In response to inflammatory stimuli, neutrophils (PMN) extrude neutrophil extracellular traps (NETs), webs of negatively-charged cell-free (cf) DNA complexed with positively-charged histones, which ensnare pathogens but also damage host tissue, contributing to diseases including sepsis, in which a large burden of NETs is acutely produced causing endothelial damage and organ dysfunction, and sickle cell disease (SCD), in which there is a chronic increase in NET production that contributes to vascular inflammation and painful vaso- occlusive episodes (VOE). Unfortunately, interventions that block NET release increase bacterial dissemination, while treatments that degrade NETs can release entrapped microbes and toxic NET- degradation products (NDPs) that contribute to multisystem organ damage. I posit that NET stabilization, in which NETs are preserved but modified to reduce NDP release and enhance bacterial capture, may be therapeutic in both sepsis and SCD. Platelet factor 4 (PF4) is a positively-charged chemokine released by activated platelets that binds to and cross-aggregates polyanions like heparin and DNA. I have found that PF4 physically compacts NETs, increasing their resistance to nucleases. PF4 also binds to negatively-charged molecules on the bacterial surface and markedly enhances their capture by NETs. KKO, a human (h) PF4:heparin complex-binding monoclonal antibody (moAb), stabilizes PF4:NETs, further increasing nuclease resistance. In murine sepsis models, hPF4 and an Fc-modified, deglycosylated KKO (DG-KKO), work in concert to decrease NDP release, enhance bacterial capture, and improve outcomes. In this proposal, I will compare the effect of NET stabilization in sepsis and SCD, to clarify its mechanism of action and assess if it is protective in both acute and chronic NET release.
Specific Aim (SA) #1: Define the mechanism(s) by which hPF4 and DG-KKO stabilize NETs. I will evaluate how hPF4 and DG-KKO modify NETs, using hPF4 variants, other cations, and anti-hPF4 moAbs in vitro and in murine sepsis models. SA#2: Define the mechanism(s) of enhanced NET antimicrobial activity. I will define how PF4:NETs bind different classes of bacteria, assess if NET stabilization enhances bacterial killing, and test whether other cations can replicate these effects. SA#3: Determine whether NET stabilization is protective in SCD. I will define whether NET-targeted therapies protective in SA#1, reduce cellular injury in microfluidic channels infused with plasma from SCD patients and in a murine model of SCD. I will then measure the association between PF4, NDP levels, and disease severity in SCD patients. I will pursue these studies within the context of a career development plan that combines didactic courses, multidisciplinary mentorship, and interdepartmental collaboration. This work will enhance our understanding of NETs and support a potential novel intervention for two distinct inflammatory disorders. It will also facilitate my growth as an independent clinician-scientist with a career at an academic pediatric center focused on the basic research and clinical care of children with prothrombotic/proinflammatory diseases.
Neutrophils, a class of white blood cells that combat bacteria, can become overactive when fighting overwhelming infection in a condition called sepsis, releasing anti-bacteria compounds that contribute to life- threatening organ damage. I propose a new therapy that neutralizes these dangerous compounds without interfering with neutrophils? ability to control infections. My goal is to further understand how this strategy works and to bring it to patient care to improve survival in sepsis and other conditions complicated by neutrophil-related inflammation, such as sickle cell disease.
