > LEA > 1-LG in presence of 13-HODE-G hydrolysis inhibition with methyl-arachidonoyl-fluorophosphonate. Human neutrophils also metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was maximal after 15-30 s. Substrate preference was LA ≫ 1-LG > LEA. Importantly, 13-HODE-G was found in humans and mouse tissue samples. In conclusion, our data show that human eosinophils and neutrophils metabolize 1-LG and LEA into the novel endogenous 15-lipoxygenase metabolites 13-HODE-G and 13-HODE-EA. The full biological importance of 13-HODE-G and 13-HODE-EA remains to be explored.

Background: Diagnosis of eosinophilic esophagitis (EoE) requires manual quantification of tissue eosinophils. Eosinophil peroxidase (EPX) is an eosinophil-specific, cytoplasmic granule protein released during degranulation., Aims: The objective of this study was to evaluate image analysis of EPX immunohistochemistry as an automated method for histologic diagnosis of EoE., Methods: We performed a secondary analysis of prospectively collected esophageal biopsies obtained from adult subjects with EoE and controls. Tissue sections were stained with hematoxylin and eosin (H&E) and evaluated for peak eosinophils per high power field (eos/hpf). The same slides were de-stained and re-stained to detect EPX for direct comparison. Slides were digitized, and EPX staining area/mm 2 was quantified using image analysis. Paired samples were compared for changes in EPX staining in treatment responders and non-responders., Results: Thirty-eight EoE cases and 49 controls were analyzed. Among EoE subjects, matched post-treatment biopsies were available for 21 responders and 10 non-responders. Baseline EPX/mm 2 was significantly increased in EoE subjects and decreased in treatment responders. EPX quantification correlated strongly with eos/hpf (r = 0.84, p < 0.0001) and identified EoE subjects with high diagnostic accuracy (AUC 0.95, p < 0.0001). The optimal diagnostic EPX-positive pixel/area threshold was 17,379 EPX/mm 2 . Several controls (5/49) with < 15 eos/hpf on H&E staining exceeded this cutoff., Conclusions: EPX/mm 2 correlates strongly with eos/hpf, accurately identifies subjects with EoE, and decreases in treatment responders. Automated quantification of intact eosinophils and their degranulation products may enhance pathologic assessment. Future studies are needed to correlate EPX/mm 2 with symptoms, endoscopic findings, and esophageal distensibility.

The control of antimicrobial resistance requires the development of novel antimicrobial alternatives and naturally occurring peroxidase-catalyzed systems may be of great value in this era of emerging antimicrobial resistance. In the peroxidase system, a peroxidase enzyme catalyzes the oxidation of a halide/pseudohalide, at the expense of hydrogen peroxide, to generate reactive products with broad antimicrobial properties. The appropriate use of peroxidase systems needs a better understanding of the identities and properties of the generated antimicrobial oxidants, specific targets in bacterial cells, their mode of action and the factors favoring or limiting their activity. Here, the ABCs (antibacterial activity, bacterial "backtalk" and cytotoxicity) of these systems and their mimics are discussed. Particular attention is paid to the concomitant use of thiocyanate and iodide dual substrates in peroxidase/peroxidase-free systems with implications on their antimicrobial activity. This review also provides a summary of actual applications of peroxidase systems as bio-preservatives in oral healthcare, milk industry, food/feed specialties and related products, mastitis and wound treatment; lastly, this review points to opportunities for further research and potential applications., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Autoimmune diseases are defined by an immune response against a specific autoantigen, driven by antigen-specific T cells or antibodies. While the mechanisms resolving brief episodes of acute inflammation elicited by microbial components or tissue injury are well understood, the mechanisms resolving tissue inflammation in autoimmune diseases are still largely elusive. We have, therefore, addressed the mechanisms of resolution in IgG-mediated autoimmune diseases using a mouse model of the pemphigoid disease "bullous pemphigoid-like epidermolysis bullosa acquisita" (BP-like EBA) as prototypical example. We found that 12/15-LO is induced in skin lesions of BP-like EBA and is predominantly expressed in eosinophils. Dependent on the expression of 12/15-LO, large amounts of proresolving lipid mediators, are biosynthesized in the skin by the point disease peaks. Their production is timely correlated to the gradual reversal of tissue inflammation. Genetic deficiency in Alox15, the gene encoding 12/15-LO, disrupts this process significantly protracting and aggravating disease. This protraction is associated reduced recruitment of regulatory T cells (T regs ) into lesional skin. Intriguingly, Alox15 -/- mice also exhibit reduced recruitment of eosinophils into the skin, and the chemotaxis of cultured Alox15 -/- eosinophils towards CCL11/eotaxin-1 is compromised. Finally, we demonstrate that 15-lipoxygenase-1, the human homologue of 12/15-LO is induced in granulocytes in lesional skin of patients suffering from a pemphigoid disease. Collectively, our result uncover key mechanisms resolving IgG-mediated skin inflammation. These mechanisms are orchestrated by 12/15-LO expressed in eosinophils promoting the recruitment of eosinophils and T regs , which in turn inhibit neutrophils., (Copyright © 2020. Published by Elsevier Ltd.)

