Early and Late Processes Driving NET Formation, and the Autocrine/Paracrine Role of Endogenous RAGE Ligands

Neutrophil extracellular trap (NET) formation has emerged as an important response against various pathogens; it also plays a role in chronic inflammation, autoimmunity, and cancer. Despite a growing understanding of the mechanisms underlying NET formation, much remains to be elucidated. We previously showed that in human neutrophils activated with different classes of physiological stimuli, NET formation features both early and late events that are controlled by discrete signaling pathways. However, the nature of these events has remained elusive. We now report that PAD4 inhibition only affects the early phase of NET generation, as do distinct signaling intermediates (TAK1, MEK, p38 MAPK). Accordingly, the inducible citrullination of residue R2 on histone H3 is an early neutrophil response that is regulated by these kinases; other arginine residues on histones H3 and H4 do not seem to be citrullinated. Conversely, elastase blockade did not affect NET formation by several physiological stimuli, though it did so in PMA-activated cells. Among belated events in NET formation, we found that chromatin decondensation is impaired by the inhibition of signaling pathways controlling both early and late stages of the phenomenon. In addition to chromatin decondensation, other late processes were uncovered. For instance, unstimulated neutrophils can condition themselves to be poised for rapid NET induction. Similarly, activated neutrophils release endogenous proteic factors that promote and largely mediate NET generation. Several such factors are known RAGE ligands and accordingly, RAGE inbibition largely prevents both NET formation and the conditioning of neutrophils to rapidly generate NETs upon stimulation. Our data shed new light on the cellular processes underlying NET formation, and unveil unsuspected facets of the phenomenon that could serve as therapeutic targets. In view of the involvement of NETs in both homeostasis and several pathologies, our findings are of broad relevance.

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The Signaling Pathways in Nitric Oxide Production by Neutrophils Exposed to N-nitrosodimethylamine

Letters in Drug Design & Discovery ◽

10.2174/1570180815666180426121503 ◽

2018 ◽

Vol 16 (2) ◽

pp. 194-199

Author(s):

Wioletta Ratajczak-Wrona ◽

Ewa Jablonska

Keyword(s):

Nitric Oxide ◽

Signaling Pathways ◽

Molecular Mechanisms ◽

Polymorphonuclear Neutrophils ◽

Microbial Pathogens ◽

Human Neutrophils ◽

No Production ◽

Oxide Synthase ◽

Inducible Nitric Oxide

Background: Polymorphonuclear neutrophils (PMNs) play a crucial role in the innate immune system’s response to microbial pathogens through the release of reactive nitrogen species, including Nitric Oxide (NO). </P><P> Methods: In neutrophils, NO is produced by the inducible Nitric Oxide Synthase (iNOS), which is regulated by various signaling pathways and transcription factors. N-nitrosodimethylamine (NDMA), a potential human carcinogen, affects immune cells. NDMA plays a major part in the growing incidence of cancers. Thanks to the increasing knowledge on the toxicological role of NDMA, the environmental factors that condition the exposure to this compound, especially its precursors- nitrates arouse wide concern. Results: In this article, we present a detailed summary of the molecular mechanisms of NDMA’s effect on the iNOS-dependent NO production in human neutrophils. Conclusion: This research contributes to a more complete understanding of the mechanisms that explain the changes that occur during nonspecific cellular responses to NDMA toxicity.

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Mast Cell Tryptase Potentiates Neutrophil Extracellular Trap Formation

Journal of Innate Immunity ◽

10.1159/000520972 ◽

2021 ◽

pp. 1-14

Author(s):

Gunnar Pejler ◽

Sultan Alanazi ◽

Mirjana Grujic ◽

Jeremy Adler ◽

Anna-Karin Olsson ◽

...

