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Neutrophils are the most abundant innate immune cells in the circulation and they are the first cells recruited to sites of infection or inflammation. Almost half of the intracellular protein content in neutrophils consists of S100A8 and S100A9, though there has been controversy about their actual localization. Once released extracellularly, these proteins are thought to act as damage-associated molecular patterns (DAMPs), though their mechanism of action is not well understood. These S100 proteins mainly form heterodimers (S100A8/A9, also known as calprotectin) and this heterocomplex is recognized as a useful biomarker for several inflammatory diseases. We observed that S100A8/A9 is highly present in the cytoplasmic fraction of neutrophils and is not part of the granule content. Furthermore, we found that S100A8/A9 was not released in parallel with granular content but upon the formation of neutrophil extracellular traps (NETs). Accordingly, neutrophils of patients with chronic granulomatous disease, who are deficient in phorbol 12-myristate 13-acetate (PMA)-induced NETosis, did not release S100A8/A9 upon PMA stimulation. Moreover, we purified S100A8/A9 from the cytoplasmic fraction of neutrophils and found that S100A8/A9 could induce neutrophil activation, including adhesion and CD11b upregulation, indicating that this DAMP might amplify neutrophil activation.

Introduction: Developing prognostic markers can be useful for clinical decision-making. Peripheral blood (PB) examination is simple and basic that can be performed in any facility. We aimed to investigate whether PB examination can predict prognosis in coronavirus disease (COVID-19)., Methods: Complete blood count (CBC) and PB cell morphology were examined in 38 healthy controls (HCs) and 40 patients with COVID-19. Patients with COVID-19, including 26 mild and 14 severe cases, were hospitalized in Juntendo University Hospital (Tokyo, Japan) between April 1 and August 6, 2020. PB examinations were performed using Sysmex XN-3000 automated hematology analyzer and Sysmex DI-60 employing the convolutional neural network-based automatic image-recognition system., Results: Compared with mild cases, severe cases showed a significantly higher incidence of anemia, lymphopenia, and leukocytosis (P < .001). Granular lymphocyte counts were normal or higher in mild cases and persistently decreased in fatal cases. Temporary increase in granular lymphocytes was associated with survival of patients with severe infection. Red cell distribution width was significantly higher in severe cases than in mild cases (P < .001). Neutrophil dysplasia was consistently observed in COVID-19 cases, but not in HCs. Levels of giant neutrophils and toxic granulation/Döhle bodies were increased in severe cases., Conclusion: Basic PB examination can be useful to predict the prognosis of COVID-19, by detecting SARS-CoV-2 infection-induced multi-lineage changes in blood cell counts and morphological anomalies. These changes were dynamically correlated with disease severity and may be associated with disruption of hematopoiesis and the immunological system due to bone marrow stress in severe infection., (© 2021 John Wiley & Sons Ltd.)

Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22 phox and p47 phox at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils., (©2021 Society for Leukocyte Biology.)

Cholesterol has been implicated in the clinical progression of breast cancer, a disease that continues to be the most commonly diagnosed cancer in women. Previous work has identified the cholesterol metabolite 27-hydroxycholesterol (27HC) as a major mediator of the effects of cholesterol on breast tumor growth and progression. 27HC can act as an estrogen receptor (ER) modulator to promote the growth of ERα+ tumors, and as a liver X receptor (LXR) ligand in myeloid immune cells to establish an immune-suppressive program. In fact, the metastatic properties of 27HC require the presence of myeloid cells with neutrophils (polymorphonuclear neutrophils; PMNs) being essential for the increase in lung metastasis in murine models. In an effort to further elucidate the mechanisms by which 27HC alters breast cancer progression, we made the striking finding that 27HC promoted the secretion of extracellular vesicles (EVs), a diverse assortment of membrane bound particles that includes exosomes. The resulting EVs had a size distribution that was skewed slightly larger than EVs generated by treating cells with vehicle. The increase in EV secretion and size was consistent across 3 different subtypes: primary murine PMNs, RAW264.7 monocytic cells, and 4T1 murine mammary cancer cells. Label-free analysis of 27HC-EVs indicated that they had a different metabolite composition to those from vehicle-treated cells. Importantly, 27HC-EVs from primary PMNs promoted tumor growth and metastasis in 2 different syngeneic models, demonstrating the potential role of 27HC-induced EVs in the progression of breast cancer. EVs from PMNs were taken up by cancer cells, macrophages, and PMNs, but not T cells. Since EVs did not alter proliferation of cancer cells, it is likely that their protumor effects are mediated through interactions with myeloid cells. Interestingly, RNA-seq analysis of tumors from 27HC-EV-treated mice do not display significantly altered transcriptomes, suggesting that the effects of 27HC-EVs occur early on in tumor establishment and growth. Future work will be required to elucidate the mechanisms by which 27HC increases EV secretion, and how these EVs promote breast cancer progression. Collectively, however, our data indicate that EV secretion and content can be regulated by a cholesterol metabolite, which may have detrimental effects in terms of disease progression, important findings given the prevalence of both breast cancer and hypercholesterolemia., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) has recently been shown to have a critical role in granulopoiesis in humans, mice, and zebrafish. Our patient presented with delayed cord separation, failure to thrive, and sepsis. Retrospective whole-exome sequencing confirmed a homozygous splice-site mutation in SMARCD2., Objective: We sought to provide the second description of human SMARCD2 deficiency and the first functional analysis of human primary SMARCD2-deficient cells., Methods: Heparinized venous blood and bone marrow were collected from the patient after obtaining informed consent. Patient leukocytes and CD34 + cells were then isolated, phenotyped, and assessed functionally., Results: Circulating neutrophils appeared phenotypically immature, lacking multilobed nuclei, and neutrophil granules lacked lactoferrin but showed normal levels of myeloperoxidase. Neutrophil oxidative burst was preserved in response to phorbol 12-myristate 13-acetate. Patient bone marrow-derived neutrophils and white blood cells showed a severely impaired chemotactic response. Furthermore, white blood cells showed defective in vitro killing of Staphylococcus aureus, consistent with a specific granule deficiency. Finally, patient bone marrow-derived CD34 + cells showed markedly impaired in vitro expansion and differentiation toward the neutrophil lineage. Before her molecular diagnosis, our patient underwent hematopoietic stem cell transplantation and is well 8 years later., Conclusions: This report highlights an important role for SMARCD2 in human myelopoiesis and the curative effect of hematopoietic stem cell transplantation for the hematopoietic features of SMARCD2 deficiency., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Cellular heterogeneity and diversity are recognized to contribute to the functions of neutrophils under homeostatic and pathological conditions. We previously suggested that the chronic inflammatory responses associated with hypertension (HTN) are related to the participation of different subpopulations of neutrophils. Two populations of neutrophils can be obtained by density gradient centrifugation: normal-density neutrophils (NDN) and low-density neutrophils (LDN). However, the lack of standardized functional protocols has limited phenotypic characterization and functional comparisons of LDN and NDN. Based on their capability to incorporate Na + , maturity and activation stage, we characterized NDN and LDN in blood samples from ten patients with HTN and ten healthy individuals (HI) using flow cytometry. We compared the levels of reactive oxygen species (ROS), generation of neutrophil extracellular traps (NETs) and levels of apoptosis in NDN and LDN. In general, the NDN and LDN subpopulations from patients with HTN exhibited higher levels of sodium influx and ROS, and lower levels of apoptosis than the corresponding NDN and LDN subsets from HI. Transmission electron microscopy revealed NDN and LDN from patients with HTN exhibited alterations to mitochondrial morphology and fewer cytoplasmic granules than the corresponding HI subpopulations. Our results indicate both the NDN and LDN subpopulations enhance the effects of inflammation that contribute to the pathophysiology of HTN. Further detailed studies are required to characterize the events during ontogeny of the myeloid lineage that result in the diverse phenotypic characteristics of each subpopulation of LDN and NDN., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs) are important elements of intercellular communication. A plethora of different, occasionally even opposite, physiologic and pathologic effects have been attributed to these vesicles in the last decade. A direct comparison of individual observations is however hampered by the significant differences in the way of elicitation, collection, handling, and storage of the investigated vesicles. In the current work, we carried out a careful comparative study on 3, previously characterized types of EVs produced by neutrophilic granulocytes. We investigated in parallel the modulation of multiple blood-related cells and functions by medium-sized vesicles. We show that EVs released from resting neutrophils exert anti-inflammatory action by reducing production of reactive oxygen species (ROS) and cytokine release from neutrophils. In contrast, vesicles generated upon encounter of neutrophils with opsonized particles rather promote proinflammatory processes as they increase production of ROS and cytokine secretion from neutrophils and activate endothelial cells. EVs released from apoptosing cells were mainly active in promoting coagulation. We thus propose that EVs are "custom made," acquiring selective capacities depending on environmental factors prevailing at the time of their biogenesis., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)