Despite many hypothesized benefits of dietary isoflavone genistein (GEN) deriving from soy-based products, questions surrounding GEN's developmental effects are increasing. To understand if in utero GEN exposure modulated postnatal respiratory allergies in the middle age, we conducted a time course study in the B6C3F1 offspring (PND 240-330) using a common household allergen (house dust mites: HDM; 10 μg/mouse for PND 240 and 290, and 50 μg/mouse for PND 330, a middle age in mice) following intranasal instillation, a physiological route of allergen exposure. GEN was administered to dams by gavage from gestational day 14 to parturition at a physiologically relevant dose (20 mg/kg body weight). Female and male offspring were sensitized with HDM allergens beginning about one month prior to sacrifice followed by challenges with three weekly dosings of HDM extracts, and they were euthanized at day 3 following the final HDM exposure. In utero exposure to GEN decreased HDM allergen-induced respiratory allergy in male B6C3F1 offspring at PND 330 as reflected by decreases in airway hyperresponsiveness (e.g., Penh value), HDM-specific IgG 1 (a Th2 type Ab) and the activity of eosinophil peroxidase in the lung (an indication of eosinophil recruitment to the lungs). However, in utero exposure to GEN had minimal effects on HDM allergen-induced respiratory allergy in the middle-aged female offspring. Changes in serum total IgE, HDM-specific IgE, and lung histopathology scores in both male and female offspring were not biologically significant. Overall, in utero GEN exposure exerted a protective effect on respiratory allergy in the middle-aged male, but not female, B6C3F1 offspring following later-life HDM exposures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Phosphatidylinositol-specific phospholipase C (PI-PLC) and cytosolic phospholipase A 2 (cPLA 2 ) regulate both eosinophil degranulation and leukotriene (LT) synthesis via PI-PLC-mediated calcium influx and cPLA 2 activation. Phosphatidylcholine-specific phospholipase C (PC-PLC) likely plays a key role in cellular signaling, including the eosinophilic allergic inflammatory response. This study examined the role of PC-PLC in eosinophil LT synthesis and degranulation using tricyclodecan-9-yl-xanthogenate (D609), a PC-specific PLC inhibitor. D609 inhibited N-formyl-met-leu-phe + cytochalasin B (fMLP/B)-induced arachidonic acid (AA) release and leukotriene C 4 (LTC 4 ) secretion. However, at concentrations that blocked both AA release and LTC 4 secretion, D609 had no significant inhibitory effect on stimulated cPLA 2 activity. D609 also partially blocked fMLP/B-induced calcium influx, indicating that inhibition of AA release and LTC 4 secretion by D609 is due to inhibition of calcium-mediated cPLA 2 translocation to intracellular membranes, not inhibition of cPLA 2 activity. In addition, D609 inhibited fMLP/B-stimulated eosinophil peroxidase release, indicating that PC-PLC regulates fMLP/B-induced eosinophil degranulation by increasing the intracellular calcium concentration ([Ca 2+ ] i ). Overall, our results showed that PC-PLC is critical for fMLP/B-stimulated eosinophil LT synthesis and degranulation. In addition, degranulation requires calcium influx, while PC-PLC regulates LTC 4 synthesis through calcium-mediated cPLA 2 activation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lipid mediators play important roles in regulating inflammatory responses and tissue homeostasis. Since 12/15-lipoxygenase (12/15-LOX)-derived lipid mediators such as lipoxin A 4 (LXA 4 ) and protectin D1 (PD1) protect against corneal epithelial cell damage, the major cell types that express 12/15-LOX and contribute to the corneal wound healing process are of particular interest. Here, we found that eosinophils were the major cell type expressing 12/15-LOX during the corneal wound healing process. Eosinophils were recruited into the conjunctiva after corneal epithelium wounding, and eosinophil-deficient and/or eosinophil-specific 12/15-LOX knockout mice showed delayed corneal wound healing compared with wild-type mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based mediator lipidomics revealed that a series of 12/15-LOX-derived mediators were significantly decreased in eosinophil-deficient mice and topical application of 17-hydroxydocosahexaenoic acid (17-HDoHE), a major 12/15-LOX-derived product, restored the phenotype. These results indicate that 12/15-LOX-expressing eosinophils, by locally producing pro-resolving mediators, significantly contribute to the corneal wound healing process in the eye., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The Charcot-Leyden crystal protein (CLC-P), a constituent of human and not mouse eosinophils, is one of the most abundant proteins within human eosinophils. It has a propensity to form crystalline structures, Charcot-Leyden crystals, which are hallmarks in their distinctive extracellular crystalline forms as markers of eosinophilic inflammation. The functions of CLC-P within eosinophils have been uncertain. Although the action of CLC-P as a lysophospholipase has been questioned, assays of chromatographically purified CLC-P and crystal-derived CLC-P as well as studies of transfected recombinant CLC-P have consistently documented that CLC-P endogenously expresses lysophospholipase activity, releasing free palmitate from substrate lysopalmitoylphosphatidylcholine. Rather than acting solely as a hydrolytic enzyme to release palmitate from a lysolipid substrate, some other lysophospholipases function more dominantly as acyl-protein thioesterases (APTs), enzymes that catalyze the removal of thioester-linked, long chain fatty acids, such as palmitate, from cysteine residues of proteins. As such APTs participate in palmitoylation, a post-translational modification that can affect membrane localization, vesicular transport, and secretion. CLC-P has attributes of an APT. Thus, whereas CLC-P expresses inherent lysophospholipase activity, like some other lysophospholipase enzymes, it likely also functions in regulating the dynamic palmitoylation cycle, including, given its dominant subplasmalemmal location, at the human eosinophil's plasma membrane., (©2020 Society for Leukocyte Biology.)

Background: Neutrophils and eosinophils are multifunctional granulocytes derived from common myelocytic-committed progenitor cells. Severe congenital neutropenia 1 (SCN1) caused by ELANE mutations is a rare disease characterized by very low numbers of circulating neutrophils. Little is known about the functional characteristics of the SCN1 granulocytes, except that eosinophilia has been noticed in both bone marrow and peripheral blood. In this study, we profiled the number and function of granulocytes in patients suffering from SCN1., Methods: Nine patients diagnosed with SCN1 were enrolled in this study and absolute counts of eosinophils and neutrophils from bone marrow aspirates and peripheral blood samples were analysed. In addition, Ficoll-Paque enriched granulocytes from patients and healthy controls were analysed for specific eosinophil and neutrophil markers using flow cytometry and for NADPH-oxidase activity-profile by chemiluminescence., Results: Our data demonstrate a skewed granulocyte population in SCN1 patients dominated by eosinophils in both bone marrow and peripheral blood. The latter was detected only by blood smear examination, but not by automated blood analysers. Furthermore, we show that the SCN1 eosinophils exerted normal production of reactive oxygen species generated by the NADPH-oxidase, however the response was profoundly different from that of healthy control neutrophils., Conclusions: SCN1 patients with ELANE mutations suffer from neutropenia yet display eosinophilia in the bone marrow and blood, as revealed by smear examination but not by automatic blood analysers. The SCN1 eosinophils are functionally normal regarding production of reactive oxygen species (ROS). However, the ROS profile produced by eosinophils differs drastically from that of neutrophils isolated from the same blood donor, implying that the eosinophilia in SCN1 cannot compensate for the loss of neutrophils regarding ROS-mediated functions.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD + ) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD + adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD + using purified EPO and eosinophils isolated from asthmatic donors. UV-Vis spectroscopy revealed INH oxidation by EPO led to a new product (λ max  = 326 nm) in the presence of NAD + . This adduct was confirmed to be INH-NAD + using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD + , was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD + adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Renal fibrosis is the pathological hallmark of chronic kidney disease (CKD) and manifests as glomerulosclerosis and tubulointerstitial fibrosis. Reactive oxygen species contribute significantly to renal inflammation and fibrosis, but most research has focused on superoxide and hydrogen peroxide (H 2 O 2 ). The animal heme peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPX), and peroxidasin (PXDN) uniquely metabolize H 2 O 2 into highly reactive and destructive hypohalous acids, such as hypobromous and hypochlorous acid. However, the role of these peroxidases and their downstream hypohalous acids in the pathogenesis of renal fibrosis is unclear. Our study defines the contribution of MPO, EPX, and PXDN to renal inflammation and tubulointerstitial fibrosis in the murine unilateral ureteral obstruction (UUO) model. Using a nonspecific inhibitor of animal heme peroxidases and peroxidase-specific knockout mice, we find that loss of EPX or PXDN, but not MPO, reduces renal fibrosis. Furthermore, we demonstrate that eosinophils, the source of EPX, accumulate in the renal interstitium after UUO. These findings point to EPX and PXDN as potential therapeutic targets for renal fibrosis and CKD and suggest that eosinophils modulate the response to renal injury.

