Abstract
Simple SummaryNeutrophil extracellular traps (NETs) are extracellular web-like structures comprised of proteins and DNA that have been associated with the development of cancer. Citrullination of histone H3 has been implicated in the formation of NETs. The aim of our study was to develop a quick and reliable method for NET quantitation. Here, we describe a novel protocol for the quantification of NETs based on the detection of citrullinated histone H3 bound to DNA (CitH3DNA binding assay). This assay was validated by comparing the ability of neutrophils from control tumor-free and myeloma-bearing mice to form NETs in response to stimuli. We demonstrated that neutrophils from tumor-bearing mice produced more NETs than those from tumor-free counterparts following stimulation with PMA. The increase in NET production as detected by microscopy correlated with significantly higher histone H3 citrullination levels and increased measurements of CitH3DNA in our novel binding assay.Formation of neutrophil extracellular traps (NETs) has been associated with multiple pathologies including cancer. While the visualization of NETs by microscopy is a routine technique, their quantification presents a number of challenges. Commonly, as citrullination of histone H3 is required for NET formation, the presence of this modified histone along with DNA is considered to be a hallmark of NETs. Here, we describe and validate a novel assay for the quantification of NETs based on the detection of citrullinated histone H3 bound to DNA (CitH3DNA binding assay). Using this assay, we investigated the effect of phorbol 12-myristate 13-acetate (PMA) on NET formation by neutrophils isolated from the bone marrow of control and myeloma-bearing mice. We found that PMA induced citrullination of histone H3, an increase in the level of CitH3DNA, and NET formation in neutrophils from both tumor-free and myeloma-bearing mice. The levels of CitH3DNA in the NET fractions, as measured by our assay directly correlated with the citrullination of histone H3 in neutrophils, as detected by Western blotting, and were significantly higher in PMA-stimulated compared to unstimulated neutrophils. Neutrophils from tumor-bearing mice produced more NETs than those from tumor-free counterparts following stimulation with PMA. The increase in NET production correlated with significantly higher histone H3 citrullination levels and increased measurements of CitH3DNA. Thus, our data indicate that bone marrow neutrophils from myeloma-bearing hosts are prone to NET formation.
Highlights
There has been recent increased interest in the evaluation of neutrophil extracellular traps (NETs) as there is a growing body of evidence that they are involved in a number of pathologies including cancer [1,2,3,4]
We compared the ability of neutrophils isolated from the bone marrow (BM) of tumor-free and myeloma-bearing mice to form NETs in response to stimulation with phorbol 12-myristate 13-acetate (PMA)
BM neutrophils from both control and myeloma-bearing mice were able to spontaneously form NETs; the level of spontaneous NETosis was low between neutrophils from myeloma-bearing and tumor-free mice (Figure 1)
Summary
There has been recent increased interest in the evaluation of neutrophil extracellular traps (NETs) as there is a growing body of evidence that they are involved in a number of pathologies including cancer [1,2,3,4]. PMA-induced NETosis is initiated by the activation of protein kinase C, the release of calcium from intracellular stores, and activation of the Mitogen-activated protein kinase-Extracellular-signal-regulated kinase (Raf–MEK–ERK) pathway followed by assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and generation of reactive oxygen species including hydrogen peroxide [6]. Increased intracellular calcium levels lead to the activation of peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination. Citrullination of histone H3 at arginine residues R2, R8, and R17 by PAD4 results in a reduction of the positive charge and decondensation of chromatin [14,15,16]. The nuclei of neutrophils lose their characteristic lobular structure, round up and expand This is followed by disintegration of the nuclear membrane resulting in mixing of the nucleoplasm and cytoplasm. The cell membrane ruptures, and the contents of the neutrophil is released into the extracellular space in the form of a NET
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