Sepsis is a dysregulated response to infection that leads to life-threatening organ damage. There remains an urgent clinical need for therapeutics to ameliorate this devastating condition. At presentation, the body bears an overwhelming load of toxic substances, including bacterial components (e.g., lipopolysaccharides (LPS)), and products of the immune response [e.g., neutrophil extracellular traps (NETs), which are webs of negatively- charged, cell-free DNA (cfDNA) complexed with positively-charged histones and antimicrobial proteins]. Interventions that block NET release (NETosis) are unlikely to be effective as overwhelming NETosis occurs in many patients prior to presentation. Enhancing NET digestion may cause harm by liberating captured bacteria and inducing the release of toxic NET-degradation products (NDPs). Both strategies also decrease microbial entrapment. Platelet factor 4 (PF4) is a positively charged chemokine released in large amounts following platelet activation that binds to and aggregates polyanions like heparin and DNA. I have found that PF4 physically compacts NETs, increasing their resistance to DNase I digestion. PF4 also binds to negatively-charged molecules on the surface of bacteria, such as LPS, and I show that PF4 markedly enhances bacterial capture by NETs. KKO, a human (h) PF4/heparin complex-binding monoclonal antibody (moAb), stabilizes PF4-NET complexes, further enhancing DNase I resistance. In vivo, hPF4 and an Fc-modified, deglycosylated KKO (DG- KKO) work in concert to improve outcomes in murine LPS endotoxemia and the cecal ligation and puncture (CLP) model of sepsis. In this proposal, I will examine the processes underlying these effects, focusing on 3 interrelated mechanisms of action: i. NET stabilization, ii. enhanced NET microbial entrapment, and iii. LPS neutralization.
Specific Aim (SA) #1: Define the mechanism by which hPF4 and DG-KKO stabilize NETs. I will define how PF4 and DG-KKO modify NETs, using PF4 variants, other cations, and anti-PF4 moAbs in vitro and in the CLP polymicrobial sepsis model. I will also examine a biorepository of septic patient plasma samples for association between PF4 levels, cfDNA concentration, cfDNA fragment size, and clinical outcomes. SA#2: Define the mechanism(s) of enhanced NET antimicrobial activity. I will test whether other cations also enhance bacterial binding in vitro and reduce microbial titers in vivo. I will define how PF4-NETs binds to gram positive- and gram negative-bacteria and assess if PF4 and DG-KKO binding improve NET-mediated bacterial killing. SA#3: Determine the mechanism by which PF4 neutralizes LPS. I will examine if PF4 blocks LPS activation of leukocytes and endothelial cells, and define how PF4 impedes LPS-induced toxicity in vitro and murine sepsis. I will perform correlative studies in plasma samples from septic patients. The proposed studies will enhance our understanding of the biology of sepsis and support a potential novel therapeutic intervention. They will also facilitate my development as an independent clinician-scientist with an academic career focused on both the care of patients with prothrombotic/proinflammatory states, and in basic research in this field.
Sepsis, which develops due to an overwhelming immune response to infection, has a high risk of poor outcomes, remains a major cause of death in the United States, and is in urgent need of novel therapeutics. I examine a model of how a class of white blood cells, called neutrophils, contribute to the development of organ damage in sepsis, and propose an innovative therapeutic strategy to reduce the damage caused by the body?s response to the infection, while enhancing the capture of bacteria and neutralization of bacterial toxins. My goal is to define the ways this strategy is protective in sepsis so that we can optimize its effects and eventually bring our approach to patient care.
