Shlomit Radom-Aizik

Evidence for microRNA involvement in exercise-associated neutrophil gene expression changes

Exercise leads to a rapid change in the profile of gene expression in circulating neutrophils. MicroRNAs (miRNAs) have been discovered to play important roles in immune function and often act to attenuate or silence gene translation. We hypothesized that miRNA expression in circulating neutrophils would be affected by brief exercise. Eleven healthy men (19-30 yr old) performed 10, 2-min bouts of cycle ergometer exercise interspersed with 1-min rest at a constant work equivalent to approximately 76% of maximal oxygen uptake (Vo(2 max)). We used the Agilent Human miRNA V2 Microarray. A conservative statistical approach was used to determine that exercise significantly altered 38 miRNAs (20 had lower expression). Using RT-PCR, we verified the expression level changes from before to after exercise of seven miRNAs. In silico analysis showed that collectively 36 miRNAs potentially targeted 4,724 genes (2 of the miRNAs had no apparent gene targets). Moreover, when we compared the gene expression changes (n = 458) in neutrophils that have been altered by exercise, as previously reported, with the miRNAs altered by exercise, we identified three pathways, Ubiquitin-mediated proteolysis, Jak-STAT signaling pathway, and Hedgehog signaling pathway, in which an interaction of miRNA and gene expression was plausible. Each of these pathways is known to play a role in key mechanisms of inflammation. Brief exercise alters miRNA profile in circulating neutrophils in humans. These data support the hypothesis that exercise-associated changes in neutrophil miRNA expression play a role in neutrophil gene expression in response to physical activity.

Effects of Exercise on microRNA Expression in Young Males Peripheral Blood Mononuclear Cells

MicroRNAs are increasingly seen as targets of drug discovery because they influence gene function acting both to silence and subtly modulate protein translation. Little is known about effects of dynamic physiological states on microRNA regulation in humans. We hypothesized that microRNA expression in peripheral blood mononuclear cells (PBMCs) would be affected by brief exercise. Twelve young men performed brief bouts of heavy exercise. PBMC microRNA was analyzed before and immediately after exercise using the Agilent Human microRNA V2 Microarray. Exercise altered expression level of 34 microRNAs (FDR < 0.05). Many of them play roles in inflammatory processes (e.g., miR‐125b[↓], down‐regulated by proinflammatory factor LPS; and miR‐132[↑], 125b[↓] and let‐7e[↓] involved inTLR4 signaling). Using previous exercise data in PBMCs, we linked the microRNA changes to specific gene pathways. This analysis identified 12 pathways including the TGF‐β and MAPK signaling. We also compared exercise‐associated microRNA changes in PBMCs with the exercise‐associated microRNAs previously identified in neutrophils. Nine microRNAs were affected in both PBMCs and neutrophils, but only six changed in the same direction. A commonly occurring physiologic perturbation, brief heavy exercise, changes microRNA profiles in PBMCs, many of which are related to inflammatory processes. The pattern of change suggests that exercise differentially influences microRNAs in leukocyte subtypes. Clin Trans Sci 2012; Volume #: 1–7

Hormonal and inflammatory responses to different types of sprint interval training.

