1. Introduction
1-1. Introduction and statement of problem
A growing number of published reports on exercise immunology provide evidence that in contrast to moderate physical activity, an acute bout of prolonged (>1.5 h), exhaustive exercise such as marathon or half-marathon running can cause adverse effects on immunity as reflected by transient immunosuppression and inflammation-like reactions following the event (Abbasi et al. 2013). While the beneficial effects of moderate exercise were reported with some delay, reports on increased risks of upper respiratory tract infection (URTI) following acute exhaustive bouts of exercise have been accumulating since 2 decades (Nieman et al., 1990; Nieman, 2007).
The time of decreased host protection following exhaustive exercise may last 3-72 hours and has been called ``open window`` (Fig-1) and it is now widely accepted that in this time bacteria and viruses may gain a foothold to establish an infection (see review Nieman, 2007).
Figure 1. The ``open window theory`` established by DC. Nieman (see review Nieman DC 2007a).
Demonstrated immune parameters which were affected by exhaustive exercise comprise changes in peripheral cell numbers; decreases in granulocyte burst activity, NK cytotoxic activity and lymphocyte proliferation. Certain cytokines appear in plasma (IL-6, IL-8, IL-1, IL-10), but stimulated in vitro production of some cytokines is decreased (IL-1, TNF-α,
IFN-2
γ) (Stewart et al., 2005; Kakanis et al., 2010; Woods et al., 2000; Shephard and Shek, 1999;
Ostrowski et al., 1999; Weinstock et al., 1997). Body temperature changes, increased blood flow and dehydration, and changes in stress hormones including adrenaline and glucocorticoids have been discussed as underlying mechanisms (Nieman, 1995). In particular, corticoids are known for their broad immunosuppressive effects and have been shown to be elevated in response to prolonged exhaustive endurance exercise (Keast et al., 1988). Even so, at present, the sequence of biological reactions leading to transient post-exercise immunosuppression is not really clear. One theory focuses on the observation that the normal cytokine response in vitro is strongly suppressed following exhaustive exercise. In special IFN-γ was > 90% suppressed after a marathon (Northoff et al., 1994). In fact, these experiments have shown that many possibly important effects of exhaustive exercise could neither be detected in native plasma nor in un-stimulated blood cultures, but required in vitro stimulation to become visible. LPS is a very prominent and suitable stimulant since it mimics presence of gram negative bacteria. It will rapidly engage pattern recognition receptors (in this case TLR-4) leading to activation of NF-κB transcription factor and release of a host of pro-inflammatory cytokines including IFNs and chemokines (Beutler, 2000).
Although different aspects of the immune system including macrophage activation, Natural killer cell number and activation, lymphocyte proliferation, and cytokine production have been investigated to elucidate which exercise induced changes do occur, the molecular mechanisms by which exercise exerts its negative or positive effects on the immune system are poorly understood. Several studies have examined the effect of exercise on expression of selected individual genes (mostly cytokines and heat shock proteins, which seem to be key players of inflammation and immune reactions) in peripheral blood using RT-PCR (Fehrenbach et al. 2003; Nieman et al. 2006, Nieman 2007).
Today, the microarray technology makes it possible to evaluate large numbers of genes and to assess the pattern of gene regulation simultaneously in one tissue sample. Accordingly it is now a widely used tool for comprehensive analysis of gene expression and has also been used in several exercise related studies (Connolly et al. 2004; Zieker et al. 2005; Büttner et al.
2007; Radom-Aizik et al. 2009a,b, 2008, 2013, 2014; Königsrainer et al. 2010, 2012).
However all of the existing studies focus directly on the effect of exercise on organ tissues and cells of the peripheral blood and studies investigating the effect of exhaustive exercise on
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the early steps of the immune reaction to pathogen contact using high throughput analysis were lacking before our work.
Therefore, we decided to use LPS stimulation of whole blood cultures as a model for an in vivo infection at different time points in relation to exercise, and to analyze the effects of exercise, the effects of LPS stimulation, and the effects of their combined action using gene expression microarray technology. We therefore took blood from athletes at different time points before and after exercise and performed short term whole blood culture in presence or absence of pathogen stimulation (LPS) and compared the protein and gene expression response by microarray technology. This should lead to the identification of new pathways, candidate genes or interaction patterns of exercise and pathogen effects, ultimately helping to further understand the mechanisms underlying exercise-induced immunosuppression and thus the ``open window for infection`` phenomenon.
We also included sex-specific aspects in our studies. For some good reasons we were encouraged to consider this as a second aim of our studies. First of all, the majority of exercise studies have been done in male athletes/individuals. Therefore, it is still unclear to what extent gender influences immunological responses to exercise. Sex-specific differences in the immune response to exercise have clear implications for understanding sex-specific adaptations to exercise for athletic performance and overall health. Secondly, most of the studies which have investigated sex-specific differences in immune response under exercise conditions have not considered the sex hormones fluctuations /menstrual phases in women.
