A Novel Microfluidic System for Screening Anti-inflammatory Therapeutics

Abstract

Inflammation is a crucial physiological protective response of body to infection or injury. However, in pathological conditions such as sepsis or radiation damage, the body may exhibit a strong inflammatory response and cause organ damage. Loss of barrier function and leukocyte dysfunction plays an important role during inflammation (e.g. sepsis, radiation exposure, etc.) and induces tissue injury through release of proteases and oxygen radicals. Currently, pharmacological therapies for inflammatory conditions are supportive and there is an urgent need for specific treatments to effectively target key points in neutrophil-endothelial interaction. Our research team has developed a novel microfluidic system to study the mechanisms by which Protein Kinase C isotype delta (PKCδ) impacts neutrophil-endothelial interactions and its inhibition can protect vascular endothelial integrity and attenuate sepsis-induced tissue damage. This novel system will allow for rational design of next generation therapeutics for treating inflammation. We will utilize our novel biomimetic microfluidic assay (bMFA) to systematically delineate the mechanism by which PKCδ regulates individual steps in neutrophil recruitment to the inflamed/activated endothelium. In Specific Aim 1, we will investigate the impact of PKCδ inhibition on neutrophil interaction with endothelial cells as well as adhesion molecules expression. In Specific Aim 2, we will test the specificity of the PKCδ inhibitor in microcirculation and among different species. In Specific Aim 3, we will investigate the role of PKCδ in crosstalk between neutrophils and endothelial cells and endothelium integrity after high dose X-ray irradiation. Our findings indicate that PKCδ inhibition significantly reduces neutrophil interactions with the endothelium during acute inflammation. Our novel biomimetic microfluidic assay (bMFA) provides a rapid screening system for testing the specific response of novel therapeutics. Moreover, results indicate that in many cases the response of murine cells to inflammatory signals may be a poor predictor of response in human cells. Furthermore, our discoveries indicate a key role for PKCδ regulation of radiation-induced changes in endothelial cell barrier structure and function, expression of several key cell adhesion molecules, neutrophil-endothelial cell interaction and leukocyte migration through the endothelium. Our findings indicate that PKCδ-TAT peptide inhibitor may offer an important approach for treating inflammatory disease and we propose that PKCδ inhibition may serve as a novel medical countermeasure for treating radiation-induced vascular damage. Findings from this study will not only elucidate the mechanisms of action for this novel therapeutic but also provide a roadmap for the rational design of future therapeutics for acute inflammatory diseases. The long-term goal of this work is to establish microfluidic devices as a novel prescreening tool for screening therapeutics to allow for fast and precise prediction of response in human. This allows for efficient design of therapeutics using human cells and tissues, designing proper drug carrier, and planning possible future clinical studies.