Project description:Relatively brief bouts of exercise alter gene expression in peripheral blood mononuclear cells (PBMCs), but whether or not exercise changes gene expression in circulating neutrophils (whose numbers, like PBMCs, increase) is not known. We hypothesized that exercise would activate neutrophil genes involved in apoptosis, inflammation, and cell growth and repair, since these functions in leukocytes are known to be influenced by exercise. Blood was sampled before and immediately after 30-min of constant, heavy (about 80% peak O2 uptake) cycle-ergometer exercise in 12 healthy men (19-29 yr old) of average fitness. Neutrophils were isolated using density gradients; RNA was hybridized to Affymetrix U133+2 Genechip arrays. Using FDR<0.05 with 95% confidence a total of 526 genes were differentially expressed between before and after exercise. 316 genes had higher expression after exercise. The Jak/STAT pathway, known to inhibit apoptosis, was significantly activated (EASE score, p<0.005), but 14 genes were altered in a way likely to accelerate apoptosis as well. Similarly, both proinflammatory (e.g., IL32, TNFSF8 and CCR5) and anti-inflammatory (e.g., ANXA1) were affected. Growth and repair genes like AREG and FGF2 receptor genes (involved in angiogenesis) were also activated. Finally, a number of neutrophil genes known to be involved in pathological conditions like asthma and arthritis were altered by exercise, suggesting novel links between physical activity and disease or its prevention. In summary, brief heavy exercise leads to a previously unknown substantial and significant alteration in neutrophil gene expression. Experiment Overall Design: Twelve healthy men (19-29 yr old) participated in this study. A baseline blood sample was taken before the onset of exercise and immediately after 30-min exercise bout. Neutrophils were isolated using OptiPrep Density Gradient Medium (SIGMA). Total RNA was extracted using TRIzol. cRNA was hybridized onto Affymetrix U133+2 arrays (total of 24 chips).

Project description:The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naive neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC). In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-a, IL-1-a/b), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages. This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations. Various types of myeloid cells have been shown to promote tumor progression by direct immune suppression and by production of angiogenic factors, matrix-degrading enzymes, or growth factors. In untreated tumors, neutrophils have been reported to produce angiogenic factors and matrix-degrading enzymes, support the acquisition of a metastatic phenotype, and suppress the anti-tumor immune response. Neutrophils, like all other leukocytes, move into tissues from the blood under the influence of specific chemokines (e.g. KC/CXCL-1, MIP-2a/CXCL-2 and GCP-2/CXCL-6), cytokines (e.g. TNFa and IFN-x), and cell adhesion molecules located on their own surface (e.g. CD11b) and on the surface of endothelial cells (e.g. selectins, ICAM-1 and PECAM-1). When they traffic into tumors, they are referred to as TAN. In mice, TAN can be defined by the specific surface markers CD11b and Ly6G with low expression of macrophage markers such as F4/80. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immune suppressive cells that are produced excessively in cancer. They comprise at least two subsets -granulocytic (Ly6G+, G-MDSC) and monocytic cells (Ly6C+, M-MDSC), potentially with different immunosuppressive properties. It has been previously shown that MDSC can enter tumors and differentiate to mature macrophages (TAM) or neutrophils (TAN). However, since no definitive markers have been established, it is unknown whether intratumoral N2 neutrophils (N2 TAN) are granulocytic MDSC from spleen that are attracted to the tumor or if they are typical blood-derived neutrophils that are then converted to an N2 phenotype by the tumor microenvironment, specifically by the high local concentrations of TGF-b. The purpose of this study was to use a transcriptomics approach to gain further information about TANs by comparing the RNA profile of these cells to naive bone-marrow neutrophils (NN) and to the granulocytic fraction of myeloid derived suppressor cells (G-MDSC). We examined which pathways and gene-groups varied amongst these 3 populations of neutrophils and performed a detailed analysis on pathways related to the main functions of neutrophils, such as respiratory burst, granule proteins, phagocytosis, apoptosis, structural genes, antigen presentation and specific immune effects. Our data defines TAN as a unique population of neutrophils, quite distinct from both NN and G-MDSC.

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Effects of exercise on gene expression in human neutrophils

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