Envenoming & NETosis: defining the pathophysiological contribution and drug target validity of neutrophil extracellular traps to snakebite morbidity

Background Snakebite envenoming is a World Health Organization-listed neglected tropical disease that predominately affects rural, impoverished populations in the tropics. More than 100,000 people die each year from snakebite and perhaps as many as half a million more suffer from lifelong morbidity. Venom toxin variation among different medically important biting snake species results in diverse snakebite pathologies in victims. The signs and symptoms of envenoming are currently treated with immunoglobulin-based, toxin-specific therapies known as antivenom. However, these treatments are ineffective at combatting local envenoming, which is caused by various venom cytotoxins and commonly presents as progressive swelling, blistering and tissue destruction around the bite site, ultimately resulting in the need for surgical interventions, such as tissue removal or digit/limb amputations. There is therefore an urgent and compelling need to better understand the pathophysiology of local envenoming in order to identify more effective treatment strategies to reduce the substantial burden of snakebite-induced morbidity in the rural tropics. Rationale Existing snakebite treatments are likely ineffective against local envenoming because intravenously delivered antivenom does not effectively spread from the bloodstream into the affected bite site, and because patients often present to healthcare facilities long after venom cytotoxins have initiated host processes that contribute to the severity of envenoming pathology. Although various pathways are thought to be involved in these initial responses to envenoming, NETosis - an innate immunity process associated with disease pathology in conditions such as HIV/AIDS, malaria and sepsis - seems likely to play a major role in snakebite morbidity by locally entrapping venom toxins, blocking blood vessels and freeing cell-damaging histones, leading to tissue destruction around the bite site. Indeed, our preliminary data from experimental models of envenoming shows that venom can strongly stimulate NETosis via the formation of neutrophil extracellular traps and release of pathologically relevant levels of extracellular histones. However, the mechanisms by which snake venoms stimulate NETosis, the generality of this process across different morbidity-causing snake venoms, and the relative contribution of NETosis to the severity of local envenoming, though likely to be high, remains largely unknown. In this project, we seek to redress these important knowledge gaps and additionally test whether therapeutics targeting NETosis reduce the severity of snakebite envenoming, thereby presenting a novel adjunct treatment strategy for preventing morbidity caused by this neglected tropical disease. Objectives We will use a combination of cell-based, in vivo preclinical and clinical laboratory research strategies to address the following key questions: How do snake venoms stimulate NETosis? Which venom toxins are responsible for causing this effect? What is the relative contribution of NETosis to the severity of local snakebite pathology? Do clinical samples from human snakebite victims show evidence of NETosis? Is NETosis a valid drug target for the development of new treatments effective at preventing snakebite morbidity? Which therapeutic molecules show the most promise at preventing NETosis? Implications We anticipate that this research will define the importance and generality of NETosis to snakebite envenoming pathology, including its detection in human snakebite victims for the first time, while simultaneously identifying preclinically validated therapies that effectively disrupt this process. This project therefore has considerable potential to identify, for the first time, adjunct snakebite treatments that could prevent the disabling effects and long-term physical morbidity caused by the world’s most neglected tropical disease.