Rolling neutrophils form tethers with submicron diameters. Here, we report that these tethers detach, forming elongated neutrophil-derived structures (ENDS) in the vessel lumen. We studied ENDS formation in mice and humans in vitro and in vivo. ENDS do not contain mitochondria, endoplasmic reticulum, or DNA, but are enriched for S100A8, S100A9, and 57 other proteins. Within hours of formation, ENDS round up, and some of them begin to present phosphatidylserine on their surface (detected by annexin-5 binding) and release S100A8-S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. ENDS appear in blood plasma of mice upon induction of septic shock. Compared with healthy donors, ENDS are 10-100-fold elevated in blood plasma of septic patients. Unlike neutrophil-derived extracellular vesicles, most ENDS are negative for the tetraspanins CD9, CD63, and CD81. We conclude that ENDS are a new class of bloodborne submicron particles with a formation mechanism linked to neutrophil rolling on the vessel wall., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Marki et al.)

This study evaluated the role of endogenous and exogenous annexin A1 (AnxA1) in the activation of the NLRP3 inflammasome in isolated peritoneal neutrophils. C57BL/6 wild-type (WT) and AnxA1 knockout mice (AnxA1 -/- ) received 0.3% carrageenan intraperitoneally and, after 3 h, the peritoneal exudate was collected. WT and AnxA1 -/- neutrophils were then stimulated with lipopolysaccharide, followed by the NLRP3 agonists nigericin or ATP. To determine the exogenous effect of AnxA1, the neutrophils were pretreated with the AnxA1-derived peptide Ac 2-26 followed by the NLRP3 agonists. Ac 2-26 administration reduced NLRP3-derived IL-1β production by WT neutrophils after nigericin and ATP stimulation. However, IL-1β release was impaired in AnxA1 -/- neutrophils stimulated by both agonists, and there was no further impairment in IL-1β release with Ac 2-26 treatment before stimulation. Despite this, ATP- and nigericin-stimulated AnxA1 -/- neutrophils had increased levels of cleaved caspase-1. The lipidomics of supernatants from nigericin-stimulated WT and AnxA1 -/- neutrophils showed potential lipid biomarkers of cell stress and activation, including specific sphingolipids and glycerophospholipids. AnxA1 peptidomimetic treatment also increased the concentration of phosphatidylserines and oxidized phosphocholines, which are lipid biomarkers related to the inflammatory resolution pathway. Together, our results indicate that exogenous AnxA1 negatively regulates NLRP3-derived IL-1β production by neutrophils, while endogenous AnxA1 is required for the activation of the NLRP3 machinery.

Neutrophils are short-lived cells after isolation. The analysis of neutrophil vesicular trafficking requires rapid and gentle handling. Recently developed super-resolution microscopy technologies have generated unparalleled opportunities to help understand the molecular mechanisms regulating neutrophil vesicular trafficking, exocytosis, and associated functions at the molecular level. Here, we describe super-resolution and total internal reflection fluorescence (TIRF) microscopy approaches for the analysis of vesicular trafficking and associated functions of primary neutrophils.

Background: To reveal detailed histopathological changes, virus distributions, immunologic properties and multi-omic features caused by SARS-CoV-2 in the explanted lungs from the world's first successful lung transplantation of a COVID-19 patient., Materials and Methods: A total of 36 samples were collected from the lungs. Histopathological features and virus distribution were observed by optical microscope and transmission electron microscope (TEM). Immune cells were detected by flow cytometry and immunohistochemistry. Transcriptome and proteome approaches were used to investigate main biological processes involved in COVID-19-associated pulmonary fibrosis., Results: The histopathological changes of the lung tissues were characterized by extensive pulmonary interstitial fibrosis and haemorrhage. Viral particles were observed in the cytoplasm of macrophages. CD3 + CD4 - T cells, neutrophils, NK cells, γ/δ T cells and monocytes, but not B cells, were abundant in the lungs. Higher levels of proinflammatory cytokines iNOS, IL-1β and IL-6 were in the area of mild fibrosis. Multi-omics analyses revealed a total of 126 out of 20,356 significant different transcription and 114 out of 8,493 protein expression in lung samples with mild and severe fibrosis, most of which were related to fibrosis and inflammation., Conclusions: Our results provide novel insight that the significant neutrophil/ CD3 + CD4 - T cell/ macrophage activation leads to cytokine storm and severe fibrosis in the lungs of COVID-19 patient and may contribute to a better understanding of COVID-19 pathogenesis., (© 2020 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Eimeria ninakohlyakimovae represents a highly pathogenic coccidian parasite causing severe haemorrhagic typhlocolitis in goat kids worldwide. NETosis was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites in vitro and in vivo. In vitro interactions of caprine PMN with parasitic stages of E. ninakohlyakimovae (i. e. oocysts and sporozoites) as well as soluble oocyst antigens (SOA) were analyzed at different ratios, concentrations and time spans. Extracellular DNA staining was used to illustrate classical molecules induced during caprine NETosis [i. e. histones (H3) and neutrophil elastase (NE)] via antibody-based immunofluorescence analyses. Functional inhibitor treatments with DPI and DNase I were applied to unveil role of NADPH oxidase (NOX) and characterize DNA-backbone composition of E. ninakohlyakimovae-triggered caprine NETosis. Scanning electron microscopy (SEM)- and immunofluorescence-analyses demonstrated that caprine PMN underwent NETosis upon contact with sporozoites and oocysts of E. ninakohlyakimovae, ensnaring filaments which firmly entrapped parasites. Detailed co-localization studies of E. ninakohlyakimovae-induced caprine NETosis revealed presence of PMN-derived DNA being adorned with nuclear H3 and NE corroborating molecular characteristics of NETosis. E. ninakohlyakoimovae-induced caprine NETosis was found to be NOX-independent since DPI inhibition led to a slight decrease of NETosis. Exposure of caprine PMN to vital E. ninakohlyakimovae sporozoites as well as SOA resulted in up-regulation of IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in stimulated PMN. Since vital E. ninakohlyakimovae-sporozoites induced caprine NETosis, this effective entrapment mechanism might reduce initial sporozoite epithelial host cell invasion during goat coccidiosis ultimately resulting in less macromeront formation and reduced merozoites I production., (Copyright © 2020 Elsevier Inc. All rights reserved.)

