Abstract
Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates.
Highlights
Over 95% of animal species are invertebrates and all of them utilize only one arm of immunity, the innate response [1,2]
Coelomocytes isolated from E. andrei which were stimulated in vivo for 24 hours with various stimulants of the immune system, including bacterial (LPS) and fungal components as well as whole live bacteria (Gram-Xenorhabdus bovienii and Gram+Micrococcus luteus) released extracellular DNA when kept ex vivo (Fig 1 and S1 Fig)
It is more difficult to distinguish between necrotic cells and those that released extracellular traps (ETs) and their bodies are surrounded by extracellular DNA (extDNA), the latter commonly seen in mice [60]
Summary
Over 95% of animal species are invertebrates and all of them utilize only one arm of immunity, the innate response [1,2]. One of the killing mechanism common to all animals (SIR1) is generation of ROS which are highly cytotoxic and antimicrobial agents [3,4] Apart of this function, they can act as reversible signal transduction mediators to regulate redox-sensitive target proteins [25]. To verify if earthworms release ETs when fighting infection in their coelom, we treated the animals with immunostimulants and imaged microscopically ET formation inside of the coelom (the in vivo setting). We report that to vertebrate NETs, earthworm ET formation depends on serine protease-activity and that histones H3 are attached to ETs. We identified some of the components of earthworm ETs (other than extDNA) and showed capacity of ETs to capture bacteria. The current study adds Annelida and its exemplary terrestrial species to the list of invertebrates capable of ET release but most importantly provides insides into innate mechanisms of ET formation in lower animal taxa
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