Ochkur, Sergei I.

Abstract Eosinophils play an important role in mediating itch and inflammation in dermatitis. The role of the eosinophil granule protein eosinophil peroxidase (EPX) in mediating inflammation and itch was tested in a dermatitis mouse model. Mice were sensitized to trimellitic anhydride (TMA) and subsequently challenged chronically on the ear to establish dermatitis. Loss of EPX (in EPX−/− mice) or blocking EPX with the drug resorcinol significantly reduced dermatitis in mice exposed to TMA. Resorcinol also reduced levels of thymic stromal lymphopoietin protein (TSLP) in skin. Further studies showed that EPX increased different cytokines in keratinocytes in cell culture via 2 distinct mechanisms. EPX-induced TSLP expression requires lysophosphatidic acid signaling while EPX-induced expression of TNF-α, CSF2, CSF3, and IL1a required IL-1 signaling. We also showed that blocking IL-1 reduced inflammation in skin following TMA exposure in mice. Thus, EPX is an important mediator of inflammation and itch, that are mediated via at least 2 pathways. This suggests that both EPX and its signaling pathways may provide novel therapeutic strategies in dermatitis.

Background: Pollen is a key source of aeroallergens responsible for allergic rhinitis, conjunctivitis, and asthma. Objective: The goal of this scoping review was to summarize current available literature on the factors that affect pollen counts, allergenicity, and thresholds that induce symptoms in individuals who were sensitized. Methods: Several databases showed no published articles with a similar scope as of January 2022. A search of these data bases yielded 373 articles for assessment. These were then reviewed for relevance, and articles were selected to demonstrate the breadth of available data on pollen counts, allergenicity, and thresholds that induce symptoms in individuals who were sensitized. Additional articles were identified through examination of bibliographies of search-identified articles. Results: Several environmental factors have shown a correlation with pollen counts and allergen load, including the distance from the source, wind characteristics, pollen size, terrain, urban environments, air composition (particulate matter, CO2 levels, ozone, NO2), and weather conditions (humidity, thunderstorms, precipitation). Pollen thresholds at which symptoms were induced varied by study, pollen type, symptom, disease, and location. In addition, there was heterogeneity in study designs, threshold definition, and outcome measures. Conclusion: This scoping review demonstrates the plethora of variables that influence the relationship between pollen and the symptoms of allergic diseases. Analysis of the available data sheds light on the complex interaction between environmental and biologic factors that affect pollen's role in allergic diseases and provides guidance on multiple areas for further investigation.

Objective To evaluate eosinophil peroxidase (EPX) as a biomarker for tissue levels of eosinophilia, cytokines, and chemokines within chronic rhinosinusitis (CRS). Methods Twenty‐eight subjects undergoing sinonasal surgery were prospectively enrolled. Ethmoid tissue was analyzed with an in‐house EPX immunoassay and a 48‐plex cytokine‐chemokine array. Clinical severity was assessed using SNOT‐22 and Lund‐Mackay scores. Subjects were grouped as follows: controls, polyp status (CRS with [CRSwNP] and without nasal polyps [CRSsNP]), tissue eosinophilia (eosinophilic CRS [eCRS], non‐eosinophilic CRS [neCRS]), or combinations thereof (eCRSwNP, eCRSsNP, neCRSsNP). eCRS was defined as >10 eosinophils per high power field (HPF). Subjects without CRS or asthma were enrolled as controls. Results EPX was elevated in CRSwNP compared to control ( p = 0.007), in eCRS compared to neCRS ( p = 0.002), and in eCRSwNP along with eCRSsNP compared to neCRSsNP ( p = 0.023, p = 0.015, respectively). eCRS displayed elevated IL‐5 compared to neCRS ( p = 0.005). No significant differences in EPX or IL‐5 were observed between eCRSwNP and eCRSsNP. IL‐5 was elevated in eCRSwNP ( p = 0.019) compared neCRSsNP. Area under the receiver operator characteristic curve was 0.938 (95% CI, 0.835–1.00) for EPX and tissue eosinophilia, with an optimal cut‐point of 470 ng/mL being 100% specific and 81.25% sensitive for tissue eosinophilia. Linear regression revealed a strong correlation between EPX and IL‐5 (R 2 = 0.64, p < 0.001). Comparing EPX and IL‐5, only EPX displayed significant correlation with SNOT‐22 ( p = 0.04) and Lund‐Mackay score ( p = 0.004). Conclusion EPX is associated with tissue eosinophilia in CRS patients regardless of polyp status. EPX correlates with IL‐5 and could be potentially considered a biomarker for anti‐IL‐5 therapies. Level of Evidence 3 Laryngoscope , 134:69–78, 2024

