title: Trophoblast cells inhibit neutrophil extracellular trap formation and enhance apoptosis through vasoactive intestinal peptide-mediated pathways.
abstract: Do human trophoblast cells modulate neutrophil extracellular trap (NET) formation, reactive oxygen species (ROS) synthesis and neutrophil apoptosis through mechanisms involving vasoactive intestinal peptide (VIP)?
  
  
  
  Trophoblast cells inhibited NET formation and ROS synthesis and enhanced neutrophil apoptosis through VIP-mediated pathways in a model of maternal-placental interaction.
  
  
  
  Immune homeostasis maintenance at the maternal-placental interface is mostly coordinated by trophoblast cells. Neutrophil activation and NET formation increases in pregnancies complicated by exacerbated pro-inflammatory responses. VIP has anti-inflammatory and immunosuppressant effects and is synthesized by trophoblast cells.
  
  
  
  This is a laboratory-based observational study that sampled circulating neutrophils from 50 healthy volunteers to explore their response in vitro to factors derived from human trophoblast cells.
  
  
  
  Peripheral blood neutrophils were isolated from healthy volunteers and tested in vitro with first trimester trophoblast cell line (Swan-71 and HTR8) conditioned media (CM) or with VIP. The effect of VIP and trophoblast CM on NET formation was assessed by co-localization of elastase and DNA by confocal microscopy, DNA release and elastase activity measurement. Neutrophil apoptosis was determined by flow cytometry or fluorescence microscopy. ROS formation was assessed by flow cytometry with a fluorescent probe. VIP silencing was performed by siRNA transfection. For phagocytosis of apoptotic neutrophils, autologous monocytes were sampled, and engulfment and cytokines were assessed by flow cytometry and ELISA.
  
  
  
  Trophoblast CM and 10 nM VIP promoted neutrophil deactivation by preventing phorbol myristate acetate-induced NET formation and ROS synthesis while they increased neutrophil spontaneous apoptosis and reversed the anti-apoptotic effect of lipopolysaccharide (all P < 0.05 versus control). The effects of trophoblast CM were prevented by a VIP antagonist or when VIP knocked-down trophoblast cells were used (P < 0.05 versus control). Neutrophils driven to apoptosis by trophoblast CM could be rapidly engulfed by monocytes without increasing IL-12 production.
  
  
  
  Not applicable.
  
  
  
  The mechanisms of neutrophil deactivation by trophoblast VIP are based on the results obtained with neutrophils drawn from peripheral blood of healthy individuals interacting with trophoblast cell lines in vitro. These studies were designed to investigate biological processes at the cellular and molecular level; therefore, they have the limitations of studies in vitro and it is not possible to ascertain if these mechanisms operate similarly in vivo. We tested 50 neutrophil samples from healthy volunteers that have a normal variability in their responses. Cell lines derived from human trophoblast were used, and we cannot rule out a differential behavior of trophoblast cells in contact with neutrophils in vivo.
  
  
  
  Results presented here are consistent with an active mechanism through which neutrophils in contact with trophoblast cells would be deactivated and silently cleared by decidual macrophages throughout pregnancy. They support a novel immunomodulatory role of trophoblast VIP on neutrophils at the placenta, providing new clues for pharmacological targeting of immune and trophoblast cells in pregnancy complications associated with exacerbated inflammation.
  
  
  
  This work was funded by the National Agency of Sciences and Technology (PICT 2011-0144, 2014-0657 and 2013-2177) and University of Buenos Aires (UBACyT 20020130100040BA, 20020150100161BA and 20020130100744BA). The authors declare no competing interests.
authors.affiliation: CONICET, Facultad de Ciencias Exactas y Naturales, Instituto de Química Biológica (IQUIBICEN), Universidad de Buenos Aires, Ciudad Universitaria, Pab II. Fourth Floor, 1428 Buenos Aires, Argentina.
authors.forename: Guillermina
authors.initials: G
authors.lastname: Calo
authors.affiliation: CONICET, Facultad de Medicina, Instituto de Medicina Experimental, Academia Nacional de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Florencia
authors.initials: F
authors.lastname: Sabbione
authors.affiliation: CONICET, Facultad de Ciencias Exactas y Naturales, Instituto de Química Biológica (IQUIBICEN), Universidad de Buenos Aires, Ciudad Universitaria, Pab II. Fourth Floor, 1428 Buenos Aires, Argentina.
authors.forename: Daiana
authors.initials: D
authors.lastname: Vota
authors.affiliation: CONICET, Facultad de Ciencias Exactas y Naturales, Instituto de Química Biológica (IQUIBICEN), Universidad de Buenos Aires, Ciudad Universitaria, Pab II. Fourth Floor, 1428 Buenos Aires, Argentina.
authors.forename: Daniel
authors.initials: D
authors.lastname: Paparini
authors.affiliation: CONICET, Facultad de Ciencias Exactas y Naturales, Instituto de Química Biológica (IQUIBICEN), Universidad de Buenos Aires, Ciudad Universitaria, Pab II. Fourth Floor, 1428 Buenos Aires, Argentina.
authors.forename: Rosanna
authors.initials: R
authors.lastname: Ramhorst
authors.affiliation: CONICET, Facultad de Medicina, Instituto de Medicina Experimental, Academia Nacional de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Analía
authors.initials: A
authors.lastname: Trevani
authors.affiliation: CONICET, Facultad de Ciencias Exactas y Naturales, Instituto de Química Biológica (IQUIBICEN), Universidad de Buenos Aires, Ciudad Universitaria, Pab II. Fourth Floor, 1428 Buenos Aires, Argentina cpleiros@qb.fcen.uba.ar.
authors.forename: Claudia
authors.initials: C
authors.lastname: Pérez Leirós
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D017077:Culture Media, Conditioned / Q000494:pharmacology; D065206:Extracellular Traps / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006801:Humans; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology; D011247:Pregnancy; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014327:Trophoblasts / Q000187:drug effects / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology*
chemical_list: D017077:Culture Media, Conditioned; D017382:Reactive Oxygen Species; D014660:Vasoactive Intestinal Peptide
keywords: neutrophil apoptosis; neutrophil extracellular traps; neutrophils; phagocytosis of apoptotic cells; trophoblast cells; vasoactive intestinal peptide