The Acute Effects of Cardiorespiratory Exercise on Telomere-Associated Genes and MicroRNA Expression in Immune Cell Subsets.

The acute effects of cardiorespiratory exercise on telomere-associated genes and microRNA expression in immune cell subsets. CHILTON WL, MARQUES FZ, O’BRIEN BJ, and CHARCHAR F. School of Health Sciences; University of Ballarat; Victoria, Australia. ABSTRACT Telomeres are specialized nucleoprotein structures that protect the ends of linear chromosomes from degradation. Habitual physical activity is positively associated with longer leukocyte telomere length; however the molecular mechanisms underpinning the association are unclear. Human telomerase reverse transcriptase (hTERT) is the rate-limiting component of the telomere extending enzyme telomerase. The effective functioning of the adaptive immune system depends heavily upon the replicative potential of T cells, which is largely determined by telomere length and hTERT expression. Sirtuin 6 (SIRT6) also serves important pro-telomeric functions via an interaction with telomeric chromatin and regulatory roles in genome stabilization and DNA repair. It is unknown if cardiorespiratory exercise acutely regulates mRNA levels of hTERT, SIRT6 or other telomere-associated genes in white blood cells in general and T cell subsets in particular. Additionally, the exercise-induced regulation of microRNAs (short, non-coding RNA molecules that negatively regulate gene expression) with potential telomeric functions is unknown. Twenty-three healthy males (mean age=23.96 ±1.49 years) undertook 30min of treadmill running at 80% of previously determined VO2peak. Blood samples were taken before exercise, immediately post-exercise and 60min post-exercise. White blood cells and flow cytometry-sorted T cell subsets were assessed via quantitative polymerase chain reaction for differential regulation of telomeric genes and microRNAs. Expression levels of hTERT and SIRT6 mRNA were up-regulated following exercise in white blood cells and various T cell subsets (CD4+ naïve, CD4+ memory, CD8+ naïve, and CD8+ memory). Additionally, exercise differentially regulated several genes associated with telomere structure. A total of 56 microRNAs were differentially regulated post-exercise, six of which were investigated for potential telomeric functions. MicroRNAs-186, 636, 15a, and 96 showed significant up-regulation 60min post-exercise. MicroRNAs-186 and 636 showed detectable differential regulation in naïve and memory subsets. Intense cardiorespiratory exercise differentially regulated a host of telomeric genes in white blood cells and T cell subsets. Furthermore, it resulted in differential regulation of 56 microRNAs, some of which have binding potential to telomeric genes. Importantly, we demonstrated cell type-specific expression patterns in telomeric genes and microRNA. These results could have important implications for T cell-dependent immune functions and telomere homeostasis

Acute exercise leads to regulation of Telomere-Associated genes and MicroRNA expression in immune Cells

Telomeres are specialized nucleoprotein structures that protect chromosomal ends from degradation. These structures progressively shorten during cellular division and can signal replicative senescence below a critical length. Telomere length is predominantly maintained by the enzyme telomerase. Significant decreases in telomere length and telomerase activity are associated with a host of chronic diseases; conversely their maintenance underpins the optimal function of the adaptive immune system. Habitual physical activity is associated with longer leukocyte telomere length; however, the precise mechanisms are unclear. Potential hypotheses include regulation of telomeric gene transcription and/or microRNAs (miRNAs). We investigated the acute exercise-induced response of telomeric genes and miRNAs in twenty-two healthy males (mean age = 24.1±1.55 years). Participants undertook 30 minutes of treadmill running at 80% of peak oxygen uptake. Blood samples were taken before exercise, immediately post-exercise and 60 minutes post-exercise. Total RNA from white blood cells was submitted to miRNA arrays and telomere extension mRNA array. Results were individually validated in white blood cells and sorted T cell lymphocyte subsets using quantitative real-time PCR (qPCR). Telomerase reverse transcriptase (TERT) mRNA (P = 0.001) and sirtuin-6 (SIRT6) (P<0.05) mRNA expression were upregulated in white blood cells after exercise. Fifty-six miRNAs were also differentially regulated post-exercise (FDR <0.05). In silico analysis identified four miRNAs (miR-186, miR-181, miR-15a and miR-96) that potentially targeted telomeric gene mRNA. The four miRNAs exhibited significant upregulation 60 minutes post-exercise (P<0.001). Telomeric repeat binding factor 2, interacting protein (TERF2IP) was identified as a potential binding target for miR-186 and miR-96 and demonstrated concomitant downregulation (P<0.01) at the corresponding time point. Intense cardiorespiratory exercise was sufficient to differentially regulate key telomeric genes and miRNAs in white blood cells. These results may provide a mechanistic insight into telomere homeostasis and improved immune function and physical health. Funding NHMR

Differential regulation of selected miRNAs in unsorted WBCs.

