Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Sabulski, Mary J.; Fura, Jonathan M.; Pires, Marcos M.

doi:10.3791/52114

Cited by 2 publications

(2 citation statements)

References 37 publications

(28 reference statements)

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“…After 45 min incubation in the dark, final 5 µL of a trypsin solution (10 mg/mL bovine trypsin in 100 mM EDTA) was added to each well. After another 20 min of incubation in the dark, fluorescence (λ ex = 355 nm and λ em = 460 nm) was measured with the Omega Plate Reader [ 23 ].…”

Section: Methodsmentioning

confidence: 99%

Nicotine and Cotinine Induce Neutrophil Extracellular Trap Formation—Potential Risk for Impaired Wound Healing in Smokers

Aspera-Werz¹,

Mück²,

Herbst³

et al. 2022

Antioxidants

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Smoking undoubtedly affects human health. Investigating 2318 representative patients at a level 1 trauma center identified delayed wound healing, tissue infections, and/or sepsis as main complications in smokers following trauma and orthopedic surgery. Therefore, smoking cessation is strongly advised to improve the clinical outcome in these patients, although smoking cessation often fails despite nicotine replacement therapy raising the need for specific interventions that may reduce the complication rate. However, the underlying mechanisms are still unknown. In diabetics, delayed wound healing and infections/sepsis are associated with increased neutrophilic PADI4 expression and formation of neutrophil extracellular traps (NETs). The aim was to investigate if similar mechanisms hold for smokers. Indeed, our results show higher PADI4 expression in active and heavy smokers than non-smokers, which is associated with an increased complication rate. However, in vitro stimulation of neutrophils with cigarette smoke extract (CSE) only moderately induced NET formation despite accumulation of reactive oxygen species (ROS). Physiological levels of nicotine and its main metabolite cotinine more effectively induced NET formation, although they did not actively induce the formation of ROS, but interfered with the activity of enzymes involved in anti-oxidative defense and NET formation. In summary, we propose increased formation of NETs as possible triggers for delayed wound healing, tissue infections, and/or sepsis in smokers after a major trauma and orthopedic surgery. Smoking cessation might reduce this effect. However, our data show that smoking cessation supported by nicotine replacement therapy should be carefully considered as nicotine and its metabolite cotinine effectively induced NET formation in vitro, even without active formation of ROS.

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Section: Methodsmentioning

confidence: 99%

Nicotine and Cotinine Induce Neutrophil Extracellular Trap Formation—Potential Risk for Impaired Wound Healing in Smokers

Aspera-Werz¹,

Mück²,

Herbst³

et al. 2022

Antioxidants

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show abstract

“…The in vitro PAD4 activity assay was performed colorimetrically as described previously 21 . BMDN (1 × 10 6 cells) were lysed in lysis solution [50 mM Tris, 50 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF, MW 174.94 g/mol) and 1 mM dithiothreitol (DTT, MW 154.25 g/mol), pH = 8.0] by sonication.…”

Section: Methodsmentioning

confidence: 99%

Cold-inducible RNA-binding Protein Induces Neutrophil Extracellular Traps in the Lungs during Sepsis

Ode

Aziz

Jin

et al. 2019

Sci Rep

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Extracellular cold-inducible RNA-binding protein (CIRP) exaggerates inflammation and tissue injury in sepsis. Neutrophil extracellular traps (NETs) are released by activated neutrophils during sepsis. NETs contribute to pathogen clearance, but excessive NET formation (NETosis) causes inflammation and tissue damage. Peptidylarginine deiminase 4 (PAD4) is associated with NETosis by increasing histone citrullination and chromatin decondensation. We hypothesized that CIRP induces NETosis in the lungs during sepsis via upregulating PAD4 expression. Sepsis was induced in C57BL/6 wild-type (WT) and CIRP −/− mice by cecal ligation and puncture (CLP). After 20 h of CLP induction, NETs in the lungs of WT and CIRP −/− mice were quantified by flow cytometry by staining the single cell suspensions with MPO and CitH3 Abs. PAD4 expression in the lungs of WT and CIRP −/− mice after sepsis was assessed by Western blotting. In vitro effects of recombinant mouse (rm) CIRP for NETosis and PAD4 expression in the bone marrow-derived neutrophils (BMDN) were assessed by flow cytometry and Western blotting, respectively. After 20 h of CLP, NETosis in the lungs was significantly decreased in CIRP −/− mice compared to WT mice, which also correlated with the decreased PAD4 expression. Intratracheal administration of rmCIRP into WT mice significantly increased NETosis and PAD4 expression in the lungs compared to vehicle-injected mice. In vitro culture of BMDN with rmCIRP significantly increased NETosis and PAD4 expression compared to PBS-treated control. Fluorescence microscopy revealed typical web-like structures consistent with NETs in rmCIRP-treated BMDN. Thus, CIRP serves as a novel inducer of NETosis via PAD4 during sepsis.

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Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Cited by 2 publications

References 37 publications

Nicotine and Cotinine Induce Neutrophil Extracellular Trap Formation—Potential Risk for Impaired Wound Healing in Smokers

Nicotine and Cotinine Induce Neutrophil Extracellular Trap Formation—Potential Risk for Impaired Wound Healing in Smokers

Cold-inducible RNA-binding Protein Induces Neutrophil Extracellular Traps in the Lungs during Sepsis

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