Figure 4Figure 4
Neutrophil-derived C5a enhances NET production and activates microglia via C5aR1 signaling. ( A and B ) Gray matter of AQP4-IgG recipient mice, C5a immunoreactivity was not seen in GFAP + astrocytes ( A ) and rarely seen in IBA1 + microglia/macrophages ( B ). ( C ) Triple immunostaining of lumbar cord of AQP4-IgG recipient mice revealed C5a in neutrophils (MPO + , left) and monocytes (CCR2-GFP + , enhanced by anti-GFP-IgG, right). ( D ) The percentage of spinal cord cells expressing C5a in AQP4-IgG-infused mice ( n = 4 mice per group). ( E ) Colocalization analysis of C5a and MPO signals in C using Zen software. ( F and G ) Confocal images of neutrophil (Ly6G, red) containing C5a (green in F ), and C5 (green in G ) after TNF-α incubation in vitro. Granule protein, MPO + , magenta; nuclear segmentation, DAPI + , blue. BF, bright-field. ( H ) Lumbar cords from IgG-infused mice were dissociated enzymatically and subjected to high parametric flow cytometric analysis. ( I ) t-SNE