Functional Immune Phenotyping of Sepsis Patients: Integrating Microphysiological Assays, Omics and In Silico Modeling

Abstract Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection with over 1.7 million cases/year and >350,000 deaths in the US. Neutrophils are important contributors to the dysregulated immune response and play a critical role in sepsis-induced organ failure through interactions with the vascular endothelium resulting in barrier disruption and increased neutrophil trafficking into vital organs. While neutrophils are vital to host defense, neutrophil dysregulation has a critical role in organ damage through release of proteases, neutrophil extracellular traps (NETs), and reactive oxygen species (ROS), which damage host tissue leading to organ failure. Drug development has been hindered for multiple reasons including the heterogenous nature of sepsis. There is now a consensus that the host response to sepsis is highly diverse among patients, and this heterogeneity impacts immune function and response to infection. To develop more effective and targeted therapeutics, a better understanding of neutrophil characteristics and identification of distinct neutrophil subpopulations is needed. To our knowledge, no studies have examined the impact of different neutrophil phenotypes on the functional consequences of neutrophil-endothelial interactions in sepsis or linked these functional consequences to altered omics. We hypothesize that functional neutrophil phenotypes in sepsis patients regulate their ability to interact with the vascular endothelium and traffic into critical organs. Employing organ-on- chip analysis, we identified three neutrophil functional phenotypes (Hyperimmune, Hypoimmune and Hybrid) in sepsis patients based on ex vivo neutrophil adherence/migration patterns. These functional phenotypes were associated with distinct proteomic signatures and differentiated sepsis patients by important clinical parameters related to disease severity. The Hyperimmune group had increased oxygen requirements, increased mechanical ventilation, and longer ICU length of stay compared to the Hypoimmune and Hybrid groups. We will use a synergistic combination of our organ-on-chip, clinical data, proteomics, and in silico modeling to provide important mechanistic insight into neutrophil functional phenotypes in sepsis. The aims of this study are: 1) Characterize the temporal progression of neutrophil functional phenotypes in patients to ascertain whether neutrophil functional phenotypes shift as sepsis progresses, 2) Test the hypothesis that neutrophil functional phenotypes are organ specific in their interactions with different organ-specific endothelial cells, 3) Determine the impact of sepsis patient phenotypes on response to therapeutics. We will identify FDA-approved drugs that target differentially expressed proteins within and across phenotypes. Using human patient neutrophils offers a unique platform to identify omic and phenotypic differences in drug responses between neutrophil phenotypes. The proposed studies will determine the impact of neutrophil phenotypes on therapeutic responses.