Introduction

Exercise is an immunomodulatory stimulus with both acute and chronic exercise modulating aspects of the innate and adaptive immune system [40]. Notably, moderate exercise has been associated with improved immune function [22], whereas high volumes of training and competing in endurance events can negatively affect the risk of infection [26]. Prolonged exercise has been linked to an increased risk of upper respiratory symptoms [7] and decreased salivary immunoglobulin A (IgA) secretion interpreted as acute immunosuppression [25].

Intense exercises such as wrestling [21], resistance exercise [23], or cycling [20] can also have a significant impact on the expression and quantity of immune cells. Even though many athletes adopt a polarized training model and dedicate a significant amount of time to high-intensity training [31, 35], less research effort has focused on the immunosuppressive effects of intense or exhaustive training modalities. The impact on immune function resultant from high-intensity exercise has been suggested to be due to the release of catecholamines [29] and stress hormones that are known to have potent immunosuppressive effects [6].

Nutritional adjuncts to counter the negative sequelae of exercise-induced fluctuations in immune functioning include carbohydrate, whey protein, vitamin, and caffeine supplementation [11, 24]. While caffeine does demonstrate antioxidant properties [1], it is also known to elevate post-exercise cortisol in a dose-dependent manner [2]. Thus, there is the potential for immunosuppressive effects either directly via adenosine receptor antagonism, or indirectly via an enhanced catecholamine and/or cortisol release [9, 16]. Direct increases in activation of natural-killer at caffeine doses of 6 mg kg−1 have also been reported [10]. Currently, an interaction between caffeine and high-intensity exercise involving incremental exercise until exhaustion on immune function is not well characterized. Here we tested the hypothesis that caffeine would upregulate anti-inflammatory and immunoregulatory cytokine release (IL-4, IL-10, and TGF-β1) during short incremental exercise until exhaustion.

Methods and Materials

Seven healthy male physical education students (age 22.1 ± 0.6; height: 1.83 ± 0.08 m; body mass: 72.8 ± 13.0 kg) volunteered to participate in this randomized, double-blinded crossover study with at least seven days between trials. The only inclusion criteria were that the participants were free from injury or any contraindications to performing exhaustive exercise. All participants reported being low caffeine consumers with daily dietary consumption of < 60 mg per day. After signing to acknowledge informed consent and refraining from strenuous exercise and all forms of caffeine intake for 48 h, each participant performed an exhaustive exercise protocol on a cycle ergometer. The initial workload for the exercise protocol was set at 100 W and the intensity was increased by 50 W every two minutes until either volitional exhaustion or the time point at which the participant was unable to maintain a pedaling cadence of 60 rpm. Sixty minutes before each exercise protocol the participants were randomly assigned to ingest either caffeine (6 mg/kg) or a maltodextrin placebo (6 mg/kg) administered in an indistinguishable gelatin capsule via simple random allocation using a computer software program to generate the random sequence. Blood samples were collected pre-, post-, and 1-h post-exercise and analyzed for serum levels of IL-4, IL-10, and TGF-β1 following the manufacturer’s instructions (Immunoready, Kampenhout, Belgium) and reported in pg/mL. Seated venous blood samples were collected in suitable vacutainers. Within 30 min of blood collection, plasma was obtained via centrifugation (15 min, 1000 g, 4 °C) and all samples were stored at − 80 °C until measurements were performed. The experimental protocol was approved by the University of Kurdistan Review Board in accordance with the latest version of the Declaration of Helsinki.

Changes in the mean of each measure with and without caffeine treatment were used to assess magnitudes of effects by dividing the changes by the appropriate between-participant standard deviations. Pairwise comparisons were made between conditions, and differences were interpreted in relation to the likelihood of exceeding the smallest worthwhile effects with individual change thresholds for each variable. Hormonal data were log-transformed to reduce non-uniformity of error, with effects derived by back transformation as percentage changes Magnitudes of the standardized effects were interpreted using thresholds of 0.2, 0.6 and 1.2 for small, moderate, and large, respectively [15]. Standardized effects of between − 0.19 and 0.19 were termed trivial. To make inferences about the large-sample value of an effect, the uncertainty in the effect was expressed as 90% confidence limits. An effect was deemed unclear if the confidence interval overlapped the thresholds for both small positive and negative effects; otherwise, the effect was deemed clear and substantial. The significance level was set at P < 0.05.

Results

Caffeine ingestion increased time to exhaustion by 14.1% ± 6.1% (P = 0.005; Effect Size [ES] = 1.33; Fig. 1; Mean ± SD). Specifically, caffeine ingestion improved the time to exhaustion from 413 ± 25.5 s to 472 ± 41.4 s. IL-4 increased during exercise with a 22.3% ± 10.5% greater increase following caffeine ingestion (P = 0.004; ES = 2.34). A substantial difference in IL-4 concentration persisted between the caffeine and placebo interventions one hour after exercise cessation (P = 0.027; 10.1% ± 7.1%; ES = 1.11; Fig. 2A). Very large increases in IL-10 were observed following exercise and again these increases were 10% ± 15.1% greater following caffeine ingestion (P = 0.047; ES = 0.41). A substantial difference persisted in IL-10 concentration between the caffeine and placebo interventions one hour after exercise cessation (13.9% ± 13.3%; ES = 0.81; Fig. 2B). TGF-β1 increased to a greater extent following exercise in the caffeine condition (16.9% ± 16.7%; P = 0.013; ES = 0.76); however, there were no substantial differences between the conditions in TGF-β1 concentration at the post-exercise or 1-h post-exercise time point. Specifically, the exercise-induced increase in TGF-β1 was from 525 ± 174 to 556 ± 198 pg/mL in the control condition; whereas, it increased from 485 ± 38 to 600 ± 89 pg/mL in the caffeine condition.