An issue of the novel coronavirus infection spreading is currently in the first place among others in the list of the international medical community. Due to lack of information, conflicting research findings, multicomponent effect of the virus on the body host, as well as various consequences that the virus triggers in the body, now every medical specialty does study the viral attack pathogenesis. Recent months showed that vascular complications are the most severe in the Coronavirus Disease 2019 (COVID-19) and are the main cause of death in the patients. The mechanisms of vascular complications are complex and affect both the hemostatic system and immune responses, "inflammatory storm", disorders of the renin-angiotensin-aldosterone system, endotheliopathy, etc. Due to the leading role of vascular complications in the viral infection pathogenesis, several groups of patients are at extra risk, including pregnant women, patients with a burdened obstetric history, with hereditary thrombophilia and antiphospholipid syndrome, and patients after in vitro fertilization (IVF). In this category of pregnant women, use of low-molecular-weight heparins (LMWH) is particularly important for both prevention of vascular and obstetric complications, and for pathogenetic therapy of COVID-19.

Polymorphonuclear neutrophils (PMN) are critical for first line innate immune defence against Staphylococcus aureus . Mature circulating PMN maintain a short half-life ending in constitutive apoptotic cell death. This makes them unlikely candidates as a bacterial intracellular niche. However, there is significant evidence to suggest that S. aureus can survive intracellularly within PMN and this contributes to persistence and dissemination during infection. The precise mechanism by which S. aureus parasitizes these cells remains to be established. Herein we propose a novel mechanism by which S. aureus subverts both autophagy and apoptosis in PMN in order to maintain an intracellular survival niche during infection. Intracellular survival of S. aureus within primary human PMN was associated with an accumulation of the autophagic flux markers LC3-II and p62, while inhibition of the autophagy pathway led to a significant reduction in intracellular survival of bacteria. This intracellular survival of S. aureus was coupled with a delay in neutrophil apoptosis as well as increased expression of several anti-apoptotic factors. Importantly, blocking autophagy in infected PMN partially restored levels of apoptosis to that of uninfected PMN, suggesting a connection between the autophagic and apoptotic pathways during intracellular survival. These results provide a novel mechanism for S. aureus intracellular survival and suggest that S. aureus may be subverting crosstalk between the autophagic and apoptosis pathways in order to maintain an intracellular niche within human PMN., (Copyright © 2020 Mulcahy, O’Brien, O’Keeffe, Vozza, Leddy and McLoughlin.)

Acquisition of a lipid-laden phenotype by immune cells has been defined in infectious diseases and atherosclerosis but remains largely uncharacterized in cancer. Here, in breast cancer models, we found that neutrophils are induced to accumulate neutral lipids upon interaction with resident mesenchymal cells in the premetastatic lung. Lung mesenchymal cells elicit this process through repressing the adipose triglyceride lipase (ATGL) activity in neutrophils in prostaglandin E2-dependent and -independent manners. In vivo, neutrophil-specific deletion of genes encoding ATGL or ATGL inhibitory factors altered neutrophil lipid profiles and breast tumor lung metastasis in mice. Mechanistically, lipids stored in lung neutrophils are transported to metastatic tumor cells through a macropinocytosis-lysosome pathway, endowing tumor cells with augmented survival and proliferative capacities. Pharmacological inhibition of macropinocytosis significantly reduced metastatic colonization by breast tumor cells in vivo. Collectively, our work reveals that neutrophils serve as an energy reservoir to fuel breast cancer lung metastasis.

The glycans on the plasma membrane of cells manifest as the glycocalyx, which serves as an information-rich frontier that is directly in contact with its immediate milieu. The glycoconjugates (GCs) that adorn most of the mammalian cells are also abundant in gametes, especially the spermatozoa where they perform unique reproduction-specific functions e.g., inter-cellular recognition and communication. This study aimed to implicate the sperm glycosylation pattern as one of the factors responsible for low conception rates observed in buffalo bulls. We hypothesized that a differential abundance of glycans exists on the spermatozoa from bulls of contrasting fertilizing abilities endowing them with differential immune evasion abilities. Therefore, we investigated the role of glycan abundance in the phagocytosis and NETosis rates exhibited by female neutrophils (PMNs) upon exposure to such spermatozoa. Our results indicated that the spermatozoa from high fertile (HF) bulls possessed a higher abundance of O-linked glycans e.g., galactosyl (β-1,3)N-acetylgalactosamine and N-linked glycans like [GlcNAc]1-3, N-acetylglucosamine than the low fertile (LF) bull spermatozoa. This differential glycomic endowment appeared to affect the spermiophagy and NETosis rates exhibited by the female neutrophil cells (PMNs). The mean percentage of phagocytizing PMNs was significantly different ( P < 0.0001) for HF and LF bulls, 28.44 and 59.59%, respectively. Furthermore, any introduced perturbations in the inherent sperm glycan arrangements promoted phagocytosis by PMNs. For example, after in vitro capacitation the mean phagocytosis rate (MPR) rate in spermatozoa from HF bulls significantly increased to 66.49% ( P < 0.01). Likewise, the MPR increased to 70.63% ( p < 0.01) after O-glycosidase & α2-3,6,8,9 Neuraminidase A treatment of spermatozoa from HF bulls. Moreover, the percentage of PMNs forming neutrophil extracellular traps (NETs) was significantly higher, 41.47% when exposed to spermatozoa from LF bulls vis-à-vis the spermatozoa from HF bulls, 15.46% ( P < 0.0001). This is a pioneer report specifically demonstrating the role of O-linked glycans in the immune responses mounted against spermatozoa. Nevertheless, further studies are warranted to provide the measures to diagnose the sub-fertile phenotype thus preventing the losses incurred by incorrect selection of morphologically normal sperm in the AI/IVF reproduction techniques., (Copyright © 2020 Batra, Dagar, Nayak, Kumaresan, Kumar and Datta.)

