MicroRNA Profiling in Human Neutrophils during Bone Marrow Granulopoiesis and In Vivo Exudation

et al. (2013) MicroRNA Profiling in Human Neutrophils during Bone Marrow Granulopoiesis and In Vivo Exudation. PLoS ONE 8(3): e58454. doi:10.1371/journal.pone.0058454 MicroRNA Profiling in Human Neutrophils during Bone Marrow Granulopoiesis and In Vivo Exudation Maria T. Larsen 0 Christoffer Hother 0 Mattias Ha ger 0 Corinna C. Pedersen 0 Kim Theilgaard-Mo nch 0 Niels Borregaard 0 Jack B. Cowland 0 Helene F. Rosenberg, NIAID, United States of America 0 1 The Granulocyte Research Laboratory, Department of Hematology, National University Hospital, University of Copenhagen , Copenhagen , Denmark , 2 The Epigenome Research Laboratory, Department of Hematology, National University Hospital, University of Copenhagen , Copenhagen , Denmark The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, polymorphonuclear neutrophil (PMNs) from peripheral blood, and extravasated PMNs from skin windows using the Affymetrix 2.0 platform. Our data reveal 135 miRNAs differentially regulated during bone marrow granulopoiesis. The majority is differentially regulated between the myeloblast/ promyelocyte (MB/PM) and myelocyte/metamyelocyte (MC/MM) stages of development. These 135 miRNAs were divided into six clusters according to the pattern of their expression. Several miRNAs demonstrate a pronounced increase or reduction at the transition between MB/PM and MC/MM, which is associated with cell cycle arrest and the initiation of terminal differentiation. Seven miRNAs are differentially up-regulated between peripheral blood PMNs and extravasated PMNs and only one of these (miR-132) is also differentially regulated during granulopoiesis. The study indicates that several different miRNAs participate in the regulation of normal granulopoiesis and that miRNAs might also regulate activities of extravasated neutrophils. The data present the miRNA profiles during the development and activation of the neutrophil granulocyte in healthy humans and thus serves as a reference for further research of normal and malignant granulocytic development. - Differentiation of the PMN is a tightly regulated process in which the myeloid progenitor cells, the myeloblasts (MBs), divide and mature along a well defined path in the bone marrow (granulopoiesis). The different stages of maturation are characterized by distinct morphological features such as cell size, nuclear shape, and granular content [1]. Promyelocytes (PM) and myelocytes (MC) are also capable of undergoing cell division and cell cycle arrest occurs between the myelocyte and metamyelocyte (MM) stages [24]. Terminal differentiation proceeds through the band cell (BC) and segmented cell (SC) stages ending with the release of mature PMNs to peripheral blood [1]. We have previously described the in vivo mRNA and protein profiles of granule proteins [5], transcription factors [6], and cellcycle regulatory proteins [2] during neutrophil development in the bone marrow. Those studies were based on neutrophil precursors isolated at different stages of maturation from normal human bone marrow. Furthermore, cells purified by this technique have been used for a global description of mRNA expression profiles during granulopoiesis by mRNA microarray analysis [3]. We have also collected peripheral blood PMNs and PMNs from skin window chambers and examined the changes in their transcription profile by mRNA microarray analysis [7]. The main function of neutrophils is to detect and destroy invading microorganisms in tissues [8]. This involves attachment to the blood vessel wall at sites of inflammation, activation of the PMNs, mobilization of their secretory vesicles, and up-regulation of extracellular adhesion molecules [8]. Attachment to the blood vessel wall and subsequent extravasation are succeeded by migration through tissue to the inflammatory focus. During this process, PMNs are exposed to chemotactic stimuli, inflammatory cytokines, and anti-apoptotic growth factors which influence the gene expression profile of the cells [911]. The temporal expression of the genes encoding granule proteins, which can be divided into those stored in azurophil granules (formed in MB&PMs), in specific granules (formed in MC&MMs), and in gelatinase granules (formed in BCs) [8], is tightly regulated during granulopoiesis in the bone marrow by the specific up- and down-regulation of essential transcription factors such as RUNX1, PU1, C/EBP-a, and C/EBP-e [6,12]. C/EBP-a and C/EBP-e have furthermore been shown to be important for cell cycle arrest and the initiation of terminal differentiation, underscoring the importance of a strictly regulated temporal expression of transcription factors for correct neutrophil development. Different factors play a role in the fine-tuning of transcription factor expression and miRNAs are potential candidates by virtue of their ability to regulate protein synthesis. Regulation of protein expression by miRNAs may also be essential in neutrophils which have migrated to an inflammatory site in order to adapt to their new environment. MiRNAs are a group of small non-coding RNAs (1923 nt) that mainly regulate protein expression post-transcriptionally by binding to complementary sequences of the 39 UTR of mRNAs thereby destabilizing the mRNA or inhibiting its translation. A regulatory role for miRNAs has been demonstrated for several biological processes, such as proliferation, differentiation, and apoptosis, and the dysregulation of miRNA expression has been shown to contribute to the development and progression of cancer [1316]. This also applies to the development of neoplastic myeloid diseases such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [1719] and points to an important role of miRNAs as regulators of normal granulopoiesis. The purpose of this study was to determine the miRNA profile during normal human granulopoiesis starting with the first identifiable granulocytic precursor cell in the bone marrow (the myeloblast) and ending with activated neutrophils that have migrated into the tissue. We found 135 differentially expressed miRNAs during neutrophil development in the bone marrow. These could be divided into six clusters according to their expression pattern. In addition, we found seven miRNAs that were up-regulated in neutrophils that had migrated into the tissue compared to neutrophils in peripheral blood. The expression patterns of the miRNAs were verified by quantification of one or two representative miRNAs from each cluster using real-time PCR. Materials and Methods Ethics Statement All blood and bone marrow donors as well as participants in skin window experiments gave written informed consent before giving donations according to the guidelines established by the local (...truncated)