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Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma

Published on Jun 1, 2017in Immunologic Research2.61
· DOI :10.1007/s12026-017-8905-3
Charlotte Thålin4
Estimated H-index: 4
(KI: Karolinska Institutet),
Maud Daleskog6
Estimated H-index: 6
(KI: Karolinska Institutet)
+ 7 AuthorsMélanie Demers20
Estimated H-index: 20
(KI: Karolinska Institutet)
Sources
Abstract
There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.
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References39
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ABSTRACTNeutrophils play a major role in cancer biology and both pro- and antitumoral functions of tumor-infiltrating neutrophils have been described. We have shown that tumors, by releasing G-CSF into the bloodstream, prime circulating neutrophils to form neutrophil extracellular traps (NETs) and we have detected the presence of NETs within the tumor microenvironment. Here, we report, using PAD4-deficient mice with a defect in neutrophil chromatin decondensation and NET formation, that the prim...
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#1Marloes HoeksemaH-Index: 2
#2Martin van EijkH-Index: 21
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Histones are known for their ability to bind to and regulate expression of DNA. However, histones are also present in cytoplasm and extracellular fluids where they serve host defense functions and promote inflammatory responses. Histones are a major component of neutrophil extracellular traps that contribute to bacterial killing but also to inflammatory injury. Histones can act as antimicrobial peptides and directly kill bacteria, fungi, parasites and viruses, in vitro and in a variety of animal...
21 CitationsSource
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#2Mélanie Demers (Harvard University)H-Index: 20
Last. Sara Aspberg (KI: Karolinska Institutet)H-Index: 8
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Abstract Introduction Large elevations of high sensitive Troponin T (hsTnT) in ischemic stroke patients is associated with a poor outcome. In a pilot study we found a high prevalence of malignancies among these patients. Since neutrophil extracellular traps (NETs) have been linked to cancer-associated thrombosis, we hypothesized that the concomitant cerebral and myocardial ischemia could be the result of a NET-induced hypercoagulable state. Materials and methods Clinical assessments, plasma anal...
46 CitationsSource
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