It has been reported that neutrophil extracellular traps (NETs) impair wound healing in diabetes and that inhibiting NET generation (NETosis) improves wound healing in diabetic mice. Gonadotropin-releasing hormone (GnRH) agonists are associated with a greater risk of diabetes. However, the role of GnRH in diabetic wound healing is unclear. We determined whether GnRH-promoted NETosis and induced more severe and delayed diabetic wound healing. A mouse model of diabetes was established using five injections with streptozotocin. Mice with blood glucose levels >250 mg/dL were then used in the experiments. GnRH agonist treatment induced delayed wound healing and increased NETosis at the skin wounds of diabetic mice. In contrast, GnRH antagonist treatment inhibited GnRH agonist-induced delayed wound healing. The expression of NETosis markers PAD4 and citrullinated histone H3 were increased in the GnRH-treated diabetic skin wounds in diabetic mice and patients. In vitro experiments also showed that neutrophils expressed a GnRH receptor and that GnRH agonist treatment increased NETosis markers and promoted phorbol myristate acetate (PMA)-induced NETosis in mouse and human neutrophils. Furthermore, GnRH antagonist treatment suppressed the expression of NETosis markers and PMA-induced NETosis, which were increased by GnRH treatment. These results indicated that GnRH-promoted NETosis and that increased NETosis induced delayed wound healing in diabetic skin wounds. Thus, inhibition of GnRH might be a novel treatment of diabetic foot ulcers.

Neutrophil extracellular trap (NET) is one of the first-line defenses against microbes. Under certain circumstances, however, it also plays an aggravating factor in diverse inflammation-related diseases including cancers and vascular diseases. Our aim is to develop a new method to detect NET in cells and tissues using a DNA-specific fluorescence probe CDr15. CDr15 was characterized to be impermeable to the cell membranes and to emit a strong fluorescence in association with extracellular DNAs in NET. Due to these properties, CDr15 was successfully shown to quantify NETs in vitro and to be applicable for real-time monitoring NET formation in PMA-stimulated neutrophils. Even in formaldehyde-fixed tumor specimens, CDr15 could detect NETs spreading around cancer cells. Compared with DAPI and SYTOX DNA dyes, CDr15 showed a lower level of background fluorescence and a higher specificity in NET detection. Based on these results, we propose CDr15 as a novel marker of NET to be applicable in experimental and clinical studies., Competing Interests: Declaration of competing interest The authors declare no conflict of interest that might be construed to influence the results or interpretation of their manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Talc, a hydrous magnesium silicate, often used for genital hygiene purposes, is associated with ovarian carcinoma in case-control studies. Its potential to cause inflammation, injury, and functional changes in cells has been described. A complication of such studies is that talc preparations may be contaminated with other materials. A previous study by (Beck et al. Toxicol Appl Pharmacol 87:222-34, 1987) used a hamster model to study talc and granite dust exposure effects on various biochemical and cellular inflammatory markers. Our current study accessed key materials used in that 1987 study; we re-analyzed the original talc dust with contemporary scanning electron microscopy and energy dispersive x-ray analysis (SEM/EDX) for contaminants. We also examined the original bronchoalveolar lavage (BAL) cells with polarized light microscopy to quantify cell-associated birefringent particles to gain insight into the talc used., Results: SEM/EDX analyses showed that asbestos fibers, quartz, and toxic metal particulates were below the limits of detection in the original talc powder. However, fibers with aspect ratios ≥3:1 accounted for 22% of instilled material, mostly as fibrous talc. Talc (based on Mg/Si atomic weight % ratio) was the most abundant chemical signature, and magnesium silicates with various other elements made up the remainder. BAL cell counts confirmed the presence of acute inflammation, which followed intratracheal instillation. Measurements of cell associated birefringent particles phagocytosis revealed significant differences among talc, granite, and control exposures with high initial uptake of talc compared to granite, but over the 14-day experiment, talc phagocytosis by lavaged cells was significantly less than that of granite. Phagocytosis of talc fibers by macrophages was observed, and birefringent particles were found in macrophages, neutrophils, and multinucleate giant cells in lavaged cells from talc-exposed animals., Conclusion: Our data support the contention that talc, even without asbestos and other known toxic contaminants, may elicit inflammation and contribute to lung disease. Our findings support the conclusions of (Beck et al. Toxicol Appl Pharmacol 87:222-34, 1987) study. By analyzing particulate exposures with polarized light microscopy and SEM/EDX, fibrous talc was identified and a distinctive pattern of impaired particulate ingestion was demonstrated.

Competing Interests: Competing interests: None declared.

