Original Research Article Caffeine downregulates antibody production in a mouse model Miroslav Pohanka a,b, * a Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic b Karel English College in Brno, Brno, Czech Republic Introduction There are few legally available stimulants on the market. Caffeine (proper chemical name 1,3,7-trimethyl-1 H -purine- 2,6(3 H ,7 H )-dione; also designated trimethylxanthine) is con- tained in numerous foods, drugs and drinks without legal restriction making it an exception to other stimulants. Easy accessibility is due not only to its popularity but also because it has minimal adverse health effects (Cizkova et al., 2008; Seifert et al., 2013). In the body, caffeine competitively antagonizes adenosine at adenosine receptors. This is considered its major pharmacodynamic mechanism (Ribeiro and Sebastiao, 2010; Chen et al., 2013). Neurons with adenosine receptors are implicated in pro-in fl ammatory responses and caffeine can inhibit the initiation of in fl ammation j o u r n a l o f a p p l i e d b i o m e d i c i n e x x x ( 2 0 1 4 ) x x x – x x x a r t i c l e i n f o Article history: Received 13 June 2014 Received in revised form 3 September 2014 Accepted 4 September 2014 Available online xxx Keywords: Caffeine Cholinergic anti-in fl ammatory pathway Antibody Interleukin Keyhole limpet hemocyanin Vaccination Adjuvants Coffee Immunity a b s t r a c t Caffeine is a secondary plant metabolite found in coffee and tea. Its major pathway is interaction with adenosine receptors. Minor pathways are also known. An effect of caffeine on immunity has been proposed. In this paper, the role of caffeine on the immune system was studied, using a BALB/c mouse model. The animals received saline (controls), keyhole limpet hemocyanin (KLH) 1 mg/kg or caffeine (alone or in combination with KLH) in doses of 1 – 16 mg/kg. The mice were sacri fi ced 1 – 7 days later and plasma levels of interleukin (IL) 2, 4, 6, 10, 12 and antibodies against KLH were determined by enzyme linked immunosorbent assay (ELISA). Caffeine caused a signi fi cant decrease in KLH-stimulated antibody produc- tion. The effect was dose dependent. There were similar fi ndings for IL-2 and IL-4 but not for IL-6, IL-10 and IL-12. The signi fi cance of the fi ndings is discussed with extrapolation to humans based on caffeine doses used in the study and the amount of caffeine in available beverages. # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. * Correspondence to : Faculty of Military Health Sciences, University of Defence, Trebesska 1575, CZ-50001 Hradec Kralove, Czech Republic. Tel.: +420 973253091. E-mail address: miroslav.pohanka@gmail.com JAB-40; No. of Pages 6 Please cite this article in press as: Pohanka, M., Caffeine downregulates antibody production in a mouse model. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.09.001 Available online at www.sciencedirect.com ScienceDirect journal homepage: http://www.elsevier.com/locate/jab 1214-021X/$ – see front matter # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.jab.2014.09.001 (Antonioli et al., 2013; Peacock et al., 2013; Samsel et al., 2013; Sun et al., 2013). Beside the major pathway, caffeine is known to involve minor pathways as well. For example, it can interfere with secondary messengers via inhibition of phosphodiesterase (Bhaskara et al., 2008; Herman and Herman, 2013) causing alteration to intracellular Ca 2+ levels via ryanodine receptors (Hotta et al., 2007; Puttachary et al., 2010; Skiteva et al., 2012; Gerasimova et al., 2013; Gaburjakova and Gaburjakova, 2014). Caffeine interacts with a number of enzymes too. Weak inhibition of acetylcholinesterase (AChE) is an example (Okello and Leylabi, 2012). In some papers, a link between caffeine and the immune response has been proposed; however, the mechanism of this effect remains obscure. Immunomodula- tion via the cholinergic system has been extensively reviewed (Pohanka, 2014a). This review was aimed at the effect of caffeine on multiple targets in the body and the cholinergic anti-in fl ammatory pathway was considered one possible