Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation
Christian C. Yost, … , Andrew S. Weyrich, Guy A. Zimmerman
Christian C. Yost, … , Andrew S. Weyrich, Guy A. Zimmerman
Published September 6, 2016
Citation Information: J Clin Invest. 2016;126(10):3783-3798. https://doi.org/10.1172/JCI83873.
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Research Article Inflammation

Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation

Abstract

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation.

Authors

Christian C. Yost, Hansjörg Schwertz, Mark J. Cody, Jared A. Wallace, Robert A. Campbell, Adriana Vieira-de-Abreu, Claudia V. Araujo, Sebastian Schubert, Estelle S. Harris, Jesse W. Rowley, Matthew T. Rondina, James M. Fulcher, Curry L. Koening, Andrew S. Weyrich, Guy A. Zimmerman

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Figure 1

A NET- inhibitory factor is present in human umbilical cord blood.

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A NET- inhibitory factor is present in human umbilical cord blood. (A) N...
(A) Neutrophils from 7 preterm neonates were longitudinally examined over the first 28 days after birth for NET formation in response to LPS (100 ng/ml, 1 hour) assessed by live cell imaging (NETs, red fluorescence, yellow arrows; nuclear DNA, gray) and release of NET-associated histone H3 (fold change over baseline; mean ± SEM). Original magnification, ×20. Scale bar: 100 μm. One-way ANOVA with Tukey’s post hoc testing. *P < 0.05; **P < 0.01, compared with control histone H3 release arbitrarily set at 1 (red dashed line). (B) Neutrophils isolated from cord blood of a healthy term neonate on the day of delivery (left panel) or from venous blood on day 2 after birth (right panel) were stimulated with LPS (100 ng/ml, 1 hour) and imaged as in A. Analysis of NET formation by neutrophils from a second-term neonate yielded the same pattern. (C) Neutrophils isolated from venous blood of a healthy pregnant woman on the day of delivery were incubated in medium alone or stimulated with LPS (100 ng/ml) for 1 hour and imaged as in A. Neutrophils from a second healthy term mother also robustly formed NETs in response to LPS. Original magnification, ×60. Scale bar: 100 μm. (D) Neutrophils were isolated from venous blood of 60-day-old preterm neonates (n = 5), preincubated for 1 hour with day-60 autologous plasma or with stored autologous cord blood plasma, stimulated with LPS, and assessed for NET formation as in A. Original magnification, ×60. Scale bar: 100 μm. Neutrophils isolated from venous blood of healthy adults and preincubated in autologous or stored cord blood plasma were studied in parallel. One-way ANOVA with Tukey’s post hoc testing. *P < 0.05, LPS/adult vs. LPS/neonatal; **P < 0.01, neonatal PMNs in autologous plasma vs. cord blood plasma; †P < 0.001, adult PMNs in autologous plasma vs. cord blood plasma.