Nardhy Gomez-Lopez1,2,3, Roberto Romero1,4,5,6, Yi Xu1,2, Derek Miller1,2,3, Ronald Unkel1,2, Majid Shaman1,2, Suzanne M Jacques1,7, Bogdan Panaitescu1,2, Valeria Garcia-Flores1,2, Sonia S Hassan1,2. 1. 1 Perinatology Research Branch, NICHD/NIH/DHHS, Bethesda, MD, USA and Detroit, MI, USA. 2. 2 Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA. 3. 3 Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI, USA. 4. 4 Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA. 5. 5 Department of Epidemiology and Biostatistics, Michigan State University, East Lansing, MI, USA. 6. 6 Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA. 7. 7 Department of Pathology, Hutzel Women's Hospital/Harper University Hospital, Wayne State University School of Medicine, Detroit, MI, USA.
Abstract
OBJECTIVE: 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.
OBJECTIVE: 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.
Authors: Jose Galaz; Roberto Romero; Yi Xu; Derek Miller; Dustyn Levenson; Robert Para; Aneesha Varrey; Richard Hsu; Anna Tong; Sonia S Hassan; Chaur-Dong Hsu; Nardhy Gomez-Lopez Journal: J Perinat Med Date: 2020-09-25 Impact factor: 1.901
Authors: Roberto Romero; Nardhy Gomez-Lopez; Andrew D Winters; Eunjung Jung; Majid Shaman; Janine Bieda; Bogdan Panaitescu; Percy Pacora; Offer Erez; Jonathan M Greenberg; Madison M Ahmad; Chaur-Dong Hsu; Kevin R Theis Journal: J Perinat Med Date: 2019-11-26 Impact factor: 1.901
Authors: N Gomez-Lopez; R Romero; M Arenas-Hernandez; G Schwenkel; D St Louis; S S Hassan; T N Mial Journal: Clin Exp Immunol Date: 2017-05-10 Impact factor: 4.330