Abstract Body (Do not enter title and authors here): Background: Many patients with sustained hypereosinophilia develop cardiac complications, and eosinophilic heart disease (EHD) is the major cause of morbidity and mortality in these patients. The mechanism of EHD is poorly understood. Since eosinophil granule proteins can cause cardiomyocyte damage and one mechanism of their release is regulated necrosis, this study tested the hypothesis that eosinophils undergo regulated necrosis in vitro and that this is damaging to cardiomyocytes in a mouse model of EHD.
Methods: To develop a model of EHD, hypereosinophilic mice were challenged with cardiac myosin; biomarkers and histologic outcomes were assessed. For in vitro studies, we used bone marrow derived eosinophils and cell death assessed by biochemical and morphologic outcomes (annexin V binding, membrane permeabilization, citrullinated histone 3 (citH3), extracellular DNA and granule protein).
Results: EHD mice showed eosinophil-rich inflammation in the heart associated with cardiomyocyte necrosis and later fibrosis. Heart inflammation was decreased in eosinophil-deficient mice. Release of cell-free DNA and troponin was detectable in plasma. Immunohistochemistry for citH3, a marker of regulated necrosis, highlighted eosinophils in the tissue, suggesting they are undergoing regulated necrosis. To study regulated necrosis of eosinophils in vitro, eosinophil were stimulated with known to induce different types of cell death and stimuli that mimic activation by conditions in the inflammatory microenvironment. While all stimuli induced dose and time-dependent cell death, we noted two different patterns: while some stimuli (e.g. CD95 ligation) caused eosinophils to transition through annexinV+/7AAD- stage (early apoptosis), other stimuli (e.g. PMA and ionomycin) had increased ratio of 7AAD+ cells, suggesting they directly enter necrotic pathway. By microscopy, cells stimulated with CD95, PMA and ionomycin were positive for citH3, while only the ones stimulated with PMA had NET-like structures. This was further seen with staining for DNA and an eosinophil granule protein.
Conclusion: Together, our data show that mouse eosinophils undergo specific and distinct cell death pathways, and this may have varying effects on disease outcomes.