Summary Background Eosinophils are immunomodulatory leucocytes that contribute to the pathogenesis of Th2‐driven asthma and allergic lung diseases. Objective Our goal was to identify unique properties of eosinophils recruited to the lungs and airways of mice in response to challenge with asthma‐associated fungal allergens. Methods Mice were challenged intranasally on days 0, 3 and 6 with a filtrate of Alternaria alternata. Recruited eosinophils were enumerated in bronchoalveolar lavage fluid. Eosinophils were also isolated from lungs of mice sensitized and challenged with Aspergillus fumigatus and evaluated ex vivo in tissue culture. Results Eosinophils persist in the airways for several weeks in response to brief provocation with A. alternata in wild‐type, Gm‐csf‐ and eotaxin‐1‐gene‐deleted mice, while eosinophils are recruited but do not persist in the absence of IL‐13. Eosinophils isolated from the lungs A. alternata‐challenged mice are cytokine‐enriched compared to those from IL5tg mice, including 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. TNF‐α (but not IL5, IL‐3, eotaxin‐1 or GM‐CSF) was detected in supernatants of ex vivo eosinophil cultures from the lungs of fungal allergen–challenged wild‐type mice. However, neither TNF‐α gene deletion nor anti‐TNF‐α neutralizing antibodies had any impact sustained eosinophil survival ex vivo. Conclusion and Clinical Relevance Eosinophils are phenotypically and functionally heterogeneous. As shown here, eosinophils from fungal allergen–challenged wild‐type mice maintain a distinct cytokine profile, and, unlike eosinophils isolated from IL5tg mice, they survive ex vivo in the absence of exogenous pro‐survival cytokine support. As treatments for asthma currently in development focus on limiting eosinophil viability via strategic cytokine blockade, the molecular mechanisms underlying differential survival merit further investigation.

Flow cytometry protocols designed to identify mouse eosinophils typically target Siglec F, an α-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 IL5Rα (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 IL5tg mice. Purity by visual inspection of stained cytospin preparations and by Siglec F-diagnostic flow cytometry was 98 − 99% and 97 − 99%, respectively. Eosinophils isolated by this method (yield, ~ 4 × 106 / mouse) generated high-quality RNA suitable for gene expression analysis.

No longer regarded simply as end‐stage cytotoxic effectors, eosinophils are now recognized as complex cells with unique phenotypes that develop in response stimuli in the local microenvironment. In our previous study, we documented eosinophil infiltration in damaged muscle characteristic of dystrophin‐deficient (mdx) mice that model Duchenne muscular dystrophy. Specifically, we found that eosinophils did not promote the generation of muscle lesions, as these persisted in eosinophil‐deficient mdx.PHIL mice. To obtain additional insight into these findings, we performed RNA sequencing of eosinophils isolated from muscle tissue of mdx, IL5tg, and mdx.IL5tg mice. We observed profound up‐regulation of classical effector proteins (major basic protein‐1, eosinophil peroxidase, and eosinophil‐associated ribonucleases) in eosinophils isolated from lesion‐free muscle from IL5tg mice. By contrast, we observed significant up‐regulation of tissue remodeling genes, including proteases, extracellular matrix components, collagen, and skeletal muscle precursors, as well as the immunomodulatory receptor, Trem2, in eosinophils isolated from skeletal muscle tissue from the dystrophin‐deficient mdx mice. Although the anti‐inflammatory properties of Trem2 have been described in the monocyte/macrophage lineage, no previous studies have documented its expression in eosinophils. We found that Trem2 was critical for full growth and differentiation of bone marrow‐derived eosinophil cultures and full expression of TLR4. Immunoreactive Trem2 was also detected on human peripheral blood eosinophils at levels that correlated with donor body mass index and total leukocyte count. Taken together, our findings provide important insight into the immunomodulatory and remodeling capacity of mouse eosinophils and the flexibility of their gene expression profiles in vivo.