## 4 Discussion
However, and in the same line as IL-10, no effect of caffeine was observed on IL-12 production [24, 25]. The mechanisms leading to the differential effect of caffeine on cytokine production remains to be elucidated. However, similar effects have been reported for other methylxanthines such as theophylline or pentoxifylline [24, 36]. On the other hand, it was shown in an animal model that adenosine receptor agonists increased LPS-induced plasma IL-10 levels [37]. Therefore, it was suggested that although caffeine did not modify IL-10 and IL-12 production in vitro, these interleukins may be modulated in vivo by caffeine and other adenosine receptor agonists [25]. Since oxidative stress is involved in pro-inflammatory cytokine production, mainly through the activation of the redox-sensitive Nuclear Factor \(\kappa\)B (NF-\(\kappa\)B) [38, 39], MDA was measured in cell culture supernatants. However, no effect of caffeine or 5-CQA was observed on MDA levels. Despite antioxidant effects having been attributed to caffeine [40], this result is mainly relevant for the 5-CQA experiments. In this regard, chlorogenic acids have been characterized as important antioxidants [3, 41], exerting, among other effects, an attenuation of NF-\(\kappa\)B [17, 18, 42]. In agreement with this observation, two studies carried out with mouse macrophages demonstrated how the presence of 5-CQA (the most used CGA in studies), in cell culture with LPS-stimulation suppressed the expression of pro-inflammatory compounds such as cyclooxygenase [18] as well as IL-1\(\beta\), TNF-\(\alpha\) and IL-6 [17]. In these studies, the lower pro-inflammatory expression was associated with attenuated NF-\(\kappa\)B [17, 18]. However, it should be noted that in both studies 5-CQA concentrations in the range of \(\upmu\)M, much higher than physiological ones, were used. In the present study, 5-CQA influenced only IL-6 production, with suppressed levels, but only for the highest 5-CQA concentrations tested, 2 \(\upmu\)M and 20 \(\upmu\)M. This result is in agreement with the effect observed in cultures of mouse macrophages using similar high 5-CQA concentrations and with the role of 5-CQA in attenuating NF-\(\kappa\)B activation [17]. On the other hand, when physiological concentrations were considered, no effect of 5-CQA was observed. Due to the relationship between oxidative stress and activation of the NF-\(\kappa\)B [38], the limited effects of 5-CQA on cytokine production in the present study may be related to the lack of effects on culture MDA concentrations. However, it cannot be discarded that chlorogenic acids play an important role in vivo as has been suggested from studies focused on the health effects of coffee consumption [3, 14, 16]. In this regard, studies in animal models have shown that the administration of 5-CQA lowers the circulating concentrations of pro-inflammatory cytokines such as TNF-\(\alpha\) or IL-1\(\beta\)[19, 20, 21]. On the contrary, supplementations in humans using coffee preparation have shown little (mainly increasing adiponectin concentrations) or no effect on inflammatory markers (reviewed by [PERSON] et al. [43]). Moreover, only a higher increase in total plasma antioxidant capacity, without affecting the inflammatory response, was observed in response to exercise after supplementation with high-chlorogenic-acid coffee [44]. The authors acknowledge some of the limitations that are present in this study. No intracellular measurements, mainly cAMP but also MDA, were performed. The concentrations of cAMP and MDA were measured only in culture supernatants. Furthermore, NF-\(\kappa\)B was not measured. The effects of caffeine and 5-CQA were not tested together. Since in coffee, as well as in other products such as tea, they are found in combination, it would be of interest to test the combined effects of both substances.