Breakthroughs in understanding and treating eosinophilic gastrointestinal diseases presented at the CEGIR/TIGERs Symposium at the 2022 American Academy of Allergy, Asthma & Immunology Meeting.

The Consortium of Eosinophilic Gastrointestinal Diseases and The International Gastrointestinal Eosinophil Researchers organized a day-long symposium at the 2022 Annual Meeting of the American Academy of Allergy, Asthma & Immunology. The symposium featured a review of recent discoveries in the basic biology and pathogenesis of eosinophilic gastrointestinal diseases (EGIDs) in addition to advances in our understanding of the clinical features of EGIDs. Diagnostic and management approaches were reviewed and debated, and clinical trials of emerging therapies were highlighted. Herein, we briefly summarize the breakthrough discoveries in EGIDs.

Developing a standardized approach for assessing mast cells and eosinophils on tissue biopsies: A Work Group Report of the AAAAI Allergic Skin Diseases Committee.

Mast cells and eosinophils are commonly found, expectedly or unexpectedly, in human tissue biopsies. Although the clinical significance of their presence, absence, quantity, and quality continues to be investigated in homeostasis and disease, there are currently gaps in knowledge related to what constitutes quantitatively relevant increases in mast cell and eosinophil number in tissue specimens for several clinical conditions. Diagnostically relevant thresholds of mast cell and eosinophil numbers have been proposed and generally accepted by the medical community for a few conditions, such as systemic mastocytosis and eosinophilic esophagitis. However, for other mast cell- and eosinophil-associated disorders, broad discrepancies remain regarding diagnostic thresholds and how samples are processed, routinely and/or specially stained, and interpreted and/or reported by pathologists. These discrepancies can obfuscate or delay a patient's correct diagnosis. Therefore, a work group was assembled to review the literature and develop a standardized consensus for assessing the presence of mast cells and eosinophils for a spectrum of clinical conditions, including systemic mastocytosis and cutaneous mastocytosis, mast cell activation syndrome, eosinophilic esophagitis, eosinophilic gastritis/enteritis, and hypereosinophilia/hypereosinophilic syndrome. The intent of this work group is to build a consensus among pathology, allergy, dermatology, hematology/oncology, and gastroenterology stakeholders for qualitatively and quantitatively assessing mast cells and eosinophils in skin, gastrointestinal, and bone marrow pathologic specimens for the benefit of clinical practice and patients.

Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 in patients with eosinophilic disorders: Receptor expression and targeting using chimeric antibodies.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 is selectively expressed on eosinophils, mast cells, and basophils and, when engaged on eosinophils, can cause cell death. OBJECTIVE: We sought to characterize surface and soluble Siglec-8 (sSiglec-8) levels in normal donors (NDs) and eosinophilic donors (EOs) and assess the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. METHODS: Eosinophil expression of Siglec-8 was assessed by using flow cytometry and quantitative PCR. Serum sSiglec-8 levels were measured by means of ELISA. Induction of eosinophil death by IgG4 (chimeric 2E2 IgG4) and afucosylated IgG1 (chimeric 2E2 IgG1 [c2E2 IgG1]) anti-Siglec-8 antibodies was evaluated in vitro by using flow cytometry and in vivo in humanized mice. RESULTS: Siglec-8 was consistently expressed on eosinophils from NDs and EOs and did not correlate with absolute eosinophil count or disease activity. sSiglec-8 levels were measurable in sera from most donors unrelated to absolute eosinophil counts or Siglec-8 surface expression. c2E2 IgG1 and chimeric 2E2 IgG4 were equally effective at inducing cell death (Annexin-V positivity) of purified eosinophils from NDs and EOs after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EOs, and natural killer cell-mediated eosinophil killing was seen only with c2E2 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. CONCLUSIONS: Siglec-8 is highly expressed on blood eosinophils from EOs and NDs and represents a potential therapeutic target for eosinophilic disorders. Enhanced killing of eosinophils in the presence of IL-5 might lead to increased efficacy in patients with IL-5-driven eosinophilia.

Heart disease in a mutant mouse model of spontaneous eosinophilic myocarditis maps to three loci.

