Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates

Abstract

Neutrophils are highly specialized innate effector cells that have evolved for killing of pathogens. Human neonates have a common multifactorial syndrome of neutrophil dysfunction that is incompletely characterized and contributes to sepsis and other severe infectious complications. We identified a novel defect in the antibacterial defenses of neonates: inability to form neutrophil extracellular traps (NETs). NETs are lattices of extracellular DNA, chromatin, and antibacterial proteins that mediate extracellular killing of microorganisms and are thought to form via a unique death pathway signaled by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-generated reactive oxygen species (ROS). We found that neutrophils from term and preterm infants fail to form NETs when activated by inflammatory agonists-in contrast to leukocytes from healthy adults. The deficiency in NET formation is paralleled by a previously unrecognized deficit in extracellular bacterial killing. Generation of ROSs did not complement the defect in NET formation by neonatal neutrophils, as it did in adult cells with inactivated NADPH oxidase, demonstrating that ROSs are necessary but not sufficient signaling intermediaries and identifying a deficiency in linked or downstream pathways in neonatal leukocytes. Impaired NET formation may be a critical facet of a common developmental immunodeficiency that predisposes newborn infants to infection.

Figures

Figure 1

Stimulated PMNs isolated from newborn term or preterm human infants fail to form NETs. (A) NET formation was detected by live cell imaging with confocal microscopy using 2 DNA dyes, one cell impermeable (Sytox Orange, which stains extracellular DNA red in these images) and the other cell permeable (Syto Green, which identifies nuclear DNA) after activation of PMNs with LPS (100 ng/mL) for 60 minutes. Experiments were performed on poly-l-lysine– or fibrinogen-coated (top row, third panel) glass coverslips. DNase (2.5 Units/mL) was added following LPS stimulation (60 minutes) to dismantle extracellular NETs (top row, far right panel). The images are representative of experiments performed with LPS-stimulated PMNs isolated from 6 different donors from each subject group (adult, term, and preterm). Incubations on immobilized fibrinogen and treatment of LPS-stimulated PMNs with DNase were performed with neutrophils from 3 different adult subjects in each case. Control and LPS stimulation, 20× objective. (B) NET formation was detected as in panel A following stimulation of adult, term, and preterm PMNs with PAF (10 nM) for 60 minutes. These images are representative of separate experiments using PMNs isolated from 6 different donors in each subject group (adult, term, and preterm). Control and PAF stimulation, 60× objective. (C) NET formation was examined by scanning electron microscopy following stimulation of PMNs with LPS (100 ng/mL) for 60 minutes. These images are representative of 3 different experiments with PMNs from different donors in each subject group.

Figure 2

PMNs from term and preterm neonates respond to LPS and PAF, excluding developmental deficiency in these signaling systems as a mechanism of defective NET formation. (A) Expression of mRNA for TLR4, the PAF receptor, and GAPDH in PMNs isolated from each subject group was determined by semiquantitative RT-PCR. (B) FITC-labeled LPS uptake was determined in each subject group by immunocytochemistry and confocal microscopy. (C) Intracellular Ca++ release was determined by fluorometry following preincubation with Fluo4, a Ca++-dependent fluorochrome, and stimulation with the indicated concentrations of PAF. The data bars indicate mean plus or minus SEM fluorescence in 3 separate experiments with PMNs from adult, term, and preterm subjects. (D) Secretion of IL-8 at baseline and at 240 minutes was determined by ELISA following stimulation of PMNs with PAF (10 nM) or LPS (100 ng/mL). The data indicate mean plus or minus SEM supernatant IL-8 concentrations in 3 separate experiments with PMNs from each subject group. A similar pattern was seen after a 120-minute incubation time (not shown).

Figure 3

Stimulated PMNs from term and preterm infants extrude less DNA and release less neutrophil elastase, a surrogate marker of NET formation, than do PMNs from adults. (A) Supernatant DNA content was determined via fluorometry by quantitation of a non–cell-permeable DNA dye (Sytox Orange) in samples from PMNs stimulated with LPS (100 ng/mL) for 1 hour. The data are expressed as mean plus or minus SEM values for extracellular DNA. The asterisk indicates a significant difference (P < .001) between values from adult PMNs and those from term and preterm neutrophils. (B) NET-associated neutrophil elastase was examined by immunocytochemical analysis of PAF-stimulated and control PMNs from healthy adults and term infants. Stimulated PMNs from adults (left) extruded NETs that stained robustly for NE (magenta fluorescence; white arrows). Stimulated PMNs isolated from term infants (right) failed to form NET lattices containing extracellular NE. NE was detected in primary granules in PMNs from both term neonates and adults (yellow arrows). Extracellular and intracellular DNA counterstaining as in Figure 1 demonstrated both nuclear and, depending on the source of PMNs (adult vs neonate), extracellular NET-associated DNA. (C) NET-associated NE activity was determined in DNase-treated incubations of control PMNs and neutrophils stimulated with LPS (100 ng/mL) for 120 minutes. Mean plus or minus SEM NE concentrations are shown. The asterisk indicates a significant difference (P < .05) in NE concentration in DNase-treated samples from neonatal PMNs compared with the DNase-treated samples from adult PMNs. The images and data in Figure 3 are from a minimum of 3 separate experiments in each case.

Figure 4

Extracellular bacterial killing associated with NET formation is deficient in neonatal PMNs. NET trapping of S aureus (A) and E coli (B) was examined by scanning electron microscopy following incubation of the bacteria with unstimulated PMNs from adult donors for 60 minutes. These images are representative of 3 different experiments. (C) Total bacterial killing by PMNs isolated from adults and term infants following stimulation with LPS (100 ng/mL) for 1 hour was determined (left bars). To determine NET-associated extracellular bacterial killing, phagocytosis and intracellular bacterial killing were blocked by pretreatment with cytochalasins B and D (right bars). DNase treatment was used to degrade NETs formed following LPS stimulation and block NET-associated bacterial killing to thus determine phagocytotic, intracellular bacterial killing (middle bars). See “Bacterial killing assay” for additional details. The bars indicate mean bacterial killing plus or minus SEM in 3 separate experiments. An asterisk indicates a significant difference (P < .05) in total bacterial killing and phagocytotic, intracellular bacterial killing by PMNs isolated from term infants compared with PMNs isolated from healthy adults. A double asterisk indicates a significant difference (P < .001) in extracellular, NET-associated bacterial killing by PMNs isolated from term infants compared with PMNs isolated from adults.

Figure 5

ROSs induce NET formation by adult PMNs but do not complement defective NET formation by PMNs isolated from newborn infants. NET formation was detected by live cell imaging with confocal microscopy (20× objective) using cell-permeant (green fluorescence) and impermeant (red fluorescence) DNA dyes as in Figure 1. All images are representative of 4 different experiments using PMNs from each subject group. (A) PMNs isolated from adults were incubated with buffer alone (control) or stimulated with LPS (100 ng/mL) for 60 minutes. (B) Adult PMNs were pretreated with DPI (20 μM) and then incubated for 60 minutes with either LPS (100 ng/mL) or exogenous ROSs generated by treatment with glucose/GO (1 mM/200 mU/mL). DPI inhibited endogenous ROS generation. (C) Adult PMNs were treated with glucose/GO (1 mM/200 mU/mL) alone without LPS or other agonist stimulation. (D) PMNs isolated from term (left panel) or preterm (right panel) infants were stimulated with exogenous ROSs by treatment with glucose/GO (1 mM/1000 mU/mL). Additional incubations using GO in concentrations from 200 mU/mL (ie, as with adult PMNs in panels B and C) to 1000 mU/mL yielded similar results (not shown).