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.

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.

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.

Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice.

Neutrophil extracellular traps (NETs; webs of DNA coated in antimicrobial proteins) are released into the vasculature during sepsis where they contribute to host defense, but also cause tissue damage and organ dysfunction. Various components of NETs have also been implicated as activators of coagulation. Using multicolor confocal intravital microscopy in mouse models of sepsis, we observed profound platelet aggregation, thrombin activation, and fibrin clot formation within (and downstream of) NETs in vivo. NETs were critical for the development of sepsis-induced intravascular coagulation regardless of the inciting bacterial stimulus (gram-negative, gram-positive, or bacterial products). Removal of NETs via DNase infusion, or in peptidylarginine deiminase-4-deficient mice (which have impaired NET production), resulted in significantly lower quantities of intravascular thrombin activity, reduced platelet aggregation, and improved microvascular perfusion. NET-induced intravascular coagulation was dependent on a collaborative interaction between histone H4 in NETs, platelets, and the release of inorganic polyphosphate. Real-time perfusion imaging revealed markedly improved microvascular perfusion in response to the blockade of NET-induced coagulation, which correlated with reduced markers of systemic intravascular coagulation and end-organ damage in septic mice. Together, these data demonstrate, for the first time in an in vivo model of infection, a dynamic NET-platelet-thrombin axis that promotes intravascular coagulation and microvascular dysfunction in sepsis.

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.

Decreased expression of the ß2 integrin on tumor cells is associated with a reduction in liver metastasis of colorectal cancer in mice.

BACKGROUND: Lymphocyte Function-Associated Antigen-1 (LFA-1; CD18/CD11a) is one of the main adhesion molecules used by immune cells to infiltrate the liver under inflammatory conditions. Recently, the expression of this integrin has also been reported on several solid tumors, including colorectal cancer. However, its functional role in the metastatic progression to the liver remains unknown. Using in vitro assays and an experimental orthotopic in vivo model of liver metastasis, we aimed to elucidate the role of tumor LFA-1 in the metastatic progression by means of the partial depletion of the ß2 subunit of LFA-1, required for integrin activation, firm adhesion and signaling. METHODS: To do so, we evaluated the effects of ß2 reduction on the murine colon carcinoma C26 cell line on their pro-metastatic features in vitro and their metastatic potential in vivo in a mouse model of colon carcinoma metastasis to the liver. RESULTS: The reduction in ß2 integrin expression correlated with a slower proliferation, and a reduced adhesion and migration of C26 cells in an in vitro setting. Additionally, tumor cells with a reduced in ß2 integrin expression were unable to activate the liver sinusoidal endothelial cells (LSECs). This resulted in a recovery of the cytotoxic potential of liver lymphocytes which is compromised by LSECs activated by C26 cells. This was related to the abrogation of RNA expression of inflammatory and angiogenic cytokines by C26 cells after their activation with sICAM-1, the main ligand of ß2αL. Furthermore, in vivo tumor cell retention and metastasis were profoundly reduced, along with a decrease in the recruitment and infiltration of myeloid derived suppressor cells (MDSCs) and lymphocytes to the liver. CONCLUSION: Taken together, our findings uncovered the modulatory role for the tumor ß2 subunit of the LFA-1 integrin in the metastatic progression of colorectal cancer to the liver by impairing activation of liver endothelium and thus, the local immune response in the liver. Besides, this integrin also showed to be critical in vivo for tumor cell retention, cytokine release, leukocyte recruitment and metastasis development. These data support a therapeutical potential of the integrin LFA-1 as a target for the treatment of colorectal liver metastasis.

Imaging the dynamic platelet-neutrophil response in sterile liver injury and repair in mice.

UNLABELLED: Although platelets have been extensively studied in hemostasis and inflammation, their role is not well understood in sterile liver injury and repair. Using a thermally induced focal liver injury and repair model and multichannel spinning disk confocal microscopy allowed visualization of the dynamic behavior of platelets and neutrophils in this insult. Platelets instantaneously adhered to molecularly altered sinusoidal endothelium adjacent to the afflicted area, paving approximately 200 µm abutting the injury. Platelets remained adherent for at least 4 hours, but dissipated by 8 hours. The early recruitment occurred by GPIIbIIIa (CD41) and the later recruitment was dependent upon both GPIIbIIIa and GPIb (CD42B). Platelets did not occlude the vessels, but rather paved the altered endothelium. Endothelin-induced vasoconstriction by hepatic stellate cells, and not platelet accumulation or coagulation, was responsible for temporarily restricted perfusion around the injury. Neutrophils crawled into the injury from significant distances through the sinusoids. The crawling neutrophils required the platelet-paved endothelium given that very little neutrophil recruitment was noted in thrombocytopenic or CD41-deficient mice. As platelets slowly dissipated, neutrophil recruitment was also halted. Previous work suggested that platelets binding to immobilized neutrophils induced neutrophil extracellular trap (NET) formation in response to infection as well as during thrombosis and other forms of sterile injury. In this model of neutrophils crawling on immobilized platelets, very few NETs were observed and no additional injury was noted. In fact, GPIIbIIIa-deficient mice had delayed repair. CONCLUSION: In a liver model of sterile injury and repair, platelets play a critical role in forming a substratum and pave the way for neutrophils to enter the injured site for subsequent repair.

