Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement

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.

Earthworm coelomocyte extracellular traps: structural and functional similarities with neutrophil NETs

“…Regardless of the adopted cell names of coelomocytes and hemocytes, their killing mechanisms are similar to each other and pathogen destruction is based on phagocytosis, enzyme activation (e.g., lysozyme), and formation of reactive oxygen species (ROS) and antimicrobial proteins (e.g., defensins) (Bilej et al 2010 ; Söderhäll 2010 ). Recent papers also confirm the possibility that invertebrate phagocytes are capable to produce extracellular traps (ETs) (e.g., Homa et al 2016a ; Robb et al 2014 ).…”

“…The latest reports indicate that the cells of simpler organisms, e.g., the social amoeba ( Dictyostelium discoideum ), also have an ability to release extracellular DNA with the formation of structures similar to NETs (Zhang et al 2016 ; Zhang and Soldati 2016 ). Earthworm coelomocytes show a similar mechanism (Homa et al 2016a ).…”

“…EFE degrades fibrinogen, elastin and fibrin, but also partially converts plasminogen into active plasmin (Zhao et al 2007 ). In our experiments on earthworm ETs, we found that protease inhibitors including serine proteases and elastase inhibit ET formation while the inhibitors of autophagy and the inhibitors of apoptosis-promoting caspases did not hinder this process (Homa et al 2016a ). Surprisingly, it was shown that NET formation in human neutrophils is dependent on autophagy (Remijsen et al 2011a ).…”

“…In some studies of the structure of ETs released from invertebrate immunocompetent cells, only the presence of extracellular DNA (extDNA) was found after cell immunological stimulation (Koiwai et al 2016 ). Other studies have revealed that histones (Ng et al 2013 ; Robb et al 2014 ; Homa et al 2016a ), hsp 27 (Homa et al 2016a ) and c-type lysozyme (Koiwai et al 2016 ) are also attached to extDNA. The most detailed characteristic of ETs was revealed in shrimp hemocytes (Ng et al 2013 , 2015 ).…”

“…Moreover, it was revealed that the coelomocyte ETs are built, among others, of nuclear DNA, H3 histones (Fig. 2 a–g) and conserved heat shock proteins HSP27 (Homa et al 2016a ). However, it should be mentioned that the lack of specific antibodies makes studies of invertebrate ETs very difficult.…”