Human Vascular Endothelial Cell Derived Exosomes Contribute To The Excessive Inflammatory Response Observed In Sepsis Through A Dysregulated MicroRNA Expression Profile.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection and is a leading cause of mortality worldwide. The pathophysiology of sepsis is characterised by endothelial dysfunction, hypercoagulation, and sustained hyperinflammation and are key events that lead to multi-organ failure and death. A growing body of literature now suggests that the vascular endothelium plays a critical role in driving early events of sepsis progression. Upon bacterial infection, the endothelium releases a specific class of extracellular vesicle called exosomes. Exosomes are plasma membrane-derived lipid nanovesicles (30 – 120nm) capable of transporting biological material from donor to recipient cells. Biological material including DNA, protein, mRNA, and microRNA (miRNA). While the nature of the signals that drive the sustained hyperinflammatory response observed in sepsis are unknown, there are suggestions that genetic elements may be playing a role. miRNAs are short ~22 nucleotide posttranscriptional and translational repressors of gene expression. They bind mRNA with partial or complete complementarity, which results in translational inhibition of mRNA or its’s catalytic cleavage. Therefore, miRNAs can alter cell signalling pathways, gene expression and physiological responses including inflammation. An increased exosomal load containing a dysregulated miRNome released into the bloodstream by an infected endothelium may be driving the sustained an excessive inflammatory response observed in sepsis. There were three primary objectives of the research presented in this thesis. First, to demonstrate whether Staphylococcus aureus (S. aureus) infection of endothelial cells affected exosome production and exosomal miRNA packaging and expression. Secondly, to deliver S. aureus infected endothelial exosomes to monocytes and observe alterations to the inflammatory phenotype of these cells. Thirdly, to identify the specific miRNA pathways involved in driving the proinflammatory phenotype in monocytes. In this study, we demonstrated that S. aureus infection does not alter exosome production in endothelial cells. However, the small RNA (Delivery of S. aureus infected endothelial exosomes to monocytes increased CD11b and MHCII expression. Both CD11b and MHCII are upregulated in monocytes in response to proinflammatory stimuli and are, therefore, markers of monocyte activation. Increased proinflammatory marker expression correlated with heightened production of the proinflammatory cytokine IL-6 and dampened production of the antinflammatory cytokine IL-10. Serum levels of both IL-6 and IL-10 in sepsis have the highest correlation with mortality ¬in-vivo (3). Interestingly, mTOR protein expression was elevated following delivery of S. aureus infected endothelial exosomes. We demonstrated that inhibition of mTOR using miR-99a and miR-99b mimetics heightened IL-6 and dampened IL-10 production following S. aureus infection. Whereas inhibition of miR-99a and miR-99b using antagomirs facilitated increased mTOR activity which had the opposing effects on IL6 and IL-10 production. Collectively, we have demonstrated that endothelial cells release exosomes containing dysregulated miRNA expression profile following S. aureus infection. These exosomes shift monocytes into a proinflammatory phenotype resulting in heightened IL-6 production, a key proinflammatory cytokine that mediates the initial events leading to sepsis. We demonstrated that this increase in IL-6 production was directly correlated with mTOR activity. Overall, we have identified a novel pathway by which the endothelium drives the proinflammatory phenotype of monocytes in response to infection. Our data suggest that blocking miR-99a and miR-99b inhibition of mTOR following infection may be appropriate treatment strategies for critically ill sepsis patients. Further work is required to assess the effects endothelial exosomes have on other innate immune cells such as macrophages, neutrophils, dendritic cells and to identify the exact mTOR signalling cascade involved. </div

Early host interactions that drive the dysregulated response in sepsis.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. While many individual cells and systems in the body are involved in driving the excessive and sometimes sustained host response, pathogen engagement with endothelial cells and platelets early in sepsis progression, are believed to be key. Significant progress has been made in establishing key molecular interactions between platelets and pathogens and endothelial cells and pathogens. This review will explore the growing number of compensatory connections between bacteria and viruses with platelets and endothelial cells and how a better understanding of these interactions are informing the field of potential novel ways to treat the dysregulated host response during sepsis

Intra-vital imaging of mesenchymal stromal cell kinetics in the pulmonary vasculature during infection

Mesenchymal stem/stromal cells (MSCs) have demonstrated efficacy in pre-clinical models of inflammation and tissue injury, including in models of lung injury and infection. Rolling, adhesion and transmigration of MSCs appears to play a role during MSC kinetics in the systemic vasculature. However, a large proportion of MSCs become entrapped within the lungs after intravenous administration, while the initial kinetics and the site of arrest of MSCs in the pulmonary vasculature are unknown. We examined the kinetics of intravascularly administered MSCs in the pulmonary vasculature using a microfluidic system in vitro and intra-vital microscopy of intact mouse lung. In vitro, MSCs bound to endothelium under static conditions but not under laminar flow. VCAM-1 antibodies did not affect MSC binding. Intravital microscopy demonstrated MSC arrest at pulmonary micro-vessel bifurcations due to size obstruction. Retention of MSCs in the pulmonary microvasculature was increased in Escherichia coli-infected animals. Trapped MSCs deformed over time and appeared to release microvesicles. Labelled MSCs retained therapeutic efficacy against pneumonia. Our results suggest that MSCs are physically obstructed in pulmonary vasculature and do not display properties of rolling/adhesion, while retention of MSCs in the infected lung may require receptor interaction