Abstract
The molecular mechanisms driving specific regulation of neutrophils are not completely understood to date. In order to characterize fundamental granulocyte features on protein level, we analyzed changes in proteome composition as reaction to stress from cell activation processes. For this purpose, we isolated primary granulocytes from equine whole blood through density gradient centrifugation followed by sodium chloride lysis and stimulated cells for 30 min with interleukin-8 (IL8) due to its role as a chemotactic factor for neutrophils. We additionally used phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), which are primarily associated to neutrophil extracellular trap formation and release of reactive oxygen species. From mass spectrometry analysis, we identified a total of 2,032 proteins describing the whole granulocyte proteome, including 245 proteins (12% of identified proteome) newly associated to in vivo expression in primary equine granulocytes (hypothetical proteins). We also found distinct and different changes in protein abundance (ratio ≥ 2) after short stimulation of cells with various stimuli, pointing to rapid and differentiated reaction pattern. IL8 stimulation resulted in increased protein abundance of 58 proteins (3% of proteome), whereas PMA induced changed protein abundance of 207 (10 % of proteome) and LPS of 46 proteins (2% of proteome). Enrichment analyses clearly showed fundamental differences between stimuli, with primary association of IL8 stimulation to processes in immune response, receptor signaling and signal transduction. Top enrichment for PMA on the other hand pointed to vesicle mediated transport and exocytosis. Stimulation with LPS did not result in any significant enrichment. Although we detected 43% overlap of enrichment categories for IL8 and PMA stimulation, indicating that activation of neutrophils with different stimuli partly induces some similar biological processes and pathways, hierarchical clustering showed clear differences in distribution and biological relevance of clusters between the chosen stimuli. Our studies provide novel information on the granulocyte proteome and offer insights into early, differentiated granulocyte reaction to stimuli, which contribute to a better understanding of molecular mechanisms involved in activation and recruitment of neutrophils, through inflammatory stimuli.
Highlights
Granulocytes have initially been labeled as short-lived, terminally differentiated cells, driving innate immune response through phagocytosis, degranulation, ROS release and, as described more recently, NETosis [1, 2]
We identified the equine whole granulocyte proteome, comprising a total of 2,032 proteins
After stimulation with three different stimulating agents, we found distinct changes in granulocyte protein abundance compared to medium controls
Summary
Granulocytes have initially been labeled as short-lived, terminally differentiated cells, driving innate immune response through phagocytosis, degranulation, ROS release and, as described more recently, NETosis [1, 2]. Still relatively little is known about specific, differentiated regulation mechanisms in early granulocyte activation involved in subsequent innate immune responses. For this reason, we investigated fundamental granulocyte features by analyzing changes in proteome composition as reaction to cell activation and allocating these changes to different biological processes and pathways in an equine model. The granulocytes analyzed in these studies most likely represent the subpopulation of low density neutrophils (LDN), which were recently discovered [15]. We were especially interested in the impact of initial activation on downstream innate immune response and the pathways switched on in course of activation-induced cell stress in order to provide fundamental knowledge on granulocyte activation mechanisms
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