Keyword(s):

Neutrophil Extracellular Trap ◽

Human Neutrophils ◽

Functional Communication ◽

Extracellular Milieu ◽

Extracellular Traps ◽

Inflammatory Conditions ◽

Histone 3 ◽

Activated Neutrophils ◽

Core Histones

Previous research has indicated an intimate functional communication between mast cells (MCs) and neutrophils during inflammatory conditions, but the nature of such communication is not fully understood. Activated neutrophils are known to release DNA-containing extracellular traps (neutrophil extracellular traps [NETs]) and, based on the known ability of tryptase to interact with negatively charged polymers, we here hypothesized that tryptase might interact with NET-contained DNA and thereby regulate NET formation. In support of this, we showed that tryptase markedly enhances NET formation in phorbol myristate acetate-activated human neutrophils. Moreover, tryptase was found to bind vividly to the NETs, to cause proteolysis of core histones and to cause a reduction in the levels of citrullinated histone-3. Secretome analysis revealed that tryptase caused increased release of numerous neutrophil granule compounds, including gelatinase, lactoferrin, and myeloperoxidase. We also show that DNA can induce the tetrameric, active organization of tryptase, suggesting that NET-contained DNA can maintain tryptase activity in the extracellular milieu. In line with such a scenario, DNA-stabilized tryptase was shown to efficiently degrade numerous pro-inflammatory compounds. Finally, we showed that tryptase is associated with NET formation in vivo in a melanoma setting and that NET formation in vivo is attenuated in mice lacking tryptase expression. Altogether, these findings reveal that NET formation can be regulated by MC tryptase, thus introducing a novel mechanism of communication between MCs and neutrophils.

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Adenosine Triphosphate Neutralizes Pneumolysin-Induced Neutrophil Activation

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa277 ◽

2020 ◽

Vol 222 (10) ◽

pp. 1702-1712 ◽

Cited By ~ 1

Author(s):

Fabian Cuypers ◽

Björn Klabunde ◽

Manuela Gesell Salazar ◽

Surabhi Surabhi ◽

Sebastian B Skorka ◽

...

Keyword(s):

Adenosine Triphosphate ◽

Extracellular Space ◽

Cytolytic Activity ◽

Neutrophil Activation ◽

Human Neutrophils ◽

Pneumococcal Infections ◽

Pneumococcal Strain ◽

Neutrophil Degranulation ◽

Neutrophil Response

Abstract Background In tissue infections, adenosine triphosphate (ATP) is released into extracellular space and contributes to purinergic chemotaxis. Neutrophils are important players in bacterial clearance and are recruited to the site of tissue infections. Pneumococcal infections can lead to uncontrolled hyperinflammation of the tissue along with substantial tissue damage through excessive neutrophil activation and uncontrolled granule release. We aimed to investigate the role of ATP in neutrophil response to pneumococcal infections. Methods Primary human neutrophils were exposed to the pneumococcal strain TIGR4 and its pneumolysin-deficient mutant or directly to different concentrations of recombinant pneumolysin. Neutrophil activation was assessed by measurement of secreted azurophilic granule protein resistin and profiling of the secretome, using mass spectrometry. Results Pneumococci are potent inducers of neutrophil degranulation. Pneumolysin was identified as a major trigger of neutrophil activation. This process is partially lysis independent and inhibited by ATP. Pneumolysin and ATP interact with each other in the extracellular space leading to reduced neutrophil activation. Proteome analyses of the neutrophil secretome confirmed that ATP inhibits pneumolysin-dependent neutrophil activation. Conclusions Our findings suggest that despite its cytolytic activity, pneumolysin serves as a potent neutrophil activating factor. Extracellular ATP mitigates pneumolysin-induced neutrophil activation.

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Neutrophils: back in the thrombosis spotlight

Blood ◽

10.1182/blood-2018-10-862243 ◽

2019 ◽

Vol 133 (20) ◽

pp. 2186-2197 ◽

Cited By ~ 20

Author(s):

Denis F. Noubouossie ◽

Brandi N. Reeves ◽

Brian D. Strahl ◽

Nigel S. Key

Keyword(s):