Hydrogen sulfide (H 2 S) is a toxic air pollutant that causes immune damage. Recent studies have found that neutrophil extracellular trap (NET) formation is one way in which neutrophils exert immune functions. In addition, the formation of NETs is also related to thrombosis and autoimmune diseases. Recent studies have shown that miRNAs are involved in the regulation of a variety of pathophysiological processes. Here, we investigated the role of H 2 S in regulating the formation of NETs by affecting miR-16-5p. Our study established an in vitro H 2 S exposure model for neutrophils using phorbol-myristate-acetate (PMA) to induce NET formation. We observed the morphological changes of cells with scanning electron microscopy and fluorescence microscopy. Then, the content of extracellular DNA and the expression of MPO and NE in each group were detected. The results showed that H 2 S inhibited the formation of NETs. The expression of miR-16-5p and its target genes PiK3R1 and RAF1 was then measured by qRT-PCR. H 2 S upregulated miR-16-5p and inhibited expression of the target genes PiK3R1 and RAF1, and it subsequently inhibited the Pi3K/AKT and ERK pathways and decreased respiratory burst levels. Furthermore, H 2 S attenuated inositol 1,4,5-trisphosphate receptor (IP3R)-mediated endoplasmic reticulum calcium outflow as well as autophagy caused by PMA. This study enriches H 2 S immunotoxicity research and provides a possible solution for the treatment of NET-related diseases., Competing Interests: Declaration of competing interest No conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Neutrophils release neutrophil extracellular traps (NETs) in response to numerous pathogenic microbes as the last suicidal resource (NETosis) in the fight against infection. Apart from the host defense function, NETs play an essential role in the pathogenesis of various autoimmune and inflammatory diseases. Therefore, understanding the molecular mechanisms of NETosis is important for regulating aberrant NET release. The initiation of NETosis after the recognition of pathogens by specific receptors is mediated by an increase in intracellular Ca 2+ concentration, therefore, the use of Ca 2+ ionophore A23187 can be considered a semi-physiological model of NETosis. Induction of NETosis by various stimuli depends on reactive oxygen species (ROS) produced by NADPH oxidase, however, NETosis induced by Ca 2+ ionophores was suggested to be mediated by ROS produced in mitochondria (mtROS). Using the mitochondria-targeted antioxidant SkQ1 and specific inhibitors of NADPH oxidase, we showed that both sources of ROS, mitochondria and NADPH oxidase, are involved in NETosis induced by A23187 in human neutrophils. In support of the critical role of mtROS, SkQ1-sensitive NETosis was demonstrated to be induced by A23187 in neutrophils from patients with chronic granulomatous disease (CGD). We assume that Ca 2+ -triggered mtROS production contributes to NETosis either directly (CGD neutrophils) or by stimulating NADPH oxidase. The opening of the mitochondrial permeability transition pore (mPTP) in neutrophils treated by A23187 was revealed using the electron transmission microscopy as a swelling of the mitochondrial matrix. Using specific inhibitors, we demonstrated that the mPTP is involved in mtROS production, NETosis, and the oxidative burst induced by A23187., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest concerning this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Extracellular DNA (eDNA) plays an important role in both the aggregation of bacteria and in the interaction of the resulting biofilms with polymorphonuclear leukocytes (PMNs) during an inflammatory response. Here, transmission electron and confocal scanning laser microscopy were used to examine the interaction between biofilms of Pseudomonas aeruginosa and PMNs in a murine implant model and in lung tissue from chronically infected cystic fibrosis patients. PNA FISH, DNA staining, labeling of PMN DNA with a thymidine analogue and immunohistochemistry were applied to localize bacteria, eDNA, PMN-derived eDNA, PMN-derived histone H3 (H3), neutrophil elastase (NE) and citrullinated H3 (citH3). Host-derived eDNA was observed surrounding bacterial biofilms but not within the biofilms. H3 localized to the lining of biofilms while NE was found throughout biofilms. CitH3, a marker for neutrophil extracellular traps (NETs) was detected only sporadically indicating that most host-derived eDNA in vivo was not a result of NETosis. Together these observations show that, in these in vivo biofilm infections with P. aeruginosa, the majority of eDNA is found external to the biofilm and derives from the host., (© FEMS 2020.)

Purpose : To evaluate whether glucocorticoids affect the prognosis of fungal keratitis by inhibiting the formation of neutrophil extracellular traps (NETs). Methods : A mouse model of Candida albicans ( C.albicans ) keratitis was established. Animals were randomly assigned to treatment with 0.1% dexamethasone (DXM) eye drops and normal saline (3 times each day for 3 days). The effects of DXM on fungal keratitis were assessed using clinical scores, immunofluorescence staining, histopathological examination, scanning electron microscopy (SEM), and pathogen burden assay. All the analyses were performed using SPSS software version 17.0 (Chicago, IL). Results : NETs formation was noteworthy in the cornea lesions of fungal keratitis. The clinical score of the DXM-treated group was significantly higher than that of the control group ( P < .05). During the measured period, corneas from DXM-treated group contained more C.albicans than those from the control group by histology and pathogen burden assay. Compared with the control group, the DXM treatment group had a higher depth of infiltration of C.albicans . Histological and immunofluorescence staining showed that there were fewer neutrophils in the cornea focus of DXM-treated group ( P < .05）, and the number of NETs formed in scrapings from control group was higher than that in the DXM treatment group on day 3 ( P < .05, Z = -3.56)) and day 5 ( P < .05, Z = -3.69). In a similar amount of cell scraping, the NETs of neutrophils formation from the DXM-treated group were also less than that from the control group. Conclusion : Our results indicated that NETs were involved in the immune response in C.albicans keratitis. Glucocorticoids may exacerbate fungal keratitis not only by increasing fungal aggressivity and reducing the infiltration of neutrophils but also by inhibiting the formation of NETs.

Objective: Neutrophils are key immune cells participating in host defense through several mechanisms, including the formation of neutrophil extracellular traps (NETs). This study was undertaken to investigate the role of neutrophils in juvenile dermatomyositis (JDM)., Methods: Electron microscopy was used to identify neutrophils in tissue. NETs were also imaged using fluorescence microscopy and quantified using a myeloperoxidase-DNA enzyme-linked immunosorbent assay (ELISA) in plasma obtained from healthy children (n = 20), disease controls (n = 29), JDM patients (n = 66), and JDM patients with history of calcifications (n = 20). Clinical data included disease activity scores and complement C4 levels. Levels of immune complexes (ICs) and calprotectin were analyzed using ELISA., Results: Using electron microscopy, neutrophils were found to infiltrate affected muscle tissue, engulfing deposited calcium crystals. Uptake of the crystals led to neutrophil activation (P < 0.01) and subsequent phosphatidylinositol 3-kinase- and NADPH oxidase-dependent but peptidylarginine deiminase 4-independent formation of NETs, which contained mitochondrial DNA (P < 0.05), as confirmed in vivo (P < 0.001) and in vitro (P < 0.01). Peripheral NET levels were associated with calcinosis (P = 0.01), ICs (P = 0.008), and interleukin-8 levels (P = 0.004). Children with JDM had impaired NET clearance (P = 0.01), associated with autoantibody profiles including melanoma differentiation-associated protein 5 (P = 0.005), and depressed complement C4 levels (r = -0.72, P = 0.002). Furthermore, children with JDM showed evidence of neutrophil activation, with elevated levels of peroxidase activity (P = 0.02) and calprotectin (P < 0.01), which were associated with disease activity (P = 0.007), and dyslipidemia (odds ratio 4.7, P < 0.05)., Conclusion: We found novel mechanisms of both calcium crystal-mediated neutrophil activation and cell death in JDM pathophysiology. Targeting this pathway may reduce the frequency and extent of calcinosis, as well as prevent long-term development of comorbidities, including atherosclerosis., (© 2019, American College of Rheumatology.)

The process of neutrophil apoptosis has an important role in the resolution of acute inflammation. Apoptotic cell death is characterized by a coordinated sequence of cellular alterations that serve to uncouple neutrophil effector functions whilst maintaining plasma membrane integrity. In this way the release on neutrophil intracellular contents, including proteases, glycosidases, and reactive oxygen species, is limited during apoptosis. In addition, plasma membrane alterations associated with neutrophil apoptosis provide molecular cues that enable recognition by phagocytic cells, including macrophages. The recognition and uptake of apoptotic neutrophils by macrophages dampens proinflammatory responses to pathogen- or damage-associated molecular patterns and triggers release of proresolution mediators, that further promote resolution of inflammation. The key cellular and molecular events that act to control neutrophil apoptosis and subsequent macrophage phagocytosis have been characterized by in vitro studies, unveiling potential therapeutic targets for the manipulation of these regulatory pathways. In this chapter, we outline some of the key assays that are used to assess neutrophil apoptosis in vitro, together with methods to assess activation of the apoptotic machinery and phagocytic clearance of apoptotic neutrophils.

