Minimal inhibitor concentrations used for NET experiments are shown in blue. of independent experiments with 3 different Glycerol 3-phosphate donors. (C) Increasing doses of inhibitors were added to murine neutrophils before stimulation with LPS. Flavopiridol: 0.025 M; 0.05 M; Glycerol 3-phosphate 0.1 M; 0.2 M; 1M, CAS 577784-91-9: 1 M, CHX: 0.5 g/ ml; 1 g/ ml; 5 g/ ml. Minimal concentrations blocking production of Mip-1 and subsequently used for NET experiments are shown in blue. Efficiency of inhibitors was analyzed by measuring production of Mip-1 in response to LPS stimulation (200 ng/ ml) for 20h.(EPS) pone.0157454.s001.eps (1.0M) GUID:?9A45D1D6-B606-479C-98F9-D96ED7C64009 S2 Fig: No effect of individual or combined inhibitors on NET formation. Inhibitors were used at the following concentrations: Actinomycin D (1 g/ ml), flavopiridol (0.05 M), CAS 577784-91-9 (10 M), CHX (1 g/ ml). (A, B) Human primary neutrophils were treated with transcription/ translation inhibitors as indicated. (A) After inhibitor treatment, cells were Rabbit Polyclonal to HSF2 treated with 50 nM SYTOX green, stimulated with 100 nM PMA and analyzed by measuring emission of SYTOX green every hour in a fluorometer. (B) After inhibitor treatment, neutrophils were treated with 50 nM SYTOX green and infected with opsonized at MOI 5. SYTOX emission was measured every hour in a fluorometer. (C) Representative immunofluorescence pictures of human primary neutrophils treated with combinations of transcription/ translation inhibitors as indicated and stimulated with 100 nM PMA. Cells were stained with PL2-3 (chromatin, red), NE (Neutrophil elastase, green) and Hoechst (blue). (D) Representative pictures of SYTO green/ SYTOX orange assay in human primary neutrophils stimulated with (MOI 5). SYTO green stains all cells, SYTOX orange stains dead cells and extracellular traps.(EPS) pone.0157454.s002.eps (21M) GUID:?71F76911-7B55-409A-B5CF-94E1BEB15811 S1 Table: Numerical values used for quantification. (XLSX) pone.0157454.s003.xlsx (36K) GUID:?C61D6B04-FDC2-4B37-BD17-3FC8AD771F40 S2 Table: Numerical values used for quantification of supplementary data. (XLSX) pone.0157454.s004.xlsx (44K) GUID:?1D16EEF7-A958-4139-90BE-8EAB6AFE9CDC Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Microscopy pictures which are not included in the figures or supplemental files due to file size limitations are accessible from Figshare: https://figshare.com/s/6da319b9cc911d775bc6, https://figshare.com/s/882d7cf3ee140a102ef8. Abstract Neutrophils are essential innate immune cells whose responses are crucial in the clearance of invading pathogens. Neutrophils can respond to infection by releasing neutrophil extracellular traps (NETs). NETs are formed of chromatin and specific granular proteins and are released after execution of a poorly characterized cell death pathway. Here, we show that NET formation induced by PMA or is independent of RNA polymerase II and III-mediated transcription as well as of protein synthesis. Thus, neutrophils contain all the factors required for NET formation when Glycerol 3-phosphate they emerge from the bone marrow as differentiated cells. Introduction Neutrophils (also called polymorphonuclear leukocytes, PMNs) are essential for innate immune defense because they are directly antimicrobial and can shape adaptive immunity [1, 2]. Neutropenic individuals are prone to infections, underscoring the key role of neutrophils in fighting pathogens. PMNs differentiate in the bone marrow and are released in high numbers into the circulation as terminally differentiated cells. During infections neutrophils are rapidly recruited to inflammatory sites where they activate different antimicrobial programs, such as phagocytosis, production of reactive oxygen species (ROS), degranulation or the formation of neutrophil extracellular traps (NETs). NETs are released by PMNs after the activation of a specialized cell death pathway and consist of chromatin bound to cytoplasmic proteins [3, 4, 5]. Microorganisms as well as chemical compounds trigger NET formation, however, the molecular mechanism leading to release of NETs is only poorly characterized. Many NET inducers trigger MAP kinase signaling [6], activate NADPH oxidase (Nox2) and involve the subsequent production of ROS. This leads to granule rupture mediated by a protein complex called azurosome, translocation of neutrophil elastase (NE) to the nucleus, chromatin decondensation and NET production [7]. NETs sequester and immobilize pathogenic organisms, thus contributing to immune defense. Furthermore, NETs are dyresgulated in several auto-immune and inflammatory diseases, making them an important target for potential therapeutic interventions [5]. Most proteins required for neutrophil antimicrobial activity are transiently synthesized during development and packed in specialized granules that are deployed upon PMN activation. Indeed, essential neutrophil antimicrobial defense proteins like NE, Proteinase 3, Cathepsin G or Myeloperoxidase (MPO) are only produced during a neutrophil precursor stage and not in circulating cells [8]. Despite this, PMNs respond to bacteria by markedly changing gene expression patterns [9]. Most prominently, they produce chemokines like Interleukin (IL)-8 or Macrophage Inflammatory Protein (Mip) -1. Furthermore, the MAP kinase pathways, which are known to be essential for NET induction, can Glycerol 3-phosphate also induce transcription. We therefore tested whether transcription or translation are.