Abstract Eosinophils comprise only 1-3% of circulating leukocytes but are often a diagnostic feature associated with a variety of inflammatory and disease states, including parasitic and fungal infections, allergic diseases (e.g., asthma, rhinitis and sensitivities to specific foods), cancer, and transplant rejection. The accurate evaluations of eosinophilia as well as the release of stored eosinophil granule proteins are important for the monitoring disease progression and/or assessing the effectiveness of a given treatment strategy(ies). Despite the availability of detection methods for each of the eosinophil granule proteins logistical difficulties limiting either the assays specificity and/or sensitivity have prevented their extensive use. We have generated unique eosinophil peroxidase specific monoclonal antibodies (EPX-mAb) and a high throughput sandwich ELISA assay as a means of overcoming these difficulties. Our EPX-mAb based ELISA is ~10 times more sensitive than traditional OPD based activity assays and allows detection of EPX in mouse BAL fluid after an acute OVA protocol as well as EPX release from mouse eosinophils stimulated ex vivo. The assay is highly specific and gives no signal in samples from EPX deficient mice even with massive eosinophilia (e.g., EPX deficient IL-5 transgenic mice). More significantly, this assay detects EPX in human tissue extracts and biological fluids and thus represents novel diagnostic assay previously unavailable in clinical settings.

Abstract Eosinophil infiltration into tumors has been associated with positive and negative growth human cancers and in mouse models of tumorigenesis. Our previous studies have shown that eosinophils infiltrate tumors and differentially accumulate in the necrotic and capsule regions of tumors. Using various eosinophil-deficient mouse models, we demonstrate that in the absence of eosinophils tumor growth is attenuated. Methods: Tumor growth kinetics were assessed using a B16F10 melanoma subcutaneous cell injection model. Eosinophil chemotaxis was assessed using an ex vivo transwell insert assay system. We compared tumor growth in wild type animals vs. various eosinophil-deficient mouse models that are transgenic line of mice (PHIL) and MBP-/-/EPO-/- double knockout mice that are deficient in eosinophil-specific proteins (major basic protein (MBP) and eosinophil peroxide (EPO)) resulting in a blockade of eosinophilpoiesis. Conclusions: The migration of eosinophils was inhibited through blocking the sphingosine-1-phosphate pathway, suggesting recruitment and accumulation is primarily an inflammatory response. Tumor growth was decreased by ~40-60% in PHIL and in MBP-/-/EPO-/- double knockout mice relative to tumors in wild type animals. In addition, EPO-/- mice, and not MBP-/-mice, replicate this ~40% reduction in tumor size implicating a significant role for EPO in tumor growth kinetics. These data suggest eosinophils respond to inflammatory mediators and mediate augment tumor growth.

Abstract Products of the 5-lipoxygenase (5-LO) pathway (i.e., cys-leukotrienes and LTB4), are contributors to lung inflammation, airway remodeling, and hyperresponsiveness associated with allergic respiratory inflammation. Eosinophils are capable of leukotriene synthesis/secretion and chemotactically respond to these compounds. However, the in vivo role of eosinophils in leukotriene mediated events during asthmatic airway inflammation is not defined. To study the link between eosinophils and leukotrienes we used the I5/E2 double transgenic mouse model of severe asthma, over-expressing IL-5 systemically and eotaxin-2 in airways, in which lung pathologies are entirely dependent on the presence of eosinophils. We show that airway levels of cysteinyl leukotrienes and LTB4 are significantly elevated in I5/E2 mice. Crosses of I5E2 mice with 5-LO knockout animals abolished AHR in the I5/E2 parental model without significant effects on lung histopathology. Administration of a cys-leukotriene receptor antagonist (Montelukast®) to I5/E2 mice replicates the effects of the total leukotriene loss in I5/E2/5-LO-/- animals. In contrast, I5/E2 mice crossed with BLT-1-/- mice which are deficient of the LTB4 receptor fail to display effects on the pulmonary pathologies occurring in the I5/E2 parental model. These data demonstrate a unique role for cys-leukotrienes in the development of AHR independent of lung remodeling events occurring in this transgenic model of severe asthma.

Abstract Atopic dermatitis and allergic contact dermatitis are two of the most common skin diseases. In both diseases, eosinophils have been suggested as potential contributors of the pathologies linked with these skin conditions. Our goal was to establish a causative relationship between the inflammatory response of chemically induced contact hypersensitivity and eosinophil-mediated activities. Wild type and eosinophil-less PHIL mice were sensitized and challenged with 2,4-dinitrochlorobenzene (DNFB) or alternatively trimellitic anhydride (TMA). Time lapsed videography was used to capture the number of itching events associated with treatment. Immunohistochemistry for eosinophils and nerves was performed to correlate the effect of dermatitis on innervation and eosinophil accumulation in these mice. Our studies showed that in both the DNFB and TMA models a robust eosinophil infiltrate occurred that was accompanied by copious levels of eosinophil degranulation. More importantly, these studies demonstrated that metrics of inflammation (e.g., induced ear swelling (i.e., thickness)) was reduced in PHIL mice relative to wild type. Our observations were also directly correlative with a loss of nerve growth and branching in PHIL mice. These data implicate eosinophils as contributors to the pathologies linked with chemically-induced contact hypersensitivity, including the induced itch responses associated with these models.

