Activation and Cell Death of Mouse Eosinophils in Response to Different Microenvironmental Stimuli

In inflammatory states, eosinophils are exposed to stimuli leading to activation, increased survival, and/or different cell death subroutines, which have differing effects on tissue inflammation. The mechanisms of signal integration are poorly understood. In this manuscript, we investigated cell death types in response to stimuli mimicking the inflammatory microenvironment. Mouse bone marrow-derived eosinophils (BMDeos) were stimulated with cytokines, cell-cell interaction mimics, pathogen-associated molecular patterns (PAMP), and broad cell activation stimuli. Both PMA and crosslinking of CD95 (cCD95) induced cell death of BMDeos. However, cCD95-induced cell death was consistent with apoptosis, while activation with PMA lead to EETosis. Both stimuli lead to caspase 3 activation and increased total level of histone H3 citrullination, indicating that these outcomes are not able to discriminate between the two cell death types. Flow cytometry for annexinV/7AAD pattern at early time points, and morphologic assessment by immunofluorescence (for DNA, eosinophil granule protein and citH3) were the most reliable outcomes for distinguishing the cell death subtypes. While LPS alone did not decrease BMDeos viability, LPS in the presence of caspase inhibition (zVAD) caused delayed cell death, which did not conform to either of the two cell death types. Finally, LPS and LPS/zVAD led to an increased level of surface expression of CD274 (type 1 activation), while both cCD95 and PMA increased the surface expression of CD101 (type 2 activation). In summary, at least three different activation-associated cell death pathways are seen in BMDeos activated with microenvironment-mimicking stimuli. Crosslinking CD95 leads to type 2 activation and apoptotic cell death. PMA also leads to type 2 activation but EETosis-associated cell death. LPS and LPS/zVAD are associated with type 1 activation, and only LPS/zVAD lead to cell death via a subtype different from both apoptosis and EETosis.