NETs: Protection or Harm in Neonatal Inflammation and Infection

Utah State Higher Education System--University Of Utah, Salt Lake City UT

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Linked publications & trials

Abstract

We have successfully used our novel Neutrophil Extracellular Trap (NET)-Inhibitory Peptides (NIPs) to show that in experimental models of neonatal sepsis NETs increase inflammatory injury and mortality. Neutrophils extrude NETs, extracellular lattices of decondensed chromatin decorated with histones and antimicrobial proteins, to protect against infection by trapping and killing microbes. NIPs, however, inhibit NETs, decrease inflammatory injury, and increase survival in models of experimental sepsis whether used alone or in combination with antimicrobial agents like meropenem. The receptor for NIPs and the cellular mediators of NIP-induced NET inhibition remain unknown. This project will determine whether proteinase 3 (PR3), a serine protease present on the outer membrane of a subset of unstimulated neutrophils, serves as that receptor and whether NIPs must bind PR3 to inhibit NET formation. Using NIPs as tools for discovery, we will evaluate PR3 as a novel endogenous regulatory checkpoint that limits NET-mediated inflammatory tissue damage in sepsis. PR3 represents a novel NIP binding partner which complexes CD177 and phospholipid scramblase 1 (PLSCR-1) on the neutrophil surface. CD177 activates the Mac-1 integrin on neutrophils leading to reactive oxygen species generation and NET formation. PLSCR-1 mediates neutrophil apoptosis and facilitates membrane flipping in neutrophil lipid rafts. Using surrogate neutrophils differentiated from human hematopoietic stem cells and CRISPR/cas9 gene editing, our preliminary data show that NIP-mediated NET inhibition in neutrophils requires PR3 but NET formation does not. Further, PR3 knockout mouse pups fail to respond to NIPs and show decreased survival compared to wild type NIP- treated pups, suggesting that NIPs require PR3 to inhibit NET formation in vivo. Additional data show that NIP binding to PR3 may alter CD177- and PLSCR-1-dependent effects on NET formation and neutrophil apoptosis. This proposal outlines three specific aims that will provide novel insights into PR3’s role as an anti-inflammatory mediator of NET inhibition by NIPs in neonatal sepsis. We will: 1) determine whether PR3 is a/the required receptor of NIPs; 2) determine whether the PR3-complexed proteins CD177 and PLSCR-1 facilitate PR3- dependent NIP-mediated NET inhibition and apoptosis, respectively; and 3) determine whether neutrophil PR3 expression protects or harms in neonatal sepsis. These studies will significantly advance the field by identifying the receptor for NIPs on neutrophils, determining the downstream mechanistic effects of NIP binding to PR3, and evaluating the importance of neutrophil PR3 expression in preclinical models of neonatal sepsis. Our laboratory is uniquely poised to answer these innovative, translational, and mechanistic questions regarding PR3 and NIPs in neonatal sepsis. As demonstrated during our initial award, the impact of these studies will extend beyond neonatal sepsis to other inflammatory syndromes of prematurely born infants and adults, e.g., necrotizing enterocolitis, ischemic stroke, and COVID-19.