Building a Better NET: Neutrophil Extracellular Trap Stabilization to Enhance Microbe Entrapment

Kandace Gollomp, M.D.
The Children’s Hospital of Philadelphia
Philadelphia, U.S.

Sepsis is a dysregulated response to infection that leads to life-threatening organ damage. This process induces the release of neutrophil extracellular traps (NETs), webs of negatively charged cell-free DNA (cfDNA) complexed with positively charged histones and antimicrobial proteins, such as myeloperoxidase (MPO), that capture and kill pathogens. However, when present in high concentrations, NETs injure the microvasculature and induce organ dysfunction. Recently, NET degradation products (NDPs), including cfDNA, histones, and MPO, have been identified in patients with COVID-19, and higher levels have been associated with increased disease severity. This finding has increased interest in targeting NETs to improve outcomes in both viral and bacterial sepsis. The most commonly proposed strategy is to eliminate NETs by either preventing their release or degrading them with nucleases. However, by the time patients with sepsis become symptomatic, they have already released a large burden of NETs, and further NET blockade is unlikely to be effective. On the other hand, therapies that accelerate NET degradation liberate captured bacteria and release toxic NDPs such as histones that can induce further tissue damage.

We propose NET stabilization as an alternative therapy for the treatment of sepsis. We have shown that the platelet chemokine platelet factor 4 (PF4, CXCL4), a highly positively charged protein that aggregates polyanions, compacts NETs and causes them to become resistant to DNase lysis. Moreover, PF4 compaction markedly enhances NET’s ability to capture bacteria. When human PF4 binds to polyanions, it changes conformation, revealing antigenic sites that bind the monoclonal antibody KKO. KKO further enhances PF4-NET complex resistance to DNase I, preventing the release of NDPs and entrapped bacteria upon exposure to DNases. These in vitro observations led us to hypothesize that infused PF4 and/or KKO may serve as a targeted therapeutic in sepsis. However, unmodified KKO stimulates leukocytes and induces a prothrombotic state. We therefore modified KKO using an IgG-specific endoglycosidase to create deglycosylated KKO (DG-KKO) that retains the ability to bind to PF4-NET complexes but has little capacity to activate platelets or complement. In a murine model of polymicrobial sepsis, DG-KKO decreased the severity of thrombocytopenia, reduced NDP release, decreased bacterial dissemination, and improved survival. These studies provide mechanistic insights into how NETs contribute to end-organ damage in sepsis and offer a targeted and novel therapeutic that minimizes their harmful effects while enhancing their protective properties.