Current Challenges and Limitations in Antibody-Based Detection of Citrullinated Histones

Abstract

Studies on NETosis demand reliable and convenient markers to monitor the progress of this form of cell death. Because a determining step in the release of nuclear chromatin NETs requires the conversion of arginine residues to citrulline residues in histones by peptidylarginine deiminase, citrullinated histones can provide such a marker. Here, we evaluate antibody reagents for the detection of citrulline residues in histones and observe alarming differences between commercial antisera and mouse and rabbit monoclonal antibodies in their ability to detect their nominal target residues. Differences between antibodies that are currently used to detect citrulline residues in histones could jeopardize efforts to reach a scientific consensus and instead lead to inconsistent and even conflicting conclusions regarding the regulation of histone deimination. Our results will assist others in planning their initial or ongoing studies on peptidylarginine deiminase activity with the use of currently available antibodies. Furthermore, we argue that, along with the careful attention to experimental conditions and calcium concentrations, validated antibody reagents are urgently needed to avoid possible setbacks in the research on NETosis.

Citrullinated Histone H3 as a Therapeutic Target for Endotoxic Shock in Mice

“…We found that injection of CitH3 mAb (4 Cit) markedly improves survival following LPSinduced lethal endotoxic shock and attenuates pro-inflammatory responses, ALI, as well as NET formation. Our antibody fills the gap in the field of citrullinated histone that was reported by Neeli and Radic, "current challenges and limitations in antibody-based detection of citrullinated histones, " in Frontiers in Immunology (28). In addition, we believe that CitH3 can be a potential target for directed therapies in endotoxic patients in the future.…”

“…As such, CitH3 increases HUVEC permeability and can induce NET formation ex vivo. Although there are some commercially available antibodies that bind to citrulline residues in histones, their efficacy has been found to be inconsistent (28), and there is a need for more reliable anti-CitH3 antibodies. In addition, the commercially available anti-CitH3 mAb only recognizes histone H3 citrullinated R2+R8+R17 [CitH3 mAb (3 Cit)], for which PAD4 is responsible (29).…”

Abnormal neutrophil signature in the blood and pancreas of presymptomatic and symptomatic type 1 diabetes

“…Additional proof of tissue-residing NETting neutrophils was also obtained by the identification of decondensed DNA decorated with MPO and citrullinated histones. Staining with anti-human citrullinated histone antibodies is known to be cumbersome and highly dependent on organ preservation (19,20). This staining was therefore only possible in a proportion of the available pancreas sections (i.e., 21 sections from 13 subjects across the 4 cohorts).…”

The origin of extracellular DNA in bacterial biofilm infectionsin vivo

“…Histone citrullination in vitro depends on stimulation of PMNs [46, 47]; indeed, an increase in histone citrullination is associated with chromatin de-condensation during NETs formation and LPS-induced early responses to inflammatory stimuli of PMNs [25, 26]. However, one widely used stimulus, PMA, has resulted in conflicting results in the literature and has been shown to induce NETs with and without co-localized citH3 and H3 [17, 47, 48]. In addition citrullination of H3 is strongly reduced when PMNs are stimulated during anoxic conditions [49], which is present in the endobronchial secretion in infected CF lungs [50, 51].…”