NETosis in Neonates: Evidence of a Reactive Oxygen Species-Independent Pathway in Response to Fungal Challenge

Abstract

Release of neutrophil extracellular traps (NETs) is a significant antimicrobial host defense mechanism in adults. In neonates, fungal sepsis is a frequent cause of morbidity and mortality and may be a consequence of inadequate neutrophil defense functions. Like neutrophils from adult donors, we found that neutrophils from neonates formed robust cellular aggregates and released NETs in response to fungal β-glucan and Candida albicans hyphae when presented with extracellular matrix. Therefore, in response to fungal stimulation, neonatal neutrophils are capable of NETosis. Neonate susceptibility to fungal infections may not be due to an inability of their neutrophils to produce NETs.

Keywords: NETs; fungi; glucan; neonates; neutrophils.

Figure 1.

Neonatal neutrophils form CR3-mediated clusters and rapid neutrophil extracellular traps (NETs) in response to fibronectin (Fn) plus β-glucan, which is independent of respiratory burst. Neonatal neutrophils on fibronectin plus β-glucan form aggregates and NETs. A, Micrographs show adult and neonatal polymorphonuclear leukocytes (PMNs) that were adhered to wells precoated with Fn alone (left columns) or Fn supplemented with β-glucan (right columns). PMNs were pretreated with 10⁻⁹ M fMLF and 1 mM Mn⁺² immediately before adding cells to the coated wells and incubating for 30 minutes at 37°C. Data represent at least 5 independent experiments done using neutrophils from different individual donors. All images were obtained at 20× original magnification (bar, 100 µm). B, Adult and neonatal PMNs prepared as described above were pretreated with CR3-blocking monoclonal antibody (clone 44abc; left columns) or the respiratory burst inhibitor DPI (right columns) before they were adhered to wells coated with Fn plus β-glucan. Images were taken at 20× original magnification (bar, 100 µm). These results represent at least 4 independent experiments, using neutrophils from different individual donors. C, Quantification of NET formation as the percentage of the total imaged field. Data represent 4–20 wells per condition. Error bars represent standard errors of the mean (SEM). *P < .01 vs cells treated with Fn alone and those treated with CR3-blocking monoclonal antibody. D, Scanning electron microscopy images of neonatal PMNs obtained at 40× original magnification, demonstrating NET elaboration. Neonatal neutrophils were prepared as described above and were then fixed and prepared for scanning electron microscopy. Abbreviation: NS, nonsignificant.

Figure 2.

Neonatal neutrophils form CR3-mediated clusters and rapid NETs in response to Candida albicans hyphae in the context of fibronectin (Fn), which is independent of respiratory burst. Neonatal neutrophils form aggregates and NETs in response to C. albicans hyphae elaborated on Fn. A, Micrographs show adult and neonatal polymorphonuclear leukocytes (PMNs) that were adhered to wells precoated with 10 µg/mL Fn, upon which C. albicans hyphae were grown. PMNs were pretreated with 10⁻⁹ M fMLF and 1 mM Mn⁺² before adding cells to the coated wells and incubating for 30 minutes at 37°C. Data represent at least 5 independent experiments done using neutrophils from different individual donors. All images were obtained at 20× original magnification (bar, 100 µm). B, Adult and neonatal PMNs prepared as described above were pretreated with CR3-blocking monoclonal antibody (clone 44abc; left columns) or the respiratory burst inhibitor DPI (right columns) before they were adhered to wells coated with Fn and hyphae, as described above, and incubated at 37°C for 30 minutes. Images were obtained at 20× original magnification (bar, 100 µm). These results represent at least 4 independent experiments performed using neutrophils from different individual donors. C, Quantification of NET formation as the percentage of the total imaged field. Data represent 4–20 wells per condition. Error bars represent standard errors of the mean. *P < .01 vs both untreated PMNs and PMNs treated with DPI. Abbreviation: NS, nonsignificant.