Fig. 5. Leukadherin-1 allows NET formation but attenuates cluster formation. XVA143 blocks both cluster formation and NETosis.

(A) Human neutrophils assayed as in Fig. 2. Where indicated, neutrophils were additionally pretreated either with CR3 agonist LA1, β₂ allosteric antagonist XVA, or vehicle control. After 30 min incubation, NET formation was visualized using Sytox green, imaged, and scored for NETs. Bright field and FITC images acquired at 20x; bar = 100μm. (B) Quantification of NET formation. NETs were visualized with Sytox green and multiple images were taken per well. Images were thresholded and gated to include NETs and exclude nuclei. NET formation was quantified as a percent area of the total imaged field. Well averages were ensemble averaged to generate this data. * p<0.001 XVA vs. all other conditions; ANOVA full factorial, post hoc Newman-Keuls; Error bars represent SEM (C) Neutrophil cluster formation was quantified using ImageJ and custom MatLab software and plotted as average clusters per mm (x-axis) vs. average cluster area in μm² (y-axis) per condition. Ellipses represent 2D-SEM. Cells on Fn + β-glucan pretreated with the XVA β₂ antagonist (green) have a significant decrease in both cluster number and area vs. cells on Fn + β-glucan (blue). Cells on Fn + β-glucan pretreated with vehicle control (purple) showed no significant difference in clustering parameters vs. Fn + β-glucan (blue). Cells on Fn + β-glucan pretreated with the CR3 agonist LA1 (red) showed an intermediate phenotype with a significant decrease in clustering parameters vs. Fn + β-glucan (blue) but a significant increase in clustering parameters vs. cells pretreated with the XVA blocking peptide (green). p < 0.001; ANOVA full factorial, post hoc Newman-Keuls.