Eosinophil peroxidase (EPX) is a major constituent of the large cytoplasmic granules of both human and mouse eosinophilic leukocytes. Human EPX deficiency is a rare, autosomal-recessive disorder limited to the eosinophil lineage. Our intent was to explore the impact of EPX gene deletion on eosinophil content, structure, and function. In response to repetitive intranasal challenge with a filtrate of the allergen, Alternaria alternata, we found significantly fewer eosinophils peripherally and in the respiratory tracts of EPX -/- mice compared to wild-type controls; furthermore, both the major population (Gr1 -/lo ) and the smaller population of Gr1 hi eosinophils from EPX -/- mice displayed lower median fluorescence intensities (MFIs) for Siglec F. Quantitative evaluation of transmission electron micrographs of lung eosinophils confirmed the relative reduction in granule outer matrix volume in cells from the EPX -/- mice, a finding analogous to that observed in human EPX deficiency. Despite the reduced size of the granule matrix, the cytokine content of eosinophils isolated from allergen-challenged EPX -/ - and wild-type mice were largely comparable to one another, although the EPX -/- eosinophils contained reduced concentrations of IL-3. Other distinguishing features of lung eosinophils from allergen-challenged EPX -/- mice included a reduced fraction of surface TLR4 + cells and reduced MFI for NOD1. Interestingly, the EPX gene deletion had no impact on eosinophil-mediated clearance of gram-negative Haemophilus influenzae from the airways. As such, although no clinical findings have been associated with human EPX deficiency, our findings suggest that further evaluation for alterations in eosinophil structure and function may be warranted., (©2018 Society for Leukocyte Biology.)

Galectin-1 (Gal-1) is a β-galactoside-binding protein with diverse biological activities in the pathogenesis of inflammation, however the mechanisms by which Gal-1 modulates cellular responses in allergic inflammatory processes have not been fully determined. In this study, we evaluated the therapeutic potential of Gal-1 eye drops in an experimental model of conjunctivitis. Wistar rats received a topical application of compound (C)48/80 (100 mg/ml) into right eyes and a drop of vehicle into the contralateral eye. Another group of rats received Gal-1 (0.3 or 3 μg/eye) or sodium cromoglycate (SCG; 40 mg/ml) in both eyes and, after 15 min, right eye was challenged with C48/80. Conjunctivitis-induced by C48/80 was characterized by severe eyelid oedema and tearing, but clinical signs were ameliorated by eye drop doses of both Gal-1 (0.3/3 μg) and SCG. As expected, an increased proportion of degranulated mast cells (62%, P < 0.01) and lower histamine levels were observed after 6 h of C48/80 challenge, compared to control (32%). This effect was abrogated by Gal-1 and SCG, which reduced mast cell degranulation (31-36%), eosinophil migration and eosinophil peroxidase levels in the eyes. Gal-1 (3 μg) and SCG treatments also decreased IL-4 levels, as well as activation of mitogen activated protein kinases compared to untreated C48/80 eyes. Our findings suggest that Gal-1 eye drops represent a new therapeutic strategy for ocular allergic inflammation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Lungs allografts have worse long-term survival compared with other organ transplants. This is most likely due to their unique immunoregulation that may not respond to traditional immunosuppression. For example, local NO generation by inducible NOS (iNOS) is critical for lung allograft acceptance but associates with rejection of other solid organs. The source of NO in accepting lung allografts remains unknown. Here, we report that, unlike the case for other pulmonary processes in which myeloid cells control NO generation, recipient-derived eosinophils play a critical and nonredundant role in iNOS-mediated lung allograft acceptance. Depletion of eosinophils reduces NO levels to that of recipients with global deletion of iNOS and leads to a costimulatory blockade-resistant form of rejection. Furthermore, NO production by eosinophils depends on Th1 polarization by inflammatory mediators, such as IFN-γ and TNF-α. Neutralization of such mediators abrogates eosinophil suppressive capacity. Our data point to what we believe to be a unique and previously unrecognized role of eosinophil polarization in mediating allograft tolerance and put into perspective the use of high-dose eosinophil-ablating corticosteroids after lung transplantation.

Background and Aim: Eosinophils are markers of the eosinophilic esophagitis (EoE) disease, and this work aimed to assess whether activation of eosinophils could be a noninvasive test to contribute for EoE diagnosis., Methods: The activation state of peripheral blood eosinophils in EoE patients and control subjects was assessed based on the morphological aspects of the eosinophil after adherence to slide. Cyclooxygenase-2 and 5-lipoxygenase expressions were evaluated by means of immunofluorescence microscopy to verify if and which eicosanoid pathway is triggered in eosinophils in blood in EoE., Results: The eosinophils of patients with EoE were significantly more activated than those of control individuals. The lowest percentage of normal eosinophils for control subjects was 40%, while the highest percentage of eosinophils of normal aspect for patients with EoE was 32%. Considering 36% as a cutoff for normal eosinophils, this value differentiated all individuals with EoE from individuals without the disease with a sensitivity of 100%, considering the diagnosis of EoE as currently defined. Eosinophils of EoE patients showed higher expression of cyclooxygenase-2 than those of control subjects., Conclusions: The quantification of morphological changes in eosinophils is a feasible, easy, and reliable manner to identify EoE patients. Therefore, patients with symptoms of esophageal dysfunction showing higher than 36% activated eosinophils in peripheral blood could be a useful way to help definition and diagnostic criterion for EoE., (© 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

OBJECTIVE To evaluate a method for identifying intact and degranulated eosinophils in the small intestine of dogs with inflammatory bowel disease (IBD) by use of a monoclonal antibody (mAb) against eosinophil peroxidase (EPX). ANIMALS 11 untreated dogs with IBD, 5 dogs with IBD treated with prednisolone, and 8 control dogs with no clinical evidence of gastrointestinal tract disease and no immunosuppressive treatment. PROCEDURES 4-μm-thick sections of paraffin-embedded tissues from necropsy specimens were immunostained with EPX mAb. Stained intact and degranulated eosinophils in consecutive microscopic fields (400X magnification) of the upper (villus tips) and lower (between the muscularis mucosae and crypts) regions of the lamina propria of the jejunum were manually counted. RESULTS Compared with control and treated IBD dogs, untreated IBD dogs had a significantly higher number of degranulated eosinophils in the lower region of the lamina propria. However, no significant differences were detected in the number of intact eosinophils in this region among groups. In the upper region of the lamina propria, untreated IBD dogs had a significantly higher number of degranulated and intact eosinophils, compared with control and treated IBD dogs. Number of degranulated and intact eosinophils did not differ significantly between control and treated IBD dogs. CONCLUSIONS AND CLINICAL RELEVANCE Immunohistologic analysis with EPX mAb yielded prominent granule staining that allowed reliable morphological identification of degranulated and intact eosinophils, which may provide a strategy for quantitative and selective evaluation of eosinophils in gastrointestinal biopsy specimens and a potential method to diagnose IBD and evaluate treatment outcome.

