In FY2018, we contributed three (3) original research manuscripts and one (1) review on this topic as follows: Manuscript #1: Title: Eosinophil persistence in vivo and sustained viability ex vivo in response to respiratory challenge with fungal allergens. In this study, our intent was to identify unique properties of eosinophils recruited to the lungs and airways of mice in response to challenge with asthma associated allergens. Mice were challenged intranasally with a filtrate of Alternaria alternata, a fungal allergen of the family Pleosporaceae which has been associated with the development of asthma and respiratory allergies. We found that eosinophils recruited in response to A. alternata persist in the airways for several weeks wildtype, GM CSF and eotaxin 1 gene deleted mice, while eosinophils are recruited but do not persist in the absence of IL 13. Furthermore, eosinophils isolated from the lungs A. alternata challenged mice are cytokine enriched compared to those from IL-5 transgenic hypereosinophilic mice, and contain 800 fold higher levels of eotaxin 1. Furthermore, eosinophils from the lungs and spleen of fungal allergen challenged wild type mice are capable of prolonged survival ex vivo, in contrast to eosinophils from both untreated and fungal allergen challenged IL5tg mice, which undergo rapid demise in the absence of exogenous cytokine support. From these studies, we conclude that eosinophils are phenotypically and functionally heterogeneous. As shown here, eosinophils from fungal allergen challenged mice maintain a distinct cytokine profile, and, unlike eosinophils isolated from IL5 hypereosinophilic transgenic mice, they survive ex vivo in the absence of exogenous prosurvival cytokine support. Critical point: As treatments for asthma currently in development focus on limiting eosinophil viability via strategic cytokine blockade, the molecular mechanisms underlying differential survival in tissue merit further investigation. Ref: WE Geslewitz, CM Percopo, HF Rosenberg. 2018. Clin. Exp. Allergy, 48: 29-38 Manuscript #2: Title: FACS isolation of live mouse eosinophils at high purity via a protocol that does not target Siglec F. Flow cytometry protocols designed to identify mouse eosinophils typically target Siglec F, an -2,3-sialic acid binding transmembrane protein expressed universally on cells of this lineage. While a convenient target, antibody-mediated ligation of Siglec F induces eosinophil apoptosis, which limits its usefulness for isolations that are to be followed by functional and/or gene expression studies. We present here a method for FACS isolation which does not target Siglec F and likewise utilizes no antibodies targeting IL5R (CD125) or CCR3. Single cell suspensions are prepared from lungs of mice that were sensitized and challenged with Aspergillus fumigatus antigens; eosinophils were identified and isolated by FACS as live SSChi/FSChi CD11c Gr1/loMHCII cells. This strategy was also effective for eosinophil isolation from the lungs of IL5 transgenic mice. Purity by visual inspection of stained cytospin preparations and by Siglec F-diagnostic flow cytometry was 98 to 99% and 97 to 99%, respectively. Eosinophils isolated by this method (yield, 4e6 /mouse) generated high-quality RNA suitable for gene expression analysis. Critical point: We present a FACS isolation protocol that does not target the eosinophil-specific antigen, Siglec F. Eosinophils are isolated at high yield, 99% purity and full viability. These eosinophils yield high quality RNA that is suitable for RNA-seq analysis. Ref: WE Geslewitz, CM Percopo, HF Rosenberg. 2018. Journal of Immunol. Methods. 454:27-31 Manuscript #3: Title: Alternaria alternata challenge at the nasal mucosa results in eosinophilic inflammation and increased susceptibility to influenza infection. Eosinophils in the nasal mucosa are an elemental feature of allergic rhinitis. Our original objective was to explore eosinophilic inflammation and its impact on respiratory virus infection at the nasal mucosa. Eosinophilic inflammation in the nasal mucosae of mice was established by repetitive stimulation with strict intranasal volumes of a filtrate of Alternaria alternata. Mice were then challenged with influenza virus (A/HK/1/68). Repetitive stimulation with A. alternata resulted in eosinophil recruitment to the nasal passages in association with elevated levels of IL-5, IL-13 and eotaxin-1; eosinophil recruitment was diminished in eotaxin-1-/- mice, and abolished in Rag1-/- mice. A. alternata also resulted in elevated levels of nasal wash IgA in both wild-type and eosinophil deficient dblGATA mice. Interestingly and unexpectedly, A. alternata-treated mice responded to an influenza virus infection with profound weight