## 4 Discussion
Regarding 5-CQA, concentrations around the ones observed in plasma after coffee drinking, 5-12 nM [22], were tested. Furthermore, to allow the comparison with similar studies using much higher concentrations, values in the uM range were also considered. The results from the present study indicate an anti-inflammatory effect of caffeine, characterized by a less pronounced production of in vitro pro-inflammatory TNF-\(\alpha\). Two previous studies reported inhibitory effects of caffeine on stimulated in vitro TNF-\(\alpha\) production in whole blood cultures [24; 25]. In the present study, the inhibitory effects of caffeine were observed for the 50 uM concentration. Therefore, as has been indicated above, it is unlikely that this effect could be observed commonly in vivo due to the high caffeine intake required to achieve this concentration. Actually, in previous studies the inhibitory effect of caffeine was observed for higher concentrations, 200 ug/mL (around 1 mM), which supposes a concentration 20-fold higher than the highest concentration tested in the present study, and also using _S. pneumoniae_ rather than LPS as a stimulant [24], and 100 uM [25]. It is difficult to ascertain the reasons for the different caffeine concentrations required to produce significant decreases in TNF-\(\alpha\) production. In fact, the only differences that can be surmised are methodological, such as different blood dilution, different LPS concentrations, or blood donors as in the study from [PERSON], a low number (eight), only female participants were considered. However, as observed in the present study, [PERSON] et al. [25] reported, albeit non-significant, a downward trend from the lower concentration tested (10 uM). The results could not be considered essentially different because in the present study, a non-significant downward trend from the lower concentration tested (10 uM) was also observed. It is also noteworthy that a wide range of caffeine concentrations have been suggested to inhibit TNF-\(\alpha\) production when different cell cultures have been used. In this sense, a study using human adipose tissue primary cultures, found an inhibitory effect of caffeine on TNF-\(\alpha\) spontaneous production with a caffeine concentration as low as 0.5 uM [33]. Less pronounced levels in TNF-\(\alpha\) production have been attributed to increased levels of the immunomododulator cAMP [34] and mediated by the cAMP/ protein kinase A (PKA) pathway [25]. In this regard, a significant effect of caffeine increasing cAMP concentration was observed, with significantly higher values for the highest caffeine concentration. It should be considered that in the present study cAMP levels were measured only in supernatants; however, because of their intracellular role, the concentrations in supernatants may not properly reflect intracellular cAMP concentrations. Changes in cAMP concentrations in the presence of caffeine could be expected because caffeine, acting as an adenosine antagonist, influences cAMP concentrations. Fur thermore, caffeine, as an inhibitor of cAMP-phosphodiesterase, induces higher cAMP concentrations [23]. Actually, the anti-inflammatory effects of caffeine, at least in in vitro studies, have been mainly attributed to the inhibition of cAMP-phosphodiesterase (PDE) [23]. It is worth noting that a previous study reported a significant inhibition of PDE following a coffee intervention, highlighting that this inhibition was not directly dependent on caffeine consumption [35]. Therefore, while these authors recognized a role for caffeine, they concluded that other components of coffee could play an important role in the modulation of cAMP contributing to the potential health benefits of coffee [35]. In this regard, cAMP has been reported to stimulate the synthesis of the anti-inflammatory IL-10 [34]. However, in agreement with previous studies [24, 25], no significant effects of caffeine were observed on IL-10 concentrations. On the other hand, cAMP has been suggested to inhibit IL-12.