Human Vascular Endothelial Cell Derived Exosomes Contribute To The Excessive Inflammatory Response Observed In Sepsis Through A Dysregulated MicroRNA Expression Profile.
thesisposted on 10.03.2022, 16:09 authored by Glenn FitzpatrickGlenn Fitzpatrick
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 (<200 nucleotides) concentration of S. aureus infected endothelial exosomes was significantly heightened. This increased small RNA packaging corresponded to 14 down- and 17 upregulated miRNAs being identified in endothelial exosomes following S. aureus infection. This included the upregulation of two miR-99 family members - miR-99a and miR-99b. Both miR-99a and miR-99b are putative downstream targets of mechanistic target of rapamycin (mTOR), a protein associated with the antinflammatory response to microbes.
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.
Irish Research Council (GOIPG/2016/1094)
First SupervisorProf. Steve W. Kerrigan
CommentsSubmitted for the Award of Doctor of Philosophy to the Royal College of Surgeons in Ireland, 2021.
Published CitationFitzpatrick G. Human Vascular Endothelial Cell Derived Exosomes Contribute To The Excessive Inflammatory Response Observed In Sepsis Through A Dysregulated MicroRNA Expression Profile. [PhD Thesis] Dublin: Royal College of Surgeons in Ireland; 2021
Degree NameDoctor of Philosophy (PhD)
Date of award31/05/2021
- Doctor of Philosophy (PhD)
- Vascular Biology