Fig. 3

Neutrophils of HD patients show dramatically diminished NOX-independent NETosis with a significant interruption in chromatin decondensation. A Western blot analysis of steady-state levels of the PADI-4 enzyme in HC and HD donors neutrophils. B NETosis assay and visualization of NET deployment by neutrophils of HC (left) or HD (right) stimulated with Calcium Ionophore A23187 for the stimulation of the NOX-independent pathway visualized by Sytox green in fluorescence microscopy. C Quantification of supernatant cfDNA of neutrophils of HC and HD donors stimulated with A23187 for 3 h. The remaining cells were plated, and supernatant cfDNA was quantified by a rapid fluorometric assay using Sytox green. Results are presented as means ± S.E. of RFU n=6. D The machine learning image analysis tool for quantifying NETosis and chromatin architecture shows sample snapshots of the four neutrophil nuclear stages of NETosis as lobulated, delobulated, diffused, and spread NETs. E Overall image analysis of 100 cells per donor out of 6 total donors for each group (e.g., HC or HD). With no stimulation, HC and HD cells show no difference in distribution when 85% of the nuclei are defined as Lobulated. After 3 h stimulation with A23187, 82% of HC cells were defined as spread NETs in HC while only 40% in HD cells. Importantly, in HD cells, a large portion of cells is defined as Delobulated, Diffused as an indication of interruption in chromatin decondensation during NOX-independent NET formation