Abstract
Background: Mature human neutrophils are characterised by their multilobed nuclear morphology. Neutrophil hypersegmentation, a pathologic nuclear phenotype, has been described in the alveolar compartment of patients with acute respiratory distress syndrome and in several other contexts. This study aimed to characterise the transcriptional changes associated with neutrophil hypersegmentation. Methods: A model of hypersegmentation was established by exposing healthy peripheral blood neutrophils to the angiotensin converting enzyme inhibitor (ACEi) captopril. Laser capture microdissection (LCM) was then adapted to isolate a population of hypersegmented neutrophils. Transcriptomic analysis of microdissected hypersegmented neutrophils was undertaken using ribonucleic acid (RNA) sequencing. Differential gene expression (DEG) and enrichment pathway analysis were conducted to investigate the mechanisms underlying hypersegmentation. Results: RNA-Seq analysis revealed the transcriptomic signature of hypersegmented neutrophils, with five genes differentially expressed. VCAN, PADI4 and DUSP4 were downregulated, while LTF and PSMC4 were upregulated. Modulated pathways included histone modification, protein-DNA complex assembly and antimicrobial humoral response. The role of PADI4 was further validated using the small molecule inhibitor, Cl-amidine. Conclusions: Hypersegmented neutrophils display a marked transcriptomic signature, characterised by the differential expression of five genes. This study provides insights into the mechanisms underlying neutrophil hypersegmentation and describes a novel method to isolate and sequence neutrophils based on their morphologic subtype.
Highlights
Neutrophils are the most numerous white blood cells in humans (Rosales 2018) and the principal effectors of the innate immune system
Captopril induces neutrophil hypersegmentation in primary human neutrophils To further our understanding of neutrophil hypersegmentation, we first had to overcome the lack of availability of hypersegmented neutrophils from patients
Having determined that 300 μM was the optimum concentration of captopril, we explored the time-dependent effects of angiotensin converting enzyme inhibitor (ACEi) on hypersegmentation
Summary
Neutrophils are the most numerous white blood cells in humans (Rosales 2018) and the principal effectors of the innate immune system. To fulfil this role, neutrophils are able to readily migrate to sites of inflammation where they recognise, phagocytose, and kill invading microorganisms[1]. Mature human neutrophils are characterised by their multilobed nuclear morphology. Methods: A model of hypersegmentation was established by exposing healthy peripheral blood neutrophils to the angiotensin converting enzyme inhibitor (ACEi) captopril. Results: RNA-Seq analysis revealed the transcriptomic signature of hypersegmented neutrophils, with five genes differentially expressed. Conclusions: Hypersegmented neutrophils display a marked transcriptomic signature, characterised by the differential expression of five genes. This study provides insights into the mechanisms underlying neutrophil hypersegmentation and describes a novel method to isolate and sequence neutrophils based on their morphologic subtype
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