Abstract Eosinophil effector functions have been hypothesized to be mediated in part by the release of proteins stored in the secondary granules of mature cells of this leukocyte lineage. Major basic protein-1 (MBP-1) and eosinophil peroxidase (EPO) comprise the majority of protein in these granules. No role has been speculated for these proteins in eosinophilopoiesis and/or the accumulation of mature cells in circulation. Methods: Eosinophilopoiesis and eosinophil accumulation in the periphery were assessed in single and double knockout (MBP-1-/- and/or EPO-/-) mice. Bone marrow and peripheral blood leukocytes were assessed by IHC, FACS and in vitro cell culture assays. Mice were subjected to an acute OVA protocol assessing induced pulmonary pathologies in each of the strains of granule protein knockout mice. Results: We demonstrate that, unlike the single deficiency of MBP-1 or EPO, the absence of both granule proteins resulted in the loss of peripheral blood eosinophils. IHC assessments of bone marrow and spleen demonstrate that eosinophil lineage commitment occurs in these mice. However, these assessments and FACS studies demonstrated a blockade in the maturation of eosinophil-lineage committed cells. This blockade is not rescued by ex vivo culture or by bone marrow engraftment into wild type recipient mice. Similar to other eosinophil-less mouse models, MBP-1-/-/EPO-/- mice also fail to develop pulmonary inflammation in an OVA protocol.

OBJECTIVES: Eosinophilic esophagitis (EoE), a chronic food allergic disease, lacks sensitive and specific peripheral biomarkers. We hypothesized that levels of EoE-related biomarkers captured using a 1-hour minimally invasive Esophageal String Test (EST) would correlate with mucosal eosinophil counts and tissue concentrations of these same biomarkers. We aimed to determine whether a 1-hour EST accurately distinguishes active from inactive EoE or a normal esophagus. METHODS: In a prospective, multisite study, children and adults (ages 7–55 years) undergoing a clinically indicated esophagogastroduodenoscopy performed an EST with an esophageal dwell time of 1 hour. Subjects were divided into 3 groups: active EoE, inactive EoE, and normal esophageal mucosa. Eosinophil-associated protein levels were compared between EST effluents and esophageal biopsy extracts. Statistical modeling was performed to select biomarkers that best correlated with and predicted eosinophilic inflammation. RESULTS: One hundred thirty-four subjects (74 children, 60 adults) with active EoE (n = 62), inactive EoE (n = 37), and patient controls with a normal esophagus (n = 35) completed the study. EST-captured eosinophil-associated biomarkers correlated significantly with peak eosinophils/high-power field, endoscopic visual scoring, and the same proteins extracted from mucosal biopsies. Statistical modeling, using combined eotaxin-3 and major basic protein-1 concentrations, led to the development of EoE scores that distinguished subjects with active EoE from inactive EoE or normal esophagi. Eighty-seven percent of children, 95% of parents, and 92% of adults preferred the EST over endoscopy if it provided similar information. DISCUSSION: The 1-hour EST accurately distinguishes active from inactive EoE in children and adults and may facilitate monitoring of disease activity in a safe and minimally invasive fashion.

SUMMARYEosinophil peroxidase is an eosinophil-specific, cytoplasmic protein stored in the secondary granules of eosinophils. While eosinophil peroxidase deposition is increased in the esophagus in eosinophilic esophagitis (EOE), its potential role as a peripheral marker is unknown. This study aims to examine the relationship between serum eosinophil peroxidase and esophageal eosinophilia in eosinophilic esophagitis. Prospectively collected serum from 19 subjects with incident EoE prior to treatment and 20 non-EoE controls were tested for serum eosinophil peroxidase, eosinophilic cationic protein, and eosinophil derived neurotoxin using ELISA. Matching esophageal tissue sections were stained and assessed for eosinophil peroxidase deposition using a histopathologic scoring algorithm. Mean peripheral blood absolute eosinophil counts in eosinophilic esophagitis subjects were significantly elevated compared to controls (363 vs. 195 cells/μL,P = 0.008). Absolute median serum eosinophil peroxidase, eosinophil cationic protein, and eosinophil derived neurotoxin did not differ between groups; however, when normalized for absolute eosinophil counts, eosinophilic esophagitis subjects had significantly lower median eosinophil peroxidase levels (2.56 vs. 6.96 ng/mL per eos/μL,P = 0.002, AUC 0.79 (0.64, 0.94 95% CI)). Multivariate analysis demonstrated this relationship persisted after controlling for atopy. Esophageal biopsies from eosinophilic esophagitis subjects demonstrated marked eosinophil peroxidase deposition (median score 46 vs. 0,P &lt; 0.0001). Normalized eosinophil peroxidase levels inversely correlated with esophageal eosinophil density (r = −0.41,P = 0.009). In contrast to marked tissue eosinophil degranulation, circulating eosinophils appear to retain their granule proteins in EoE. Investigations of normalized serum eosinophil peroxidase levels as a biomarker of EoE are ongoing.