Formation of polymorphonuclear neutrophils (PMN) is a tightly regulated process where the myeloid progenitor cells, myeloblasts (MBs), divide and mature in the bone marrow, along a well defined path. The cells pass through six well defined stages in differentiation ending up with the release of mature PMNs to peripheral blood (granulopoiesis). Expression of essential transcription factors such as RUNX1, C/EBP-a, and C/EBP-e during granulopoiesis has been shown to have great importance for correct neutrophil development. microRNAs (miRNAs) could be important players in the fine-tuning of transcription factor expression due to their ability to regulate protein synthesis.
The function of neutrophils is to detect and destroy invading microorganisms. This involves activation of the PMNs in the blood stream causing a release of secretory vesicles and up-regulation of extracellular adhesion molecules followed by migration in the tissue towards the focus of inflammation. Expression of miRNAs might also be regulated during activation and diapedesis of the neutrophils in order to adapt the neutrophil to its new environment and function.
A regulatory role for miRNAs has been demonstrated for several biological processes, such as proliferation, differentiation, inflammation and cancer, and dysregulation of miRNA expression has been shown to contribute to disease development.
The purpose of this study was to determine the miRNA expression profiles during normal human granulopoiesis starting with the first identifiable granulocytic precursor cell (MB) and ending with activated neutrophils that have migrated into the tissue using an Affymetrix 2,0 miRNA microarray platform. We isolated four populations of cells: Myeloblasts (MB) and promyelocytes (PM), myelocytes (MC) and metamyelocytes (MM), and band cells (BC) and segmented cells (SC) from the bone marrow and PMNs from peripheral blood from three different donors. We found 135 differentially expressed miRNAs in granulopoiesis, which could be divided into six clusters according to their expression pattern. 87% of the 135 miRNAs were differentially regulated between the MB/PM (dividing cells) and the MC/MM stages (cessation of cell proliferation and initiation of terminal differentiation) and could imply a need for miRNA-mediated regulation of the many proteins involved in regulating this process. Interestingly, we also found two distinct clusters of miRNAs that were either up- or down-regulated only in the MC/MM population, indicating the importance of a specific temporary regulation of some proteins during neutrophil development.
To determine miRNA expression profiles in activated granulocytes, we examined PMNs and activated neutrophils from skin window (i.e. PMNs migrated to a site of inflammation). We found seven differentially expressed miRNAs, - all of them up regulated in the activated neutrophils.
Using microRNA target-prediction software, we found that miRNAs 155, 146a and 130a, all of which are strongly up-regulated in the MB/PM stage, have several targets in the IL1-receptor signalling cascade, indicating the importance of miRNA of dampening an innate immune response in immature neutrophil precursors.
miR-146a, 155 and 130a also have predicted targets in either the TGF-βI or the TGF-βII receptor which inhibits proliferation when binding to TGF-β. This finding supports the proliferating profile for the MB/PM cells, and the shift towards cell cycle arrest when the cells differentiate to the next stage, where expression of these three miRNAs is low. miRNA-34c-3p is highly expressed only in the MC/MM stage and has verified targets in many different mRNAs involved in the regulation of cell cycle arrest. All the miRNAs that were up-regulated in the activated neutrophils have several predicted targets in the IL1R pathway, and some of them (miR-212, −132 and −297) have previously been shown to be important in regulating the inflammatory response.
The study indicates that several different miRNAs have important roles in the regulation of normal granulopoiesis, and that miRNAs also might be part of a possible negative feed back loop in the inflammatory response in activated neutrophils.
Grant acknowledgments: The Danish Cancer Society, Lundbeck foundation, Danish Medical Research Council, Brøchner Mortensen foundation
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.