Sepsis is the leading cause of death in intensive care unit (ICU) and the most expensive condition treated in the United States. Over one-third of children who die in tertiary care pediatric ICU (PICU) in the United States have severe sepsis. Furthermore, recent reports suggest that its prevalence is on the rise. The Sepsis, Prevalence, Outcomes and Therapies (SPROUT) study, the first world-wide prospective study of pediatric sepsis showed that the hospital mortality of severe sepsis was significantly high (25%), hinting the immediate need to improve our care of pediatric sepsis. Given that the mortality of pediatric sepsis is associated with the presence of multiple organ dysfunction (MODS), it is critical to understand the mechanism of how MODS develops in pediatric sepsis, which will pave a way for therapeutic intervention. Neutrophils are double-edged swords in sepsis; they function to eradicate microbes, but also play a significant role in causing MODS in sepsis. Neutrophils were previously considered homogeneous but a growing literature supports that they form heterogeneous subpopulations. Our preliminary data in specific pathogen free (SPF) mice with sepsis induced by cecal ligation and puncture (CLP) model showed the development of organ injury in a time-dependent manner. In parallel, neutrophils developed heterogeneity by flow cytometry. The immunological profile of SPF mice is considered representative of immune system in children. Thus this model fits for studying pediatric sepsis. How neutrophil phenotype correlates with organ injury has not been studied in depth. Here we hypothesize that 1) neutrophils will show heterogeneous population in pediatric sepsis and there will be the distinct subpopulation associated with the degree of organ injury during sepsis, and 2) the subpopulation observed in both septic mice and pediatric patients share common features. We will test these hypotheses in mice and pediatric patients. Single cell RNA sequencing (scRNAseq) is an extremely robust technology to delineate population in an unbiased matter and has been increasingly used to define cellular heterogeneity in immunology field. Understanding the gene signature using scRNAseq will provide us an in-depth knowledge about neutrophil phenotypes. With dimensional reduction with T-distributed stochastic neighbor embedding, neutrophil subpopulations (clusters) will be presented. Pathway analysis for each cluster will be performed. We will determine the correlation between neutrophil subpopulation associated and organ injury in mice and patients. In addition, we will study in vitro neutrophil functions using blood, and determine their correlation with neutrophil subpopulations. In mice experiments, we will also perform scRNAseq of neutrophils recruited to liver, lung and kidney to delineate neutrophil population involved in tissue infiltration. At the completion of this study, we expect that we identify common neutrophil subpopulation and gene transcripts in murine sepsis and pediatric sepsis associated with organ injury. Future step is to develop therapeutic approach using mice model as a platform. The ultimate goal is to intervene pediatric sepsis early to prevent or mitigate organ injury.
The first world-wide prospective study of pediatric sepsis showed a significantly high mortality of 25%, alerting the immediate need to understand its pathophysiology and perform intervention. Because the degree of organ injury in sepsis is associated with pediatric sepsis mortality and neutrophils play a significant role in the development of organ injury in mice and pediatric patients, we will study the phenotype of neutrophils using single cell RNA sequencing and we will combine this data with organ injury analysis. Following the completion of this study, we expect that we will find genes associated with organ injury in sepsis, which will serve to understand its pathophysiology and develop potential therapeutic intervention.