(G) Representative images of human neutrophils stained with PL2-3 (chromatin, red), NE (Neutrophil Elastase, green) and Hoechst (blue) after treatment with transcription or translation inhibitors and PMA induction. inhibitors were added to murine neutrophils before stimulation with LPS. Flavopiridol: 0.025 M; 0.05 M; 0.1 M; 0.2 M; 1M, CAS 577784-91-9: 1 M, CHX: 0.5 g/ ml; 1 g/ ml; 5 g/ ml. Minimal concentrations blocking production of Mip-1 and subsequently used for NET experiments are shown in blue. Efficiency of inhibitors was analyzed by measuring production of Mip-1 in response to LPS stimulation (200 ng/ ml) for 20h.(EPS) pone.0157454.s001.eps (1.0M) GUID:?9A45D1D6-B606-479C-98F9-D96ED7C64009 S2 Fig: No effect of individual or combined inhibitors on NET formation. Inhibitors were used at the following concentrations: Actinomycin D (1 g/ ml), flavopiridol (0.05 M), CAS 577784-91-9 (10 M), CHX (1 g/ ml). (A, B) Human primary neutrophils were treated with transcription/ translation inhibitors as indicated. (A) After inhibitor treatment, cells were treated with 50 nM SYTOX green, stimulated with 100 nM PMA and analyzed by measuring emission of SYTOX green every hour in a fluorometer. (B) After inhibitor treatment, neutrophils were treated with 50 nM SYTOX green and infected with opsonized at MOI 5. SYTOX emission was measured every hour in a fluorometer. (C) Representative immunofluorescence pictures of human primary neutrophils treated with combinations of transcription/ translation inhibitors as indicated and stimulated with 100 nM PMA. Cells were stained with PL2-3 (chromatin, red), NE (Neutrophil elastase, green) and Hoechst (blue). (D) Representative pictures of SYTO green/ SYTOX orange assay in human primary neutrophils stimulated with (MOI 5). SYTO green stains all cells, SYTOX orange stains dead cells and extracellular traps.(EPS) pone.0157454.s002.eps (21M) GUID:?71F76911-7B55-409A-B5CF-94E1BEB15811 S1 Table: Numerical values used for quantification. (XLSX) pone.0157454.s003.xlsx (36K) GUID:?C61D6B04-FDC2-4B37-BD17-3FC8AD771F40 S2 Table: Numerical values used for quantification of supplementary data. (XLSX) pone.0157454.s004.xlsx (44K) GUID:?1D16EEF7-A958-4139-90BE-8EAB6AFE9CDC Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Microscopy pictures which are not included in the figures or supplemental files due to file size limitations are accessible from Figshare: https://figshare.com/s/6da319b9cc911d775bc6, https://figshare.com/s/882d7cf3ee140a102ef8. Abstract Neutrophils are essential innate immune cells whose responses are crucial in the clearance of invading pathogens. Neutrophils can respond to infection by releasing neutrophil extracellular traps (NETs). NETs are formed of chromatin and specific granular proteins and are released after execution of a poorly characterized cell death pathway. Here, we show that NET formation induced by PMA or is independent of RNA polymerase II and III-mediated transcription as well as of protein synthesis. Therefore, neutrophils contain all the factors required for NET formation when they emerge from your bone marrow as differentiated cells. Intro Neutrophils (also called polymorphonuclear leukocytes, PMNs) are essential for innate immune defense because they are directly antimicrobial and may shape adaptive immunity [1, 2]. Neutropenic individuals are prone to infections, underscoring the key part of neutrophils in fighting pathogens. PMNs differentiate in the bone marrow and are released in high figures into the blood circulation as terminally differentiated cells. During infections neutrophils are rapidly recruited to inflammatory sites where they activate different antimicrobial programs, such as phagocytosis, production of reactive oxygen varieties (ROS), degranulation or the formation of neutrophil extracellular traps (NETs). NETs are released by PMNs after the activation of a specialized cell death pathway and consist of chromatin bound to cytoplasmic proteins [3, 4, 5]. Microorganisms as well as chemical compounds trigger NET formation, however, the molecular mechanism leading to launch of NETs is only poorly characterized. Many NET inducers result in MAP kinase signaling [6], activate NADPH oxidase (Nox2) and involve the subsequent production of ROS. This prospects to granule rupture mediated by a protein complex called azurosome, translocation of neutrophil elastase (NE) to the nucleus, chromatin Glycerol 3-phosphate decondensation and NET production [7]. NETs sequester and immobilize pathogenic organisms, thus contributing to immune defense. Furthermore, NETs are dyresgulated in several auto-immune and inflammatory diseases, making them an.