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ELife Jun 2017Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways...
Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin, and Group B Streptococcus. We studied NET production in neutrophils from healthy donors with inhibitors of molecules crucial to PMA-induced NETs including protein kinase C, calcium, reactive oxygen species, the enzymes myeloperoxidase (MPO) and neutrophil elastase. Additionally, neutrophils from chronic granulomatous disease patients, carrying mutations in the NADPH oxidase complex or a MPO-deficient patient were examined. We show that PMA, and GBS use a related pathway for NET induction, whereas ionophores require an alternative pathway but that NETs produced by all stimuli are proteolytically active, kill bacteria and composed mainly of chromosomal DNA. Thus, we demonstrate that NETosis occurs through several signalling mechanisms, suggesting that extrusion of NETs is important in host defence.
Topics: Calcimycin; Candida albicans; Extracellular Traps; Granulomatous Disease, Chronic; Healthy Volunteers; Humans; Metabolic Networks and Pathways; Neutrophils; Nigericin; Streptococcus; Tetradecanoylphorbol Acetate
PubMed: 28574339
DOI: 10.7554/eLife.24437 -
Cell Nov 2021Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental...
Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.
Topics: Animals; Antibody Specificity; Bacteria; Bacterial Proteins; Biofilms; Cell Line; Chinchilla; DNA, Bacterial; DNA, Cruciform; Deoxyribonucleases; Extracellular Matrix; Extracellular Space; Extracellular Traps; Humans; Tetradecanoylphorbol Acetate
PubMed: 34735796
DOI: 10.1016/j.cell.2021.10.010 -
PloS One 2023THP-1 monocyte, which can be differentiated into macrophages by PMA, is widely used in researches on pathogen infection and host innate immunity, but reports on the...
THP-1 monocyte, which can be differentiated into macrophages by PMA, is widely used in researches on pathogen infection and host innate immunity, but reports on the induction methods of PMA are different and lack a unified standard, and the transcriptome characteristics of macrophage compared with THP-1 cells remains unclear. In this research, we examined the differentiation effect of three factors including induction time, cell seeding density and PMA concentration by detecting the positive rate of CD14 expression. The concentration of 80ng/ml of PMA, the induction time of 24h, and the cell seeding density of 5×105 cells/ml, could respectively facilitates a relatively higher CD14 positive rate in THP-1 cells. Under this optimized conditions, the CD14 positive rate of THP-1 cells can reach 66.52%. Transcriptome sequencing showed that after the above induction, the mRNA expression of 3113 genes which were closely related to cell communication, signal transduction, cell response to stimulus, signaling receptor binding and cytokine activity were up-regulated, and the top 10 genes were RGS1, SPP1, GDF15, IL-1B, HAVCR2, SGK1, EGR2, TRAC, IL-8 and EBI3. While the mRNA expression of 2772 genes which were associated with cell cycle process, DNA binding and replication and cell division, were down-regulated, and the top genes were SERPINB10, TRGC2, SERPINB2, TRGC1, MS4A3, MS4A4E, TRGJP1, MS4A6A, TRGJP2, MS4A4A. This research optimized the induction method on THP-1 cell differentiation from three aspects and delineated the transcriptomic profile of PMA-induced THP-1 cells, laying a foundation for the construction method of cell model and for the functional study of macrophage.
Topics: Humans; Transcriptome; THP-1 Cells; Tetradecanoylphorbol Acetate; Macrophages; Monocytes; Cell Differentiation; RNA, Messenger
PubMed: 37459313
DOI: 10.1371/journal.pone.0286056 -
Journal of Visualized Experiments : JoVE Apr 2015Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil's antimicrobial armamentarium. Apart from their role in fighting...
Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil's antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions. In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves. A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained. We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs.
Topics: Extracellular Traps; Humans; Neutrophil Activation; Neutrophils; Tetradecanoylphorbol Acetate
PubMed: 25938591
DOI: 10.3791/52687 -
Immunology and Cell Biology Nov 2022For cell-based assays studying monocytes and macrophages, the immortalized monocyte cell line THP-1 is widely used and stimulated with phorbol 12-myristate 13-acetate,...