Metallothionein 2 and Heat Shock Protein 72 Protect Allolobophora chlorotica from Cadmium But Not Nickel or Copper Exposure: Body Malformation and Coelomocyte Functioning.

Earthworms serve as good indicators of heavy metal contamination due to their innate sensitivity towards soil pollution. However, to date, not many studies have focused on endogeic earthworms, such as the omnipresent Allolobophora chlorotica. The current study was designed to verify whether this earthworm could serve as a novel distinctively susceptible species for environmental contamination studies. We show that the dermal exposure to Cu, Ni, and Cd affected the mortality and morphology of A. chlorotica, and the number and functioning of coelomocytes. These features particularly were pronounced in animals treated with Ni and Cu and interestingly to a lesser extend with Cd. In contrast, Cd induced a strong expression of metallothioneins (MT-2) and heat shock proteins (HSP72). The presence of MT-2 was detected not only in coelomocytes but also in the intestine, blood vessels, and epidermis. In conclusion, Allolobophora chlorotica coelomocytes are adopted to respond differentially to various heavy metals, generating powerful response towards potentially most dangerous exogenous non-essential elements.

Phagocytes & granulocytes. Angiogenic neutrophils: a novel subpopulation paradigm.

In this issue of Blood, Christoffersson et al provide data indicating the existence of a new neutrophil subset with angiogenic characteristics.

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.

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 poly(L-lactide) versus poly(L-lactide-co-trimethylene carbonate) on biological characteristics of fibroblasts and osteoblasts.

Bone tissue loss due to injury or disease often requires application ofautologous tissue grafts or artificial biomaterials to fill the fracture. Synthetic biomaterials provide temporary structural support for bone tissue and can be subsequently colonized by host tissue-specific cells. One of the most investigated groups of biomaterials are degradable polymers that naturally decompose in tissues with time. In particular aliphatic polyesters such as polylactides were reported to fulfill biocompatibility requirements as they induce a minor or lack an immune response and integrate with the surrounding tissue. Here we report on the biological effects of two polymers: poly(L-lactide) (PLLA) and a copolymer of L-lactide and trimethylene carbonate (PLTMC) on osteoblasts (MG-63) and fibroblasts (L-929). Osteoblasts are bone forming cells that are in the closest contact with the potential implant while fibroblasts produce the stroma forming the extracellular matrix (ECM) and along with macrophages initiate inflammation. We detected that both types of cells adhered better to PLLA than to PLTMC which might be related to the more rough surface of the former. However, both polymers, but especially PLTMC, increased apoptotic death of both cell types. Moreover, in contrast to PLLA, PLTMC modulated the production of some immune-related mediators by fibroblasts: it increased nitric oxide production and synthesis of numerous pro-inflammatory factors, cytokines (TNF-a and IL-6) activating leukocytes, and ECM-degrading MMP-9 which facilitates leukocyte migration. Thus, overall, our data suggest that PLTMC is less cytocompatible than PLLA.

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.

Morphine-modulated mast cell migration and proliferation during early stages of zymosan-induced peritonitis in CBA mice.

We have previously shown that supplementation of inflammation-inducing zymosan with a high dose ofmorphine inhibits peritoneal influx ofleukocytes in Swiss, C57C3H, Balb/c, and C57BL/6 strains but not in CBA mice. We have also reported that the different pattern of the response to morphine treatment might be, at least partially, due to the inter-strain differences in the peritoneal mast cell (P-MC) number (high in CBA mice versus other strains) and P-MC specific features (high sensitivity to degranulation upon morphine treatment in CBA mice). The aim of the present study was to investigate the mechanism of morphine action on P-MC in CBA mice. In particular, the effects of morphine on the proliferation and migration of P-MC in CBA mice with ongoing zymosan-induced peritonitis modulated by morphine were studied. Morphine alone acted as a strong chemoattractant for P-MC of CBA mice and this effect was opioid receptor-independent. Moreover, flow cytometric analysis showed that i.p. morphine injection induced significant proliferation of P-MC in CBA mice. Therefore, we conclude that the lack of anti-inflammatory effects of morphine during peritonitis in CBA mice might result not only from a unique sensitivity of CBA mast cells to morphine-induced degranulation but also from the fact that mast cell numbers increase at the inflammatory focus. The latter might be due to morphine-induced mast cell proliferation and/or migration.