BACKGROUND: Heart disease (HD) is the major cause of morbidity and mortality in patients with hypereosinophilic diseases. Due to a lack of adequate animal models, our understanding of the pathophysiology of eosinophil-mediated diseases with heart complications is limited. We have discovered a mouse mutant, now maintained on an A/J inbred background, that spontaneously develops hypereosinophilia in multiple organs. Cellular infiltration into the heart causes an eosinophilic myocarditis, with affected mice of the mutant line (i.e., A/JHD) demonstrating extensive myocardial damage and remodeling that leads to HD and premature death, usually by 15-weeks old. RESULTS: Maintaining the A/JHD line for many generations established that the HD trait was heritable and implied the mode of inheritance was not too complex. Backcross and intercross populations generated from mating A/JHD males with females from four different inbred strains produced recombinant populations with highly variable rates of affected offspring, ranging from none in C57BL/6 J intercrosses, to a few mice with HD using 129S1/SvImJ intercrosses and C57BL/6 J backcrosses, but nearly 8% of intercrosses and > 17% of backcrosses from SJL/J related populations developed HD. Linkage analyses of these SJL/J derived recombinants identified three highly significant loci: a recessive locus mapping to distal chromosome 5 (LOD = 4.88; named Emhd1 for eosinophilic myocarditis to heart disease-1); and two dominant variants mapping to chromosome 17, one (Emhd2; LOD = 7.51) proximal to the major histocompatibility complex, and a second (Emhd3; LOD = 6.89) that includes the major histocompatibility region. Haplotype analysis identified the specific crossovers that defined the Emhd1 (2.65 Mb), Emhd2 (8.46 Mb) and Emhd3 (14.59 Mb) intervals. CONCLUSIONS: These results indicate the HD trait in this mutant mouse model of eosinophilic myocarditis is oligogenic with variable penetrance, due to multiple segregating variants and possibly additional genetic or nongenetic factors. The A/JHD mouse model represents a unique and valuable resource to understand the interplay of causal factors that underlie the pathology of this newly discovered eosinophil-associated disease with cardiac complications.

Characterization of a mouse model of hypereosinophilia-associated heart disease.

Hypereosinophilic syndrome is characterized by sustained and marked eosinophilia leading to tissue damage and organ dysfunction. Morbidity and mortality occur primarily due to cardiac and thromboembolic complications. Understanding the cause and mechanism of disease would aid in the development of targeted therapies with greater efficacy and fewer side effects. We discovered a spontaneous mouse mutant in our colony with a hypereosinophilic phenotype. Mice develop peripheral blood eosinophilia; infiltration of lungs, spleen, and heart by eosinophils; and extensive myocardial damage and remodeling. This ultimately leads to heart failure and premature death. Histopathological assessment of the hearts revealed a robust inflammatory infiltrate composed primarily of eosinophils and B-lymphocytes, associated with myocardial damage and replacement fibrosis, consistent with eosinophilic myocarditis. In many cases, hearts showed dilatation and thinning of the right ventricular wall, suggestive of an inflammatory dilated cardiomyopathy. Most mice showed atrial thrombi, which often filled the chamber. Protein expression analysis revealed overexpression of chemokines and cytokines involved in innate and adaptive immunity including IL-4, eotaxin, and RANTES. Disease could be transferred to wild-type mice by adoptive transfer of splenocytes from affected mice, suggesting a role for the immune system. In summary, the pathologies observed in the mutant lines are reminiscent of those seen in patients with hypereosinophilia, where cardiac-related morbidities, like congestive heart failure and thrombi, are the most common causes of death. As such, our model provides an opportunity to test mechanistic hypotheses and develop targeted therapies.NEW & NOTEWORTHY This article describes a new model of heart disease in hypereosinophilia. The model developed as a spontaneous mouse mutant in the colony and is characterized by peripheral blood eosinophilia and infiltration of lungs, spleen, and heart by eosinophils. In the heart, there is extensive myocardial damage, remodeling, fibrosis, and thrombosis, leading to heart failure and death. The immune microenvironment is one of increased innate and adaptive immunity, including Th1 and Th2 cytokines/chemokines. Finally, adoptive transfer of splenocytes transfers disease to recipient mice. In summary, this model provides an opportunity to test mechanistic hypotheses and develop targeted therapies for this rare but devastating disease.

