Haemocyte extracellular chromatin facilitates encapsulation reactions in a decapod host

The controlled release of chromatin meshes from the nuclei of neutrophils and other inflammatory cells (a process known as ETosis) is now recognised as an important microbial-trapping strategy in mammals. However, it is also associated with a number of pathological states, so its role in host protection in higher vertebrates remains ambiguous. After demonstrating that invertebrate phagocytes also exhibit a true ETotic response, we hypothesised that ETosis is an ancient process beneficial in lower taxa without a tubular vascular system. To test this, we studied the response in vivo in a protostome invertebrate, the crab, Carcinus maenas. We found that extracellular chromatin decorated with histone H2A is present within haemocyte capsules formed in the gill filaments 24 h after injection of lipopolysaccharide. Further in vitro analyses showed that similar encapsulation reactions are stimulated by phorbol myristate acetate (PMA), an inducer of ETosis and ROS production, in haemocytes in suspension culture. The response was inhibited by diphenylene iodonium, (DPI), an inhibitor of ETosis and ROS through its blocking effect on NADPH oxidase. Crucially DNAse-1 prevented PMA-stimulated cells from forming fully compacted capsules in vitro, showing that externalized chromatin serves as a scaffold upon which non-ETotic haemocytes assemble during the cell aggregation process. As encapsulation is a major defence reaction responsible for clearing high numbers of infective microbes from the haemocoel, ETosis is clearly beneficial to hosts without complex tubular circulatory systems and must therefore be an ancient cellular phenomenon that has been conserved throughout evolution.