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Blood, Vol. 113, Issue 25, 6419-6427, June 18, 2009
Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates
Blood Yost et al.
113: 6419
Supplemental materials for: Yost et al
Files in this Data Supplement:
- Table S1. Clinical characteristics and infectious complication of preterm infants (PDF, 14 KB)
- Figure S1. Neonatal PMNs generate ROS but do not form NETs in response to PMA (JPG, 655 KB)
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(A) Adult, term, or preterm PMNs were incubated with PMA (20 nM) for 30 minutes. They were then incubated with nitroblue tetrazolium (NBT) (Nathan et al. 1969. J Clin Invest 48:1895). The blue cytoplasmic staining represents ROS-mediated reduction of NBT, indicating ROS generation. (B) PMNs from all three donor groups were stimulated with PMA (20 nM) for 60 minutes and imaged as in Figure 1 via confocal microscopy.
- Figure S2. Co-incubation of adult PMNs with increasing ratios of preterm PMNs fails to inhibit NET formation by adult PMNs (JPG, 418 KB)
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Preterm PMNs and adult PMNs were preincubated with cell permeable DNA dyes with different fluorescent emissions (preterm, blue staining; adult, green staining), the labeled cells were mixed at increasing ratios of neonatal PMNs to adult PMNs (0.5, 1, 3 neonatal PMNs : 1 adult PMN), and imaging via confocal microscopy with staining for NET formation using Sytox orange (red fluorescence) was done as in Fig. 1. LPS-stimulated preterm and adult PMNs incubated separately are shown as controls in panels A and B respectively. Panel C demonstrates NET formation when equal numbers of preterm PMNs (blue) and adult PMNs (green) were incubated together. NET formation also occurred when preterm and adult PMNs in different ratios were stimulated together, and when a similar experiment was done with term PMNs (not shown).
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