Hypereosinophilic syndrome in the differential diagnosis of pulmonary infiltrates with eosinophilia.

OBJECTIVE: To describe key diagnostic considerations in patients with pulmonary infiltrates with eosinophilia, with a special emphasis on raising awareness of hypereosinophilic syndrome (HES), a disease that often involves the lungs and prompts investigation for clonal neoplastic processes that determine prognosis and treatment. DATA SOURCES: Studies and review articles were selected from PubMed and Scopus for relevance to pertinent topics. STUDY SELECTIONS: The literature was screened for studies that described lung eosinophilia and HES. Studies relevant to the topic were included in this review. RESULTS: Pulmonary eosinophil infiltrates in lung biopsy specimens present a broad differential diagnosis, including eosinophilic pneumonia; hypersensitivity reactions, such as allergic bronchopulmonary fungal disease; and pulmonary manifestations of systemic diseases, such as eosinophilic granulomatosis with polyangiitis. An additional important consideration in the differential diagnosis is pulmonary involvement by HES. HES is a rare syndrome that comprises a heterogeneous group of conditions characterized by persistent blood and/or tissue eosinophilia associated with organ dysfunction. Approximately one-third of HES cases are caused by neoplastic diseases, with the remaining cases classified as reactive or idiopathic. Lung involvement is seen in up to 67% of cases and may be the presenting manifestation of the disorder. CONCLUSION: The differential diagnosis of pulmonary eosinophilia is broad and requires a multidisciplinary approach with clinicopathologic-radiologic correlation.

Cyclophilin D regulates necrosis, but not apoptosis, of murine eosinophils.

Eosinophil degranulation and clusters of free extracellular granules are frequently observed in diverse diseases, including atopic dermatitis, nasal polyposis, and eosinophilic esophagitis. Whether these intact granules are released by necrosis or a biochemically mediated cytolysis remains unknown. Recently, a peptidyl-prolyl isomerase located within the mitochondrial matrix, cyclophilin D (PPIF), was shown to regulate necrotic, but not apoptotic, cell death in vitro in fibroblasts, hepatocytes, and cardiomyocytes. Whether cyclophilin D regulates necrosis in hematopoietic cells such as eosinophils remains unknown. We used PPIF-deficient (Ppif(-/-)) mice to test whether cyclophilin D is required for regulating eosinophil necrosis. PPIF deficiency did not affect eosinophil development or maturation at baseline. After in vitro ionomycin or H2O2 treatment, Ppif(-/-) eosinophils were significantly protected from Ca(2+) overload- or oxidative stress-induced necrosis. Additionally, Ppif(-/-) eosinophils demonstrated significantly decreased necrosis, but not apoptosis, in response to Siglec-F cross-linking, a stimulus associated with eosinophil-mediated processes in vitro and in vivo. When treated with apoptosis inducers, Ppif(+/+) and Ppif(-/-) eosinophils exhibited no significant difference in apoptosis or secondary necrosis. Finally, in a dextran sodium sulfate-induced colitis model, although levels of colitogenic cytokines and eosinophil-selective chemokines were comparable between Ppif(+/+) and Ppif(-/-) mice, the latter exhibited decreased clinical outcomes. This correlated with significantly reduced eosinophil cytolysis in the colon. Collectively, our present studies demonstrate that murine eosinophil necrosis is regulated in vitro and in vivo by cyclophilin D, at least in part, thus providing new insight into the mechanism of eosinophil necrosis and release of free extracellular granules in eosinophil-associated diseases.

Carbonic anhydrase IV is expressed on IL-5-activated murine eosinophils.