Respiratory viruses cause asthma exacerbations. Because eosinophils are the prominent leukocytes in the airways of 60-70% of patients with asthma, we evaluated the effects of eosinophils on a common respiratory virus, parainfluenza 1, in the lung. Eosinophils recruited to the airways of wild-type mice after ovalbumin sensitization and challenge significantly decreased parainfluenza virus RNA in the lungs 4 days after infection compared with nonsensitized animals. This antiviral effect was also seen in IL-5 transgenic mice with an abundance of airway eosinophils (NJ.1726) but was lost in transgenic eosinophil-deficient mice (PHIL) and in IL-5 transgenic mice crossed with eosinophil-deficient mice (NJ.1726-PHIL). Loss of the eosinophil granule protein eosinophil peroxidase, using eosinophil peroxidase-deficient transgenic mice, did not reduce eosinophils' antiviral effect. Eosinophil antiviral mechanisms were also explored in vitro. Isolated human eosinophils significantly reduced parainfluenza virus titers. This effect did not involve degradation of viral RNA by eosinophil granule RNases. However, eosinophils treated with a nitric oxide synthase inhibitor lost their antiviral activity, suggesting eosinophils attenuate viral infectivity through production of nitric oxide. Consequently, eosinophil nitric oxide production was measured with an intracellular fluorescent probe. Eosinophils produced nitric oxide in response to virus and to a synthetic agonist of the virus-sensing innate immune receptor, Toll-like receptor (TLR) 7. IFNγ increased expression of eosinophil TLR7 and potentiated TLR7-induced nitric oxide production. These results suggest that eosinophils promote viral clearance in the lung and contribute to innate immune responses against respiratory virus infections in humans.

The objective of the study was to compare nasal, pharyngeal, and sputum eosinophil peroxidase (EPX) levels with induced sputum eosinophil percentage in 10 adults with poorly controlled asthma and 10 normal controls. EPX was measured using an ELISA and normalized for grams of protein for nasal and pharynx specimens and for mL-gram of protein for sputum. Sputum EPX levels were statistically different between asthma and control subjects (P = 0.024). EPX levels measured in the nasal and pharyngeal swab samples derived from the same patients were also different between asthma and control subjects, each displaying a high degree of significance (P = 0.002). Spearman's correlation coefficients for nasal EPX and pharyngeal EPX levels compared to induced sputum eosinophil percentage were 0.81 (P = 0.0007) and 0.78 (P = 0.0017), respectively. Thus, there is a strong association in a given patient between both nasal and pharyngeal EPX levels and the eosinophil percentage of induced sputum., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The aim of the present work was to evaluate the number of apoptotic eosinophils in the livers of sheep experimentally infected with Fasciola hepatica during the migratory and biliary stages of infection. Four groups (n=5) of sheep were used; groups 1-3 were orally infected with 200 metacercariae (mc) and sacrificed at 8 and 28 days post-infection (dpi), and 17 weeks post-infection (wpi), respectively. Group 4 was used as an uninfected control. Apoptosis was detected using immunohistochemistry with a polyclonal antibody against anti-active caspase-3, and transmission electron microscopy (TEM). Eosinophils were identified using the Hansel stain in serial sections for caspase-3, and by ultrastructural features using TEM. At 8 and 28 dpi, numerous caspase-3(+) eosinophils were mainly found at the periphery of acute hepatic necrotic foci. The percentage of caspase -3(+) apoptotic eosinophils in the periphery of necrotic foci was high (46.1-53.9) at 8 and 28 dpi, respectively, and decreased in granulomas found at 28 dpi (6%). Transmission electron microscopy confirmed the presence of apoptotic eosinophils in hepatic lesions at 8 and 28 dpi. At 17 wpi, apoptotic eosinophils were detected in the infiltrate surrounding some enlarged bile ducts containing adult flukes. This is the first report of apoptosis induced by F. hepatica in sheep and the first study reporting apoptosis in eosinophils in hepatic inflammatory infiltrates in vivo. The high number of apoptotic eosinophils in acute necrotic tracts during the migratory and biliary stages of infection suggests that eosinophil apoptosis may play a role in F. hepatica survival during different stages of infection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Studies have shown that eosinophils are closely related to pathogenesis of bronchial asthma. Eosinophils release eosinophil cationic protein (ECP), which plays an important role in infection and allergic reactions. Serum ECP mRNA expression in children with bronchial asthma has not been adequately investigated. We analyzed serum ECP mRNA expression in 63 children with bronchial asthma and 21 healthy children by using reverse-transcriptase polymerase chain reaction to understand the role of ECP in children with bronchial asthma. The children with bronchial asthma were segregated into acute-phase and stable-phase groups, based on the severity of the illness. Serum ECP mRNA expression in children with bronchial asthma (0.375 ± 0.04) was significantly higher than that in healthy controls (0.20 ± 0.02; P < 0.05). Additionally, children in the acute-phase group showed higher ECP mRNA expression level (0.44 ± 0.06) than those in the stable-phase (0.31 ± 0.03) and healthy control groups (0.20 ± 0.02; P < 0.05), while the level in the stable-phase (0.31 ± 0.03) was markedly higher than that in the healthy control group (0.20 ± 0.02; P < 0.05). Detection of serum ECP mRNA expression level has possible applications in the diagnosis and treatment of children with bronchial asthma.