Rapid activation of resident glia occurs after spinal cord injury. Somewhat later, innate and adaptive immune responses occur with the invasion of peripheral immune cells into the wound site. The activation of resident and peripheral immune cells has been postulated to play harmful as well as beneficial roles in the regenerative process. Mauthner cells, large identifiable neurons located in the hindbrain of most fish and amphibians, provided the opportunity to study the morphological relationship between reactive cells and Mauthner axons (M-axons) severed by spinal cord crush or by selective axotomy. After crossing in the hindbrain, the M-axons of adult goldfish, Carassius auratus, extend the length of the spinal cord. Following injury, the M-axon undergoes retrograde degeneration within its myelin sheath creating an axon-free zone (proximal dieback zone). Reactive cells invade the wound site, enter the axon-free dieback zone and are observed in the vicinity of the retracted M-axon tip as early as 3 hr postinjury. Transmission electron microscopy allowed the detection of microglia/macrophages and granulocytes, some of which appear to be neutrophil-like, at each of these locations. We believe that this is the first report of the invasion of such cells within the myelin sheath of an identifiable axon in the vertebrate central nervous system (CNS). We speculate that microglia/macrophages and granulocytes that are attracted within a few hours to the damaged M-axon are part of an inflammatory response that allows phagocytosis of debris and plays a role in the regenerative process. Our results provide the baseline from which to utilize immunohistochemical and genetic approaches to elucidate the role of non-neuronal cells in the regenerative process of a single axon in the vertebrate CNS., (© 2019 Wiley Periodicals, Inc.)

Neutrophils respond differently to violations of the body's physiological barriers during infections. Extracellular traps comprise one of the mechanisms used by these cells to reduce the spread of pathogens to neighboring tissues, as well as ensure a high concentration of antimicrobial agents at the site of infection. To date, this innate defense mechanism has not been previously demonstrated in neutrophils of cats exposed to Toxoplasma gondii. The aim of this study was to characterize the in vitro release of neutrophil extracellular traps (NETs) when neutrophils isolated from cats were exposed to T. gondii. First, cellular viability was tested at different time points after parasite exposure. The production of reactive oxygen species (ROS) and lactate dehydrogenase and the amount of extracellular DNA were quantified. In addition, the number of parasites associated with neutrophils was determined, and the observed NETs formed were microscopically characterized. Results showed that (i) in culture, neutrophils isolated from cats presented diminished cellular viability after 4 h of incubation, and when neutrophils were incubated with T. gondii, they displayed cytotoxic effects after 3 h of interaction; (ii) neutrophils were able to release structures composed of DNA and histones, characterized as NETs under optical, immunofluorescence, and electron scanning microscopy, when stimulated with T. gondii; (iii) only 11.4% of neutrophils were able to discharge NETs during 3 h of incubation; however, it was observed through extracellular quantification of DNA that this small number of cells were able to display different behavior compared to a negative control (no parasite) group; (iv) significant differences in ROS production were observed in neutrophils exposed to T. gondii. In conclusion, our results showed that neutrophils isolated from cats exposed to T. gondii release structures composed of DNA and histones, similar to what has already been described in other neutrophil species infected with the parasite., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Neutrophils contribute to low oxygen availability at inflammatory sites through the generation of reactive oxidants. They are also functionally affected by hypoxia, which delays neutrophil apoptosis. However, the eventual fate of neutrophils in hypoxic conditions is unknown and this is important for their effective clearance and the resolution of inflammation. We have monitored the survival and function of normal human neutrophils exposed to hypoxia over a 48 h period. Apoptosis was delayed, and the cells remained intact even at 48 h. However, hypoxia promoted significant changes in neutrophil morphology with the appearance of many new cytoplasmic vesicles, often containing cell material, within 5 hours of exposure to low O 2 . This coincided with an increase in LC3B-II expression, indicative of autophagosome formation and an autophagy-like process. In hypoxic conditions, neutrophils preferentially lost myeloperoxidase, a marker of azurophil granules. Short-term (2 h) hypoxic exposure resulted in sustained potential to generate superoxide when O 2 was restored, but the capacity for oxidant production was lost with longer periods of hypoxia. Phagocytic ability was unchanged by hypoxia, and bacterial killing by neutrophils in both normoxic and hypoxic conditions was substantially diminished after 24 hours. However, pre-exposure to hypoxia resulted in an enhanced ability to kill bacteria by oxidant-independent mechanisms. Our data provide the first evidence for hypoxia as a driver of neutrophil autophagy that can influence the function and ultimate fate of these cells, including their eventual clearance and the resolution of inflammation., (©2019 Society for Leukocyte Biology.)

The last few decades of protease research has confirmed that a number of important biological processes are strictly dependent on proteolysis. Neutrophil elastase (NE) is a critical protease in immune response and host defense mechanisms in both physiological and disease-associated conditions. Particularly, NE has been identified as a promising biomarker for early diagnosis of lung inflammation. Recent studies have shown an increasing interest in developing methods for NE activity imaging both in vitro and in vivo. Unlike anatomical imaging modalities, functional molecular imaging, including enzymatic activities, enables disease detection at a very early stage and thus constitutes a much more accurate approach. When combined with advanced imaging technologies, opportunities arise for measuring imbalanced proteolytic activities with unprecedented details. Such technologies consist in building the highest resolved and sensitive instruments as well as the most specific probes based either on peptide substrates or on covalent inhibitors. This review outlines strengths and weaknesses of these technologies and discuss their applications to investigate NE activity as biomarker of pulmonary inflammatory diseases by imaging.