Meckel, Y, Nemet, D, Bar-Sela, S, Radom-Aizik, S, Cooper, DM, Sagiv, M, and Eliakim, A. Hormonal and inflammatory responses to different types of sprint interval training. J Strength Cond Res 25(8): 2161-2169, 2011—We evaluated the effect of different types of sprint interval sessions on the balance between anabolic and catabolic hormones and circulating inflammatory cytokines. Twelve healthy elite junior handball players (17-25 years) participated in the study. Exercise consisted of increasing distance (100 m, 200 m, 300 m, 400 m) and decreasing distance (400 m, 300 m, 200 m, 100 m) sprint interval runs on a treadmill (at random order), at a constant work rate of 80% of the personal maximal speed (calculated from the maximal speed of a 100 m run). The total rest period between the runs in the different interval sessions were similar. Blood samples were collected before, after each run, and after 1-hour recovery. Both types of sprint interval trainings led to a significant (p < 0.05) increase in lactate and the anabolic factors growth hormone, insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3), and testosterone levels. Both types of sprint interval sessions led to a significant (p < 0.05) increase in the circulating pro- and anti-inflammatory mediators IL-1, IL-6, and IL1ra. IL-6 remained elevated in both sessions after 1-hour recovery. Area under the curve was significantly greater (p < 0.05) for lactate and growth hormone (GH) in the decreasing distance session. In contrast, rate of perceived exertion was higher in the increasing distance session, but this difference was not statistically significant (p = 0.07). Changes in anabolic-catabolic hormones and inflammatory mediators can be used to gauge the training intensity of anaerobic-type exercise. Changes in the GH-IGF-I axis and testosterone level suggest exercise-related anabolic adaptations. Increases in inflammatory mediators may indicate their important role in muscle tissue repair after anaerobic exercise. The decreasing distance interval was associated with a greater metabolic (lactate) and anabolic (GH) response but not with a higher rate of perceived exertion. Coaches and athletes should be aware of these differences, and as a result, of a need for specific recovery adaptations after different interval training protocols.

Impact of brief exercise on peripheral blood NK cell gene and microRNA expression in young adults

Natural killers (NK) cells are unique innate immune cells that increase up to fivefold in the circulating blood with brief exercise and are known to play a key role in first-response defense against pathogens and cancer immunosurveillance. Whether exercise alters NK cell gene and microRNA (miRNA) expression is not known. Thirteen healthy men (20-29 yr old) performed ten 2-min bouts of cycle ergometer exercise at a constant work equivalent to an average of 77% of maximum O2 consumption interspersed with 1-min rest. Blood was drawn before and immediately after the exercise challenge. NK cells were isolated from peripheral blood mononuclear cells using a negative magnetic cell separation method. We used Affymetrix U133+2.0 arrays for gene expression and Agilent Human miRNA V2 Microarray for miRNAs. A stringent statistical approach (false discovery rate < 0.05) was used to determine that exercise significantly altered the expression of 986 genes and 23 miRNAs. Using in silico analysis, we found exercise-related gene pathways where there was a high likelihood of gene-miRNA interactions. These pathways were predominantly associated with cancer and cell communication, including p53 signaling pathway, melanoma, glioma, prostate cancer, adherens junction, and focal adhesion. These data support the hypothesis that exercise affects the gene and miRNA expression pattern in the population of NK cells in the circulation and suggest mechanisms through which physical activity could alter health through the innate immune system.

A brief bout of exercise alters gene expression and distinct gene pathways in peripheral blood mononuclear cells of early- and late-pubertal females.

Recent studies show that brief exercise alters circulating neutrophil and peripheral blood mononuclear cell (PBMC) gene expression, ranging from cell growth to both pro-and anti-inflammatory processes. These initial observations were made solely in males, but whether PBMC gene expression is altered by exercise in females is not known. Ten early-pubertal girls (8-11 yr old) and 10 late-pubertal girls (15-17 yr old) performed ten 2-min bouts of cycle ergometry ( approximately 90% peak heart rate) interspersed with 1-min rest intervals. Blood was obtained at rest and after exercise, and microarrays were performed in each individual subject. RNA was hybridized to Affymetrix U133+2.0 Arrays. Exercise induced significant changes in PBMC gene expression in early (1,320 genes)- and late (877 genes)-pubertal girls. The expression of 622 genes changed similarly in both groups. Exercise influenced a variety of established gene pathways (EASE < 0.04) in both older (6 pathways) and younger girls (11 pathways). Five pathways were the same in both groups and were functionally related to inflammation, stress, and apoptosis, such as natural killer cell-mediated cytotoxicity, antigen processing and presentation, B cell receptor signaling, and apoptosis. In summary, brief exercise alters PBMC gene expression in early- and late-pubertal girls. The pattern of change involves diverse genetic pathways, consistent with a global danger-type response, perhaps readying PBMCs for a range of physiological functions from inflammation to tissue repair that would be useful following a bout of physical activity.

