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 6

NRPs selectively inhibit NET formation.

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NRPs selectively inhibit NET formation. Isolated adult neutrophils or pl...
Isolated adult neutrophils or platelets were preincubated with buffer or with CRISPP, nNIF, or CRISPP-SCR (1 nM, 1 hour for each), followed by measurement of functional responses. A minimum of 3 separate assays were completed for each response. Error bars represent SEM. Tukey’s post hoc testing was applied in B, D, E, and F. (A) After preincubation, neutrophils were stimulated with LPS (100 ng/ml) to trigger NET formation and incubated with a pathogenic isolate of E. coli. Total, phagocytic, and NET-mediated bacterial killing were measured (28). One-way ANOVA with Bonferroni’s post hoc testing. *P < 0.05; **P < 0.01. (B) Reactive oxygen species generation was measured by dihydrorhodamine detection after LPS stimulation (100 ng/ml, 1 hour). (C) Phagocytosis of fluorescently labeled E. coli bioparticles was measured by microscopy after a 4 hour incubation. Treatment with cytochalasin B and D served as a control for inhibition of phagocytosis. Student’s t test. *P < 0.05. (D) Chemotaxis in response to IL-8 (2 ng/ml) was examined in a Boyden chamber assay. The dashed line indicates the response to IL-8 alone arbitrarily set at 1. (E) Surface translocation of P selectin on platelets activated by thrombin (0.1 U/ml) was measured by flow cytometry. The dashed line indicates surface P selectin on unstimulated platelets. (F) Formation of platelet-neutrophil aggregates was measured after mixing of platelets activated with thrombin (0.1 U/ml) and neutrophils activated with LPS (100 ng/ml). The dashed line indicates control aggregate formation in response to LPS stimulation of the PMNs alone. *P < 0.05, CRISPP and CRISPP-SCR compared with control.