Employing Organ-on-Chip Technology for the Study of Sepsis and Drug Screening

Abstract

Inflammation is a crucial physiological defense mechanism of the human body to injury or infection. However, dysregulation of the magnitude or duration of inflammation response underlies multiple disease pathologies and may cause organ damage. Sepsis is a severe inflammatory disease now known as a clinical syndrome defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis patients often die of organ failure and the endothelium and neutrophil-endothelial cell (EC) interactions play an active role in the regulation of the systemic inflammatory response. Systemic inflammatory disease often results in alterations in vascular endothelium barrier function, increased permeability, excessive leukocyte trafficking, and reactive oxygen species production, leading to organ damage. While neutrophils are critical 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 can damage host tissue leading to organ failure. To date therapeutic approaches are largely supportive and therapeutics targeting endothelium inflammation and immune cell dysregulation are urgently needed. However, strong concerns regarding the level of phenotypic heterogeneity of microvascular ECs between different organs have been expressed. Microvascular EC heterogeneity in different organs and organ-specific variations in EC structure and function are regulated by intrinsic signals that are differentially expressed across organs and species, as a result of which neutrophil recruitment to discrete organs may be regulated differently. In addition, therapeutic development is hindered due to the heterogeneous nature of sepsis and the presence of multiple distinct immune phenotypes that can impact function and response to infection. In fact, clinically sepsis is a heterogeneous syndrome and diagnosis is complicated due to the broad spectrum of non-specific clinical features. Patients with similar clinical symptoms can be associated with distinct immune cell phenotypes ranging from excessive immune activation to immunosuppression, which means different therapeutics are required. In this work, the morphological and functional variations of differently originated microvascular endothelium are discussed and how these variances affect systemic function in response to inflammation. Emerging in vivo and in vitro models and techniques including microphysiological devices, proteomics, and RNA-Sequencing used to study the cellular and molecular heterogeneity of endothelium from different organs will also be discussed. Our group have developed a novel Organ-on-Chip, the biomimetic microfluidic assay (bMFA) that mimics physiological conditions, allowing us to observe real-time neutrophil-endothelial interactions, including rolling, adhesion, and migration, and to study endothelial barrier function under physiologically relevant conditions including the effect of shear forces and vascular geometry. The bMFA enables the quantification of leukocyte-EC interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, EC permeability, adhesion molecule expression and other important variables. Furthermore, by using human related samples, such as human ECs and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability. In this work, a protocol was developed to study endothelium function and neutrophil-endothelial interactions during inflammation in the bMFA. Lastly, to develop targeted therapeutics, immunophenotyping is needed to identify distinct immune cell functional phenotypes. We have developed a methodology to classify ICU sepsis patients into three phenotypes using patient data, Organ-on-Chip-based neutrophil functional analysis and proteomics. The findings of the study will help identify different sepsis patient immune-phenotypes and personalize treatment accordingly.