Eosinophilia and its cellular activation are hallmark features of asthma, as well as other allergic/Th2 disorders, yet there are few, if any, reliable surface markers of eosinophil activation. We have used a FACS-based genome-wide screening system to identify transcriptional alterations in murine lung eosinophils recruited and activated by pulmonary allergen exposure. Using a relatively stringent screen with false-positive correction, we identified 82 candidate genes that could serve as eosinophil activation markers and/or pathogenic effector markers in asthma. Carbonic anhydrase IV (Car4) was a top dysregulated gene with 36-fold induction in allergen-elicited pulmonary eosinophils, which was validated by quantitative PCR, immunohistochemistry, and flow cytometry. Eosinophil CAR4 expression was kinetically regulated by IL-5, but not IL-13. IL-5 was both necessary and sufficient for induction of eosinophil CAR4. Although CAR4-deficient mice did not have a defect in eosinophil recruitment to the lung, nor a change in eosinophil pH-buffering capacity, allergen-challenged chimeric mice that contained Car4(-/-) hematopoietic cells aberrantly expressed a series of genes enriched in biological processes involved in epithelial differentiation, keratinization, and anion exchange. In conclusion, we have determined that eosinophils express CAR4 following IL-5 or allergen exposure, and that CAR4 is involved in regulating the lung transcriptome associated with allergic airway inflammation; therefore, CAR4 has potential value for diagnosing and monitoring eosinophilic responses.

Role of siglecs and related glycan-binding proteins in immune responses and immunoregulation.

Virtually all cells and extracellular material are heavily decorated by various glycans, yet our understanding of the structure and function of these moieties lags behind the understanding of nucleic acids, lipids, and proteins. Recent years have seen a tremendous acceleration of knowledge in the field of glycobiology, revealing many intricacies and functional contributions that were previously poorly appreciated or even unrecognized. This review highlights several topics relevant to glycoimmunology in which mammalian and pathogen-derived glycans displayed on glycoproteins and other scaffolds are recognized by specific glycan-binding proteins (GBPs), leading to a variety of proinflammatory and anti-inflammatory cellular responses. The focus for this review is mainly on 2 families of GBPs, sialic acid-binding immunoglobulin-like lectins (siglecs) and selectins, that are involved in multiple steps of the immune response, including distinguishing pathogens from self, cell trafficking to sites of inflammation, fine-tuning of immune responses leading to activation or tolerance, and regulation of cell survival. Importantly for the clinician, accelerated rates of discovery in the field of glycoimmunology are being translated into innovative medical approaches that harness the interaction of glycans and GBPs to the benefit of the host and might soon lead to novel diagnostics and therapeutics.

Differential eosinophil and mast cell regulation: mast cell viability and accumulation in inflammatory tissue are independent of proton-sensing receptor GPR65.

Extracellular acidification has been observed in allergic inflammatory diseases. Recently, we demonstrated that the proton-sensing receptor G protein-coupled receptor 65 (GPR65) regulates eosinophil survival in an acidic environment in vitro and eosinophil accumulation in an allergic lung inflammation model. For mast cells, another inflammatory cell type critical for allergic responses, it remains unknown whether GPR65 is expressed and/or regulates mast cell viability. Thus, in the present study, we employed in vitro experiments and an intestinal anaphylaxis model in which both mastocytosis and eosinophilia can be observed, particularly in the gastrointestinal tract, to enable us to directly compare the effect of GPR65 expression on these two cell types. We identified GPR65 expression on mast cells; however, unlike eosinophil viability, mast cell viability in vitro is not affected by acidification or GPR65 expression. Mechanistically, we determined that mast cells do not respond to extracellular acidification with increased cAMP levels. Furthermore, in the intestinal anaphylaxis model, we observed a significant reduction of eosinophils (59.1 ± 9.2% decrease) in the jejunum of allergen-challenged GPR65-deficient mice compared with allergen-challenged wild-type mice, despite the degree of antigen sensitization and the expression levels of Th2 cytokines (Il4, Il13) and eosinophil chemokines (Ccl11, Ccl24) in the jejunum being comparable. In contrast, the accumulation of mast cells in allergen-challenged mice was not affected by GPR65 deficiency. In conclusion, our study demonstrates differential regulation of eosinophils and mast cells in inflammatory tissue, with mast cell viability and accumulation being independent of GPR65.

