We investigated the transcriptional changes in the complete genome of peripheral blood leukocytes in young endurance athletes as compared with non-athlete controls. Gene-level testing and pathway analysis revealed that genes involved in mitochondrial OXPHOS and gene translation and ribosomal protein synthesis were significantly up-regulated in endurance athletes as compared to their non-athletic counterparts. The pathway analysis also revealed that the biological processes linked to inflammation were downregulated in athletes.
We observed extensive moderate changes (transcript changes ≤2 fold) in the leukocyte transcriptome of athletes. These moderate changes were expected. In our previous work as well as the research by others, we observed changes in transcript abundance in response to acute exercise stimuli that are largely transient [9, 12, 13], and most of these changes return to basal levels within 48 h [12, 13]. It is conceivable that genomic expression adapts over time to a new steady-state level, with small differences in transcript abundance, as found in yeast cells subjected to various environmental changes . The coordinated changes we observed, albeit moderate, in the transcription levels of multiple genes within a particular biological process or a signaling pathway, may be critical to the alteration of immunological state and immune function in highly trained individuals.
Blood, a fluid tissue functioning to connect the entire biological system at the physical level, expresses over 80% of the genes in the human genome. It has been found that the expression profiles of circulating blood cells contain a specific signature in response to various physiological, pathological and environmental changes [15, 16]. Overall, the findings from the present study support this notion. The upregulation of mitochondrial OXPHOS and ribosomal protein synthesis, and downregulation of inflammation, as a consequence of endurance exercise training, have been frequently reported in skeletal muscle [17–19] and adipose tissue . Thus, the results of the present study support the idea that peripheral blood can serve as a surrogate tissue to assess the effect of exercise training on the whole system.
The alterations in the athlete’s leukocyte transcriptome may not only reflect cellular changes occurring in other tissue types, such as skeletal muscle and adipose tissue, but may also reflect alterations in immune function, since blood cells constitute the first line of the immune defense system . In the present study, we found that genes implicated in the cellular translation machinery were consistently upregulated in athletes. This included genes involved in RNA processing (e.g., SNORD14E, SNORD4B, MIR15B, SNHG12, NCRNA00188), and ribosome biogenesis (e.g., RPL10A, RPL21, RPS27, RPS19, RPLP0, RPL23). Vigorous exercise exerts a heavy assault on the biological system of participants, such as alterations in energy substrates, accumulation of metabolites, increases in body temperature, and changes in neuro-endocrine activity, etc. Living cells, including blood cells, exposed to these environmental changes, may respond with activation of protein synthesis, and accordingly activation of transcription, pre-mRNA processing, alternative splicing, etc. It is conceivable that the upregulation of genes involved in these processes is part of the molecular basis associated with the adaptation to long-term exercise training. Based on our results, the leukocyte transcriptional profile suggests that endurance athletes have a higher translation capacity and thus, protein production rate. Presumably, a higher protein turnover rate should be linked to an improved immune function due to the replacement of defective proteins with newly synthesized functioning proteins. However, the evidence linking this transcriptional change to immune function is lacking. Interestingly, a downregulation of these pathways and the pathways related to mitochondrial OXPHOS, has been identified as a key feature of aging immune cells (i.e., immunosenescence) . Therefore, the results of our study suggests that transcriptional upregulation of leukocyte mitochondrial OXPHOS and ribosomal protein synthesis may be implicated as a protective effect of endurance exercise on immunosenescence.
In the present study, genes involved in mitochondrial OXPHOS and biogenesis were upregulated in athletes. They included those encoding electron transport chain proteins (such as UQCR10, COX4I1, NDUFA12, ATP5J, ATP5H), and genes encoding mitochondria ribosomal proteins (such as MRPL51, MRPL28, MRPS33). A similar finding has been made previously, in which genes encoding enzymes in the oxidative cycle had an upregulation in blood leukocytes following six months of high volume endurance exercise training . Research over the past several years provides evidence that mitochondria play a fundamental role in the innate immune response involved in pattern-recognition, anti-bacterial immunity and sterile inflammation [23, 24]. Further, leukocyte mitochondria dysfunction, manifested by a decrease in mitochondria O2 consumption and an increase in the production of reactive oxygen species, has been implicated in the pathology of various diseases such as neurodegenerative disease , insulin resistance , type II diabetes , and cancer . Accordingly, the data from our study suggest that intense exercise training can augment individual innate immunity and resistance to certain types of diseases via upregulation of mitochondrial energetics in circulating leukocytes. It is also plausible that this transcriptional change in leukocytes reflects a low metabolic and inflammatory stress from the whole system in athletes as compared with non-athlete controls.