Fig. 1
figure 1

Time to exhaustion. A solid line represents means response; Dotted lines represent individual data

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Fig. 2
figure 2

Exercise-induced cytokine response. A IL-4; B IL-10; *Significantly different to control condition

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Discussion

Here we present data that caffeine demonstrates both an acute exercise performance enhancement and an immunoregulatory effect with an amplified response of the cytokines IL-4 and IL-10. This modulation may have important ramifications as these cytokines have potent anti-inflammatory and immunoregulatory properties, and can enhance the production of salivary immunoglobulin A [32, 36]. Thus, caffeine may enhance the protective effect of intense regular exercise on diseases associated with chronic inflammation [40].

The large improvement in time-to-exhaustion performance observed in the current study is consistent with other research demonstrating improved cycling performance [12]. The mechanism behind the impact of caffeine on performance is likely related to a decrease in perceived exertion, a decrease in the neuronal activation threshold of motor neurons, and/or an enhanced energy contribution from anaerobic metabolism [3, 8, 33]. The results of the current study confirm previous work demonstrating that caffeine can have a positive effect on incremental, time to exhaustion exercise protocols [34].

IL-4 is a classic Th-2 (T lymphocyte helper cells expressing cell surface molecule CD4) type cytokine and has been labeled as a “prototypic immunoregulatory cytokine” [32]. While in vitro studies have reported that methylxanthines such as caffeine inhibit the Th-2 immune response [28], in vivo human research has shown an increase in lymphocyte count and activation of CD4+ cells following exercise combined with 6 mg/kg caffeine ingestion [4]. These contrasting results demonstrate that the immune response to exercise is part of a complex network, under the control of a range of interactive regulatory and feedback mechanisms not easily recapitulated in an in vitro environment. The accentuated response of IL-4 resultant from our incremental exercise until exhaustion protocol is consistent with prior work demonstrating an enhanced anti-inflammatory physiological milieu. Given the large magnitude of the accentuated response, we believe it is reasonable to assume that caffeine could have a meaningful impact on the overall anti-inflammatory response.

Similar to IL-4, IL-10 is produced by Th-2 cells and is a potent anti-inflammatory agent involved in the production of IgA, the main immunoglobulin in salivary immunity [32]. IL-10 is a “key immunoregulator” during infection [5], and a depressed post-exercise level of IgA has been associated with upper respiratory tract infections in endurance runners [25]. While an appropriate pro-inflammatory response is required upon immune challenge, a positive feedback loop coordinated by IL-10 allows the resolution of infection. Of note, Northoff and colleagues [27] compared the immune response to an acute bout of exercise to that induced by infection. In a previous exercise study that assessed an interaction between caffeine and IL-10, accentuated responses consistent with our data have been observed following a 15 km run of ~ 68 min duration [39] using an identical dose to our protocol. These authors had earlier suggested that the higher IL-10 response following caffeine ingestion was mediated by inhibition of cAMP-phosphodiesterase [38]. While small, the significant and clear amplification of the IL-10 response in the caffeine condition is again indicative of an enhanced anti-inflammatory response.

TGF-β is a pleiotropic cytokine with immunosuppressive effects that works in concert with IL-10 and the production and function of these two cytokines may be interdependent [18, 30]. The resultant activity ensures a controlled inflammatory response that inhibits T-cell-mediated immunopathology [19]. As the IL-10 response to exercise was accentuated by caffeine in the current exhaustive protocol, it is unsurprising that a similar, if somewhat shorter time course of elevation was observed in TGF-β1. It is worth noting though that this accentuated response contrasts that observed in in vitro work [14, 37].

Here we present novel data that addresses the interaction between caffeine and incremental cycling exercise until exhaustion on specific aspects of immune function. We acknowledge that a distinction must be made between acute and chronic cytokine responses and that the “contextual dependence” of cytokines must be taken into consideration [40]. While we did not directly assess caffeine or catecholamine levels post-ingestion, we did use an identical dosage to previous work that elicited increases in these measures; albeit in longer [4, 38] and shorter [13, 17] exercise protocols. Additionally, other biomarkers, such as immunoglobulin A would add insight into the immune response. We also note that the relative contribution of the bioactive caffeine metabolites paraxanthine, theobromine, and theophylline to the observed results is not elucidated in the current work. It is also worth acknowledging that there is no sport or competition where the outcome is determined by time to exhaustion; thus, the physiological responses to the exercise protocol adopted herein is reflective only of a brief incremental exercise until exhaustion. The large variability in responses is also noteworthy, and previous research has identified individual responsiveness to caffeine intake [14]. Regardless, the 6 mg/kg caffeine dose protocol improved exercise performance, and the cytokine data are indicative of an enhancement of the anti-inflammatory properties of exercise proposed earlier [39].