Neutrophil extracellular traps or NETs are released by highly activated neutrophils in response to infectious agents, sterile inflammation, autoimmune stimuli and cancer. In the cells, the nuclear envelop disintegrates and decondensation of chromatin occurs that depends on peptidylarginine deiminase 4 (PAD4) and neutrophil elastase (NE). Subsequently, proteins from neutrophil granules (e.g., NE, lactoferrin and myeloperoxidase) and the nucleus (histones) bind to decondensed DNA and the whole structure is ejected from the cell. The DNA decorated with potent antimicrobials and proteases can act to contain dissemination of infection and in sterile inflammation NETs were shown to degrade cytokines and chemokines via serine proteases. On the other hand, overproduction of NETs, or their inadequate removal and prolonged presence in vasculature or tissues, can lead to bystander damage or even initiation of diseases. Considering the pros and cons of NET formation, it is of relevance if the stage of neutrophil maturation (immature, mature and senescent cells) affects the capacity to produce NETs as the cells of different age-related phenotypes dominate in given (pathological) conditions. Moreover, the immune system of neonates and elderly individuals is weaker than in adulthood. Is the same pattern followed when it comes to NETs? The overall importance of individual and neutrophil age on the capacity to release NETs is reviewed in detail and the significance of these facts is discussed.

Systemic inflammation is a detrimental condition associated with high mortality. However, obese individuals seem to have higher chances of surviving sepsis. To elucidate what immunological differences exist between obese and lean individuals we studied the course of endotoxemia in mice fed high-fat diet (HFD) and ob/ob animals. Intravital microscopy revealed that neutrophil extracellular trap (NET) formation in liver vasculature is negligible in obese mice in sharp contrast to their lean counterparts (ND). Unlike in lean individuals, neutrophil influx is not driven by leptin or interleukin 33 (IL-33), nor occurs via a chemokine receptor CXCR2. In obese mice less platelets interact with neutrophils forming less aggregates. Platelets transfer from ND to HFD mice partially restores NET formation, and even further so upon P-selectin blockage on them. The study reveals that in obesity the overexaggerated inflammation and NET formation are limited during sepsis due to dysfunctional platelets suggesting their targeting as a therapeutic tool in systemic inflammation.

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.

Obesity manifests itself with low-grade chronic inflammation that shapes immune responses during infection. Albeit obese individuals are at risk of higher mortality due to comorbidities, they are better protected from systemic inflammation. Recently, we showed that in the vasculature of obese mice kept on high-fat diet (HFD), neutrophils produce less neutrophil extracellular traps (NETs) than in lean controls (normal diet, ND). NETs are used by neutrophils to counteract severe infection, but they also cause collateral damage. Hardly anything is known about metabolic requirements for their formation, especially in the context of obesity and/or sepsis. Thus, we aimed to study the immunometabolism of NET formation by application of ex vivo neutrophil analyses (Seahorse analyzer, selective inhibitors, confocal imaging) and intravital microscopy. The obtained data show that glycolysis and/or pentose phosphate pathway are involved in NETs release by ND neutrophils in both physiological and inflammatory conditions. In contrast, such cells of septic HFD mice utilize these routes only to spontaneously cast NETs, while after secondary ex vivo activation they exhibit so called “exhausted phenotype”, which manifests itself in diminished NET release despite high glycolytic potential and flexibility to oxidize fatty acids. Moreover, impact of ATP synthase inhibition on NET formation is revealed. Overall, the study shows that the neutrophil potential to cast NETs depends on both the metabolic and inflammatory state of the individual.

Neutrophil extracellular traps (NETs) contribute to pathological disorders, and their release was directly linked to numerous diseases. With intravital microscopy (IVM), we showed previously that NETs also contribute to the pathology of systemic inflammation and are strongly deposited in liver sinusoids. Over a decade since NET discovery, still not much is known about the metabolic or microenvironmental aspects of their formation. Copper is a vital trace element essential for many biological processes, albeit its excess is potentially cytotoxic; thus, copper levels are tightly controlled by factors such as copper transporting ATPases, ATP7A, and ATP7B. By employing IVM, we studied the impact of copper on NET formation during endotoxemia in liver vasculature on two mice models of copper excess or deficiency, Wilson (ATP7B mutants) and Menkes (ATP7A mutants) diseases, respectively. Here, we show that respective ATP7 mutations lead to diminished NET release during systemic inflammation despite unaltered intrinsic capacity of neutrophils to cast NETs as tested ex vivo. In Menkes disease mice, the in vivo effect is mostly due to diminished neutrophil infiltration of the liver as unmutated mice with a subchronic copper deficiency release even more NETs than their controls during endotoxemia, whereas in Wilson disease mice, excess copper directly diminishes the capacity to release NETs, and this was further confirmed by ex vivo studies on isolated neutrophils co-cultured with exogenous copper and a copper-chelating agent. Taken together, the study extends our understanding on how microenvironmental factors affect NET release by showing that copper is not a prerequisite for NET release but its excess affects the trap casting by neutrophils.

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