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.

Impact of endogenous and exogenous nitrogen species on macrophage extracellular trap (MET) formation by bone marrow-derived macrophages.

Macrophage extracellular traps (METs) represent a novel defense mechanism in the antimicrobial arsenal of macrophages. However, mechanisms of MET formation are still poorly understood and this is at least partially due to the lack of reliable and reproducible models. Thus, we aimed at establishing a protocol of MET induction by bone marrow-derived macrophages (BMDMs) obtained from cryopreserved and then thawed bone marrow (BM) mouse cells. We report that BMDMs obtained in this way were morphologically (F4/80+) and functionally (expression of inducible nitric oxide (NO) synthase and NO production) differentiated and responded to various stimuli of bacterial (lipopolysaccharide, LPS), fungal (zymosan) and chemical (PMA) origin. Importantly, BMDMs were successfully casting METs composed of extracellular DNA (extDNA) serving as their backbone to which proteins such as H2A.X histones and matrix metalloproteinase 9 (MMP-9) were attached. In rendered 3D structure of METs, extDNA and protein components were embedded in each other. Since studies had shown the involvement of oxygen species in MET release, we aimed at studying if reactive nitrogen species (RNS) such as NO are also involved in MET formation. By application of NOS inhibitor - L-NAME or nitric oxide donor (SNAP), we studied the involvement of endogenous and exogenous RNS in traps release. We demonstrated that L-NAME halted MET formation upon stimulation with LPS while SNAP alone induced it. The latter phenomenon was further enhanced in the presence of LPS. Taken together, our findings demonstrate that BMDMs obtained from cryopreserved BM cells are capable of forming METs in an RNS-dependent manner.

Synergistic Action of MCL-1 Inhibitor with BCL-2/BCL-XL or MAPK Pathway Inhibitors Enhances Acute Myeloid Leukemia Cell Apoptosis and Differentiation.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by excessive proliferation of abnormal myeloid precursors accompanied by a differentiation block and inhibition of apoptosis. Increased expression of an anti-apoptotic MCL-1 protein was shown to be critical for the sustained survival and expansion of AML cells. Therefore, herein, we examined the pro-apoptotic and pro-differentiating effects of S63845, a specific inhibitor of MCL-1, in a single-agent treatment and in combination with BCL-2/BCL-XL inhibitor, ABT-737, in two AML cell lines: HL-60 and ML-1. Additionally, we determined whether inhibition of the MAPK pathway had an impact on the sensitivity of AML cells to S63845. To assess AML cells' apoptosis and differentiation, in vitro studies were performed using PrestoBlue assay, Coulter electrical impedance method, flow cytometry, light microscopy and Western blot techniques. S63845 caused a concentration-dependent decrease in the viability of HL-60 and ML-1 cells and increased the percentage of apoptotic cells. Combined treatment with S63845 and ABT-737 or MAPK pathway inhibitor enhanced apoptosis but also induced differentiation of tested cells, as well as altering the expression of the MCL-1 protein. Taken together, our data provide the rationale for further studies regarding the use of MCL-1 inhibitor in combination with other pro-survival protein inhibitors.

Biological Properties of Heparins Modified with an Arylazopyrazole-Based Photoswitch.

Unfractionated heparin (UFH) and enoxaparin (Enox) were substituted with a photoswitch (PS) showing quantitative trans-cis and cis-trans photoisomerizations. Long half-life of the cis photoisomer enabled comparison of the properties of heparins substituted with both PS photoisomers. Hydrodynamic diameter, Dh, of UFH-PS decreased upon trans-cis photoisomerization, the change being more pronounced for UFH-PS with a higher degree of substitution (DS), while Dh of Enox-PS did not significantly change. The anticoagulative properties of substituted heparins were significantly attenuated compared to non-substituted compounds. The interaction of UFH-PS with HSA, lysozyme, and protamine was studied with ITC. Under serum-free conditions, UFH-PS-trans with a high DS stimulated proliferation of murine fibroblasts, while UFH-PS-cis decreased the viability of these cells. Under serum conditions, both UFH-PS-cis and UFH-PS-trans decreased cell viability, the reduction for UFH-PS-cis being higher than that for UFH-PS-trans. Neither Enox-PS-trans nor Enox-PS-cis influenced the viability at concentrations prolonging aPTT, while at higher concentrations their cytotoxicity did not differ.