Mechanism of Siglec-8-mediated cell death in IL-5-activated eosinophils: role for reactive oxygen species-enhanced MEK/ERK activation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is expressed on human eosinophils, where its ligation induces cell death. Paradoxically, Siglec-8-mediated cell death is markedly enhanced by the presence of the activation and survival factor IL-5 and becomes independent of caspase activity. OBJECTIVE: In this report we investigate the mechanism of Siglec-8-mediated cell death in activated eosinophils. METHODS: Human peripheral blood eosinophils were treated with agonistic anti-Siglec-8 antibody and IL-5, and cell death was determined by using flow cytometry and morphology. Phosphorylation of mitogen-activated protein kinase (MAPK) was determined by using phosphoLuminex, flow cytometry, and Western blotting. Reactive oxygen species (ROS) accumulation was determined by using dihydrorhodamine fluorescence. RESULTS: Costimulation with anti-Siglec-8 and IL-5 significantly increased the rate and proportion of cell death by means of necrosis accompanied by granule release compared with that seen after stimulation with anti-Siglec-8 alone, in which apoptosis predominated. Together with the caspase-independent mode of cell death in costimulated cells, these findings suggest the activation of a specific and distinct biochemical pathway of cell death during anti-Siglec-8/IL-5 costimulation. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and MAPK-ERK kinase (MEK) 1 was significantly enhanced and sustained in costimulated cells compared with that seen in cells stimulated with IL-5 alone; anti-Siglec-8 alone did not cause ERK1/2 phosphorylation. MEK1 inhibitors blocked anti-Siglec-8/IL-5-induced cell death. ROS accumulation was induced by Siglec-8 ligation in a MEK-independent manner. In contrast, an ROS inhibitor prevented the anti-Siglec-8/IL-5-induced enhancement of ERK phosphorylation and cell death. Exogenous ROS mimicked stimulation by anti-Siglec-8 and was sufficient to induce enhanced cell death in IL-5-treated cells. Collectively, these data suggest that the enhancement of ERK phosphorylation is downstream of ROS generation. CONCLUSIONS: In activated eosinophils ligation of Siglec-8 leads to ROS-dependent enhancement of IL-5-induced ERK phosphorylation, which results in a novel mode of biochemically regulated eosinophil cell death.

Standardized Quantification of Mast Cells in the Gastrointestinal Tract in Adults.

BACKGROUND: Current data on the normal quantity of mast cells throughout the adult gastrointestinal tract are limited in several domains. These include microanatomic localization of mast cells, standardization of staining and counting methods, and reporting of microscope field of view. OBJECTIVE: To address this lack of reliable reference ranges to facilitate the study of and diagnosis of emerging mast cell-mediated diseases. METHODS: We examined biopsies obtained from the esophagus, stomach, duodenum, and colon from an unselected cohort. Mean and peak mast cell density were determined on slides stained for tryptase and CD117, and were expressed per high power field (hpf) and surface area (mm2), thus deriving reference ranges (average ± 2 SDs). RESULTS: For the most common hpf surface area (0.238 mm2), upper limits of the derived reference ranges for average/peak mast cells were 0.15/3.67 (esophagus, tryptase), 0.70/5.98 (esophagus, CD117), 22.56/35.30 (stomach, tryptase), 31.32/53.10 (stomach, CD117), 30.28/49.77 (duodenal crypts, tryptase), 41.96/65.26 (duodenal crypts, CD117), 4.98/11.56 (duodenal villi, tryptase), 8.38/14.17 (duodenal villi, CD117), 26.58/41.08 (colon, tryptase), and 35.57/57.92 (colon, CD117). Interobserver variability was moderate to good. There was significant correlation between average and peak mast cell counts. CONCLUSIONS: These data help standardize mast cell reference ranges throughout the gastrointestinal tract in adults, which can be used to determine whether abnormal levels of mast cells are present in patients with suspected mast cell-mediated disease. Our data show that the commonly used cutoff of 20 mast cells per hpf irrespective of the gastrointestinal tract segment is an underestimate of an appropriate cutoff in stomach, duodenum (crypt area), and colon.

Pathophysiology of hypereosinophilia-associated heart disease.