For cell-based assays studying monocytes and macrophages, the immortalized monocyte cell line THP-1 is widely used and stimulated with phorbol 12-myristate 13-acetate, lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ), after which it differentiates and polarizes into proinflammatory M1-like macrophages. For the quantification of this and the effect of different factors affecting these processes, the expression levels of various maturation markers are determined using reverse transcription-quantitative PCR. For this purpose, stably expressed reference genes are crucial. However, no studies evaluating the stability of reference genes in THP-1 cells stimulated with LPS and IFN-γ have been performed. Therefore, this paper describes the selection of the most used reference genes [RPL37A (ribosomal protein L37a), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), UBC (ubiquitin C), B2M (0β2-microbulin), ACTB (β-actin) and PPIA (cyclophilin A)], the in silico primer design, the analysis and the validation of these in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and more recent recommendations for the validation of the stability of reference genes. Using the RefFinder platform, including the four most popular algorithms for reference gene validation, the Delta CT, BestKeeper, NormFinder and geNorm, we find the reference genes GAPDH and UBC to be the most stable. Furthermore, we demonstrate that the normalization of gene expression data using the least stable reference genes, ACTB and B2M, dramatically affects the interpretation of experimental data. Taken together, it is vital to validate the stability of reference genes under the specific experimental conditions used when utilizing the THP-1 monocyte model system.
Topics: Humans; Real-Time Polymerase Chain Reaction; THP-1 Cells; Lipopolysaccharides; Macrophages; Monocytes; Tetradecanoylphorbol Acetate; Gene Expression Profiling
PubMed: 36184577
DOI: 10.1111/imcb.12590 -
Cytometry. Part a : the Journal of the... Aug 2017Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from...
Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. NETosis is the metamorphosis of neutrophils with NET formation that follows decondensation of DNA and rupture of the plasma membrane. Although NETs play important roles in innate immunity, excessive formation of NETs can be harmful to the hosts. Until now, various methods for evaluation of NETs have been reported. Although each has a virtue, the gold standard has not been established. Here we demonstrate a simple, objective, and quantitative method to detect NETs using flow cytometry. This method uses a plasma membrane-impermeable DNA-binding dye, SYTOX Green. SYTOX Green-positive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12-myristate 13-acetate (PMA). The number of SYTOX Green-positive cells was increased depending on the exposure duration and concentrations of PMA. Furthermore, co-localization of MPO and plasma membrane-appendant DNA of SYTOX Green-positive cells was demonstrated. Moreover, a NET inhibitor, diphenylene iodonium, could significantly reduce the number of SYTOX Green-positive cells induced by PMA. The collective evidence suggests that SYTOX Green-positive cells include neutrophils that formed NETs. The established method could detect neutrophils that underwent NETosis but not early apoptosis with equivalence in quantification to another well-used image analysis, which is based on fluorescent staining. Additionally, NETs that were formed in vivo were also detectable by this method. It is conceivable that the established method will bring us better understanding of the relation between NETosis and human diseases. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.
Topics: Apoptosis; DNA; Extracellular Traps; Flow Cytometry; Humans; Neutrophils; Peroxidase; Tetradecanoylphorbol Acetate
PubMed: 28715618
DOI: 10.1002/cyto.a.23169 -
Molecules (Basel, Switzerland) Jul 2018Inflammation is considered the root cause of various inflammatory diseases, including cancers. Decursinol angelate (DA), a pyranocoumarin compound obtained from the...