Cancer-related inflammation impacts significantly on cancer development and progression. From early stages, neutrophils and macrophages are drawn to pre-neoplastic cells in the epidermis, but before directly interacting, they must first breach the underlying extracellular matrix barrier layer that includes the basement membrane. Using several different skin cancer models and a collagen I-GFP transgenic zebrafish line, we have undertaken correlative light and electron microscopy (CLEM) to capture the moments when immune cells traverse the basement membrane. We show evidence both for active proteolytic burrowing and for the opportunistic use of pre-existing weak spots in the matrix layer. We show that these small holes, as well as much larger, cancer cell-generated or wound-triggered gaps in the matrix barrier, provide portals for immune cells to access cancer cells in the epidermis and thus are rate limiting in cancer progression., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Objective: Neutrophil extracellular traps (NETs) are a recently discovered antimicrobial mechanism used by neutrophils that have been proposed as an intervention in the pathogenesis of periodontitis. The objective of our study was to characterize the expression of NETs in gingival tissues with periodontitis and controls and to compare the expression of these traps in gingival tissue samples of patients with gingivitis and periodontitis., Material and Methods: An observational cross-sectional study was conducted on patients with periodontitis, gingivitis, and controls that needed tooth extraction. Gingival tissue biopsies were gathered after clinical examination and tooth extraction. Electron microscopy and immunofluorescence were performed to characterize NETs, comparing periodontitis and control patients. Immunohistochemical analysis was performed to quantify neutrophil extracellular trap expression through extracellular citrullinated histone H3 and myeloperoxidase in biopsies from patients with gingivitis and periodontitis., Results: Thirteen biopsies were gathered from 13 patients: five gingivitis, six periodontitis, and two controls. Electron microscopy and immunofluorescence imaging showed greater expression of neutrophils present in periodontal inflamed tissue compared with controls. Release of nuclear content to the extracellular space was observed, compatible with the formation of NETs. The expression of citrullinated histone H3 was higher in gingivitis samples than periodontitis samples (P = 0.0106). Myeloperoxidase expression was higher in periodontitis than gingivitis, but without achieving statistical significance., Conclusion: Neutrophil extracellular traps were found in tissue samples of periodontitis as extracellular components of chromatin, along with neutrophil enzymes, that were not present in healthy controls. The comparison of NETs expression in periodontitis and gingivitis showed higher expression in gingivitis, associating them to acute phases of the periodontal inflammatory process., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Precise spatiotemporal regulation of leukocyte extravasation is key for generating an efficient immune response to injury or infection. The integrins LFA-1(CD11a/CD18) and Mac-1(CD11b/CD18) play overlapping roles in neutrophil migration because they bind the same as well as different ligands in response to extracellular signaling. Using two-photon intravital imaging and transmission electron microscopy, we observed the existence of preferred sites for neutrophil entrance into the endothelial cell monolayer and exit from the basement membrane and pericyte sheath during neutrophil extravasation, namely, hotspots I and II, by elucidating distinctive roles of LFA-1 and Mac-1. To penetrate the vascular endothelium, neutrophils must first penetrate the endothelial cell layer through hotspot I (i.e., the point of entry into the endothelium). Neutrophils frequently remain in the space between the endothelial cell layer and the basement membrane for a prolonged period (>20 min). Subsequently, neutrophils penetrate the basement membrane and pericyte sheath at hotspot II, which is the final stage of exiting the vascular endothelium. To further investigate the roles of LFA-1 and Mac-1, we newly generated LFA-1 FRET (CD11a-YFP/CD18-CFP) mice and Mac-1 FRET (CD11b-YFP/CD18-CFP) mice. Using both FRET mice, we were able to determine that LFA-1 and Mac-1 distinctly regulate the neutrophil extravasation cascade. Our data suggest that the vascular endothelium functions as a double-layered barrier in the steps of neutrophil extravasation. We propose that the harmonized regulation of neutrophil penetration through the endothelium via hotspots I and II may be critical for vascular homeostasis during inflammation.

Analysis of trisomy 8 cells and the chromosome-specific fluorescence in situ hybridization (FISH) signals on the ring-shaped nucleus of a neutrophil reveal that homologue chromosomes orient in diametrical opposition to each other. This positioning results in a separation of the two haploid sets of parental chromosomes organized as two exclusive groups. These two groups impart the nucleus a symmetry that fortifies immune protection by accelerating chemotaxis. The ring form of the nucleus is a legacy of the orientation of chromosomes as a rosette during metaphase and telophase stages. A dual control maintains this spatial order: (1) chromosomes are tethered to the centriole all through the cell cycle, and (2) during their circular orientation in telophase the chromosomes bind to each other with lamins, which reorganize the nuclear membrane of the daughter nuclei, generating an additional anchorage. Here, chromosomes serve as temporary packets to assure proper distribution of the nuclear DNA during mitosis. The remainder time of the cell cycle the chromosomes are chained together across the telomeres, allowing a continuous sequence of genes of the two genomes, maternal and paternal, thus facilitating easy reading of the gene sequence. Exceptions to these orders are either physiological and temporary, or pathological and disease causing.

Hypertension (HTN), i.e. abnormally high blood pressure, is a major risk factor for heart attack, stroke, and kidney failure. The Epithelial Sodium Channel (ENaC), one of the main transporters regulates blood pressure by tightly controlling the sodium reabsorption along the nephron. Recently, we have shown an α-ENaC overexpression in platelets from hypertensive patients compared to platelets from normotensive subjects, suggesting it makes a contribution to the activation state of platelets and the physiopathology of hypertension. However, the involvement of the α-ENaC localized in neutrophils to this disease remains unknown. Neutrophils are the first leukocytes to be recruited to an inflammatory site and are equipped with a strong ability to eliminate intra- or extracellular pathogens using reactive oxygen species or antibacterial proteins contained in their granules. Using the Western blotting (Wb), flow cytometry, and qRT-PCR approaches; we determined α-ENaC neutrophil overexpression at the protein and messenger RNA (mRNA) levels. By confocal and cytometry analysis, we determined the α-ENaC distribution and the heterogeneity of HTN neutrophils population, respectively. Immunoprecipitation and Wb assays demonstrated the presence of both α-ENaC and caveolin-1 phosphorylated forms, compared with neutrophils from healthy individuals. Although neutrophils from hypertensive subjects circulating in an activated state were exhibiting important oxidative stress and modifications registered by confocal, atomic force, and scanning electron microscope, they conserved their defense capabilities. The features described above for neutrophils from hypertensive patients could be attributed to α-ENaC overexpression, as its drug inhibition diminished their activation state modulating the actin cytoskeleton reorganization triggered during the activation process., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chronic inflammation drives the progression of colorectal cancer (CRC). Increased expression of interleukin (IL)-17A is associated with poor prognosis, and IL-17A blockade curbs tumor progression in preclinical models of CRC. Here we examined the impact of IL-1 signaling, a key regulator of the IL-17 pathway, in different cell types within the CRC microenvironment. Genetic deletion of the IL-1 receptor (IL-1R1) in epithelial cells alleviated tumorigenesis in the APC model of CRC, demonstrating a cell-autonomous role for IL-1 signaling in early tumor seed outgrowth. T cell specific ablation of IL-1R1 decreased tumor-elicited inflammation dependent on IL-17 and IL-22, thereby reducing CRC progression. The pro-tumorigenic roles of IL-1 were counteracted by its effects on myeloid cells, particularly neutrophils, where IL-1R1 ablation resulted in bacterial invasion into tumors, heightened inflammation and aggressive CRC progression. Thus, IL-1 signaling elicits cell-type-specific responses, which, in aggregate, set the inflammatory tone of the tumor microenvironment and determine the propensity for disease progression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Determination of the structure of human neutrophil (PMN) flavocytochrome b (Cytb) is a necessary step for the understanding of the structure-function essentials of NADPH oxidase activity. This understanding is crucial for structure-driven therapeutic approaches addressing control of inflammation and infection. Our work on purification and sample preparation of Cytb has facilitated progress toward the goal of structure determination. Here we describe exploiting immunoaffinity purification of Cytb for initial examination of its size and shape by a combination of classical and cryoelectron microscopic (EM) methods. For these evaluations, we used conventional negative-stain transmission electron microscopy (TEM) to examine both detergent-solubilized Cytb as single particles and Cytb in phosphatidylcholine reconstituted membrane vesicles as densely packed random, partially ordered, and subcrystalline arrays. In preliminary trials, we also examined single particles by cryoelectron microscopy (cryoEM) methods. We conclude that Cytb in detergent and reconstituted in membrane is a relatively compact, symmetrical protein of about 100 Å in maximum dimension. The negative stain, preliminary cryoEM, and crude molecular models suggest that the protein is probably a heterotetramer of two p22 phox and gp91 phox subunits in both detergent micelles and membrane vesicles. This exploratory study also suggests that high-resolution 2D electron microscopic approaches may be accessible to human material collected from single donors.