Impact of brief exercise on circulating monocyte gene and microRNA expression: Implications for atherosclerotic vascular disease

Physical activity can prevent and/or attenuate atherosclerosis, a disease clearly linked to inflammation. Paradoxically, even brief exercise induces a stress response and increases inflammatory cells like monocytes in the circulation. We hypothesized that exercise would regulate the expression of genes, gene pathways, and microRNAs in monocytes in a way that could limit pro-inflammatory function and drive monocytes to prevent, rather than contribute to, atherosclerosis. Twelve healthy men (22-30year old) performed ten 2-min bouts of cycle ergometer exercise at a constant work equivalent to an average of 82% of maximum O2 consumption interspersed with 1-min rest. Blood was drawn before and immediately after the exercise. Monocytes were isolated from peripheral blood mononuclear cells. Flow cytometry was used to identify monocyte subtypes. We used Affymetrix U133 + 2.0 arrays for gene expression and Agilent Human miRNA V2 Microarray for miRNAs. A stringent statistical approach (FDR <0.05) was used to determine that exercise significantly altered the expression of 894 annotated genes and 19 miRNAs. We found distinct gene alterations that were likely to direct monocytes in an anti-inflammatory, anti-atherogenic pathway, including the downregulation of monocyte TNF, TLR4, and CD36 genes and the upregulation of EREG and CXCR4. Exercise significantly altered a number of microRNAs that likely influence monocytes involvement in vascular health. Exercise leads to a novel genomic profile of circulating monocytes, which appears to promote cardiovascular health despite the overall stress response.

Brief bout of exercise alters gene expression in peripheral blood mononuclear cells of early- and late-pubertal males.

Peripheral blood mononuclear cells (PBMCs) are stimulated by exercise and contribute not only to host defense, but also to growth, repair, and disease pathogenesis. Whether PBMC gene expression is altered by exercise in children is not known. Ten early pubertal boys (8–12 y) and 10 late pubertal boys (15–18 y) performed ten 2-min bouts of strenuous, constant work rate exercise with 1-min rest intervals. PBMCs were isolated before and after exercise and microarray (Affymetrix U133 + 2 chips) analyzed. Statistical criterion to identify gene expression changes was less than 5% false discovery rate (FDR) with 95% confidence interval. One thousand two hundred forty-six genes were altered in older boys (517 up, 729 down), but only 109 were altered in the younger group (79 up, 30 down). In older boys, 13 gene pathways (using Expression Analysis Systematic Explorer, p < 0.05) were found (e.g. natural killer cell cytotoxicity, apoptosis). Epiregulin gene expression (EREG, a growth factor involved in wound healing) increased in older boys. In older boys exercise altered genes such as TBX21, GZMA, PGTDR, and CCL5 also play roles in pediatric inflammatory diseases like asthma. Sixty-six genes were changed significantly in both groups. The pattern of PBMC gene expression suggests the initiation of an immunologic “danger” signal associated with a sudden change in energy expenditure.

The effect of brief exercise on circulating CD34+ stem cells in early and late pubertal boys.