Cysteinyl leukotrienes (cys-LTs) are lipid mediators of inflammation. The enzyme catalyzing synthesis of cys-LTs, leukotriene C4 synthase (LTC4S), is considered an important drug target. Here we report the synthesis and characterization of three tandem benzophenone amino pyridines as inhibitors of LTC4S in vitro and in vivo. The inhibitors were characterized in vitro using recombinant human LTC4S, MonoMac 6 cells, and a panel of peripheral human immune cells. In vivo, the compounds were tested in the Zymosan A-induced peritonitis mouse model. The molecules, denoted TK04, TK04a, and TK05, were potent and selective inhibitors of LTC4S with IC50 values of 116, 124, and 95 nM, respectively. Molecular docking revealed binding in a hydrophobic crevice between two enzyme monomers and interaction with two catalytic residues, Arg104 and Arg31. The TK compounds potently inhibited cys-LT biosynthesis in immune cells. In coincubations of platelets and polymorphonuclear leukocytes, inhibition of LTC4S led to shunting of LTA4 toward anti-inflammatory lipoxin A4, which was significantly enhanced by simultaneous inhibition of LTA4H. Finally, we found that TK05 (6 mg⋅kg(-1)⋅body weight) reduces LTE4 levels in peritoneal lavage fluid by 88% and significantly decreases vascular permeability in vivo. Our findings indicate that the TK compounds are valuable experimental tools in eicosanoid research in vitro and in vivo. Their chemical structures may serve as leads for further inhibitor design. Novel drugs depleting cys-LT production could be beneficial for treatment of inflammatory diseases associated with overexpression of LTC4S., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation., (© 2014 John Wiley & Sons Ltd.)

By combining easy and fast leukocyte enrichment with aminophenyl-fluorescein (APF) staining we developed a method to quickly and specifically address the halogenating activity of the immunological relevant blood heme peroxidases myeloperoxidase and eosinophil peroxidase, respectively. For leukocyte enrichment a two-fold hypotonic lysis procedure of the blood with Millipore water was chosen which represents a cheap, fast and reliable method to diminish the amount of erythrocytes in the samples. This procedure is shown to be suitable both to human and murine blood micro-samples, making it also applicable to small animal experiments with recurring blood sampling. As all types of leukocytes are kept in the sample during the preparation, they can be analysed separately after discrimination during the flow cytometry analysis. This also holds for all heme peroxidase-containing cells, namely neutrophils, eosinophils and monocytes. Moreover additional parameters (e.g. antibody staining) can be combined with the heme peroxidase activity determination to gain additional information about the different immune cell types. Based on previous results we applied APF for specifically addressing the halogenating activity of leukocyte peroxidases in blood samples. This dye is selectively oxidized by the MPO and EPO halogenation products hypochlorous and hypobromous acid. This approach may provide a suitable tool to gain more insights into the immune-physiological role of the halogenating activity of heme peroxidases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Eosinophils are potent inflammatory cells with numerous immune functions, including antigen presentation and exacerbation of inflammatory responses through their capacity to release a range of largely preformed cytokines and lipid mediators. Thus, timely regulation of eosinophil activation and apoptosis is crucial to develop beneficial immune response and to avoid tissue damage and induce resolution of inflammation. Natural Killer (NK) cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and eosinophils. Co-culture experiments revealed that human NK cells could trigger autologous eosinophil activation, as shown by up-regulation of CD69 and down-regulation of CD62L, as well as degranulation, evidenced by increased CD63 surface expression, secretion of eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN). Moreover, NK cells significantly and dose dependently increased eosinophil apoptosis as shown by annexin V and propidium iodide (PI) staining. Direct contact was necessary for eosinophil degranulation and apoptosis. Increased expression of phosphorylated extracellular signal-regulated kinase (ERK) in cocultured eosinophils and inhibition of eosinophil CD63 expression by pharmacologic inhibitors suggest that MAPK and PI3K pathways are involved in NK cell-induced eosinophil degranulation. Finally, we showed that NK cells increased reactive oxygen species (ROS) expression by eosinophils in co-culture and that mitochondrial inhibitors (rotenone and antimycin) partially diminished NK cell-induced eosinophil apoptosis, suggesting the implication of mitochondrial ROS in NK cell-induced eosinophil apoptosis. Pan-caspase inhibitor (ZVAD-FMK) only slightly decreased eosinophil apoptosis in coculture. Altogether, our results suggest that NK cells regulate eosinophil functions by inducing their activation and their apoptosis.

Previous studies showed that the biological activity and the eosinophil content of eosinophil cationic protein (ECP, RNase 3) are determined by single-nucleotide polymorphisms (SNPs) in the ECP (RNase3) gene. In this study, we report the prevalence of a common SNP in the eosinophil protein x/eosinophil-derived neurotoxin (EPX/EDN, RNase2) and the association with the cellular contents of EPX/EDN and ECP. The genes were sequenced and the EPX/EDN405(G>C) rs2013109 SNPs were also determined by TaqMan 5′nuclease allelic discrimination assay. ECP and EPX/EDN in purified eosinophils or in whole blood extracts were analysed by sensitive immunoassays. The study included 379 non-allergic and allergic subjects. The genotype prevalence of the EPX/EDN405(G>C) polymorphism was GG 59%, GC 36% and CC 6%. The cellular contents of ECP and EPX/EDN were related in a reciprocal fashion with the sums of the protein contents being constant. The contents were associated with the ECP562(G>C) rs2233860 and EPX/EDN405(G>C) gene polymorphisms. The cellular content of eosinophil peroxidase (EPO) was not associated with the ECP and EPX/EDN genotypes. The prevalence of the EPX/EDN405(G>C) genotypes and the contents of the proteins were similar in non-allergic and allergic subjects.The production and storage of the two ancestral proteins, ECP and EPX/EDN likely share common regulatory mechanisms, which result in opposing productions of the two proteins.

To investigate pathogenic mechanisms of primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), and autoimmune hepatitis (AIH), serum levels of 26 chemokines and cytokines were determined and compared with patients with chronic hepatitis C or in healthy controls. The chemokine eotaxin-3 (E3; CCL26), which recruits eosinophils to sites of inflammation, was found to be highly elevated in all PSC, PBC, and AIH patients compared with HCV patients and healthy controls. Eotaxin-1 (E1; CCL11), another eosinophil-specific chemokine, was elevated in PSC but reduced in PBC and AIH, while the macrophage-derived chemokine (MDC; CCL22) was lower in all PSC, PBC, and AIH patients compared with HCV patients and controls. By incorporating levels of the interleukin (IL)-15 into a diagnostic algorithm, PSC, PBC, and AIH patients could each be differentiated with good sensitivity and specificity. These findings represent the first study to compare the level of serum cytokine/chemokine levels among these related autoimmune-like liver diseases. Furthermore, our data indicate that the measurement of serum E3, E1, CCL22, and IL-15 levels can aid in the diagnosis of these clinically challenging diseases and shed light on the potential pathogenic mechanisms underlying these diseases. By suggesting a potential role for an allergic phenomenon involving eosinophils, which may define them as liver-specific allergic diseases, this may open up potential new therapeutic avenues by abrogating the action of these disease-associated immune modulators.

