Transcriptomic analysis of peripheral blood mononuclear cells reveals age specific differences at baseline and response to acute exercise
Aging is associated with alterations in immune cell function, contributing to age-related diseases and frailty. As key drivers of the immune response, we investigated the transcriptome of peripheral blood mononuclear cells (PBMCs) using RNA-sequencing before and after high-intensity single-leg knee-extension exercise in young (Young; n=7, 23±4 years) and older individuals (Old; n=8, 65±7 years). Bioinformatic analyses revealed biological processes and pathways that were altered with aging and response to exercise. At baseline, 665 out of 19,762 genes differed in PBMCs with notable differences relating to pathways involved in DNA Damage/Telomere Stress Induced Senescence, NAD Signaling Pathway, and Oxidative Stress Induced Senescence. After exercise, 53 out of 13,695 genes were differentially expressed in the Young, while 1,026 out of 19,101 genes changed in the Old. In Young, the enriched processes and predicted pathways were linked to natural killer cells, while in Old, they focused on cell signaling immune responses. Lastly, 26 genes exhibited similar responses to exercise between groups, enriching the biological process of natural killer cell-mediated immunity regulation. Our findings indicate that PBMC gene expression is altered with aging and in response to acute exercise in older adults, with exercise having a more substantial modulating effect on the immune system response with aging. The underlying causes and functional consequences of the observed transcriptional differences require additional investigation. Overall design: Experimental Design At visit 1, an informed consent and health history questionnaire were completed, and participants were given an accelerometer to wear for a minimum of seven days. At visit 2, participants performed a graded single-leg knee extension exercise test to determine maximal work rate. Lastly, during visit 3, participants reported to the lab following an overnight fast, height and weight were recorded, and venous blood was obtained via venipuncture prior to and immediately following a single bout of a 60-minute high-intensity aerobic exercise. Max Test During visit 2, participants completed testing to determine their single leg maximal work rate on a custom knee extension ergometer, as previously described (18, 19). After a brief bout of familiarization, participants performed an incremental repeated knee extensor test composed of consecutive 1-min stages at 60 rpm starting at zero watts. Resistance was added by the application of friction to the flywheel to increase the work rate, which increased by five watts every stage until the participant reached volitional exhaustion. Before the completion of each stage, blood pressure, heart rate, and ratings of perceived exertion were recorded. Maximal work rate was determined by the highest work rate maintained for greater than 30 seconds. After completing the test, participants rested for 10 min before completing 3 minutes of single-leg knee extensions at 80% of their maximal work rate as a verification to ensure that participants could achieve this work rate on visit 3. Testing Protocol Participants performed single-leg knee extension exercise. This small muscle mass exercise paradigm isolates the quadriceps and minimizes central limitations to exercise that may be present when comparing young and older adults. Single-leg knee extension exercise started at 20% of maximal work rate and increased by 20% every three minutes. Following this initial progression, 3-minute intervals were performed at 80% of maximal work rate. Three minutes of passive recovery separated each high intensity interval. A total of nine intervals were performed and the total duration of the exercise bout was ~1 hour. Collection of Peripheral Blood Mononuclear Cells Prior to and immediately following the testing protocol venous blood was obtained in two, 10ml BD K2EDTA vacutainers (Cat No: BD-366643; VWR Avantor, Radnor PA) for fresh PBMC isolation. PBMCs were separated by density-gradient centrifugation using SepMateTM -50 (Cat No: 85450; STEMCELL technologies, Vancouver, Canada) tubes with 15ml of a density gradient medium, Histopaque (Cat No: 10771; Sigma-Aldrich, St. Louis, MO). Next, EDTA anti-coagulated blood was diluted 1:1 with phosphate-buffered saline (PBS) by mixing 14-16ml of whole blood with 14-16ml of PBS and centrifuged at 1200g for 10 minutes at room temperature (with the brake on). The PBMC layer was carefully transferred into a 50ml conical tube with 25ml PBS and centrifuged at 700g for 10 minutes. PBMCs were then washed by discarding the supernatant, resuspending the pellet in 1ml PBS before bringing the total volume to 45ml and centrifuging again at 700g for 10 minutes. The final PBMC pellet was resuspended in 5ml of freezing media (fetal bovine serum, supplemented with 10% dimethyl sulfoxide, Sigma-Aldrich, St. Louis, MO and EBM-2, Lonza, Basel Switzerland) to be stored at -80°C for 24 hours before the vials were transferred and stored at -150°C until RNA isolation described below. Cell count and viability were determined from a hemocytometer from a 20-fold dilution using 90µl of freezing media, 10µl of PBMCs and 10µl of trypan blue (Cat No: T10282; Thermo Fisher).