Preterm ETs Are Significantly Reduced Compared with Adults and Partially Reduced Compared with Term Infants

Abstract

The release of DNA by cells during extracellular trap (ET) formation is a defense function of neutrophils and monocytes. Neutrophil ET (NET) formation in term infants is reduced compared to adults. Objective: The aim was to quantify NET and monocyte ET (MET) release and the respective key enzymes myeloperoxidase (MPO) and neutrophil elastase (NE) in preterm infants. In this prospective explorative study, ET induction was stimulated by N-formylmethionine-leucyl-phenylalanine (fMLP), phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), and lipoteichoic acid (LTA) in the cord blood of preterm infants (n = 55, 23-36 weeks) compared to term infants and adults. METs were quantified by microscopy, and NETs by microscopy and flow cytometry. We also determined the MPO levels within NETs and the intracellular concentrations of NE and MPO in neutrophils. The percentage of neutrophils releasing ET was significantly reduced for preterm infants compared to adults for all stimulants, and with a 68% further reduction for PMA compared to term infants (p = 0.0141). The NET area was not reduced except for when fMLP was administered. The amount of MPO in NET-producing cells was reduced in preterm infants compared to term infants. For preterm infants, but not term infants, the percentage of monocytes releasing ETs was significantly reduced compared to healthy adults for LTA and LPS stimulation. Conclusion: In preterm infants, ETs are measurable parts of the innate immune system, but are released in a reduced percentage of cells compared to adults.

Keywords: extracellular traps; monocytes; neutrophils; preterm infant.

Figure 1

NET percentage and area in the preterm, term, and healthy adult groups. The NET-forming neutrophils of term (n = 10; light squares) and preterm (n = 39; light triangles) infants are shown compared to healthy adult subjects (n = 10; light dots). Isolated neutrophils were either unstimulated or treated with phorbol 12-myristate 13-acetate (PMA), N-formylmethionine-leucyl-phenylalanine (fMLP), or lipopolysaccharide (LPS). (A) NET percentage and (B) NET area (µm²) were measured by microscopy (NET >300 µm²). * p < 0.05, **** p < 0.0001. Data are presented as the median and interquartile ranges.

Figure 2

NETs were measured by flow cytometry in the preterm, term, and healthy adult groups. The NET-forming neutrophils of term (n = 19; light squares) and preterm (n = 8; light triangles) infants are shown compared to healthy adult subjects (n = 13; light dots). Isolated neutrophils were either unstimulated or treated with phorbol 12-myristate 13-acetate (PMA), N-formylmethionine-leucyl-phenylalanine (fMLP), or lipopolysaccharide (LPS). (A) The NET percentage was measured by flow cytometry. (B) The MPO amount in NETs was determined by the mean fluorescence intensity (MFI). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are presented as the median and interquartile ranges.

Figure 3

Correlation of NET percentage and area with gestational age. Preterm (n = 39) and term (n = 10) infants were investigated for a correlation by Spearman analysis. (A,B) Isolated neutrophils were either left unstimulated or (C,D) treated with phorbol 12-myristate 13-acetate (PMA), (E,F) N-formylmethionine-leucyl-phenylalanine (fMLP), or (G,H) lipopolysaccharide (LPS).

Figure 4

MET percentage and area in the preterm, term, and healthy adult groups. The MET-forming monocytes of all term (n = 16; light squares) and preterm (n = 8; light triangles) infants are shown compared to healthy adult subjects (n = 10; light dots). Isolated monocytes were either unstimulated or treated with phorbol 12-myristate 13-acetate (PMA), lipoteichoic acid (LTA), or lipopolysaccharide (LPS). (A) MET percentage and (B) MET area (µm²) were measured by microscopy; (MET >200 µm²). * p < 0.05, *** p < 0.001. Data are presented as the median and interquartile ranges.