Although eosinophils are considered useful in defense mechanisms against parasites, their exact function in innate immunity remains unclear. The aim of this study is to better understand the role of eosinophils within the gastrointestinal immune system. We show here that lipopolysaccharide from Gram-negative bacteria activates interleukin-5 (IL-5)- or interferon-gamma-primed eosinophils to release mitochondrial DNA in a reactive oxygen species-dependent manner, but independent of eosinophil death. Notably, the process of DNA release occurs rapidly in a catapult-like manner--in less than one second. In the extracellular space, the mitochondrial DNA and the granule proteins form extracellular structures able to bind and kill bacteria both in vitro and under inflammatory conditions in vivo. Moreover, after cecal ligation and puncture, Il5-transgenic but not wild-type mice show intestinal eosinophil infiltration and extracellular DNA deposition in association with protection against microbial sepsis. These data suggest a previously undescribed mechanism of eosinophil-mediated innate immune responses that might be crucial for maintaining the intestinal barrier function after inflammation-associated epithelial cell damage, preventing the host from uncontrolled invasion of bacteria.

Neutrophil extracellular traps (NETs) represent extracellular structures able to bind and kill microorganisms. It is believed that they are generated by neutrophils undergoing cell death, allowing these dying or dead cells to kill microbes. We show that, following priming with granulocyte/macrophage colony-stimulating factor (GM-CSF) and subsequent short-term toll-like receptor 4 (TLR4) or complement factor 5a (C5a) receptor stimulation, viable neutrophils are able to generate NETs. Strikingly, NETs formed by living cells contain mitochondrial, but no nuclear, DNA. Pharmacological or genetic approaches to block reactive oxygen species (ROS) production suggested that NET formation is ROS dependent. Moreover, neutrophil populations stimulated with GM-CSF and C5a showed increased survival compared with resting neutrophils, which did not generate NETs. In conclusion, mitochondrial DNA release by neutrophils and NET formation do not require neutrophil death and do also not limit the lifespan of these cells.

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SUMMARY Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly.

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