Quantification of Citrullinated Histone H3 Bound DNA for Detection of Neutrophil Extracellular Traps

Abstract

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.

Neutrophil Extracellular Traps (NETs) and Hypercoagulability in Plasma Cell Dyscrasias—Is This Phenomenon Worthy of Exploration?

“…Using a mouse model, Li et al found that neutrophils isolated from animals with multiple myeloma were able to release more NETs under the influence of PMA than neutrophils isolated from healthy mice. Such an effect was observed in mice into which different myeloma cells (DP42 and 5TGM1) were injected [38]. In vitro studies by Fagerhol et al reveal that patients suffering from multiple myeloma have a higher concentration of S100A12/calprotectin complexes than healthy subjects, and S100A12/calprotectin complexes in this study were used as markers of NET release [39].…”

The Emerging Role of Neutrophil Extracellular Traps in Arterial, Venous and Cancer-Associated Thrombosis

“…Compared with tumor-free mice, tumor-bearing mice had increased venous and arterial thrombosis, and citrulline histone H3 (8) was directly detected in the thrombus. Neutrophils isolated from tumor-bearing mice showed higher H3Cit levels upon in vitro stimulation, and NETs formed more readily than normal neutrophils (119).…”

“…Compared with tumor-free mice, tumor-bearing mice had increased venous and arterial thrombosis, and citrulline histone H3 ( 8 ) was directly detected in the thrombus. Neutrophils isolated from tumor-bearing mice showed higher H3Cit levels upon in vitro stimulation, and NETs formed more readily than normal neutrophils ( 119 ). An endotoxin injection into the abdominal cavity of tumor-bearing mice induces the formation of NETs, which in turn induces a prethrombotic state and coagulation dysfunction ( 47 ).…”

Neutrophil extracellular traps modulate inflammatory markers and uptake of oxidized LDL by human and murine macrophages

“…The authors demonstrated that citrullinated histones accelerate LDL aggregation and foam cell formation in dependence of the citrulline content [42]. As PMA, which has been used in this study to stimulate neutrophils, was found to enhance histone citrullination [43,44] too, it is likely that increased oxLDL incorporation is mediated by NETs-associated citrullinated histones.…”