Inflammation is considered the root cause of various inflammatory diseases, including cancers. Decursinol angelate (DA), a pyranocoumarin compound obtained from the roots of , has been reported to exhibit potent anti-inflammatory effects. In this study, the anti-inflammatory effects of DA on the MAP kinase and NFκB signaling pathways and the expression of pro-inflammatory cytokines were investigated in phorbol 12-myristate 13-acetate (PMA)-activated human promyelocytic leukemia (HL-60) and lipopolysaccharide (LPS)-stimulated macrophage (Raw 264.7) cell lines. PMA induced the activation of the MAP kinase-NFκB pathway and the production of pro-inflammatory cytokines in differentiated monocytes. Treatment with DA inhibited the activation of MAP kinases and the translocation of NFκB, and decreased the expression and exogenous secretion of IL-1β and IL-6. Furthermore, LPS-stimulated Raw 264.7 cells were found to have increased expression of M1 macrophage-associated markers, such as NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS), and the M2 macrophage-associated marker CD11b. LPS also activated pro-inflammatory cytokines and Erk-NFκB. Treatment with DA suppressed LPS-induced macrophage polarization and the inflammatory response by blocking Raf-ERK and the translocation of NFκB in Raw 264.7 cells. Treatment with DA also inhibited the expression of pro-inflammatory cytokines, such as IL-1β and IL-6, NOX, and iNOS in Raw 264.7 cells. These results suggest that DA has the potential to inhibit macrophage polarization and inflammation by blocking the activation of pro-inflammatory signals. These anti-inflammatory effects of DA may contribute to its potential use as a therapeutic strategy against various inflammation-induced cancers.
Topics: Animals; Anti-Inflammatory Agents; Benzopyrans; Butyrates; Cell Polarity; Cytokines; HL-60 Cells; Humans; Lipopolysaccharides; MAP Kinase Signaling System; Macrophages; Mice; NF-kappa B; Phorbol Esters; Protein Transport; RAW 264.7 Cells
PubMed: 30060484
DOI: 10.3390/molecules23081880 -
Redox Biology Aug 2023Irisin is a newly discovered myokine which links exercise to inflammation and inflammation-related diseases through macrophage regulation. However, the effect of irisin...
INTRODUCTION
Irisin is a newly discovered myokine which links exercise to inflammation and inflammation-related diseases through macrophage regulation. However, the effect of irisin on the activity of inflammation related immune cells (such as neutrophils) has not been clearly described.
OBJECTIVES
The objective of our study was to explore the effect of irisin on the neutrophil extracellular traps (NETs) formation.
METHODS
Phorbol-12-myristate-13-acetate (PMA) was used to construct a classic neutrophil inflammation model that was used to observe the formation of NETs in vitro. We studied the effect of irisin on NETs formation and its regulation mechanism. Subsequently, acute pancreatitis (AP) was used to verify the protective effect of irisin in vivo, which was an acute aseptic inflammatory response disease model closely related to NETs.
RESULTS
Our study found that addition of irisin significantly reduced the formation of NETs via regulation of the P38/MAPK pathway through integrin αVβ5, which might be the one of key pathways in NETs formation, and which could theoretically offset the immunoregulatory effect of irisin. Systemic treatment with irisin reduced the severity of tissue damage common in the disease and inhibited the formation of NETs in pancreatic necrotic tissue of two classical AP mouse models.
CONCLUSION
The findings confirmed for the first time that irisin could inhibit NETs formation and protect mice from pancreatic injury, which further elucidated the protective effect of exercise on acute inflammatory injury.
Topics: Mice; Animals; Extracellular Traps; Pancreatitis; Fibronectins; Acute Disease; Neutrophils; Inflammation; Tetradecanoylphorbol Acetate
PubMed: 37392517
DOI: 10.1016/j.redox.2023.102787 -
Journal of Neuroinflammation Oct 2022In neuromyelitis optica spectrum disorders (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), neutrophils are found in CNS lesions. We...
BACKGROUND
In neuromyelitis optica spectrum disorders (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), neutrophils are found in CNS lesions. We previously demonstrated that NMOSD neutrophils show functional deficiencies. Thus, we hypothesized that neutrophil accumulation in the CNS may be facilitated by impairments affecting mechanisms of neutrophil death.
OBJECTIVE
To evaluate cell death in blood neutrophils from aquaporin-4 (AQP4)-IgG-seropositive NMOSD and MOGAD patients as well as matched healthy controls (HC) using in vitro assays.
METHODS
Twenty-eight AQP4 + NMOSD and 19 MOGAD patients in stable disease phase as well as 45 age- and sex-matched HC were prospectively recruited. To induce cell death, isolated neutrophils were cultured with/without phorbol 12-myristate 13-acetate (PMA). Spontaneous and PMA-induced NETosis and apoptosis were analyzed using 7-AAD and annexin-V by flow cytometry. Caspase-3 was assessed by western blot. Myeloperoxidase-DNA complexes (MPO-DNA), MPO and elastase were evaluated by ELISA, and cell-free DNA (cfDNA) by a fluorescence-based assay. Reactive oxygen species (ROS) were evaluated by a dihydrorhodamine 123-based cytometric assay. Serum GM-CSF, IL-6, IL-8, IL-15, TNF-ɑ and IL-10 were evaluated by multiplex assays, and neurofilament light chain (NfL) by single-molecule array assay.
