Neutrophil-Mediated Inflammation in Response to Acute Myocardial Infarction.

<p dir="ltr"><b>Neutrophils and neutrophil extracellular traps (NETs) are essential effectors of the innate immune system and are increasingly appreciated as drivers of various human pathologies. Studies suggest that neutrophil activation and NETs are implicated in the pathophysiology of coronary artery disease (CAD) and hold prognostic value in predicting recovery after acute myocardial infarction (AMI). However, our understanding of neutrophils in these – and other – contexts has been hindered by the methodological complexities of studying these cells </b><b><i>ex vivo</i></b><b>, and investigations have largely been confined to analysis of few surrogate markers of these neutrophil functional pathways. The advancement of spectral flow cytometry has equipped us with the ability to examine co-expression of a variety of neutrophil activation, subsetting, and functional markers to comprehensively probe the role of neutrophils in human disease. Thus, this thesis developed high parameter spectral flow cytometric methodologies to accurately identify NETs and neutrophil subsets to investigate neutrophil-mediated inflammation in AMI. Additionally, we investigated the effect of dual antiplatelet therapy (DAPT) – as the mainstay therapeutic management of AMI – on neutrophil effector functions and phenotype in a healthy cohort.</b></p><p dir="ltr">Firstly, we developed a 7-marker flow cytometric methodology for the specific detection of cell-appendant NETs on neutrophils in peripheral blood. We established experimental conditions for monitoring NETosis <i>in vitro</i> and reported a conservative phenotyping strategy for the detection of NET-positive neutrophils. This panel utilised all surrogate markers of NETosis, and the gating strategy excluded necrotic cells to positively identify NETs over other cell death pathways. We then applied this methodology in a cohort of CAD patients and determined that cell-appendant NETs were elevated in AMI patients. The AMI cohort exhibited substantial variation in NET burden, although no clear clinical drivers for this variance were identified.</p><p dir="ltr">To determine how best to assess neutrophil subsets and function by flow cytometry, we conducted a review of current approaches to neutrophil phenotyping by cytometry to inform the design of a panel to examine circulating neutrophils. This review highlighted that a lack of standardised nomenclature and phenotyping approaches has limited our ability to delineate bona fide subsets from highly activated neutrophil populations. We subsequently developed a 17-marker panel, which overlaid several alternative subsetting markers, to identify eight peripheral neutrophil ‘subsets’ and interrogate neutrophil function across multiple pathways.</p><p dir="ltr">Next, we applied this panel in a CAD cohort to examine neutrophil-mediated inflammation in this context. As expected, we demonstrated that neutrophil activation was heightened in patients with AMI, compared to stable CAD. Additionally, when defined by manual gating, neutrophil maturation subsets differed between subjects with AMI and stable CAD. However, overlay of alternative markers to aid identification of these subsets was difficult due to discordant expression between these patient groups. We determined that high-dimensional analysis successfully delineated several neutrophil populations that exhibited distinct maturation and activation phenotypes. These populations could be recapitulated using a manual gating strategy for downstream applications.</p><p dir="ltr">Finally, we examined neutrophil phenotype and effector functions in a healthy cohort before and after a DAPT regime to mirror the therapy our AMI patients were on at the time of sampling in this thesis. We implemented our manual gating strategy to recapitulate neutrophil populations identified by high-dimensional analysis, and confirmed the presence of the same neutrophil populations in this cohort. Further, we demonstrated that these subsets did not change after DAPT. However, we demonstrated that neutrophil reactive oxygen species (ROS) production was altered following short-term DAPT, and some subsetting and activation markers changed following <i>in vitro</i> stimulation and short- and long-term DAPT.</p><p dir="ltr">Findings from this thesis demonstrate the complexity of examining neutrophil-mediated inflammation in healthy and disease states, such as CAD. We have demonstrated that comprehensive flow cytometric methodologies can be utilised to accurately identify circulating NETs and neutrophil subsets, and that peripheral neutrophils are phenotypically distinct in AMI compared to stable CAD. Furthermore, <i>in vivo</i> DAPT may alter the phenotype and function of circulating neutrophils after <i>in vitro</i> stimulation. This thesis provides a foundation for further investigation into neutrophil phenotyping by flow cytometry and application of these approaches in AMI will enhance our understanding of the critical role of neutrophils in this context.</p>