Detection and Quantification of Histone H4 Citrullination in Early NETosis With Image Flow Cytometry Version 4

Abstract

Neutrophil extracellular traps (NETs) formation has been implicated in an increasing number of infectious and non-infectious pathologies. NETosis is a tightly regulated process; the end-stage and read-out is the formation of DNA strands extruded from the nuclei, and traditionally assessed by fluorescence microscopy. Since NETosis has emerged as a possible biomarker of the inflammatory process, there is a need for less time-consuming, consistent, and quantitative approaches to improve its application in clinical assessment of pro-inflammatory conditions. Imaging Flow Cytometry (IFC) combines features of conventional flow cytometry with qualitative power of fluorescence microscopy and has an added advantage of the capability of assessing the early processes leading up to extrusion of the DNA-scaffolded strands. We explored the optimal imaging-based tools that can be used to measure citrullination of H4 in early NETosis. IFC identified and quantified histone 4 citrullination (H4cit3) induced with several known NETosis stimuli (Ionophore, PMA, LPS, Hemin, and IL-8) following treatment periods ranging from 2 to 60 min. Its relationship with other alterations at nuclear and cellular level, such as nuclear decondensation and super-condensation, multi-lobulated nuclei vs. 1-lobe nuclei and cell membrane damage, were also quantified. We show that the early progress of the H4cit3 response in NETosis depends on the stimulus. Our method identifies fast (Ionophore and Hemin), intermediate and slow (PMA) inducers and shows that H4cit3 appears to have a limited contribution to both early LPS-and IL-8-induced NETosis. While this method is rapid and of a higher throughput compared to fluorescence microscopy, detection and quantification is limited to H4cit3-mediated nuclear events and is likely to be stimulus-and signaling pathway dependent.

Histone citrullination: a new target for tumors

“…In addition to CitH3, the citrullination of H1.0, H1.4, H1.5 and H2B was also detected in RA NET [28]. Because of its quantifiable response to all kinds of stimuli of NETs, CitH4 was identified as an index of early NETs [129]. In conclusion, both core histones (H2, H3 and H4) and linker histone H1 present citrullination in NETs.…”

PEGylated nanoparticle albumin-bound steroidal ginsenoside derivatives ameliorate SARS-CoV-2-mediated hyper-inflammatory responses

“…In a recent study, abnormal coagulation (confirmed by D-dimer concentration) [ 56 , 57 ] and NETosis ( i.e. , cfDNA, NET, MPO activity, and NE) [ [58] , [59] , [60] , [61] ] were also confirmed in the SARS-CoV-2 ICU patient plasma, and the correlation between elevated histone H4 level and blood coagulation [ 54 , 62 ]/NETosis [ 63 , 64 ] was validated. According to previous studies, albumin exhibits the potential to inhibit the histone H4-induced platelet aggregation[ 29 ], suggesting albumin as a therapeutic candidate for managing blood coagulation.…”

Sperm induce macrophage extracellular trap formation via phagocytosis-dependent mechanism

“…During the formation of ETs resistant to pathogen invasion, MPO may drive chromatin decondensation, a critical step in ET formation [49]. In addition, H4Cit3 is a well-established NET marker [51]. Peptidylarginine deiminase 4 targets histone arginine and monomethylarginine residues for citrullination in a calcium-dependent reaction [52].…”