Fig. 5

The impaired NET formation of HD neutrophils contributes to diminished bacterial killing and can be restored by H₂O₂ or exogenic SOD. A Total bacterial killing assay (e.g., phagocytosis and NETosis) with HC and HD neutrophils mixed with either gram-positive (S. aureus) or gram-negative bacteria (E. coli). Neutrophils and bacteria were mixed in MOI of 1:1 and incubated for 1h. CFU of each bacteria were assessed with serial dilutions, and total killing was calculated compared to a parallel CFU of the control culture (bacteria with no neutrophils). B Neutrophils of HC and HD donors were incubated with cytochalasin D to halt phagocytosis. The extracellular bacterial killing (NETosis only) was performed as in A. To show that the extracellular bacterial killing derives from NETosis, the same experiment was conducted using RNASE-free DNASE-1. C Schematic diagram of ROS signaling during NOX-dependent NET formation. The red arrow indicates the rationality of adding H₂O₂ to HD neutrophils and creating ROS signaling bypass. Exogenic H₂O₂ restores the NET formation of neutrophils in HD patients. D Supernatant cfDNA was quantified by a rapid fluorometric NETosis assay using Sytox green or E visualized with fluorescence microscopy. Staining with DAPI was used to visualize nuclei, and extracellular DNA and histone H3 (red) were added to indicate the release of histone associated with the spread NETs. Representative images are shown as merges of the blue and red channels. F Pre-incubation H₂O₂ restores the bacterial killing capacity of neutrophils taken from HD patients. HD and HC neutrophils’ extracellular bacterial killing capacities were assessed as in B when cells were pre-incubated with Cytochalasin D and then exposed to 0.03% H₂O₂ to induce NET formation. The cells media were removed to avoid effects on bacterial viability, and cells were supplemented with a fresh culture medium containing either gram-positive (S. aureus) or gram-negative (E. coli). CFU of each bacteria were assessed with serial dilutions, and total killing was calculated compared to a parallel CFU of the control culture (n=6). G Exogenic recombinant SOD-1 improves the NET formation of neutrophils in HD patients. Supernatant cfDNA was quantified by a rapid fluorometric NETosis assay using Sytox green. In all NETosis, fluorometric assay results are presented as means ± S.E. of RFU n=4