loss and mortality compared to mice that received diluent alone (0% vs 100% survival, ***p < .001); the lethal response was blunted when A. alternata was heat-inactivated. Minimal differences in virus titer were detected, and eosinophils present in the nasal passages at the time of virus inoculation provided no protection against the lethal sequelae of disease. Interestingly, nasal wash fluids from mice treated with A. alternata included more neutrophils and higher levels of pro-inflammatory mediators in response to virus challenge, among these, IL6, a biomarker for disease severity in human influenza. Critical point: Repetitive administration of A. alternata resulted in inflammation of the nasal mucosae and unanticipated morbidity and mortality in response to subsequent challenge with influenza A virus. Interestingly, and in contrast to findings in the lower airways, eosinophils recruited to the nasal passages provided no protection against lethal infection. However, as increased susceptibility to influenza virus among individuals with rhinitis has been the subject of several clinical reports, this model may be used for further exploration of these observations. Ref: M Ma, JL Redes, CM Percopo, KM Druey, HF Rosenberg. 2018. Clin. Exp. Allergy 48:691-702. Manuscript #4 Title: Modeling asthma: pitfalls, promises and the road ahead. Asthma is a chronic, heterogeneous, and recurring inflammatory disease of the lower airways, with exacerbations that feature airway inflammation and bronchial hyperresponsiveness. Asthma has been modeled extensively via disease induction in both wild-type and genetically manipulated laboratory mice (Mus musculus). Antigen sensitization and challenge strategies have reproduced numerous important features of airway inflammation characteristic of human asthma, notably the critical roles of type 2 T helper cell cytokines. Recent models of disease induction have advanced to include physiologic aeroallergens with prolonged respiratory challenge without systemic sensitization; others incorporate tobacco, respiratory viruses, or bacteria as exacerbants. Nonetheless, differences in lung size, structure, and physiologic responses limit the degree to which airway dynamics measured in mice can be compared to human subjects. Other rodent allergic airways models, including those featuring the guinea pig (Cavia porcellus) might be considered for lung function studies. Finally, domestic cats (Feline catus) and horses (Equus caballus) develop spontaneous obstructive airway disorders with clinical and pathologic features that parallel human asthma. Information on pathogenesis and treatment of these disorders is an important resource. Ref: HF Rosenberg, KM Druey, in, Special Feature Issue, Research from The 10th Symposium of the International Eosinophil Society, in HF Rosenberg, PS Foster, Guest Editors, Journal of Leukocyte Biology, 2018; 104: pp. 41-48.

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Rosenberg, Helene F; Druey, Kirk M (2018) Modeling asthma: Pitfalls, promises, and the road ahead. J Leukoc Biol 104:41-48
Geslewitz, Wendy E; Percopo, Caroline M; Rosenberg, Helene F (2018) FACS isolation of live mouse eosinophils at high purity via a protocol that does not target Siglec F. J Immunol Methods 454:27-31
Ma, M; Redes, J L; Percopo, C M et al. (2018) Alternaria alternata challenge at the nasal mucosa results in eosinophilic inflammation and increased susceptibility to influenza virus infection. Clin Exp Allergy 48:691-702
Geslewitz, W E; Percopo, C M; Rosenberg, H F (2018) Eosinophil persistence in vivo and sustained viability ex vivo in response to respiratory challenge with fungal allergens. Clin Exp Allergy 48:29-38
Foster, Paul S; Maltby, Steven; Rosenberg, Helene F et al. (2017) Modeling TH 2 responses and airway inflammation to understand fundamental mechanisms regulating the pathogenesis of asthma. Immunol Rev 278:20-40
Percopo, Caroline M; Brenner, Todd A; Ma, Michelle et al. (2017) SiglecF+Gr1hi eosinophils are a distinct subpopulation within the lungs of allergen-challenged mice. J Leukoc Biol 101:321-328
Kraemer, Laura S; Brenner, Todd A; Krumholz, Julia O et al. (2017) A flow-cytometric method to evaluate eosinophil-mediated uptake of probiotic Lactobacillus reuteri. J Microbiol Methods 137:19-24
Lyons, Jonathan J; Rosenberg, Helene F; Druey, Kirk M (2017) Editorial: Stressing out mast cells via CRF1. J Leukoc Biol 102:1284-1285
Rosenberg, Helene F; Druey, Kirk M (2016) Eosinophils, galectins, and a reason to breathe. Proc Natl Acad Sci U S A 113:9139-41
Rosenberg, Helene F; Masterson, Joanne C; Furuta, Glenn T (2016) Eosinophils, probiotics, and the microbiome. J Leukoc Biol 100:881-888

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