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Frontiers in Immunology 2022Neutrophil extracellular traps (NETs)-as double-edged swords of innate immunity-are involved in numerous processes such as infection, inflammation and tissue repair....
BACKGROUND
Neutrophil extracellular traps (NETs)-as double-edged swords of innate immunity-are involved in numerous processes such as infection, inflammation and tissue repair. Research on neutrophil granulocytes is limited because of their short lifetime of only a few hours. Several attempts have been made to prolong the half-life of neutrophils using cytokines and bacterial products and have shown promising results. These long-term surviving neutrophils are reported to maintain phagocytic activity and cytokine release; however, little is known regarding their capability to release NETs.
METHODS
We analysed the prolongation of neutrophil survival under various culture conditions using granulocyte colony-stimulating factor (G-CSF), lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) by flow cytometry and a viability assay. Additionally, we assessed NET formation following stimulation with phorbol 12-myristate 13-acetate (PMA) by immunofluorescence staining, myeloperoxidase (MPO)-DNA sandwich-ELISA and fluorometric assays for cell-free DNA (cfDNA), neutrophil elastase (NE) and myeloperoxidase (MPO).
RESULTS
Untreated neutrophils could form NETs after stimulation with PMA for up to 24 h. Incubation with LPS extended their ability to form NETs for up to 48 h. At 48 h, NET release of neutrophils cultured with LPS was significantly higher compared to that of untreated cells; however, no significantly different enzymatic activity of NE and MPO was observed. Similarly, incubation with G-CSF resulted in significantly higher NET release at 48 h compared to untreated cells. Furthermore, NETs showed significantly higher enzymatic activity of NE and MPO after incubation with G-CSF. Lastly, incubation with TNF-α had no influence on NET release compared to untreated cells although survival counts were altered by TNF-α.
CONCLUSIONS
G-CSF, LPS or TNF-α each at low concentrations lead to prolonged survival of cultured neutrophils, resulting in considerable differences in NET formation and composition. These results provide new information for the use of neutrophils in long-term experiments for NET formation and provide novel insights for neutrophil behaviour under inflammatory conditions.
Topics: Cytokines; Granulocyte Colony-Stimulating Factor; Lipopolysaccharides; Neutrophils; Peroxidase; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha
PubMed: 35242132
DOI: 10.3389/fimmu.2022.815412 -
Bioorganic & Medicinal Chemistry Letters Apr 2020TRPV4 is a ubiquitously expressed, non-selective cation channel activated by a range of stimuli including hypotonicity, temperature, pH, stretch and endogenous ligands.... (Review)
Review
TRPV4 is a ubiquitously expressed, non-selective cation channel activated by a range of stimuli including hypotonicity, temperature, pH, stretch and endogenous ligands. Agents that modulate TRPV4 are sought as potential therapeutics for the treatment of many diseases including osteoarthritis, respiratory illnesses, gastrointestinal disorders, pain and congestive heart failure. In recent years, significant advances in TRPV4 drug discovery have been realized as at least seven novel TRPV4 agonist or antagonist templates were reported and the first selective TRPV4 antagonist was evaluated in early clinical trials.
Topics: Biological Products; Drug Discovery; Humans; Models, Molecular; Molecular Structure; Phorbols; TRPV Cation Channels
PubMed: 32063431
DOI: 10.1016/j.bmcl.2020.127022 -
Cells Sep 2023Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the...
Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.
Topics: Neutrophils; Calcimycin; Microscopy, Atomic Force; Extracellular Traps; Cell Nucleus; Tetradecanoylphorbol Acetate
PubMed: 37681931
DOI: 10.3390/cells12172199 -
Journal of the American Chemical Society Nov 2020Self-assembly of amphiphilic peptide-based building blocks gives rise to a plethora of interesting nanostructures such as ribbons, fibers, and tubes. However, it remains...
Self-assembly of amphiphilic peptide-based building blocks gives rise to a plethora of interesting nanostructures such as ribbons, fibers, and tubes. However, it remains a great challenge to employ peptide self-assembly to directly produce nanostructures with lower symmetry than these highly symmetric motifs. We report here our discovery that persistent and regular crescent nanostructures with a diameter of 28 ± 3 nm formed from a series of tetrapeptides with the general structure AdKKEX (Ad = adamantyl group, K = lysine residue functionalized with an -aroylthiooxime (SATO) group, E = glutamic acid residue, and X = variable amino acid residue). In the presence of cysteine, the biological signaling gas hydrogen sulfide (HS) was released from the SATO units of the crescent nanostructures, termed peptide-HS donor conjugates (PHDCs), reducing levels of reactive oxygen species (ROS) in macrophage cells. Additional studies showed that the crescent nanostructures alleviated cytotoxicity induced by phorbol 12-myristate-13-acetate more effectively than common HS donors and a PHDC of a similar chemical structure, AdKKE, that formed short nanoworms instead of nanocrescents. Cell internalization studies indicated that nanocrescent-forming PHDCs were more effective in reducing ROS levels in macrophages because they entered into and remained in cells better than nanoworms, highlighting how nanostructure morphology can affect bioactivity in drug delivery.
