Abstract
Acute myocardial infarction (AMI), otherwise known as a heart attack, is one of the leading causes of morbidity and mortality in Aotearoa New Zealand. Infarction induces cardiomyocyte death. These cells are subsequently replaced with fibrotic tissue during infarct healing. This fibrotic tissue deposition, while necessary, can contribute to the longer-term development of further coronary disease. A key part to this repair process is inflammation. Neutrophils play a crucial role in the onset and resolution of inflammation; however, excessive neutrophil mediated inflammation is thought to exacerbate the damage done to the myocardium. Previously neutrophils have been treated as a single homogenous population; neutrophil heterogeneity has largely remained understudied in coronary disease and other sterile, inflammatory diseases. Neutrophils are one of the first immune cells to infiltrate the myocardium post AMI and are heavily involved in the formation and resolution of the inflammatory response. This underlies a subsequent need to identify the potential role of neutrophils in cardiac repair. The aims of this thesis were to identify if changes to the functionality of the neutrophil occurred over time post AMI, and if there is a relationship between neutrophil function and the quality of infarct healing.
Ten AMI patients were recruited for this study. Serial blood samples were collected during the patients’ hospital stay and a cardiac magnetic resonance imaging (cMRI) was done 7-10 days post recruitment. Neutrophils were isolated from blood samples, stained with antibodies, and analysed via flow cytometry. The data was unmixed and gated on in preparation for high dimensional analysis, with the intent to investigate the expression level of eight markers: CD16, CD11b, CXCR4, CD45, CD11c, TLR2, CD42b and CCR2. Veri-Cells and PBMC’s were used as a batch control, and so were investigated for uniformity to be used for normalisation in the real time processing of the patient samples. Analysis was performed using the software OMIQ (omiq.ai).
Out of the two batch controls only cryopreserved PBMC’s showed batch-to-batch uniformity and was subsequently used for patient data normalisation. Neutrophil heterogeneity was investigated by identifying distinct expression patterns with a marker. Out of the eight markers, four showed distinct populations of varying marker expression, so were investigated beyond the level of single marker comparison. Populations of distinct, quantifiable CD16 (a neutrophil maturity marker) and CCR2 (a chemokine receptor) positive and negative cells existing simultaneously within a patient were identified, and varied changes between timepoints were observed between patients. CD11b and CD11c (both adhesion markers) showed changes closest to, and at, statistical significance respectively but were not able to be classified into defined populations. Changes were not able to be quantified but were visualised.
Due to this combination of results, relating scar volume to marker patterns was underpowered for statistical analysis. Overall, both the neutrophil populations within patients and the patterns of change occurring over time between patients displayed heterogeneity, and the investigation into marker expression and scar volume is shown to be feasible for future research.