mechanism of the action of caffeine. However, direct proof was missing. Anti-in fl ammatory effects such as reduction in tumor necrosis factor a (TNF a ), pyrogenic interleukin (IL) 1 and pro-in fl ammatory acting IL-6 were described in some studies (Horrigan et al., 2004, 2006; Bessler et al., 2012; Gordillo- Bastidas et al., 2013). However, the molecular mechanisms underlying these effects were not revealed. Some researchers have proposed that it can be mediated by toll-like receptors (Tunc et al., 2013), adenosine receptors (Ohta et al., 2006; Ohta and Sitkovsky, 2009), cAMP pathway (Rosenthal et al., 1992a), and adenosine-deaminase (Bandyopadhyay and Poddar, 1994; Tunc et al., 2013). Conclusions drawn on the effects of caffeine on immunity can be affected by congeners in natural products containing caffeine. For example, Nosal'ova et al. (2011) found a signi fi cant effect of arabinogalactan-protein in instant coffee powder. This should be taken into consideration when testing natural products containing caffeine. In the following experi- ment, pure caffeine was used, to obviate confounding by any other biologically active congeners. One hypothesis tested here is that caffeine could modulate vaccination ef fi cacy. To date, there is scant literature on the subject. For this reason, the experiment below was carried out to respond to the question whether caffeine can modulate immunity in a mouse model with keyhole limpet hemocyanin (KLH) as a standard antigen. Experimental Laboratory animals and immunization Laboratory BALB/c female mice (Velaz, Unetice, Czech Repub- lic) were used for the experiment. Female mice were chosen because of their low aggressiveness and better social tolerance compared to males (Kuchiiwa and Kuchiiwa, 2014; Tomaz et al., 2014). The results then can be more easily attributed to the caffeine variable than any other undesired effect of social stress in the animals. In total, 180 mice were kept as 18 groups of 10 animals. The mice weighed 19 2 g and they were 8 weeks old at the beginning of experiment. The animals were kept in standard conditions for breeding of laboratory rodents: temperature 22 2 8 C, humidity 50 10% and 12 h light cycle. The experiment was permitted by the Ethics Committee at the Faculty of Military Health Sciences, University of Defense (Hradec Kralove, Czech Republic; license number CZ 52760099). Caffeine and KLH were purchased from Sigma – Aldrich (Saint Louis, MO, USA) and dissolved in saline. 100 m l of the compounds were intramuscularly injected into the hind limb. KLH solution was injected into the right limb followed by application of caffeine into left limb 30 min later. Controls received saline in the same volume. The experimental design and time of euthanasia are shown in Table 1. 1 mg/kg re fl ects amount of caffeine in one cup of coffee that is reported to be approximately 100 mg (Yubero-Lahoz et al., 2012). The doses 4 and 16 mg are approximately 2.5% and 10% of median lethal dose for rodents (Warszawski et al., 1978). The dose range was chosen to cause physiological effects and no toxicity. At the end of experiment, the animals were sacri fi ced by severing the jugular vein using surgical scissors. The animals were under CO 2 narcosis when sacri fi ced. The time intervals were chosen to allow for innate immunity stimulation ( fi rst and second day after application) and the interval when antibody production can be expected (seventh day) because of antigen administration to BALB/c mice (Pohanka, 2007, 2009). Controls receiving caffeine with no KLH received 16 mg of caffeine. The control group was chosen in order neglect pertinent initiation of immunity without presence of an antigen. Lower doses of caffeine were not applied for this purpose because of effort to limit number of used laboratory animals. Blood was collected in a lithium heparin tube (Dialab, Prague, Czech Republic) and centrifuged at 1000 g for 5 min. Plasma was separated and stored at 80 8 C until assay (see next chapter). Enzyme linked immunosorbent assay (ELISA) of antibodies against KLH 100 m l per well of a 96 