Background: Cardiac complications in patients with hypereosinophilia cause significant morbidity and mortality. However, mechanisms of how eosinophilic inflammation causes heart damage are poorly understood. Methods: We developed a model of hypereosinophilia-associated heart disease by challenging hypereosinophilic mice with peptide from the cardiac myosin heavy chain. Disease outcomes were measured by histology, immunohistochemistry, flow cytometry, and measurement of cells and biomarkers in peripheral blood. Eosinophil dependence was determined by using eosinophil-deficient mice (ΔdblGATA). Single cells from heart were subjected to single cell RNA sequencing to assess cell composition, subtypes and expression profiles. Results: Mice challenged with myocarditic and control peptide had peripheral blood leukocytosis, but only those challenged with myocarditic peptide had heart inflammation. Heart tissue was infiltrated by eosinophil-rich inflammatory infiltrates associated with cardiomyocyte damage. Disease penetrance and severity were dependent on the presence of eosinophils. Single cell RNA sequencing showed enrichment of myeloid cells, T-cells and granulocytes (neutrophils and eosinophils) in the myocarditic mice. Macrophages were M2 skewed, and eosinophils had an activated phenotype. Gene enrichment analysis identified several pathways potentially involved in pathophysiology of disease. Conclusion: Eosinophils are required for heart damage in hypereosinophilia-associated heart disease. Additionally, myeloid cells, granulocytes and T-cell cooperatively or independently participate in the pathogenesis of hypereosinophilia-associated heart disease.

Biologic therapy in rare eosinophil-associated disorders: remaining questions and translational research opportunities.

Rare eosinophil-associated disorders (EADs), including hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, and eosinophilic gastrointestinal disorders, are a heterogeneous group of conditions characterized by blood and/or tissue hypereosinophilia and eosinophil-related clinical manifestations. Although the recent availability of biologic therapies that directly and indirectly target eosinophils has the potential to dramatically improve treatment options for all EADs, clinical trials addressing their safety and efficacy in rare EADs have been relatively few. Consequently, patient access to therapy is limited for many biologics, and the establishment of evidence-based treatment guidelines has been extremely difficult. In this regard, multicenter retrospective collaborative studies focusing on disease manifestations and treatment responses in rare EADs have provided invaluable data for physicians managing patients with these conditions and helped identify important questions for future translational research. During the Clinical Pre-Meeting Workshop held in association with the July 2023 biennial meeting of the International Eosinophil Society in Hamilton, Ontario, Canada, the successes and limitations of pivotal multicenter retrospective studies in EADs were summarized and unmet needs regarding the establishment of guidelines for use of biologics in rare EADs were discussed. Key topics of interest included (1) clinical outcome measures, (2) minimally invasive biomarkers of disease activity, (3) predictors of response to biologic agents, and (4) long-term safety of eosinophil depletion. Herein, we report a summary of these discussions, presenting a state-of-the-art overview of data currently available for each of these topics, the limitations of the data, and avenues for future data generation through implementation of multidisciplinary and multicenter studies.

PTPN11 Mutation Clonal Hierarchy in Acute Myeloid Leukemia.

Mutations in protein tyrosine phosphatase non-receptor type 11 ( PTPN11 ) have been considered late acquired mutations in acute myeloid leukemia (AML) development. To interrogate the ontogeny of PTPN11 mutations, we utilized single-cell DNA sequencing and identified that PTPN11 mutations can occur as initiating events in some AML patients when accompanied by strong oncogenic drivers, commonly NPM1 mutations. The co-driver role of PTPN11 mutations was confirmed in a novel murine model that exhibits an AML phenotype with early expansion of a diverse set of variably differentiated myeloid cells that engrafted into immunodeficient and immunocompetent mice. This immune diversity was reconstituted from early precursor cells when engrafted into immunodeficient mice. Moreover, immune diversity was also observed in the blast component of patient samples with NPM1 and PTPN11 mutations, providing novel antigen targets for immune based approaches in this subset of AML that is resistant to multiple targeted therapies.

Proton channel HVCN1 is required for effector functions of mouse eosinophils.

BACKGROUND: Proton currents are required for optimal respiratory burst in phagocytes. Recently, HVCN1 was identified as the molecule required for the voltage-gated proton channel activity associated with the respiratory burst in neutrophils. Although there are similarities between eosinophils and neutrophils regarding their mechanism for respiratory burst, the role of proton channels in eosinophil functions has not been fully understood. RESULTS: In the present study, we first identified the expression of the proton channel HVCN1 in mouse eosinophils. Furthermore, using HVCN1-deficient eosinophils, we demonstrated important cell-specific effector functions for HVCN1. Similar to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils produced significantly less reactive oxygen species (ROS) upon phorbol myristate acetate (PMA) stimulation compared with WT eosinophils. In contrast to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils did not show impaired calcium mobilization or migration ability compared with wild-type (WT) cells. Uniquely, HVCN1-deficient eosinophils underwent significantly increased cell death induced by PMA stimulation compared with WT eosinophils. The increased cell death was dependent on NADPH oxidase activation, and correlated with the failure of HVCN1-deficient cells to maintain membrane polarization and intracellular pH in the physiological range upon activation. CONCLUSIONS: Eosinophils require proton channel HVCN1 for optimal ROS generation and prevention of activation-induced cell death.