Itaconate Suppresses Formation of Neutrophil Extracellular Traps (NETs): Involvement of Hypoxia-Inducible Factor 1α (Hif-1α) and Heme Oxygenase (HO-1).

Neutrophil extracellular traps (NETs) immobilize pathogens during early stages of systemic inflammation but as the reaction progresses they become detrimental to endothelial cells and the organ-specific cells. For this reason it would be of importance to control their formation by either physiological or pharmacological means. Endogenously, formation of NETs is under control of cellular and whole organism metabolism as shown previously in the course of bacterial systemic inflammation, obesity or the combination of the two. Numerous leukocytes are subjected to immunometabolic regulation and in macrophages exposure to lipopolysaccharide (LPS) leads to two breaks in the Krebs cycle that impact this cell functioning. As a consequence of the first break, anti-microbial itaconic acid (itaconate) is produced whereas the second break activates hypoxia-inducible factor-1α (Hif-1α). In turn, itaconate activates transcription of the anti-inflammatory nuclear factor erythroid 2-related factor 2 (Nrf2) which upregulates cyto-protective heme oxygenase (HO-1). Here we report that exogenously added derivative of the itaconic acid, 4-octyl itaconate (4-OI), diminishes formation of NETs by neutrophils of either normal (lean) or obese mice, and independently of the age of the animals or immunoaging. Elucidating the mechanism of this inhibition we unravel that although Nrf2/HO-1 expression itself is not altered by 4-OI, it is up-regulated when compared against the NET formation while Hif-1α is downregulated in 4-OI-pre-treated LPS-stimulated neutrophils in either way. We further show that blockage of Hif-1α by its specific inhibitor diminishes NET release as does inhibition by 4-OI. Also inhibition of HO-1 activity correlates with diminished LPS-induced NET release upon pre-treatment with 4-OI albeit LPS alone induced NETs are not HO-1-dependent. In summary, we unravel that 4-OI inhibits NET formation by murine neutrophils independently of their origin (health vs. metabolically challenged animals) and the age of individuals/immunosenescence via inhibition of Hif-1α and induction of HO-1.

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.

Patients with COVID-19: in the dark-NETs of neutrophils.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

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.

On Neutrophil Extracellular Trap (NET) Removal: What We Know Thus Far and Why So Little.

Although neutrophil extracellular traps (NETs) were discovered only 16 years ago, they have already taken us from heaven to hell as we learned that apart from beneficial trapping of pathogens, they cause, or contribute to, numerous disorders. The latter is connected to their persistent presence in the blood or tissue, and we hardly know how they are removed in mild pathophysiological conditions and why their removal is impaired in multiple severe pathological conditions. Herein, we bring together all data available up till now on how NETs are cleared-from engaged cells, their phenotypes, to involved enzymes and molecules. Moreover, we hypothesize on why NET removal is challenged in multiple disorders and propose further directions for studies on NET removal as well as possible therapeutic strategies to have them cleared.

The pan-Bcl-2 inhibitor obatoclax promotes differentiation and apoptosis of acute myeloid leukemia cells.