RESULTS
In response to PMA, neutrophils from AQP4 + NMOSD but not from MOGAD patients showed an increased survival, and subsequent reduced cell death (29.6% annexin V 7-AAD) when compared to HC (44.7%, p = 0.0006). However, AQP4 + NMOSD also showed a mild increase in annexin V 7-AAD early apoptotic neutrophils (24.5%) compared to HC (20.8%, p = 0.048). PMA-induced reduction of caspase-3 activation was more pronounced in HC (p = 0.020) than in AQP4 + NMOSD neutrophils (p = 0.052). No differences were observed in neutrophil-derived MPO-DNA or serum levels of MPO, elastase, IL-6, IL-8 and TNF-ɑ. IL-15 levels were increased in both groups of patients. In AQP4 + NMOSD, an increase in cfDNA, GM-CSF and IL-10 was found in serum. A positive correlation among cfDNA and NfL was found in AQP4 + NMOSD.
CONCLUSIONS
AQP4 + NMOSD neutrophils showed an increased survival capacity in response to PMA when compared to matched HC neutrophils. Although the data indicate that the apoptotic but not the NETotic response is altered in these neutrophils, additional evaluations are required to validate this observation.
Topics: Acetates; Annexin A5; Aquaporin 4; Autoantibodies; Caspase 3; Cell Death; Cell-Free Nucleic Acids; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunoglobulin G; Interleukin-10; Interleukin-15; Interleukin-6; Interleukin-8; Myelin-Oligodendrocyte Glycoprotein; Myristates; Neuromyelitis Optica; Neutrophils; Pancreatic Elastase; Peroxidase; Phorbols; Reactive Oxygen Species; Tumor Necrosis Factor-alpha
PubMed: 36183103
DOI: 10.1186/s12974-022-02600-0 -
Journal of Natural Products Aug 2022The kernels of the Australian blushwood tree () are the source of the veterinary anticancer drug tigilanol tiglate (, Stelfonta) and contain a concentration of phorboids... (Review)
Review
The kernels of the Australian blushwood tree () are the source of the veterinary anticancer drug tigilanol tiglate (, Stelfonta) and contain a concentration of phorboids significantly higher than croton oil, the only abundant source of these compounds previously known. The oily matrix of the blushwood kernels is composed of free fatty acids and not by glycerides as found in croton oil. By active partitioning, it was therefore possible to recover and characterize for the first time a cryptic tigliane fraction, that is, the diterpenoid fraction that, because of its lipophilicity, could not be obtained by solvent partition of crude extracts. The cryptic tigliane fraction accounted for ca. 30% of the tigliane kernel titer and was quantified by H NMR spectroscopy and profiled by HPLC-MS. Long-chain (linoleates and/or oleates) 20-acyl derivatives of the epoxytigliane diesters tigilanol tiglate (EBC-46, ), EBC-47 (), EBC-59 (), EBC-83 (), and EBC-177 () were identified. By chemoselective acylation of EBC-46 () and EBC-177 () the natural triesters and and a selection of analogues were prepared to assist identification of the natural compounds. The presence of a free C-20 hydroxy group is a critical requirement for PKC activation by phorbol esters. The unexpected activity of 20-linoleoyl triester in a cytotoxicity assay based on PKC activation was found to be related mainly to its hydrolysis to tigilanol tiglate () under the prolonged conditions of the assay, while other esters were inactive. Significant differences between the esterification profile of the epoxytigliane di- and triesters exist in , suggesting a precise, yet elusive, blueprint of acyl decoration for the tigliane polyol 5-hydroxyepoxyphorbol.
Topics: Australia; Croton Oil; Euphorbiaceae; Phorbols; Trees
PubMed: 35973043
DOI: 10.1021/acs.jnatprod.2c00226