Further, only few studies have investigated the sex-specific changes in gene expression profiling using microarray technology (Northoff et al. 2008, Liu et al. 2010, 2013), and finally, there is no study available investigating sex and menstrual phase dependent gene regulation of endotoxin stimulated blood culture in response to physical exercise. Therefore, to our knowledge, our studies are the first to meet these aims using high throughput technology.
2-1. Brief summaries of the publication
Paper I is an original article that has been published in Journal Exercise Immunology Review 2013. It uses standard individual measurement technology to evaluate the capacity of whole blood to produce certain selected cytokines upon short-time (1h) exposure to endotoxin (LPS) following a half-marathon run in well-trained male and female athletes. Cytokine
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concentration was measured by ELISA technology, and LPS-dependent release (LDR) was calculated by comparison with controls. The gene expression of selected cytokines was measured by qRT-PCR. The results of this paper showed strong and significant reduction in LDR of TNF-α and slight reduction in LDR of IL-6. LDR of IL-8 was enhanced post exercise in men and TGF-β1 in women. Men showed significantly higher LDR of IL-1ra at rest and 30min post exercise as compared with women and the protein pattern was roughly paralleled by mRNA. There was a significant enhancement in the concentration and parallel mRNA expression of the anti-inflammatory cytokine 10 at 30min post exercise in both sexes. IL-10 was higher in men than in women and not influenced by LPS. The main conclusion in this paper was that changes in cytokine release could only in part be attributed to changes in mRNA, and that women in their luteal phase showed less pronounced anti-inflammatory responses than men.
Paper II was published in the journal Brain Behavior Immunity-Special issue 2014. It uses probes from the same run (half-marathon) as paper I. Its purpose was to conduct a broad scale investigation into the effect of exercise on the early steps of the immune reaction to pathogen contact on the gene expression level and compare them to exercise effects in absence of pathogen. An important aim was to avoid artifacts from preparation procedures. We therefore used short time whole blood culture ± LPS and developed a new methodology to adapt the work up procedures to analysis by microarray. It was the first (exercise related) paper to publish microarray data on pathogen stimulated cultures, and its strategy was rewarded: some genes such as TNIP3 (prominent inhibitor of the LPS/TLR signaling cascade) were strongly up-regulated in LPS-stimulated cultures only. The data in this paper confirm that there is an anti-inflammatory bias in the reaction to exercise and also prompt the authors to hypothesize that the reaction to exercise may be more of a primary, preemptive, protective anti-inflammatory reaction rather than a counteraction to exercise-induced anti-inflammatory stimuli.
Paper III which has been published in journal ``Exerc Immunol Rev. 2011`` is a huge paper to which many experts in exercise immunology have contributed. It is a position statement paper that focuses on the scientific basis of what is known, accepted and deemed to be important about the influence of exercise on immune function. This paper has different sets of authors and each author or group has its or their own part for contribution. Our main contribution to this paper was the chapter ``Omics in exercise``. Here we discuss how often and how effectively exercise studies, especially exercise immunology studies, have used Omics
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technologies, on the basis of existing data. The use of each Omics technology including transcriptomics, metabolomics, and proteomics in different tissues and different types of exercise was considered in detail. All authors had equal contribution in this review article.
Paper IV, which has been published in journal ``BMC Physiol. 2013``, is an original article investigating the effect of moderate exercise on the expression of mRNAs and miRNAs and the dynamics of miRNA-mRNA regulatory networks in circulating leukocytes. Microarray technology was used to monitor the changes in transcriptome of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. This study revealed four dynamically regulated miRNA- RNA networks following exercise and was the first study to monitor miRNAs and mRNAs in parallel into the recovery period. Controversies and future directions are also discussed in this paper, which was in cooperation with our Russian partner group.
Paper V was newly published in the Journal ``Exercise Immunology Review 2014``. This paper is a review article evaluating the role of miRNA in exercise immunology, with the focus on existing data. The biological roles of miRNAs in immune system, their expression and function in circulating leukocytes and muscles in response to physical exercise, and their possible role in the beneficial effect of exercise in different diseases are discussed in this paper. This review has also been written with the cooperation of our Russian partner group, and also contained some new original data on miRNAs in exercise.
Paper VI is an original article published in journal ``Exercise Immunology Review 2008``.
This paper investigates the role of gender and menstrual phase cycles in the reaction of the immune system to exercise. We report immune-related gene expression patterns in response to an aerobic exercise at 93% of the individual anaerobic threshold of 12 male subjects (M) and 9 women with regular menstrual cycles and no use of oral contraceptives who ran both at day 10 (follicular phase, F) and at day 25 (luteal phase, L) of their cycle. Self-produced microarrays were used to analyze and compare the differentially expressed genes between males and females, and between two different phases of the female cycle. According to this paper women in luteal phase showed a distinctly different pattern of gene regulation in response to exercise, compared with women in follicular phase or males. The overall direction of gene expression changes of women in luteal phase is clearly pro-inflammatory.
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