Animal Models ◽

Tissue Factor ◽

Contact System ◽

Neutrophil Extracellular Trap ◽

Human Neutrophils ◽

Dependent Manner ◽

Extrinsic Pathway ◽

Vessel Occlusion ◽

Activated Neutrophils

Abstract Reactive and clonal neutrophil expansion has been associated with thrombosis, suggesting that neutrophils play a role in this process. However, although there is no doubt that activated monocytes trigger coagulation in a tissue factor-dependent manner, it remains uncertain whether stimulated neutrophils can also directly activate coagulation. After more than a decade of debate, it is now largely accepted that normal human neutrophils do not synthetize tissue factor, the initiator of the extrinsic pathway of coagulation. However, neutrophils may passively acquire tissue factor from monocytes. Recently, the contact system, which initiates coagulation via the intrinsic pathway, has been implicated in the pathogenesis of thrombosis. After the recent description of neutrophil extracellular trap (NET) release by activated neutrophils, some animal models of thrombosis have demonstrated that coagulation may be enhanced by direct NET-dependent activation of the contact system. However, there is currently no consensus on how to assess or quantify NETosis in vivo, and other experimental animal models have failed to demonstrate a role for neutrophils in thrombogenesis. Nevertheless, it is likely that NETs can serve to localize other circulating coagulation components and can also promote vessel occlusion independent of fibrin formation. This article provides a critical appraisal of the possible roles of neutrophils in thrombosis and highlights some existing knowledge gaps regarding the procoagulant activities of neutrophil-derived extracellular chromatin and its molecular components. A better understanding of these mechanisms could guide future approaches to prevent and/or treat thrombosis.

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LncRNAs Regulatory Networks in Cellular Senescence

International Journal of Molecular Sciences ◽

10.3390/ijms20112615 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2615 ◽

Cited By ~ 13

Author(s):

Pavan Kumar Puvvula

Keyword(s):

Signaling Pathways ◽

Cell Fate ◽

Regulatory Networks ◽

Translation Regulation ◽

Reading Frame ◽

Downstream Signaling ◽

Cellular Processes ◽

Fate Decision ◽

Functional Mechanisms

Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides with no open reading frame. They play a key role in the regulation of cellular processes such as genome integrity, chromatin organization, gene expression, translation regulation, and signal transduction. Recent studies indicated that lncRNAs are not only dysregulated in different types of diseases but also function as direct effectors or mediators for many pathological symptoms. This review focuses on the current findings of the lncRNAs and their dysregulated signaling pathways in senescence. Different functional mechanisms of lncRNAs and their downstream signaling pathways are integrated to provide a bird’s-eye view of lncRNA networks in senescence. This review not only highlights the role of lncRNAs in cell fate decision but also discusses how several feedback loops are interconnected to execute persistent senescence response. Finally, the significance of lncRNAs in senescence-associated diseases and their therapeutic and diagnostic potentials are highlighted.

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The Potential of Telomeric G-Quadruplexes Containing Modified Oligoguanosine Overhangs in Activation of Bacterial Phagocytosis and Leukotriene Synthesis in Human Neutrophils

Biomolecules ◽

10.3390/biom10020249 ◽

2020 ◽

Vol 10 (2) ◽

pp. 249 ◽

Cited By ~ 1

Author(s):

Ekaterina A. Golenkina ◽

Galina M. Viryasova ◽

Nina G. Dolinnaya ◽

Valeria A. Bannikova ◽

Tatjana V. Gaponova ◽

...

Keyword(s):

