IMPAIRED NEUTROPHIL EXTRACELLULAR TRAP FORMATION IN PLATELET-ACTIVATING FACTOR- AND LIPOPOLYSACCHARIDE-STIMULATED POLYMORPHONUCLEAR LEUKOCYTES ISOLATED FROM PRETERM INFANTS.

Abstract

Background Activated human polymorphonuclear leukocytes (PMNs) participate in the innate immune response precipitated by tissue injury or bacterial invasion. As a recently described response to bacterial invasion, PMNs secrete lattices of DNA and antimicrobial proteins termed neutrophil extracellular traps (NETs) to effect extracellular killing of bacteria. However, PMNs isolated from infants born prematurely exhibit in vitro and in vivo functional deficits in bacterial killing. Whether these deficits result from impaired NET formation in preterm PMNs remains unknown. Hypothesis We hypothesize that preterm PMNs fail to robustly form NETs following activation in vitro. Methods We therefore stimulated human PMNs isolated from adults, term infants, and preterm infants with platelet-activating factor (PAF) (10−8M), lipopolysaccharide (LPS) (100 ng/mL), or live bacteria (Escherichia coli, 0.1-10 MOI) for 30 to 120 minutes. We visualized NET formation of live and fixed PMNs stained for extracellular DNA and myeloperoxidase expression via confocal microscopy, with and without time-lapse photography. We also obtained high-resolution images of NET formation in all three cell types via scanning electron microscopy of fixed PMNs following stimulation with PAF, LPS, or E. coli. Results We visualized limited or absent NET formation via confocal microscopy at 30 and 120 minutes in LPS- and E. coli-stimulated preterm PMNs compared with robust NET formation at 30 and 120 minutes in term and adult PMNs following the same stimuli. PAF failed to induce NET formation in preterm PMNs but did induce NET formation in term and adult PMNs at 30 minutes. Time-lapse confocal microscopy allowed visualization of NET formation in term and adult PMNs but failed to show NET formation in preterm PMN preparations. Furthermore, scanning electron microscopy confirmed robust NET formation at 30 minutes in term and adult PMNs following stimulation with PAF, LPS, and E. coli but noted minimal NET formation under those conditions in preterm PMNs. Conclusion We conclude that in vitro NET formation is impaired in preterm PMNs when compared with term and adult PMNs. Speculation We speculate that impaired NET formation may contribute to bacterial killing deficits documented in preterm infants, thus contributing to the morbidity and mortality in preterm infants associated with neonatal sepsis.