One of the key features of acute myeloid leukemia (AML) is the arrest of differentiation at the early progenitor stage of myelopoiesis. Therefore, the identification of new agents that could overcome this differentiation block and force leukemic cells to enter the apoptotic pathway is essential for the development of new treatment strategies in AML. Regarding this, herein we report the pro-differentiation activity of the pan-Bcl-2 inhibitor, obatoclax. Obatoclax promoted differentiation of human AML HL-60 cells and triggered their apoptosis in a dose- and time-dependent manner. Importantly, obatoclax-induced apoptosis was associated with leukemic cell differentiation. Moreover, decreased expression of Bcl-2 protein was observed in obatoclax-treated HL-60 cells. Furthermore, differentiation of these cells was accompanied by the loss of their proliferative capacity, as shown by G0/G1 cell cycle arrest. Taken together, these findings indicate that the anti-AML effects of obatoclax involve not only the induction of apoptosis but also differentiation of leukemic cells. Therefore, obatoclax represents a promising treatment for AML that warrants further exploration.

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).

To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps.

Since the discovery and definition of neutrophil extracellular traps (NETs) 14 years ago, numerous characteristics and physiological functions of NETs have been uncovered. Nowadays, the field continues to expand and novel mechanisms that orchestrate formation of NETs, their previously unknown properties, and novel implications in disease continue to emerge. The abundance of available data has also led to some confusion in the NET research community due to contradictory results and divergent scientific concepts, such as pro- and anti-inflammatory roles in pathologic conditions, demarcation from other forms of cell death, or the origin of the DNA that forms the NET scaffold. Here, we present prevailing concepts and state of the science in NET-related research and elaborate on open questions and areas of dispute.

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.

Challenges in 3D culturing of neutrophils: Assessment of cell viability.

Standard cell culturing on plastic plates (two dimensional (2D) cultures) does not represent the actual microenvironment where cells reside in tissues. The three dimensional (3D) systems, composed of extracellular matrix and/or pure amino acids which form a scaffold for cells, are more accurate in this respect. 3D cultures were primarily developed for cancer cells but there is also a need for their application in studies on inflammatory leukocytes. Herein we describe our approach to study neutrophil-like cells in the 3D system. We describe measures taken to establish a neutrophil-like cell line (nHL-60) and selection of 3D scaffolds (PuraMatrix alone or enriched with collagen type I) for their culturing. We focus on challenges in measurement of neutrophil viability in 3D cultures and based on our data we suggest application of resazurin, rather than tetrazolium-based dyes or trypan blue exclusion, for evaluation of neutrophil viability.

The membrane with polylactide and hyaluronic fibers for skin substitute.

PURPOSE: Skin substitutes are heterogeneous group of scaffolds (natural or synthetic) and cells. We hypothesize that nanofibers with layer composition made of polylactide (PLA) and sodium hyaluronate (HA) obtained using electrospinning method are a good matrix for cell adhesion and proliferation. METHODS: Optimal conditions of electrospinning of PLA and HA nanofibers to create layered compositions (PLA membrane covered with HA nonwovens) were determined by modifying parameters such as the appropriate amount of solvents, polymer concentration, mixing temperature and electrospinning process conditions. By changing the parameters, it was possible to control the diameter and properties of both polymer fibers. The spinning solution were characterized by surface tension and rheology. A scanning electron microscope (SEM) was used to determine the morphology and fiber diameters: PLA and HA. Structure of the PLA/HA nonwoven was analyzed using spectroscopy (FTIR/ATR). Biocompatibility of the nonwoven with fibroblasts (ECM producers) was assessed in the in vitro conditions. RESULTS: The results showed that stable conditions for the formation of submicron PLA fibers were obtained using a 13% wt. solution of the polymer, dissolved in a 3:1 mixture of DCM:DMF at 45 °C. The hyaluronic fibers were prepared from a 12% wt. solution of the polymer dissolved in a 2:1 mixture of ammonia water and ethyl alcohol. All materials were biocompatible but to a different degree. CONCLUSIONS: The proposed laminate scaffold was characterized by a hydrophobic-hydrophilic domain surface with a maintained fiber size of both layers. The material positively underwent biocompatibility testing in contact with fibroblasts.