Antimicrobial Agents ◽

Viral Infections ◽

Uv Spectroscopy ◽

Human Neutrophils ◽

First Line ◽

Cellular Effects ◽

Cellular Processes ◽

G Quadruplex ◽

New Generation

Human neutrophils are the first line of defense against bacterial and viral infections. They eliminate pathogens through phagocytosis, which activate the 5-lipoxygenase (5-LOX) pathway resulting in synthesis of leukotrienes. Using HPLC analysis, flow cytometry, and other biochemical methods, we studied the effect of synthetic oligodeoxyribonucleotides (ODNs) able to fold into G-quadruplex structures on the main functions of neutrophils. Designed ODNs contained four human telomere TTAGGG repeats (G4) including those with phosphorothioate oligoguanosines attached to the end(s) of G-quadruplex core. Just modified analogues of G4 was shown to more actively than parent ODN penetrate into cells, improve phagocytosis of Salmonella typhimurium bacteria, affect 5-LOX activation, the cytosol calcium ion level, and the oxidative status of neutrophils. As evident from CD and UV spectroscopy data, the presence of oligoguanosines flanking G4 sequence leads to dramatic changes in G-quadruplex topology. While G4 folds into a single antiparallel structure, two main folded forms have been identified in solutions of modified ODNs: antiparallel and dominant, more stable parallel. Thus, both the secondary structure of ODNs and their ability to penetrate into the cytoplasm of cells are important for the activation of neutrophil cellular effects. Our results offer new clues for understanding the role of G-quadruplex ligands in regulation of integral cellular processes and for creating the antimicrobial agents of a new generation.

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Peptidylarginine deiminase 2, 3 and 4 have distinct specificities against cellular substrates: novel insights into autoantigen selection in rheumatoid arthritis

Annals of the Rheumatic Diseases ◽

10.1136/ard.2011.151712 ◽

2011 ◽

Vol 71 (1) ◽

pp. 92-98 ◽

Cited By ~ 94

Author(s):

Erika Darrah ◽

Antony Rosen ◽

Jon T Giles ◽

Felipe Andrade

Keyword(s):

Rheumatoid Arthritis ◽

Human Neutrophils ◽

Enzyme Specificity ◽

Peptidylarginine Deiminase ◽

Protein Targets ◽

Unique Protein ◽

Activated Neutrophils ◽

Arginine Residues ◽

Intrinsic Capacity ◽

The Relationship

ObjectiveTo define the relationship between autoantigen citrullination and different peptidylarginine deiminase (PAD) enzymes in rheumatoid arthritis (RA).MethodsCitrullinated autoantigens were identified by immunoblotting control and ionomycin-activated human primary neutrophil lysate with RA sera. Autoantigen identity and citrullination sites were defined by mass spectrometry. PAD isoenzyme expression in human neutrophils was determined by immunoblotting. PAD substrate specificity was addressed in HL-60 cell lysates co-incubated with human recombinant PAD2, PAD3 and PAD4.ResultsAlthough prominent protein citrullination is observed in ionomycin-activated neutrophils, RA sera only recognised a limited number of these citrullinated molecules. Among these, the authors identified that β and γ-actins are citrullinated on at least 10 arginine residues, generating a novel 47 kDa species that is frequently recognised by RA autoantibodies. Interestingly, the authors showed that the PAD enzymes expressed in human neutrophils (ie, PAD2, PAD3 and PAD4) have unique substrate specificities, independent of their subcellular distribution. Thus, only PAD2 was able to citrullinate native β/γ-actin, while histone H3 was only citrullinated by PAD4.ConclusionThese studies identified β and γ-actins as novel citrullinated autoantigens in RA, allowing enzyme specificity against intracellular substrates to be addressed. The studies provide evidence that PAD enzymes have the intrinsic capacity to select unique protein targets. The authors propose that unique PAD specificity may play a role in autoantigen selection in RA.

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Neutrophil extracellular traps in cancer: not only catching microbes

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02036-z ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Livia Ronchetti ◽

Nouha Setti Boubaker ◽

Maddalena Barba ◽

Patrizia Vici ◽

Aymone Gurtner ◽

...

Keyword(s):