The tight regulation of granulocyte chemotaxis is crucial for initiation and resolution of inflammation. Here, we show that DAPK2, a Ca(2+)/CaM-sensitive serine/threonine kinase known to modulate cell death in various cell types, is a novel regulator of migration in granulocytes. We demonstrate that human neutrophils and eosinophils express DAPK2 but unlike other leukocytes, no DAPK1 or DAPK3 protein. When DAPK activities were blocked by inhibitors, we found that neither granulocyte lifespan nor phagocytosis was affected. However, such pharmacological inactivation of DAPK activity abolished motility of granulocytes in response to intermediary but not end-target chemoattractants ex vivo. The defect in chemotaxis in DAPK2-inactive granulocytes is likely a result of reduced polarization of the cells, mediated by a lack of MLC phosphorylation, resulting in radial F-actin and pseudopod formation. As neutrophils treated with DAPKi also showed reduced recruitment to the site of inflammation in a mouse peritonitis model, DAPK2 may be a novel target for anti-inflammatory therapies.

The roles of eosinophils in antimicrobial defense remain incompletely understood. In ovalbumin-sensitized mice, eosinophils are selectively recruited to the peritoneal cavity by antigen, eotaxin, or leukotriene(LT)B4, a 5-lipoxygenase (5-LO) metabolite. 5-LO blockade prevents responses to both antigen and eotaxin. We examined responses to eotaxin in the absence of sensitization and their dependence on 5-LO. BALB/c or PAS mice and their mutants (5-LO-deficient ALOX; eosinophil-deficient GATA-1) were injected i.p. with eotaxin, eosinophils, or both, and leukocyte accumulation was quantified up to 24 h. Significant recruitment of eosinophils by eotaxin in BALB/c, up to 24 h, was accompanied by much larger numbers of recruited neutrophils and monocytes/macrophages. These effects were abolished by eotaxin neutralization and 5-LO-activating protein inhibitor MK886. In ALOX (but not PAS) mice, eotaxin recruitment was abolished for eosinophils and halved for neutrophils. In GATA-1 mutants, eotaxin recruited neither neutrophils nor macrophages. Transfer of eosinophils cultured from bone-marrow of BALB/c donors, or from ALOX donors, into GATA-1 mutant recipients, i.p., restored eotaxin recruitment of neutrophils and showed that the critical step dependent on 5-LO is the initial recruitment of eosinophils by eotaxin, not the secondary neutrophil accumulation. Eosinophil-dependent recruitment of neutrophils in naive BALB/c mice was associated with increased binding of bacteria.

Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln(40), His(64) and Arg(105) were indicated as major contributions for the interaction. Interestingly, Gln(40) and His(64) on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln(40)-His(64) clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.

Eosinophil degranulation has been implicated in inflammatory processes associated with allergic asthma. Rab27a, a Rab-related GTPase, is a regulatory intracellular signaling molecule expressed in human eosinophils. We postulated that Rab27a regulates eosinophil degranulation. We investigated the role of Rab27a in eosinophil degranulation within the context of airway inflammation. Rab27a expression and localization in eosinophils were investigated by using subcellular fractionation combined with Western blot analysis, and the results were confirmed by immunofluorescence analysis of Rab27a and the granule membrane marker CD63. To determine the function of eosinophil Rab27a, we used Ashen mice, a strain of Rab27a-deficient animals. Ashen eosinophils were tested for degranulation in response to PAF and calcium ionophore by measuring released EPX activity. Airway EPX release was also determined by intratracheal injection of eosinophils into mice lacking EPX. Rab27a immunoreactivity colocalized with eosinophil crystalloid granules, as determined by subcellular fractionation and immunofluorescence analysis. PAF induced eosinophil degranulation in correlation with redistribution of Rab27a(+) structures, some of which colocalized with CD63(+) crystalloid granules at the cell membrane. Eosinophils from mice had significantly reduced EPX release compared with normal WT eosinophils, both in vitro and in vivo. In mouse models, Ashen mice demonstrated reduced EPX release in BAL fluid. These findings suggest that Rab27a has a key role in eosinophil degranulation. Furthermore, these findings have implications for Rab27a-dependent eosinophil degranulation in airway inflammation.

We investigated the effect of asthmatic serum on constitutive eosinophil apoptosis in normal subjects. Eosinophil apoptosis in normal subjects was inhibited by asthmatic serum but not normal serum. In a detailed analysis based on the presence of house dust mite (HDM) IgE, HDM IgE-positive (+) asthmatic serum was more effective for eosinophil apoptosis than that of HDM IgE-negative (-) asthmatic serum. HDM IgE+ asthmatic serum inhibited both HDM IgE- and HDM IgE+ normal eosinophil apoptosis, and HDM IgE- asthmatic serum suppressed eosinophil apoptosis of HDM IgE+ normal. HDM IgE- normal serum did not inhibit either HDM IgE- or HDM IgE+ normal eosinophil apoptosis, and HDM IgE+ normal serum inhibited HDM IgE+ normal eosinophil apoptosis. The kind of HDM IgE (Dermatophagoides pteronissinus-specific IgE and Dermatophagoides farinae-specific IgE) was not related to the effect of asthmatic serum on eosinophil apoptosis. Extracts of DP and DF, Der p1, and Der p2, were not effective for eosinophil apoptosis. HDM IgE+ asthmatic serum inhibited cleavage of procaspase 9 and procaspase 3. Asthmatic serum induced Akt and ERK phosphorylation, and ERK activation was suppressed by AKTi. Taken together, asthmatic serum inhibited normal eosinophil apoptosis via PI3K/Akt/ERK cascade. The novel approach taken in this study provided better insight into HDM-associated anti-apoptotic mechanism of eosinophils in patients with asthma.

Eosinophils are generally linked to innate host defense against helminths, as well as the pathologies associated with allergic diseases, such as asthma. Nonetheless, the activities of eosinophils remain poorly understood, which in turn, has prevented detailed definitions of their role(s) in health and disease. Homologous recombination in embryonic stem cells was used to insert a mammalianized Cre recombinase in the ORF encoding Epx. This knock-in strategy overcame previous inefficiencies associated with eosinophil-specific transgenic approaches and led to the development of a knock-in strain of mice (eoCRE), capable of mediating recombination of "floxed" reporter cassettes in >95% of peripheral blood eosinophils. We also showed that this Cre expression was limited exclusively to eosinophil-lineage committed cells with no evidence of Cre-mediated toxicity. The efficiency and specificity of Cre expression in eoCRE mice were demonstrated further in a cross with a knock-in mouse containing a "(flox-stop-flox)" DTA cassette at the ROSA26 locus, generating yet another novel, eosinophil-less strain of mice. The development of eoCRE mice represents a milestone in studies of eosinophil biology, permitting eosinophil-specific gene targeting and overexpression in the mouse as part of next-generation studies attempting to define eosinophil effector functions.

