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Aims: Extracellular vesicles (EVs) are membrane-derived vesicles that mediate intercellular communications. Neutrophils produce different subtypes of EVs during inflammatory responses. Neutrophil-derived trails (NDTRs) are generated by neutrophils migrating toward inflammatory foci, whereas neutrophil-derived microvesicles (NDMVs) are thought to be generated by neutrophils that have arrived at the inflammatory foci. However, the physical and functional characteristics of neutrophil-derived EVs are incompletely understood. In this study, we aimed to investigate the differences between NDTRs and NDMVs. Methods: The generation of neutrophil-derived EVs were visualized by live-cell fluorescence images and the physical characteristics were further analyzed using nanotracking analysis assay, scanning electron microscopic analysis, and marker expressions. Functional characteristics of neutrophil-derived EVs were analyzed using assays for bactericidal activity, monocyte chemotaxis, phenotype polarization of macrophages, and miRNA sequencing. Finally, the effects of neutrophil-derived EVs on the acute and chronic inflammation were examined in vivo . Results: Both EVs share similar characteristics including stimulators, surface marker expression, bactericidal activity, and chemoattractive effect on monocytes via MCP-1. However, the integrin-mediated physical interaction was required for generation of NDTRs whereas NDMV generation was dependent on PI3K pathway. Interestingly, NDTRs contained proinflammatory miRNAs such as miR-1260, miR-1285, miR-4454, and miR-7975, while NDMVs contained anti-inflammatory miRNAs such as miR-126, miR-150, and miR-451a. Although both EVs were easily uptaken by monocytes, NDTRs enhanced proinflammatory macrophage polarization whereas NDMVs induced anti-inflammatory macrophage polarization. Moreover, NDTRs showed protective effects against lethality in a murine sepsis model and pathological changes in a murine chronic colitis model. Conclusion: These results suggest that NDTR is a proinflammatory subtype of neutrophil-derived EVs distinguished from NDMV., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Rationale: Neutrophils are key effectors in the host's immune response to sepsis. Excessive stimulation or dysregulated neutrophil functions are believed to be responsible for sepsis pathogenesis. However, the mechanisms regulating functional plasticity of neutrophils during sepsis have not been fully determined., Objectives: We investigated the role of autophagy in neutrophil functions during sepsis in patients with community-acquired pneumonia., Methods: Neutrophils were isolated from patients with sepsis and stimulated with phorbol 12-myristate 13-acetate (PMA). The levels of reactive oxygen species generation, neutrophil extracellular trap (NET) formation, and granule release, and the autophagic status were evaluated. The effect of neutrophil autophagy augmentation was further evaluated in a mouse model of sepsis., Measurements and Main Results: Neutrophils isolated from patients who survived sepsis showed an increase in autophagy induction, and were primed for NET formation in response to subsequent PMA stimulation. In contrast, neutrophils isolated from patients who did not survive sepsis showed dysregulated autophagy and a decreased response to PMA stimulation. The induction of autophagy primed healthy neutrophils for NET formation and vice versa. In a mouse model of sepsis, the augmentation of autophagy improved survival via a NET-dependent mechanism., Conclusions: These results indicate that neutrophil autophagy primes neutrophils for increased NET formation, which is important for proper neutrophil effector functions during sepsis. Our study provides important insights into the role of autophagy in neutrophils during sepsis.

Hypersegmentation of nuclei is considered a distinct characteristic of the antitumoral phenotype of neutrophils. Retinoic acid, a metabolite of retinol, reorganizes and induces segmentation of the nucleus during the differentiation of neutrophils. However, the role of retinoic acid in the phenotype polarization of neutrophils has not been fully established. Here, we investigated the effect of retinoic acid on phenotype polarization of neutrophils. Retinoic acid-induced the hypersegmentation of human neutrophils via retinoic acid receptors and mTOR pathways. Retinoic acid-induced hypersegmented neutrophils enhanced neutrophil extracellular traps (NETs) formation in response to phorbol-12-myristate 13-acetate (PMA) and fMLP (N-Formylmethionine-leucyl-phenylalanine) stimulation, and increased cytotoxicity against various tumor cells. Moreover, retinoic acid treatment attenuated tumor growth in a murine model of tumor. Taken together, these results suggests that retinoic acid induces the phenotype polarization of neutrophils to exert antitumor effects., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background: The hyperinflammation that begins with sepsis is essential for eradicating infection but also causes hypoperfusion and organ failure. To understand the innate immune status of septic patients, the functional and phenotypic changes of neutrophils during sepsis and their clinical implication were studied., Methods: Seventy-four patients who were admitted to intensive care unit due to severe sepsis or septic shock were enrolled. Surface antigens of neutrophils (CD64, CD10, and CD16) were detected by flow cytometry. Respiratory burst activity (RBA) was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate and phorbol-12-myristate-13-acetate. The parameters were serially examined at Days 1 and 8 in septic shock patients., Results: High CD64 and low CD10 and CD16 on Day 1 was associated with sepsis severity (P = 0.003, 0.017, and 0.007, respectively). On Day 1, RBA and CD64 were higher in survivors than in nonsurvivors of septic shock patients (P = 0.012 and 0.027, respectively), and on Day 8, CD10 and CD16 were higher in survivors than in nonsurvivors (P = 0.019 and 0.036, respectively). High RBA and high CD64 on Day 1 showed low 28-day mortality in univariate analysis (P = 0.018 and 0.034, respectively). In multivariate analysis, RBA maintained statistical significance (P = 0.042) but CD64 revealed only a tendency (P = 0.064)., Conclusions: Neutrophil surface antigen (CD64, CD10, and CD16) could reflect sepsis severity. High CD64 expression and high RBA at early phase of sepsis might be associated with better prognosis, whereas high expression of CD10 and CD16 at late phase of sepsis might be associated with better prognosis. © 2015 International Clinical Cytometry Society., (© 2015 International Clinical Cytometry Society.)

