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 8

nNIF and CRISPP inhibit in vivo NET formation.

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nNIF and CRISPP inhibit in vivo NET formation. (A and B) C57BL/6 mice no...
(A and B) C57BL/6 mice not pretreated or pretreated with nNIF, CRISPP, or CRISPP-SCR (10 mg/kg i.p.; 1 hour) were inoculated with E. coli (4.5 × 107 bacteria i.p.). After 3 hours, animals were sacrificed and peritoneal fluid and membranes collected for analysis. (A) Peritoneal fluid NET formation (red fluorescence, yellow arrow) assessed by live cell imaging and histone H3 release (red dashed line, baseline arbitrarily set at 1). Three mice per group. Original magnification, ×60. Scale bars: 50 μm. One-way ANOVA with Tukey’s post hoc testing. ***P < 0.001, CRISPP and nNIF versus CRISPP-SCR. (B) NET formation on the surfaces of peritoneal membranes (red fluorescence, yellow arrows) was quantified by counting the number of NETs that crossed standardized grid lines in 4 random microscopic fields using ImageJ software. Original magnification, ×60. Scale bars: 50 μm. A second experiment yielded a similar pattern. (C) C57BL/6 mice not pretreated (left 2 bars) or pretreated with CRISPP, nNIF, or CRISPP-SCR were inoculated with E. coli i.p. as in A and B, and neutrophil numbers in peritoneal fluid were counted after 3 hours (3–5 mice/group). One-way ANOVA with Newman-Keul post hoc testing; †P < 0.05, CRISPP vs. CRISPP-SCR or not pretreated; *P < 0.05, control vs. all other groups. (D) C57BL/6 mice were pretreated with CRISPP or CRISPP-SCR and inoculated with E. coli as in A and B (5 animals/group). After 3 hours, bacteria CFU in the peritoneal fluid were measured. Single-tailed Mann-Whitney U test, *P < 0.05. (E and F) Swiss Webster mice not pretreated or pretreated with nNIF, CRISPP, or CRISPP-SCR were inoculated with E. coli i.p. as in A and B. After 3 hours, peritoneal fluid and membranes were collected. (E) Peritoneal fluid NET formation, imaged and measured as in A (10 mice/group). *P < 0.05, CRISPP/E. coli and nNIF/E. coli compared with CRISPP-SCR/E. coli and E. coli. (F) NET formation on peritoneal membrane surfaces, imaged and quantitated as in B (3 mice in each group). *P < 0.05, E. coli versus control (red dashed line); **P < 0.01, CRISPP-SCR/E. coli versus control; †P < 0.05, CRISPP/E. coli and nNIF/E. coli versus CRISPP-SCR/E. coli. One-way ANOVA with Tukey’s post-hoc testing applied in E and F.