In this study, we thoroughly investigated the effects of different aspects of a training program which was previously shown to allow for voluntary activation of the sympathetic nervous system and attenuation of the inflammatory response. First, we showed that, although arterial blood saturation levels and pO2 were significantly lower when subjects performed the breathing exercise with prolonged breath retention compared to that without, plasma adrenaline levels increased with a similar magnitude shortly after initiation of both breathing exercises. Second, we demonstrated that the previously observed physiological and immunological effects [1] are independent from either the length of training or the individual who provides it. Third, our data signify that the combination of the breathing exercise and cold exposure training is most effective in attenuating the inflammatory response during human endotoxaemia.
The magnitude of the initial increase in plasma adrenaline concentrations was similar for the breathing exercises with and without prolonged breath retention. The cyclic hypoxia caused by the exercise with prolonged breath retention is therefore unlikely to be an important factor in the observed adrenaline response. In accordance, hyperventilation itself and the subsequent shift in acid-base balance have been shown to increase plasma catecholamines in the absence of hypoxia, and an important role for bicarbonate has been implicated [5, 10]. Nevertheless, as catecholamine release from the adrenal chromaffin cells is dependent on a combination of neural, hormonal, redox, as well as immune signaling pathways [11, 12], the exact mechanism behind the adrenaline release induced by the breathing exercise remains elusive. The finding that neither the duration of the training, nor the trainer who provides it affected any of the measured parameters signifies that the breathing exercise is easy to learn within a time-frame of two hours. These findings may greatly facilitate uncomplicated implementation of the training program in clinical studies.
Our data clearly demonstrate that the breathing exercise plays a pivotal role in the anti-inflammatory effect of the training intervention. Nevertheless, although cold exposure training alone had minimal effects on the cytokine response, it significantly potentiated the breathing exercise-induced anti-inflammatory effects. As plasma adrenaline levels in our study were comparable between the groups practicing the breathing exercises with or without prior cold exposure training, other mechanisms are likely involved. Noteworthy, despite little effects on the cytokine response, subjects in the cold exposure training group reported remarkably less symptoms compared to the control group as well as to the other two groups. In accordance, other studies reporting symptoms during repeated exposures to cold found similar attenuation of symptoms such as discomfort and shivering [13, 14]. Symptoms, especially headache, were more pronounced during practicing of the breathing exercise, likely resulting from the hyperventilation-induced changes in pCO2 and pH. After cessation of the breathing exercise, a sharp decrease of symptoms was observed and flu-like symptoms resolved more rapidly compared to the control group.
The increase in plasma adrenaline concentrations observed shortly after initiation of the breathing exercises in both the breathing exercises and endotoxemia studies described in the present work was similar to that in our previous study [1]. Nevertheless, adrenaline levels prior to the start of the breathing exercises were higher in the past work, ultimately resulting in higher absolute plasma concentrations [1]. Effects on the cytokine response in the combined cold exposure and breathing group in the current study were largely comparable to our previous work, in which subjects were also trained in both elements [1], although the magnitude of the immunomodulatory effects was less pronounced, with the anti-inflammatory IL-10 response augmented by 44% instead of 194% in [1], and pro-inflammatory cytokines attenuated by approximately 30% as opposed to more than 50% in [1]. There are several possible explanations for this discrepancy. First, the previously mentioned higher absolute plasma adrenaline concentrations during practicing of the breathing exercises could be involved [1], which may in turn have triggered a more pronounced IL-10 release and subsequent stronger attenuation of the pro-inflammatory response. Second, the hypoxia induced by breath retention in our previous study may have directly (i.e. independently from adrenaline) modulated the inflammatory response, as our group has recently demonstrated that hypoxia enhances IL-10 release and attenuates the pro-inflammatory response via enhanced adenosine release [15]. In this light, future studies into the training intervention should still consider including the exercise with prolonged breath retention.
A striking finding from the breathing exercises study was that the profound increase in plasma adrenaline levels only occurred during the first session in the morning, not during the second session performed in the afternoon after a 1.5 hour resting period. Nevertheless, the saturation, pO2, pCO2, and pH were identical between the morning and the afternoon sessions. Therefore, the lack of a profound increase of plasma adrenaline levels during the afternoon session may be due to adaptation of the stress response, resulting in lower adrenaline release by the adrenal gland in response to repeated application of the same stressor, a phenomenon which has been described in animals [16]. Alternatively, because the synthesis and storage of catecholamines mainly takes place within chromaffin cells of the adrenal medulla, it may be speculated that the breathing exercises deplete the intravesiculair stores in the cytoplasm of these cells [11, 12]. Although animal experiments have shown that fully depleted catecholamine stores can be replenished within 2 hours [17], this may take longer in humans. In any case, if stores are indeed depleted by the breathing exercise, replenishment must occur within a relatively short timeframe (< 24 hours), as participants of this study, as well as our previous study [1] practiced the breathing exercises daily in the week leading up to the experiment in which the plasma adrenaline concentrations were measured.
Several limitations of our work need to be adressed. First, we studied groups of healthy young male adults, not (older) patients with possible comorbidities, who represent the intended target group for this intervention. Nevertheless, this study provides essential information in terms of designing the most safe and optimal training protocol for use in future clinical studies. Second, the auto-immune response observed in patients with chronic inflammatory conditions clearly differs from that elicited by LPS administration, which models an acute inflammatory response to a bacterial infection. However, several drugs currently used in patients with inflammatory conditions such as rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis are aimed at reducing the release of several pro-inflammatory cytokines [18], on which the studied intervention has a major suppressive effect. Furthermore, in vivo human efficacy of many biologics used in the treatment of auto-inflammatory disorders was first established in the experimental human endotoxaemia model [19, 20], illustrating that it has value for these diseases.