Establishing models of and characterising the genomic context of neutrophil hypersegmentation

Neutrophils are the most prominent leukocytes in the human body and represent the first line of defence against invading pathogens. Their nuclear architecture is highly dynamic, composed of chromatin clustered into lobes that are linked together by dense heterochromatin. Both the underlying mechanism of how neutrophils alter their nuclear architecture and the purpose of their multilobed nuclear morphology remain poorly understood. Neutrophil nuclear hypersegmentation was previously reported to be associated with the maturation state of a neutrophil. More recently, it has been observed in other contexts, suggesting it may not be a marker of cell maturity, but rather one of functional status. Neutrophil hypersegmentation has been described in response to a wide array of stimuli, and clinically in the context of tumour-associated neutrophils (TANs), bacterial infection and in the lung alveolar compartment of patients with acute respiratory distress syndrome (ARDS). This thesis describes the establishment of a model of hypersegmentation in primary human neutrophils, via an investigation of the impact of the angiotensin converting enzyme inhibitor (ACEi) captopril, glucose concentration, and hypoxia. Furthermore, the current work outlines the optimisation of a laser capture microdissection (LCM) technique for the isolation of neutrophils, based on their morphologic phenotype, to permit RNA sequencing. This optimisation has led to the first description of the transcriptome of microdissected neutrophils, and has identified genes and pathways associated with nuclear hypersegmentation. Furthermore, it has demonstrated a robust method for the transcriptomic analysis of this previously reported challenging cell type, and describes the first RNA-seq analysis of aged primary human neutrophils. These insights into the early ageing mechanisms of neutrophils highlight that neutrophils are multifaceted cells with an active transcriptional program. Further, this work reveals that neutrophil hypersegmentation is not a homogenous phenomenon, but rather a phenotype that occurs in response to multiple stimuli via distinct mechanisms. The thesis also describes the differentiation of the pro-myoblastic cell line, HL-60s and CD34+ hematopoietic stem cells as potential neutrophil-like cellular models for the investigation of hypersegmentation. Morphological and functional analysis has identified that these cells acquired segmented nuclear morphologies, and the ability to produce reactive oxygen species, phagocytose bacteria and upregulated neutrophil cell surface markers, validating these cells as tools for the study and genetic manipulation of neutrophils.