Aim: To study the association between inflammatory bowel disease (IBD) and genetic variations in eosinophil protein X (EPX) and eosinophil cationic protein (ECP)., Methods: DNA was extracted from ethylene diamine tetraacetic acid blood of 587 patients with Crohn's disease (CD), 592 with ulcerative colitis (UC) and 300 healthy subjects. The EPX405 (G > C, rs2013109), ECP434 (G > C, rs2073342) and ECP562 (G > C, rs2233860) gene polymorphisms were analysed, by the 5'-nuclease allelic discrimination assay. For determination of intracellular content of EPX and ECP in granulocytes, 39 blood samples was collected and extracted with a buffer containing cetyltrimethylammonium bromide. The intracellular content of EPX was analysed using an enzyme-linked immunosorbent assay. The intracellular content of ECP was analysed with the UniCAP(®) system as described by the manufacturer. Statistical tests for calculations of results were χ(2) test, Fisher's exact test, ANOVA, Student-Newman-Keuls test, and Kaplan-Meier survival curve with Log-rank test for trend, the probability values of P < 0.05 were considered statistically significant., Results: The genotype frequency for males with UC and with an age of disease onset of ≥ 45 years (n = 57) was for ECP434 and ECP562, GG = 37%, GC = 60%, CC = 4% and GG = 51%, GC = 49%, CC = 0% respectively. This was significantly different from the healthy subject's genotype frequencies of ECP434 (GG = 57%, GC = 38%, CC = 5%; P = 0.010) and ECP562 (GG = 68%, GC = 29%,CC = 3%; P = 0.009). The genotype frequencies for females, with an age of disease onset of ≥ 45 years with CD (n = 62), was for the ECP434 and ECP562 genotypes GG = 37%, GC = 52%, CC = 11% and GG = 48%, GC = 47% and CC = 5% respectively. This was also statistically different from healthy controls for both ECP434 (P = 0.010) and ECP562 (P = 0.013). The intracellular protein concentration of EPX and ECP was calculated in μg/10(6) eosinophils and then correlated to the EPX 405 genotypes. The protein content of EPX was highest in the patients with the CC genotype of EPX405 (GG = 4.65, GC = 5.93, and CC = 6.57) and for ECP in the patients with the GG genotype of EPX405 (GG = 2.70, GC = 2.47 and CC = 1.90). ANOVA test demonstrated a difference in intracellular protein content for EPX (P = 0.009) and ECP (P = 0.022). The age of disease onset was linked to haplotypes of the EPX405, ECP434 and ECP562 genotypes. Kaplan Maier curve showed a difference between haplotype distributions for the females with CD (P = 0.003). The highest age of disease onset was seen in females with the EPX405CC, ECP434GC, ECP562CC haplotype (34 years) and the lowest in females with the EPX405GC, ECP434GC, ECP562GG haplotype (21 years). For males with UC there was also a difference between the highest and lowest age of the disease onset (EPX405CC, ECP434CC, ECP562CC, mean 24 years vs EPX405GC, ECP434GC, ECP562GG, mean 34 years, P = 0.0009). The relative risk for UC patients with ECP434 or ECP562-GC/CC genotypes to develop dysplasia/cancer was 2.5 (95%CI: 1.2-5.4, P = 0.01) and 2.5 (95%CI: 1.1-5.4, P = 0.02) respectively, compared to patients carrying the GG-genotypes., Conclusion: Polymorphisms of EPX and ECP are associated to IBD in an age and gender dependent manner, suggesting an essential role of eosinophils in the pathophysiology of IBD.

Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aims: The relationship of mast cells to the pathogenesis of lung fibrosis remains undefined despite recognition of their presence in the lungs of patients with pulmonary fibrosis. This study was performed to characterize the relationship of mast cells to fibrotic lung diseases., Methods and Results: Lung tissues from patients with idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (HP), systemic sclerosis (SSc)-related interstitial lung disease (ILD) and normal individuals were subjected to chymase immunostaining and the mast cell density quantified. Eosinophils were quantified by immunostaining for eosinophil peroxidase. Changes in lung function were correlated with mast cell density. Lung tissue obtained from IPF patients had a higher density of chymase-immunoreactive mast cells than that from patients with HP, SSc-related ILD or normal lungs. IPF lung tissue had a higher density of eosinophils than normal lung. There was no correlation between mast cell density and eosinophil density in IPF lung. IPF patients with high mast cell density had a slower rate of decline in forced vital capacity (FVC) than IPF patients with low mast cell density., Conclusions: Mast cell density in IPF lungs is higher than in other fibrotic lung diseases and normal lungs. Increased mast cell density in IPF may predict slower disease progression., (© 2012 Blackwell Publishing Ltd.)

Tyrosine kinases play a critical role in transducing intracellular signals from the receptors. Many receptors do not have intrinsic tyrosine kinase activity, so they rely on cytosolic and/or membrane-associated tyrosine kinases for initial signal generation. The Src and JAK family kinases are frequently associated with receptors and generate the initial cytosolic signals. These signals are then transduced to other compartments of the cytosol and to the nucleus to elicit a specific cellular response. In this review we focus on these two families of tyrosine kinases and review their involvement in activation of cells that are involved in the pathogenesis of asthma. A Th2-type immune response dominates the processes that lead to the phenotype of asthma. For this reason we give special attention to the tyrosine kinases that are involved in a Th2 response. Further we examine the involvement of tyrosine kinases in activation of mast cells, eosinophils and other cells.

Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC(50) 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.

