Neutrophil extracellular traps (NETs) control microbial infections through their
antimicrobial activities attributed to DNA, histones, granules, and cytoplasmic proteins
(eg, elastase). Intra-amniotic infection is characterized by the influx of neutrophils
into the amniotic cavity; therefore, the aim of this study was to determine whether
amniotic fluid neutrophils form NETs in this inflammatory process.
Methods:
Amniotic fluid samples from women with intra-amniotic infection (n = 15) were stained
for bacteria detection using fluorescent dyes. Amniotic fluid neutrophils were purified
by filtration. As controls, neutrophils from maternal blood samples (n = 3) were
isolated by density gradients. Isolated neutrophils were plated onto glass cover slips
for culture with and without 100 nM of phorbol-12-myristate-13-acetate (PMA). NET
formation was assessed by 4’,6-diamidino-2-phenylindole (DAPI) staining and scanning
electron microscopy. Different stages of NET formation were visualized using antibodies
against elastase and histone H3, in combination with DAPI staining, by confocal
microscopy. Finally, maternal or neonatal neutrophils were added to amniotic fluid
samples from women without intra-amniotic infection (n = 4), and NET formation was
evaluated by DAPI staining.
Results:
(1) NETs were present in the amniotic fluid of women with intra-amniotic infection; (2)
all of the amniotic fluid samples had detectable live and dead bacteria associated with
the presence of NETs; (3) in contrast to neutrophils from the maternal circulation,
amniotic fluid neutrophils did not require PMA stimulation to form NETs; (4) different
stages of NET formation were observed by co-localizing elastase, histone H3, and DNA in
amniotic fluid neutrophils; and (5) neither maternal nor neonatal neutrophils form NETs
in the amniotic fluid of women without intra-amniotic infection.
Conclusion:
NETs are detectable in the amniotic fluid of women with intra-amniotic infection.
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