Large extracellular vesicle (EV) and neutrophil extracellular trap (NET) interaction captured in vivo during systemic inflammation.

Extracellular vesicles (EVs) and neutrophil extracellular traps (NETs) are pivotal bioactive structures involved in various processes including inflammation. Herein we report the interactions between EVs and NETs during murine endotoxemia studied in situ directly in the vasculature (cremaster muscle, liver sinusoids) using intravital microscopy (IVM). We captured NETs and EV release in real time by both non- and polarized neutrophils in liver but not in cremaster vasculature. When comparing numbers of circulating EVs of various origin (nanoparticle tracking analysis-NTA, flow cytometry) with those interacting with endothelium and NETs (IVM) we observed that whereas platelet and monocyte/macrophage-derived EVs dominate in blood and peritoneal lavage, respectively, mostly neutrophil-derived EVs interact with the vascular lining, NETs and leukocytes. Despite the interaction, NETs do not affect EV formation as NET release inhibition did not alter EV release. However, EVs inhibit NETs formation and in particular, erythrocyte-derived EVs downregulate NET release and this effect is mediated via Siglec-E-dependent interactions with neutrophils. Overall, we report that EVs are present in NETs in vivo and they do modulate their release but the process in not bidirectional. Moreover, EVs isolated from body fluids might not reflect their importance in direct endothelial- and leukocyte-related interactions.

Impact of microparticles released during murine systemic inflammation on macrophage activity and reactive nitrogen species regulation.

Microparticles (MPs) packaged with numerous bioactive molecules are essential vehicles in cellular communication in various pathological conditions, including systemic inflammation, Whereas MPs are studied mostly upon isolation, their detection in vivo is limited. Impact of MPs might depend on target cell type and cargo they carry; thus herein, we aimed at verifying MPs' impact on macrophages. Unlike neutrophils, monocytes/macrophages are rather inactive during sepsis, and we hypothesized this might be at least partially controlled by MPs. For the above reasons, we focused on the detection of MPs with intravital microscopy (IVM) and report the presence of putative neutrophil-derived MPs in the vasculature of cremaster muscle of endotoxemic mice. Subsequently, we characterized MPs isolated not only from their blood but also from the peritoneal cavity and observed differences in their size, concentration, and cargo. Such MPs were then used to study their impact on RAW 264.7 macrophage cell line performance (cell viability/activity, cytokines, oxygen, and nitrogen reactive species). Addition of MPs to macrophages with or without co-stimulation with lipopolysaccharide did not affect respiratory burst, somewhat decreased mitochondrial activity but increased inducible nitric oxide synthase (iNOS) expression, and NO production especially in case of plasma-derived MPs. The latter MPs carried more iNOS-controlling ceruloplasmin than those discharged into the peritoneal cavity. We conclude that MPs can be detected in vivo with IVM and their cellular origin identified. They are heterogeneous in nature depending on the site of their release. Consequently, microparticles released during systemic inflammation to various body compartments differentially affect macrophages.

Metabolic Pathways Involved in Formation of Spontaneous and Lipopolysaccharide-Induced Neutrophil Extracellular Traps (NETs) Differ in Obesity and Systemic Inflammation.

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.

Scrutinizing Mechanisms of the 'Obesity Paradox in Sepsis': Obesity Is Accompanied by Diminished Formation of Neutrophil Extracellular Traps (NETs) Due to Restricted Neutrophil-Platelet Interactions.

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.

Imaging of Neutrophils and Neutrophil Extracellular Traps (NETs) with Intravital (In Vivo) Microscopy.

As we have learned during recent years, neutrophils are not just simple foot soldiers of the innate immune system with a restricted set of pro-inflammatory functions, and instead, they perform sophisticated functions (some of them only recently discovered) both in innate and adaptive immune responses. Neutrophil behavior and functioning should best be studied in situ, at locations where they are executed in a living organism, especially considering that neutrophils are mobile cells, performing their functions in distal body sites and various organs. For this herein we describe an approach to detect neutrophil presence/behavior in various organs (skin, muscle, liver) of alive mice, that is, intravital imaging/microscopy. We describe all surgeries required prior to imaging and share our methods of detection of neutrophils and neutrophil extracellular traps (NETs).

Reduced Neutrophil Extracellular Trap (NET) Formation During Systemic Inflammation in Mice With Menkes Disease and Wilson Disease: Copper Requirement for NET Release.

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.

Age is the work of art? Impact of neutrophil and organism age on neutrophil extracellular trap formation.

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.

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

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.