Cancer Patients ◽

White Blood Cells ◽

Neutrophil Extracellular Traps ◽

Neutrophil Extracellular Trap ◽

Prognostic Role ◽

Extracellular Traps ◽

Activated Neutrophils ◽

Neoplastic Process ◽

Review Current

AbstractNeutrophils are the most abundant type of white blood cells circulating throughout the bloodstream and are often considered the frontline defenders in innate immunity. However, neutrophils are increasingly being recognized as having an important role in tumorigenesis and carcinogenesis due to their aberrant activation by molecules released into the tumor microenvironment. One defensive response of neutrophils that is aberrantly triggered during the neoplastic process is called NETosis, where activated neutrophils expel their DNA and intracellular contents in a web-like structure known as a neutrophil extracellular trap (NET). In cancer, NETosis has been linked to increased disease progression, metastasis, and complications such as venous thromboembolism. NET structures released by neutrophils can also serve as a scaffold for clot formation, shining new light on the role of neutrophils and NETosis in coagulation-mediated diseases.Here, we review current available knowledge regarding NET and the related NETosis process in cancer patients, with an emphasis on pre-clinical and clinical data fostering the identification and validation of biomarkers of NET with a predictive/prognostic role in cancer patients treated with immunotherapy agents. NETosis biomarkers, e.g., citH3, may integrate correlates of immunogenicity currently available (e.g., PD-L1 expression, TMB, TILs) and help select the subsets of patients who may most benefit from the use of the therapeutic weapons under discussion.

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The interrelationship between phagocytosis, autophagy and formation of neutrophil extracellular traps following infection of human neutrophils by Streptococcus pneumoniae

Innate Immunity ◽

10.1177/1753425917704299 ◽

2017 ◽

Vol 23 (5) ◽

pp. 413-423 ◽

Cited By ~ 24

Author(s):

Ihsan Ullah ◽

Neil D Ritchie ◽

Tom J Evans

Keyword(s):

Streptococcus Pneumoniae ◽

Pneumococcal Infection ◽

Neutrophil Extracellular Traps ◽

Inhibitory Effect ◽

Human Neutrophils ◽

Phosphatidylinositol 3 Kinase ◽

Cellular Processes ◽

Extracellular Traps ◽

Potent Inhibitory Effect

Neutrophils play an important role in the innate immune response to infection with Streptococcus pneumoniae, the pneumococcus. Pneumococci are phagocytosed by neutrophils and undergo killing after ingestion. Other cellular processes may also be induced, including autophagy and the formation of neutrophil extracellular traps (NETs), which may play a role in bacterial eradication. We set out to determine how these different processes interacted following pneumococcal infection of neutrophils, and the role of the major pneumococcal toxin pneumolysin in these various pathways. We found that pneumococci induced autophagy in neutrophils in a type III phosphatidylinositol-3 kinase dependent fashion that also required the autophagy gene Atg5. Pneumolysin did not affect this process. Phagocytosis was inhibited by pneumolysin but enhanced by autophagy, while killing was accelerated by pneumolysin but inhibited by autophagy. Pneumococci induced extensive NET formation in neutrophils that was not influenced by pneumolysin but was critically dependent on autophagy. While pneumolysin did not affect NET formation, it had a potent inhibitory effect on bacterial trapping within NETs. These findings show a complex interaction between phagocytosis, killing, autophagy and NET formation in neutrophils following pneumococcal infection that contribute to host defence against this pathogen.

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The role of translationally controlled tumor protein in proliferation ofDrosophilaintestinal stem cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1910850116 ◽

2019 ◽

Vol 116 (52) ◽

pp. 26591-26598 ◽

Cited By ~ 2

Author(s):

Young V. Kwon ◽

Bingqing Zhao ◽

Chiwei Xu ◽

Jiae Lee ◽

Chiao-Lin Chen ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Signaling Pathways ◽

Hippo Pathway ◽

Tissue Homeostasis ◽

Translationally Controlled Tumor Protein ◽

Cellular Processes ◽

The Hippo Pathway

Translationally controlled tumor protein (TCTP) is a highly conserved protein functioning in multiple cellular processes, ranging from growth to immune responses. To explore the role of TCTP in tissue maintenance and regeneration, we employed the adultDrosophilamidgut, where multiple signaling pathways interact to precisely regulate stem cell division for tissue homeostasis. Tctp levels were significantly increased in stem cells and enteroblasts upon tissue damage or activation of the Hippo pathway that promotes regeneration of intestinal epithelium. Stem cells with reduced Tctp levels failed to proliferate during normal tissue homeostasis and regeneration. Mechanistically, Tctp forms a complex with multiple proteins involved in translation and genetically interacts with ribosomal subunits. In addition, Tctp increases both Akt1 protein abundance and phosphorylation in vivo. Altogether, Tctp regulates stem cell proliferation by interacting with key growth regulatory signaling pathways and the translation process in vivo.

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