Chronic traffic pollution exposure is associated with eosinophilic, but not neutrophilic inflammation in older adult asthmatics.

OBJECTIVE: Airway inflammatory patterns in older asthmatics are poorly understood despite high asthma-related morbidity and mortality. In this study, we sought to define the relationship between exposure to traffic pollutants, biomarkers in induced sputum, and asthma control in older adults. METHODS: Induced sputum was collected from 35 non-smoking adults ≥65 years with a physician's diagnosis of asthma and reversibility with a bronchodilator or a positive methacholine challenge. Patients completed the Asthma Control Questionnaire (ACQ), and Elemental Carbon Attributable to Traffic (ECAT), a surrogate for chronic diesel particulate exposure, was determined. Equal numbers of subjects with high (≥0.39 µg/m(3)) versus low (<0.39 µg/m(3)) ECAT were included. Differential cell counts were performed on induced sputum, and myeloperoxidase (MPO) and eosinophil peroxidase (EPO) were measured in supernatants. Regression analyses were used to evaluate the relationship between sputum findings, ACQ scores, and ECAT. RESULTS: After adjustment for potential confounders, subjects with poorly controlled asthma based on ACQ ≥ 1.5 (n = 7) had significantly higher sputum eosinophils (median = 4.4%) than those with ACQ < 1.5 (n = 28; eosinophils = 2.6%; ß = 10.1 [95% CI = 0.1-21.0]; p = 0.05). Subjects with ACQ ≥ 1.5 also had significantly higher sputum neutrophils (84.2% versus 65.2%; ß = 7.1 [0.2-14.6]; p = 0.05). Poorly controlled asthma was associated with higher sputum EPO (ß = 2.4 [0.2-4.5], p = 0.04), but not MPO (p = 0.9). High ECAT was associated with higher eosinophils (ß = 10.1 [1.8-18.4], p = 0.02) but not higher neutrophils (p = 0.6). CONCLUSIONS: Poorly controlled asthma in older adults is associated with eosinophilic and neutrophilic inflammation. Chronic residential traffic pollution exposure may be associated with eosinophilic, but not neutrophilic inflammation in older asthmatics.

Arginase I suppresses IL-12/IL-23p40-driven intestinal inflammation during acute schistosomiasis.

Alternatively activated macrophages prevent lethal intestinal pathology caused by worm ova in mice infected with the human parasite Schistosoma mansoni through mechanisms that are currently unclear. This study demonstrates that arginase I (Arg I), a major product of IL-4- and IL-13-induced alternatively activated macrophages, prevents cachexia, neutrophilia, and endotoxemia during acute schistosomiasis. Specifically, Arg I-positive macrophages promote TGF-beta production and Foxp3 expression, suppress Ag-specific T cell proliferation, and limit Th17 differentiation. S. mansoni-infected Arg I-deficient bone marrow chimeras develop a marked accumulation of worm ova within the ileum but impaired fecal egg excretion compared with infected wild-type bone marrow chimeras. Worm ova accumulation in the intestines of Arg I-deficient bone marrow chimeras was associated with intestinal hemorrhage and production of molecules associated with classical macrophage activation (increased production of IL-6, NO, and IL-12/IL-23p40), but whereas inhibition of NO synthase-2 has marginal effects, IL-12/IL-23p40 neutralization abrogates both cachexia and intestinal inflammation and reduces the number of ova within the gut. Thus, macrophage-derived Arg I protects hosts against excessive tissue injury caused by worm eggs during acute schistosomiasis by suppressing IL-12/IL-23p40 production and maintaining the Treg/Th17 balance within the intestinal mucosa.