well microplates Maxisorp KLH solution (10 m g/ml) was injected (Nunc, Thermo Fisher Scienti fi c, Waltham, USA) and allowed to incubate at 4 8 C overnight and then twice washed by 400 m l of phosphate buffered saline with Tween 20. The wells were blocked by 100 m l of bovine Table 1 – Experimental groups and time of euthanasia after the start of the experiment. Controls (saline) 1 day (group 1) 2 days (group 7) 7 days (group 13) Caffeine 16 mg/kg 1 day (group 2) 2 days (group 8) 7 days (group 14) KLH 1 mg/kg 1 day (group 3) 2 days (group 9) 7 days (group 15) Caffeine 1 mg/kg + KLH 1 mg/kg 1 day (group 4) 2 days (group 10) 7 days (group 16) Caffeine 4 mg/kg + KLH 1 mg/kg 1 day (group 5) 2 days (group 11) 7 days (group 17) Caffeine 16 mg/kg + KLH 1 mg/kg 1 day (group 6) 2 days (group 12) 7 days (group 18) j o u r n a l o f a p p l i e d b i o m e d i c i n e x x x ( 2 0 1 4 ) x x x – x x x 2 JAB-40; No. of Pages 6 Please cite this article in press as: Pohanka, M., Caffeine downregulates antibody production in a mouse model. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.09.001 albumin 5%, w/w for 2 h and washed by the buffer with Tween. Finally, 100 m l of 100 times diluted plasma samples were applied per one well and allowed to incubate at 37 8 C for 2 h. After the washing as described above, anti-mouse immuno- globulins antibody speci fi c against IgG, IgA and IgM labeled by peroxidase (Sigma – Aldrich) diluted 10,000 times was injected in a volume of 100 m l per well and kept at 37 8 C for 2 h. The plates were washed and a fresh solution of 0.5 mg/ml 3,3 0 ,5,5 0 - tetramethylbenzidine and 5 mmol/l H 2 O 2 , allowed to react for 15 min, and then stopped by 2 mol/l H 2 SO 4 in a dose 100 m l per well. Optical density was measured by ELISA reader Sunrise (Salzburg, Austria) at 650 nm. Replacement of plasma sample by bovine albumin (1 mg/kg) was used for control purposes. ELISA of IL-2, IL-4, IL-6, IL-10, IL-12 The tested cytokines (IL-2; IL-4; IL-6; IL-10; IL-12) were assayed by indirect ELISA kit from Sigma – Aldrich. The 96-well microplates with immobilized recognition antibody were used in compliance with the manufacturer's protocol. Calibration was processed in the same way. In these tests, ELISA reader Sunrise was used for optical density measurement. Experimental data processing Statistical software Origin 8 Pro (OriginLab Corporation, Northampton, MA, USA) was used to analyze the data using two way, ANOVA with Bonferroni correction (two-sided tests) to determine whether the markers differed between groups. The alpha level was 0.05. The tests of signi fi cance are expressed for controls being sacri fi ced in the same interval like the exposed animals (i.e. groups 5 – 6 to group 1; groups 8 – 12 to group 7, and groups 14 – 16 to group 13). Results and discussion No animal perished or showed any pathology. The animals had normal behavior and no differences in the behavior were observed when the controls compared to the other animals. It should be emphasized that the controls have the tested immunochemical markers in a narrow interval and no signi fi cant differences between the control groups (1, 2 and 7 days i.e. groups 1, 7 and 13) were observed. Production of antibodies against KLH is depicted in Fig. 1. As can be seen caffeine reduced antibodyproduction triggered by KLH. The effect was signi fi cant and in a dose response manner. The data negatively correlated with coef fi cient of determination 0.856. The highest dose of caffeine (16 mg/kg) caused reduction in antibody level to values indistinguishable from controls. Antibody production appeared seven days after antigen application. Production of antibodies in the controls corre- sponded with expectations based on cited laboratory experi- ments where production of antibodies was stimulated by disparate antigens (Pohanka, 2007, 2009). However, caffeine was not used in these studies. Plasma levels of IL-2 are shown in Fig. 2. In the animals exposed to KLH only, IL-2 was signi fi cantly increased in all KLH-treated animals compared to controls. The increase was signi fi cant. The