Sulfur mustard (2,2'-bis-chloroethyl-sulfide; SM) has been a military threat since the World War I. The emerging threat of bioterrorism makes SM a major threat not only to military but also to civilian world. SM injury elicits an inflammatory response characterized by infiltration of neutrophils. Although SM was reported to prime neutrophils, the mechanism has not been identified yet. In the present study, we investigated the mechanism of SM-induced priming in human neutrophils. SM increased [Ca(2+)](i) in human neutrophils in a concentration-dependent fashion. Transient receptor potential melastatin (TRPM) 2 inhibitors (clotrimazole, econazole and flufenamic acid) and silencing of TRPM2 by shRNA attenuated SM-induced [Ca(2+)](i) increase. SM primed degranulation of azurophil and specific granules in response to activation by fMLP as previously reported. SB203580, an inhibitor of p38 MAPK, inhibited SM-induced priming. Neither PD98057, an ERK inhibitor, nor SP600215, a JNK inhibitor, inhibited SM-induced priming. In addition, SM enhanced phosphorylation of NF-kB p65 and release of TNF-α, interleukin (IL)-6 and IL-8. SB203580 inhibited SM-induced NF-kB phosphorylation and cytokine release. These results suggest the involvement of TRPM2/p38 MAPK pathway in SM-induced priming and cytokines release in neutrophils., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs) are membrane-derived heterogeneous vesicles that mediate intercellular communications. They have recently been considered as ideal vehicles for drug-delivery systems, and immune cells are suggested as a potential source for drug-loaded EVs. In this study, we investigated the possibility of neutrophils as a source for drug-loaded EVs. Neutrophil-like differentiated human promyelocytic leukemia cells (dHL-60) produced massive amounts of EVs within 1 h. The dHL-60 cells are also easily loaded with various cargoes such as antibiotics (penicillin), anticancer drug (paclitaxel), chemoattractant (MCP-1), miRNA, and Cas9. The EVs derived from the dHL-60 cells showed efficient incorporation of these cargoes and significant effector functions, such as bactericidal activity, monocyte chemotaxis, and macrophage polarization. Our results suggest that neutrophils or neutrophil-like promyelocytic cells could be an attractive source for drug-delivery EVs. [ABSTRACT FROM AUTHOR]

Tumor microenvironments polarize neutrophils to protumoral phenotypes. Here, we demonstrate that the angiotensin converting enzyme inhibitors (ACEis) and angiotensin II type 1 receptor (AGTR1) antagonist attenuate tumor growth via polarization of neutrophils toward an antitumoral phenotype. The ACEis or AGTR1 antagonist enhanced hypersegmentation of human neutrophils and increased neutrophil cytotoxicity against tumor cells. This neutrophil hypersegmentation was dependent on the mTOR pathway. In a murine tumor model, ACEis and AGTR1 antagonist attenuated tumor growth and enhanced neutrophil hypersegmentation. ACEis inhibited tumor-induced polarization of neutrophils to a protumoral phenotype. Neutrophil depletion reduced the antitumor effect of ACEi. Together, these data suggest that the modulation of Ang II pathway attenuates tumor growth via polarization of neutrophils to an antitumoral phenotype. [ABSTRACT FROM PUBLISHER]

Abstract: Phagocytic NADPH oxidase plays a critical role in superoxide generation in macrophage cells. Small GTPases, including Rac1 and Rac2, have been implicated in the regulation of NADPH oxidase activity. Rap1, which has no effect in a cell-free system of oxidase activation, recently has been proven to colocalize with cytochrome b 558. In addition, neutrophils from rap1A-/- mice reduce fMLP-stimulated superoxide production. Here, we tried to determine whether Rap1 also plays a role in the production of superoxide. IgG-opsonized zymosan (IOZ) particles treatment induced Rap1 activation and superoxide generation. Knock-down of Rap1 by si-Rap1 suppressed IOZ-induced superoxide formation. Sh-RhoA also reduced superoxide levels, but 8CPT-2Me-cAMP, an activator of Epac1 (a guanine nucleotide exchange factor (GEF) of Rap1), could recover the levels to the control value. When cells were stimulated by IOZ, Rap1 and Rac1 were translocated to the membrane, and then interacted with p22phox. 8CPT-2Me-cAMP rescued sh-RhoA-induced reduction of the interaction between Rac1 and p22phox, and enhanced lysophosphatidic acid (LPA)-induced increase of their interaction. Moreover, Rac1 activity was increased by both LPA and 8CPT-2Me-cAMP when treated with IOZ particles. Si-Vav2 impaired GTP-Rac1 levels in response to 8CPT-2Me-cAMP/IOZ. Phosphorylation of RhoA activates Rac1 in response to IOZ by the enhanced binding of phospho-RhoA to RhoGDI, leading to the release of Rac1 from the Rac1-RhoGDI complex. In conclusion, IOZ treatment induces Rap1 activation and phosphorylation of RhoA, which in turn cause Rac1 activation and promote Rac1 translocation to the membrane leading to binding with p22phox that activates NADPH oxidase and produces superoxide. [Copyright &y& Elsevier]