<p>Relative expression of each target miRNA was assessed at pre-exercise, post-exercise and 60 minutes post-exercise (n = 18). Whilst only a strong trend was observed for miR-181b (<b>A</b>), significant changes in regulation were observed for miR-186 (<b>B</b>), miR-15a (<b>C</b>), and miR-96 (<b>D</b>). All data is expressed relative to an average of RNU44 and RNU48. Error bars indicate SEM. *indicates <i>P</i><0.05 and **indicates <i>P</i><0.01, and ***indicates <i>P</i><0.001.</p

Differential regulation of selected miRNAs in sorted T cell subset pools.

<p>(n = 22): Each miRNA was assessed in T cell subset pools. miR-181b was expressed in CD4+CD45RA+ and CD4+CD45RO+ T cells (<b>A</b>) and CD8+CD45RA+ and CD8+CD45RO+ T cells (<b>B</b>). miR-186 was also expressed in CD4+CD45RA+ and CD45RO+ T cells (<b>C</b>) and CD8+CD45RA+ and CD8+CD45RO+ T cells (<b>D</b>).</p

Differential regulation of <i>TERF2IP</i> mRNA interactions.

<p>Relative expression in WBCs (n = 16) (<b>A</b>), in CD4+CD45RA+ and CD4+CD45RO+ T cell pools (n = 22) (<b>B</b>), and in CD8+CD45RA+ and CD8+CD45RO+ T cells pools (n = 22) (<b>C</b>). Gene expression data is expressed relative to endogenous reference gene (<i>GAPDH</i>). **indicates <i>P</i><0.01.</p

A schematic overview of participant blood sampling and exercise intervention.

<p>A baseline blood sample was taken 30 minutes before the onset of exercise. Participants then completed a 30 minute bout of treadmill running at 80% of previously determined V·O_2peak. Additional blood samples were taken immediately post-exercise and at 60 minutes post-exercise.</p

Treadmill ramp test and exercise intervention data.

<p>O₂ peak (highest oxygen consumption achieved in test); V′E (minute ventilation); RER (respiratory exchange ratio).</p

Differential regulation of <i>RAD50</i> mRNA.

<p>Relative expression in WBCs (n = 16) (<b>A</b>), in CD4+CD45RA+ and CD4+CD45RO+ T cell pools (n = 22) (<b>B</b>), and in CD8+CD45RA+ and CD8+CD45RO+ T cells pools relative expression (n = 22) (<b>C</b>). Gene expression data is expressed relative to endogenous reference gene (<i>GAPDH</i>).</p

Exercise-induced changes in T cell populations.

<p>T cell populations were measured at each time point and expressed as a relative percentage of CD3+ T cells (n = 22) in both CD4+ T cells (<b>A</b>) and CD8+ T cells (<b>B</b>). Relative changes in CD45RA+ and CD45RO+ phenotypes were assessed in CD4+ T cells (<b>C</b>) and CD8+ T cells (<b>D</b>) respectively. Error bars indicate SEM. <i>†</i> indicates <i>P = </i>0.05, *indicates <i>P</i><0.05, ***indicates <i>P</i><0.001.</p

Differential regulation of <i>SIRT6</i> mRNA expression.

<p>In Unsorted WBCs (n = 17) (<b>A</b>), CD4+CD45RA+ and CD4+CD45RO+ T cells (pool of n = 22) (<b>B</b>), and CD8+CD45RA+ and CD8+CD45RO+ T cells (pool of n = 22) (<b>C</b>). Gene expression data is expressed relative to endogenous reference gene (<i>GAPDH</i>). *indicates <i>P</i><0.05.</p