controls and animals exposed to caffeine only Fig. 2 – Results from ELISA for IL-2 level in plasma. Error bars indicate standard error of mean and the number inside bars indicates number of days (1, 2 or 7) following stimulation by caffeine at which euthanasia was made. Symbol * is expression for statistical difference against controls (the first three columns marked as ‘‘ controls ’’ ) at the significance level 2alpha = 0.05. When the significance tested, the same time interval (days) from either controls or the KLH groups taken into consideration. Fig. 1 – Results from ELISA for total antibodies against KLH. Error bars indicate standard error of mean and the number inside bars indicates number of days (1, 2 or 7) following stimulation by caffeine at which euthanasia was made. Symbol * respective ‘‘ a ’’ is expression for statistical difference against controls (the first three columns marked as ‘‘ control ’’ ) respective animals exposed to KLH alone (the three columns marked as ‘‘ KLH ’’ ) at the significance level 2alpha = 0.05. When the significance tested, the same time interval (days) from either controls or the KLH groups taken into consideration. j o u r n a l o f a p p l i e d b i o m e d i c i n e x x x ( 2 0 1 4 ) x x x – x x x 3 JAB-40; No. of Pages 6 Please cite this article in press as: Pohanka, M., Caffeine downregulates antibody production in a mouse model. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.09.001 had levels of IL-2 under 18 pg/ml. Caffeine caused small decrease in IL-2 in the animals immunized by KLH. However, the decrease in IL-2 was minor compared to the effect of caffeine on antibody production. Plasma IL-4 levels are presented in Fig. 3. Caffeine did not cause any alteration in IL-4. However, animals which two time intervals (2 and 7 days) than the fi rst. In the seventh day, IL-4 level in plasma was approximately doubled over controls. IL 4 levels in the KLH and the KLH plus caffeine 1 and 4 mg/kg were not signi fi cantly different. However, the upper dose of caffeine, 16 mg/kg, reduced IL-4 production in animals stimulated with KLH. IL-4 is responsible for induction of naive T cells differentiation into Th2 cells (Caubet et al., 2014; Xiang et al., 2014). It can be inferred that caffeine enhances Th2 cell maturation and the increased antibody production was a consequence of the effect on the T cells. IL-6, IL-10 and IL-12 were assayed as well. However, no signi fi cant difference in the tested cytokines was found when the markers from animals exposed to KLH and/or caffeine were compared to the controls (data not shown). The IL-6 level ranged from around 1 to 3 pg/ml, IL-10 from 0.7 to 1 pg/ml and IL-12 from 0.90 to 1.4 pg/ml, respectively. The range was equal to the expected values found in the literature (Cift et al., 2013; Brunoni et al., 2014; Kuvibidila et al., 2014). The fact that no signi fi cant response for the IL-6, IL-10 and IL-12 markers was revealed may be that assayed values were on the lower end of calibration plots in the ELISA kits. Limit of detections of the kits were not provided by the manufacture; however, it is estimated that the limits were around 0.5 pg/ml for calibration plots and con fi dence intervals of the calibration. The minor changes would disappear in value dispersions (analytical noise). On the other hand, model chosen here was not proposed to cover in fl ammation. Major focus antibody production. Effect of caffeine on in fl ammation was described by some researchers including effect on TNF a , IL-6, and IL-6 (Horrigan et al., 2004, 2006; Bessler et al., 2012; Gordillo- Bastidas et al., 2013). From the results, caffeine proved to be signi fi cantly involved in the regulation of the immune response to a protein antigen. As the antibodies as well as IL-2 and IL-4 were reduced by caffeine in a dose response manner in the animals immunized by KLH, a signi fi cant role of caffeine inhibition of speci fi c immunity maturation can be inferred. The effect on innate immunity is in a good compliance with the cited papers (Saxena et al., 1984; Mandal and Poddar, 2008) where suppression