Consistent with the majority of the studies on endurance exercise and inflammation, the anti-inflammatory effect of chronic exercise training was reflected in the leukocyte transcriptional profile of athletes. This finding was revealed through pathway analysis. The inflammation-related pathways, such as response to endogenous/external stimulus, defense response, regulation of cell proliferation, were significantly enriched among genes showing downregulation in athletes. However, the downregulation did not reach the significance level of FDR < 0.05 based on a gene-level test. The genes driving the enrichment of inflammation-related biological processes included both pro- (IL-8, IL-15) and anti-inflammatory cytokines (DUSP1), chemotactic factors (CXCL8, CXCL1, PROK2), and factors related to leukocyte migration (ACTA2, PLSCR1, IFITM3). At the protein level, the circulating immunoglobulins A, G and M were not significantly different between athletes and controls, which is consistent with some studies, suggesting that in the resting state, the plasma immunoglobulin levels of athletes and non-athletes are very similar . However, the anti-inflammatory factor, IL-1ra, was significantly lower in athletes. At the transcriptional level, IL-1RN, the gene encoding IL-1ra, was downregulated (fc = 0.82) in athletes, but did not reach significance (p = 0.12). These results are in agreement with a previous study  that reported a coordinated downregulation of pro- and anti-inflammatory cytokines (including IL-1ra) in chronically trained elite kayakers. A similar finding in former elite athletes suggested that the decrease of cytokines was associated with high volume of current leisure time physical activity . In our study, other cytokines (TNF-α, IL-1β, IL-6, IL-10) were not detectable in most of our samples likely related to the use of frozen blood samples. Overall, the transcriptional downregulation of inflammatory pathways and decreased plasma levels of IL-1ra appears to indicate a depressed inflammatory status in athletes. Interestingly, the genes associated with antigen presentation, including HLA-DPB, HLA-DPB1, HLA-DPB2, HLA-DQA1, and HLA-DRA, were upregulated in athletes. Thus, it is plausible to suggest that chronic vigorous exercise training has an anti-inflammatory effect; however, the immune function, especially the adaptive immune function, is less likely to be affected if not improved.
The clinical importance of these transcriptional changes is hard to predict because of the complexity of the immune system and the redundancy of immune functions. Additionally, the post-transcriptional regulation of gene expression might shift the profile of the end product of proteins. Nevertheless, if the actual activation status of the peripheral blood does mirror the expression data, the results of the present study suggest that chronic intense exercise training might be a double-edged sword with respect to affecting one’s health. It adversely influences participants’ efficacy of wound healing and their resistance to minor infection . It also positively reduces one’s risk for inflammation-associated chronic disease (such as cardiometabolic diseases) and autoimmune conditions.
The biological processes related to the regulation of apoptosis, transcription, and regulation of cellular metabolic process, were also enriched among the downregulated genes. However, the genes driving the enrichment of these processes significantly overlapped (>80%) with those responsible for the enrichment of inflammation-related pathways. Thus, they may not have specific implications towards the impact of exercise training on leukocytes.
In the present study, we chose to study young and healthy athletes to minimize the influence of potential confounding factors such as aging and disease, that is known to influence immune function . Also, it is worth mentioning that to avoid the potential immune dysregulation associated with intensified training and excessive emotional stress  , thus to best mimic the general population who undergo intense endurance exercise training for health and fitness purposes, the athletes were in their regular training period and were not preparing for any competition in the following three months.
Our study has a few limitations. First, we used the whole blood and did not account for the influence of changes in peripheral leukocyte subpopulations on the transcription profile. However, considering that gene expression may be influenced by manipulation inherent to the sorting procedure and the focus of the study is the overall immune status of the peripheral blood leukocytes, we believe that involvement (or not) of minor shifts in leukocyte populations/subpopulations, would not influence the valuable biological information conveyed by the results of the study. Second, due to a limited sample size, we could not examine males and females separately. However, in the design, the athlete and the control group were matched for sex. Thus, we believe that the findings of the study are the common features in both females and males. The sex effect of immune function should be investigated in a focused study in the future.