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 2

nNIF and related NRPs represent a family of NET-inhibitory peptides.

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nNIF and related NRPs represent a family of NET-inhibitory peptides. (A)...
(A) Provisional partial sequence of nNIF from mass spectroscopy and published sequences of CRISPP (35) and A1AT. (B, left panel) Samples of healthy term neonate cord blood plasma (n = 4) and adult venous plasma (n = 4) were analyzed by Western blotting using a polyclonal antibody against the carboxy terminus of A1AT (full gel shown in Supplemental Figure 1). (B, right panel) Size exclusion of full-length A1AT, quantitative Western blotting with the same polyclonal antibody against the A1AT carboxy terminus, and a standard curve generated using synthetic nNIF (Supplemental Table 2) were used to measure nNIF concentrations in cord blood plasma from preterm neonates and venous plasma from healthy adults (n = 8 in each group). Student’s t test, **P < 0.01. CB, cord blood plasma; A, adult peripheral blood plasma. (C) NET formation by LPS-stimulated (100 ng/ml, 1 hour) adult neutrophils was assessed as in Figure 1A after preincubation of the PMNs in control medium, cord blood plasma, cord blood plasma depleted of nNIF using a polyclonal carboxy terminus A1AT antibody coupled to affinity beads or eluate from the affinity beads. This result was consistent in 3 experiments with neutrophils from different donors. Original magnification, ×60. Scale bar: 50 μm. (D) Full-length A1AT, synthetic nNIF, and samples of depleted cord blood plasma and eluate studied in C were subjected to Western blotting using the carboxy terminus A1AT antibody. Full-length A1AT (52 kDa) was not detected on this 16.5% Tris-tricine gel due to its size. (E) NET formation (red fluorescence, yellow arrows; scale bar: 50 µm) by LPS-activated adult PMNs was assessed as in Figure 1A after preincubation for 1 hour in control medium (second panel), or with full-length A1AT (2 μM) or synthetic nNIF (1 nM) (n = 3). One-way ANOVA with Tukey’s post hoc test. *P < 0.05, nNIF versus both control medium/LPS and A1AT/LPS.