Neutrophil nuclear morphology has historically been used in haematology for neutrophil identification and characterisation, but its exact role in neutrophil function has remained enigmatic. During maturation, segmentation of the neutrophil nucleus into its mature, multi-lobulated shape is accompanied by distinct changes in nuclear envelope composition, resulting in a unique nucleus that is believed to be imbued with extraordinary nuclear flexibility. As a rate-limiting factor for cell migration, nuclear morphology and biomechanics are particularly important in the context of neutrophil migration during immune responses. Being an extremely plastic and fast migrating cell type, it is to be expected that neutrophils have an especially deformable nucleus. However, many questions still surround the dynamic capacities of the neutrophil nucleus, and which nuclear and cytoskeletal elements determine these dynamics. The biomechanics of the neutrophil nucleus should also be considered for their influences on the production of neutrophil extracellular traps (NETs), given this process sees the release of chromatin "nets" from nucleoplasm to extracellular space. Although past studies have investigated neutrophil nuclear composition and shape, in a new era of more sophisticated biomechanical and genetic techniques, 3D migration studies, and higher resolution microscopy we now have the ability to further investigate and understand neutrophil nuclear plasticity at an unprecedented level. This review addresses what is currently understood about neutrophil nuclear structure and its role in migration and the release of NETs, whilst highlighting open questions surrounding neutrophil nuclear dynamics.

Background and Objective: Bacterial challenge is constant in the oral cavity. To contain the commensal biofilm, partly activated neutrophils are continuously recruited as part of a normal physiologic process, without exposing the host to the harmful effect of a fully active neutrophil response. This intermediate immune state has been termed para-inflammation, as opposed to the fully activated proinflammatory state in oral disease. Directly visualizing these cells and their components via transmission electron microscopy (TEM) enhances our understanding of neutrophil activation state differences in oral health and disease, as obtained from molecular studies. The aim of this study was to describe the morphology of the para-inflammatory phenotype displayed by oral neutrophils in health, and compare it to the morphology of the naïve blood neutrophil, and the proinflammatory oral neutrophils in chronic periodontitis. This morphology was characterized by differences in granule content, phagosome content and cytoplasm and nuclear changes. We also examined the morphological changes induced in naïve neutrophils, which were stimulated in vitro by bacteria, and in oral neutrophils in full tissue samples in vivo., Material and Methods: Neutrophils were isolated from blood and saliva samples of patients with chronic periodontitis and healthy individuals. The cells were viewed under TEM and analyzed in imaging software examining granularity, cytoplasm density, euchromatin amount in the nucleus and phagosome content. A separate cohort of blood neutrophils was incubated with Streptococcus oralis and analyzed under TEM in the same manner. Gingival tissue samples were obtained from patients with chronic periodontitis and viewed under TEM, with the neutrophils present analyzed in the same manner., Results: The proinflammatory cells showed less granulation, lighter cytoplasm and higher amount of nuclear euchromatin. These changes were accentuated in the proinflammatory oral chronic periodontitis neutrophils compared to the para-inflammatory oral health neutrophils. The oral chronic periodontitis neutrophils also contained more phagosomes and had more phagosomes containing undigested bacteria. These changes were partially reproduced in the naïve blood cells after exposing them to S. oralis. The neutrophils in the gingival tissues displayed naïve morphology when viewed in the blood vessels and gradually showed proinflammatory morphological changes as they traveled through the connective tissue into the epithelium., Conclusion: Oral neutrophils display morphological changes consistent with partial or full activation, corresponding to their para- or proinflammatory states. These changes can also be induced in naïve cells by incubating them with commensal bacteria. Neutrophils change their morphology towards an activated state as they travel through the gingival tissue., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The use of fluorescent stains to visually investigate eukaryotic and/or prokaryotic cells is increasing quickly and manuscripts within all areas of research publish results using fluorescent staining techniques. However, in contrast to literature on traditional histological staining techniques, the literature on fluorescent stains and staining techniques does not offer as good an illustration of cellular morphology. The aim of this guideline is to illustrate different fluorescent stains and staining techniques for imaging immune cells, in particular the polymorphonuclear leukocytes (PMNs), in combination with infecting bacteria as seen in chronic bacterial infections. Thereby providing the first guideline for the morphology and identification of fluorescently stained PMNs, bacteria and biofilm., (© 2018 APMIS. Published by John Wiley & Sons Ltd.)

Nickel (II) nitrate hexahydrate (Ni) is a common heavy metal material in battery manufacturing, electroplating alloy parts and ceramic staining, therefore we frequently contact with Ni-related products in daily life. In this study, we aimed to investigate the effects of Ni on neutrophils extracellular traps (NETs) release by canine polymorphonuclear neutrophils (PMNs). The structure of Ni-induced NETs was observed by fluorescence confocal microscopy. Ni-triggered NETs release was quantified by Pico Green ® and fluorescence microplate reader. In addition, the inhibitors of NADPH oxidase, ERK1/2-, p38 - signaling pathways were used for preliminary inquiry into the potential mechanism of this process. The results showed that Ni markedly triggered the formation of NETs-like structures, and these structures were mainly consisted of DNA decorated with NE and MPO. Furthermore, quantification experiments showed that Ni significantly increased NETs formation compared to control groups. These results forcefully confirmed that nickel nitrate possesses the ability to induce NETs formation. However, inhibiting the NADPH oxidase, ERK1/2- and p38 MAPK-signaling pathways did not significantly change the quantitation of Ni-induced NETs release. To our knowledge, this study is the first report of Ni-triggered NETs release in vitro, which might provide an entirely new mechanism of several diseases and health issues induced by nickel overexposure., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process.

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain-like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase-independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8-dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8-dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant Staphylococcus aureus (MRSA) infection., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)