Topics: Animals; Cell Survival; Hydrogen Sulfide; Macrophages; Mice; Nanostructures; Oligopeptides; RAW 264.7 Cells; Reactive Oxygen Species; Tetradecanoylphorbol Acetate
PubMed: 33186019
DOI: 10.1021/jacs.0c09399 -
Cells Sep 2022This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs.... (Review)
Review
This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs. PMA activates PKC; however, we highlight some isoforms that contribute to specific functions. PKC α, β and δ contribute to ROS production while PKC βII and PKC ζ are involved in cytoskeleton remodeling. Actin polymerization is important for the chemotaxis of neutrophils and its remodeling is connected to ROS balance. We suggest that, although ROS and production of NETs are usually observed together in PMA-activated neutrophils, there might be a regulatory mechanism balancing both. Interestingly, we suggest that serine proteases might determine the PAD4 action. PAD4 could be responsible for the activation of the NF-κB pathway that leads to IL-1β release, triggering the cleavage of gasdermin D by serine proteases such as elastase, leading to pore formation contributing to release of NETs. On the other hand, when serine proteases are inhibited, NETs are formed by citrullination through the PAD4 pathway. This review puts together results from the last 31 years of research on the effects of PMA on the neutrophil and proposes new insights on their interpretation.
Topics: Actins; Extracellular Traps; NF-kappa B; Neutrophils; Pancreatic Elastase; Reactive Oxygen Species; Serine Proteases; Tetradecanoylphorbol Acetate
PubMed: 36139464
DOI: 10.3390/cells11182889 -
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 -
Biomedicine & Pharmacotherapy =... Jul 2023Research on transient receptor potential vanilloid-4 (TRPV4) can provide a promising potential therapeutic target in the development of novel medicines for lung... (Review)
Review
Research on transient receptor potential vanilloid-4 (TRPV4) can provide a promising potential therapeutic target in the development of novel medicines for lung disorders. TRPV4 expresses in lung tissue and plays an important role in the maintenance of respiratory homeostatic function. TRPV4 is upregulated in life-threatening respiratory diseases like pulmonary hypertension, asthma, cystic fibrosis, and chronic obstructive pulmonary diseases. TRPV4 is linked to several proteins that have physiological functions and are sensitive to a wide variety of stimuli, such as mechanical stimulation, changes in temperature, and hypotonicity, and responds to a variety of proteins and lipid mediators, including anandamide (AA), the arachidonic acid metabolite, 5,6-epoxyeicosatrienoic acid (5,6-EET), a plant dimeric diterpenoid called bisandrographolide A (BAA), and the phorbol ester 4-alpha-phorbol-12,13-didecanoate (4α-PDD). This study focused on relevant research evidence of TRPV4 in lung disorders and its agonist and antagonist effects. TRPV4 can be a possible target of discovered molecules that exerts high therapeutic potential in the treatment of respiratory diseases by inhibiting TRPV4.
Topics: Humans; Transient Receptor Potential Channels; TRPV Cation Channels; Phorbol Esters; Hypertension, Pulmonary
PubMed: 37178575
DOI: 10.1016/j.biopha.2023.114861 -
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 -
Journal of Natural Products Dec 2020During a chemical investigation of , three new [wikstrocins A-C (-)] and three known tigliane diterpenoids (-) were isolated. The structures of the new compounds were...
During a chemical investigation of , three new [wikstrocins A-C (-)] and three known tigliane diterpenoids (-) were isolated. The structures of the new compounds were elucidated from extensive physiochemical and spectroscopic analysis. The correlations between the ECD Cotton effects and B ring structures of tiglianes were also evaluated. The isolated compounds were assessed for their anti-HIV activity against HIV-1 infection of MT4 cells, and two compounds ( and ) showed potent anti-HIV activity with IC values of 3.8 and 12.8 nM, respectively.
Topics: Anti-HIV Agents; Cell Line; Diterpenes; Humans; Phorbols; Spectrum Analysis; Wikstroemia
PubMed: 33172265
DOI: 10.1021/acs.jnatprod.0c00700 -
Antiviral Research Oct 2018While combination antiretroviral therapy (cART) has successfully converted HIV to a chronic but manageable infection in many parts of the world, HIV continues to persist... (Review)
Review
While combination antiretroviral therapy (cART) has successfully converted HIV to a chronic but manageable infection in many parts of the world, HIV continues to persist within latent cellular reservoirs, which can become reactivated at any time to produce infectious virus. New therapies are therefore needed not only for HIV suppression but also for containing or eliminating HIV reservoirs. Compounds derived from plant, marine, and other natural products have been found to combat HIV infection and/or target HIV reservoirs, and these discoveries have substantially guided current HIV therapy-based studies. Here we summarize the role of natural product-derived compounds in current HIV suppression, remission, and cure strategies.
Topics: Anti-HIV Agents; Biological Products; Bryostatins; Depsipeptides; Disease Reservoirs; Diterpenes; Drug Discovery; Gene Products, tat; HIV Infections; HIV-1; Histone Deacetylase Inhibitors; Humans; Phorbol Esters; Virus Latency; Vorinostat; rev Gene Products, Human Immunodeficiency Virus
PubMed: 30063970
DOI: 10.1016/j.antiviral.2018.07.016