of the innate immune response was revealed. The suppression probably precedes maturation of speci fi c immunity response and caffeine interferes in the primary initiation of immunity. A decrease of antigen-stimulated CD4 + cells caused by caffeine is also mentioned in the literature (Fletcher and Bishop, 2012). The effect would be based on inhibition of T cell proliferation as proposed by Rosenthal et al. (Rosenthal et al., 1992b). Decline in antibody production is the consequence. The role of caffeine in immunity cannot be explained by a simple mechanism. Fletcher and Bishop stated that caffeine initiates increase of circulating NK cells and number of activated NK cells (Fletcher and Bishop, 2011a,b). Caffeine would act in immunity regulation even by inhibition of the enzyme AChE and consequent activation of the cholinergic anti-in fl ammatory pathway (Pohanka, 2012, 2013, 2014b). The link to the anti-in fl ammatory pathway should be however, further researched. The results showed that caffeine may have e impact on immunity and could affect the health of everyone drinking coffee or consuming caffeine in another form. However, the amount of caffeine should be taken into account when considering caffeine implication in immune response. The amount of caffeine consumed depends on personal habits, marketed products, etc. In the literature, the content of caffeine in a cup of coffee is approximately 90 mg (Lloret- Linares et al., 2012) or 200 mg in another source (Howards et al., 2012). If we consider the upper value, an 80 kg man will receive 2.5 mg/kg of caffeine in one cup of coffee. This value lies between the lowest and medium dose of caffeine used here. It can be inferred that the man may have e.g. less effective vaccination if he regularly takes coffee. Role of caffeine in sensitivity to infectious diseases is also highly probable but the effect is hardly predictable. Conclusions Caffeine modi fi es the immune response to a protein antigen. A signi fi cant effect of caffeine would be expected in humans if the fi ndings here apply to people drinking coffee or other caffeine containing products if the doses used here are Fig. 3 – Results from ELISA for IL-4 level in plasma. Error bars indicate standard error of mean and the number inside bars indicates number of days (1, 2 or 7) following stimulation by caffeine at which euthanasia was made. Symbol * respective ‘‘ a ’’ is expression for statistical difference against controls (the first three columns marked as ‘‘ control ’’ ) respective animals exposed to KLH alone (the three columns marked as ‘‘ KLH ’’ ) at the significance level 2alpha = 0.05. When the significance tested, the same time interval (days) from either controls or the KLH groups taken into consideration. j o u r n a l o f a p p l i e d b i o m e d i c i n e x x x ( 2 0 1 4 ) x x x – x x x 4 JAB-40; No. of Pages 6 Please cite this article in press as: Pohanka, M., Caffeine downregulates antibody production in a mouse model. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.09.001 extrapolated to humans and content of caffeine in beverages considered. The impact of caffeine should be taken into account in vaccinations and during infectious diseases. On the other hand, the extrapolation should be con fi rmed by a separate clinical test in humans. Con fl ict of interest statement None. Acknowledgements The Ministry of Education, Youth and Sports of the Czech Republic is gratefully acknowledged for project LH11023. A long-term organization development plan 1011 (Faculty of Military Health Sciences, University of Defence, Czech Repub- lic) is acknowledged as well. r e f e r e n c e s Antonioli, L., Blandizzi, C., Pacher, P., Hasko, G., 2013. Immunity, in fl ammation and cancer: a leading role for adenosine. Nat. Rev. Cancer 13, 842 – 857. Bandyopadhyay, B.C., Poddar, M.K., 1994. Caffeine-induced increase of adenosine-deaminase activity in mammalian lymphoid organs. Methods Find. Exp. Clin. Pharmacol. 16, 731 – 733. Bessler, H., Salman, H., Bergman, M., Djaldetti, M., 2012. 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