Abstract
Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease.
Highlights
Neutrophils are the most abundant white blood cell in the body, playing an essential role in the destruction of invading bacterial and fungal pathogens
Before preparation of microRNA samples, the purity of the enriched human neutrophils was assessed by cytospin to be 97.9%60.21 (n = 5, individual purities 98, 97.15, 97.95, 98.25, 98), with the very low level of contaminating cells identified as eosinophils
Elimination of monocyte contamination is critical in these experiments, since monocytes have been shown to modulate neutrophil responses to inflammatory stimuli [36] and may theoretically contribute disproportionately to the detectable microRNA population
Summary
Neutrophils are the most abundant white blood cell in the body, playing an essential role in the destruction of invading bacterial and fungal pathogens. They are rapidly recruited to sites of injury, where they extravasate into the tissues and destroy the pathogen via several mechanisms, including phagocytosis, and the release of antimicrobial substances [1]. In parallel, aged neutrophils can undergo spontaneous apoptosis, with a half-life of less than 8 hours [8] This is followed by their subsequent removal by phagocytic cells such as macrophages, a mechanism to prevent unwanted inflammation and tissue damage [9]. Neutrophil apoptosis is thought to be a major factor in the functional senescence of neutrophils [11], and the role of changes in protein expression caused by microRNAs have not to date been investigated
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