We tested the hypothesis that exercise could stimulate CD34+ peripheral blood hematopoietic stem cells (PBSC) in children. Fourteen early pubertal boys (EP, age 10.3 ± 0.3 y) and 13 late pubertal boys (LP, age 16.5 ± 0.4 y) performed 20 min of moderate-to-vigorous cycle ergometer exercise. Blood was drawn before and after exercise. Cells were stained for surface CD34+. Plasma granulocyte colony stimulating factor (G-CSF), Fms-like tyrosine kinase-3 (FLT-3), and stromal cell-derived factor-1 (SDF-1) were measured using ELISA. Exercise substantially increased PBSC (in EP from 112 ± 21 to 182 ± 30 cells/μL, p = 0.0007; in LP from 63 ± 8 to 152 ± 21, p = 0.0008), and to a smaller extent FLT-3 (in EP from 98 ± 5 to 110 ± 6 pg/mL, p < 0.0001; in LP from 73 ± 6 to 92 ± 6, p < 0.0001) and G-CSF (in EP from 26 ± 4 to 29 ± 4 pg/mL, p < 0.0001; in LP from 14 ± 1 to 18 ± 1, p < 0.0001). Baseline levels of PBSC, FLT-3, and G-CSF were significantly higher in EP. Exercise increased SDF-1α only in LP, and the FLT-3 increase was greater in LP than EP. Brief exercise affects PBSC and PBSC mediators in children.

Exercise and leukocyte interchange among central circulation, lung, spleen, and muscle.

Circulating leukocytes increase rapidly with exercise then quickly decrease when the exercise ends. We tested whether exercise acutely led to bidirectional interchange of leukocytes between the circulation and the lung, spleen, and active skeletal muscle. To accomplish this it was necessary to label a large number of immune cells (granulocytes, monocytes, and lymphocytes) in a way that resulted in minimal perturbation of cell function. Rats were injected intravenously with a single bolus of carboxyfluorescein diacetate succinamidyl ester (CFSE) dye which is rapidly and irreversibly taken up by circulating cells. The time course of the disappearance of labeled cells and their reappearance in the circulation following exercise was determined via flow cytometry. The majority of circulating leukocytes were labeled at 4h. post-injection and this proportion slowly declined out to 120 h. At both 24 and 120 h, running resulted in an increase in the proportion of labeled leukocytes in the circulation. Analysis of the skeletal muscle, spleen and lung indicated that labeled leukocytes had accumulated in those tissues and were mobilized to the circulation in response to exercise. This indicates that there is an ongoing exchange of leukocytes between the circulation and tissues and that exercise can stimulate their redistribution. Exchange was slower with muscle than with spleen and lung, but in all cases, influenced by exercise. Exercise bouts redistribute leukocytes between the circulation and the lung, spleen and muscle. The modulatory effects of exercise on the immune system may be regulated in part by the systemic redistribution of immune cells.

Dynamic Interactions of Gas Exchange, Body Mass, and Progressive Exercise in Children

PURPOSE Cardiopulmonary exercise testing (CPET) is increasingly used as a biomarker of fitness in children. Maximal or peak values remain the most common variables obtained in CPET, but these physiologically challenging high-intensity work rates (WR) are often not achieved. We hypothesized that interactions of gas exchange, heart rate (HR), and WR CPET variables (slopes) could yield useful mechanistic and clinical insights that might enhance the clinical utility of CPET in children. We further hypothesized that the dependence of the slope on body mass could be predicted by the first-principle analysis of body size and physiological response. METHODS One hundred and sixty-nine healthy participants (8-18 yr old, body mass index <95th percentile, 82 females) underwent dual x-ray absorptiometry scan to estimate lean body mass (LBM) and performed a ramp-type progressive cycle ergometry exercise protocol with a breath-by-breath measurement of gas exchange. Linear regression was used to calculate the slopes among VO2, VCO2, VE, HR, and WR. RESULTS ΔWR/ΔHR (r = 0.87) and ΔVO2/ΔHR (r = 0.96) were strongly correlated with VO2peak, whereas ΔVO2/ΔWR (r = 0.42) and ΔVE/ΔVCO2 (r = -0.51) were mildly correlated with peak values. LBM was more highly correlated with those slopes predicted to be body size dependent (P < 0.0001) compared with total body mass. CONCLUSIONS The data largely supported our original hypotheses. Unlike peak or maximal values, which are derived from no more than a few data points at the end of a progressive exercise test, the CPET slopes were calculated from a much larger data set obtained throughout the test. An analysis of these slopes might ultimately prove useful clinically and in research studies when peak values are not achieved.