Allergic asthma has emerged as an important public health problem of urban populations in developed countries. Very often herbal medicine is used to treat this widespread disease, due to the lack of efficacy and the important side effects related to the classical drugs in use. Along this line, Ocimum gratissimum (Og) is a plant widely used in Brazilian folk medicine to treat inflammatory disorders, such as asthma. In the present study we evaluated the immunomodulatory effects of Og and rosmarinic acid (RA, a polyphenolic compound) in a murine model of respiratory allergy induced by the Blomia tropicalis (Bt) mite. The respiratory allergy was induced in A/J mice by administration of Bt extract and the treatment was done using 25, 50 or 100mg/kg of an Og methanolic extract or using 2, 20 or 200mg/kg of RA. We then evaluated the changes induced by these drugs on immunological parameters related to the allergic process, which are up-regulated in this allergic model. The treatment of animals with 100mg/Kg Og and 200mg/Kg RA led to a significant reduction in the numbers of leukocytes/eosinophils in bronchoalveolar lavage (BAL); eosinophil peroxidase activity in BAL; presence of mucus in respiratory tract, histopathological changes in the lung, and IL-4 in BAL. These results suggest that the methanolic extract of Og and the polyphenol RA have therapeutic potential in this murine model of respiratory allergy to a clinically relevant human sensitizer allergen., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Atopic dermatitis (AD) skin lesions exhibit epidermal and dermal thickening, eosinophil infiltration, and increased levels of the cysteinyl leukotriene (cys-LT) leukotriene C(4) (LTC(4)). Epicutaneous sensitization with ovalbumin of WT mice but not ΔdblGATA mice, the latter of which lack eosinophils, caused skin thickening, collagen deposition, and increased mRNA expression of the cys-LT generating enzyme LTC(4) synthase (LTC(4)S). Skin thickening and collagen deposition were significantly reduced in ovalbumin-sensitized skin of LTC(4)S-deficient and type 2 cys-LT receptor (CysLT(2)R)-deficient mice but not type 1 cys-LT receptor (CysLT(1)R)-deficient mice. Adoptive transfer of bone marrow-derived eosinophils from WT but not LTC(4)S-deficient mice restored skin thickening and collagen deposition in epicutaneous-sensitized skin of ΔdblGATA recipients. LTC(4) stimulation caused increased collagen synthesis by human skin fibroblasts, which was blocked by CysLT(2)R antagonism but not CysLT(1)R antagonism. Furthermore, LTC(4) stimulated skin fibroblasts to secrete factors that elicit keratinocyte proliferation. These findings establish a role for eosinophil-derived cys-LTs and the CysLT(2)R in the hyperkeratosis and fibrosis of allergic skin inflammation. Strategies that block eosinophil infiltration, cys-LT production, or the CysLT(2)R might be useful in the treatment of AD.

Background: Nitric oxide (NO) has various roles in airway physiology and pathophysiology. Monitoring exhaled NO levels is increasingly common to measure airways inflammation and inhaled NO studied for its therapeutic value in premature infants and adult respiratory distress syndrome. NO is produced by 3 isoforms of NO synthase (NOS1, 2, 3), and each can play distinct and perhaps overlapping roles in the airways. However, the distribution, regulation, and functions of NOS in various cells in the upper airways, particularly in leukocytes, are incompletely understood., Objective: To characterize the expression of NOS isoforms in leukocytes in normal middle turbinate tissues (MT) and in inflammatory nasal tissue (nasal polyps, NP)., Methods: Normal MT tissue was collected from surgical specimens that were to be discarded. The NP samples were from surgical tissue archives of 15 patients with chronic rhinosinusitis. Isoforms of NOS in cells were identified by double immunostaining using NOS isoform-specific and leukocyte-specific (mast cell, eosinophil, macrophage, neutrophil, or T cell) antibodies., Results: The proportion of total cells below the epithelium that were positive for each isoform of NOS was higher in NP than in MT. Each isoform of NOS was found in all leukocyte populations studied, and there were significant differences in the percentage of leukocytes expressing NOS isoforms between MT and NP., Conclusion: All isoforms of NOS are expressed in leukocytes in MT and NP, and their expression varies among leukocyte types. Our data provide a basis to investigate the regulation, cell distribution, and distinct functions of NOS isoforms in normal and inflamed nasal tissues., (Copyright © 2012 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Aspirin-exacerbated respiratory disease (AERD) is a clinical syndrome associated with chronic inflammation in the airways coincident with chronic rhinitis, sinusitis, recurrent polyposis and asthma. Eosinophils are the key inflammatory cells in the development of AERD. AERD has been attributed to abnormalities of the arachidonic acid metabolism, but the pathogenesis of AERD is not fully understood. Our aim was to investigate the genetic contribution of the arachidonate 15-lipoxygenase gene (ALOX15) to the development of AERD., Methods: We enrolled 171 patients with AERD, 229 patients with aspirin-tolerant asthma, and 195 normal healthy controls in a Korean population. Three polymorphisms (-427G/A, -272C/A, -217G/C) in the promoter region of ALOX15 were genotyped. The functional variability of the promoter polymorphisms were analyzed by luciferase reporter activity assay., Result: No significant difference in the genotype frequency of the ALOX15 genetic polymorphism was found. Peripheral total eosinophil count was significantly higher in the patients carrying the GG genotype of the -427G/A polymorphism (p = 0.016). Similarly, the patients carrying haplotype 1 (ht1) (GCG) of -427G/A, -272C/A and -217G/C showed a significantly higher total eosinophil count compared to the other haplotypes (p = 0.008) in the AERD group. The promoter activity of the ht1 (GCG) construct was significantly higher compared to that of the ht3 (AGG) construct in A549 and U937 cells (both p < 0.001)., Conclusion: These results suggest that the promoter polymorphisms of the ALOX15 gene affect ALOX15 activity leading to increased eosinophil infiltration in AERD patients., (Copyright © 2012 S. Karger AG, Basel.)

Eosinophils play important roles in regulation of cellular responses under conditions of homeostasis or infection. Intestinal infection with the parasitic nematode, Trichinella spiralis, induces a pronounced eosinophilia that coincides with establishment of larval stages in skeletal muscle. We have shown previously that in mouse strains in which the eosinophil lineage is ablated, large numbers of T. spiralis larvae are killed by NO, implicating the eosinophil as an immune regulator. In this report, we show that parasite death in eosinophil-ablated mice correlates with reduced recruitment of IL-4(+) T cells and enhanced recruitment of inducible NO synthase (iNOS)-producing neutrophils to infected muscle, as well as increased iNOS in local F4/80(+)CD11b(+)Ly6C(+) macrophages. Actively growing T. spiralis larvae were susceptible to killing by NO in vitro, whereas mature larvae were highly resistant. Growth of larvae was impaired in eosinophil-ablated mice, potentially extending the period of susceptibility to the effects of NO and enhancing parasite clearance. Transfer of eosinophils into eosinophil-ablated ΔdblGATA mice restored larval growth and survival. Regulation of immunity was not dependent upon eosinophil peroxidase or major basic protein 1 and did not correlate with activity of the IDO pathway. Our results suggest that eosinophils support parasite growth and survival by promoting accumulation of Th2 cells and preventing induction of iNOS in macrophages and neutrophils. These findings begin to define the cellular interactions that occur at an extraintestinal site of nematode infection in which the eosinophil functions as a pivotal regulator of immunity.