Immunobiology xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Immunobiology
journal homepage: www.elsevier.com/locate/imbio

Review

Caffeine: Well-known as psychotropic substance, but little as

immunomodulator
⁎
Tatiana Al Reef, Esther Ghanem
Department of Sciences, Faculty of Natural and Applied Sciences, Notre Dame University, Louaize, Lebanon

A R T I C LE I N FO A B S T R A C T

Keywords: To date, numerable reviews are found in the literature prominent to the effect of caffeine on the immune system,
Caffeine with the latest review published in 2006. Database screening reveals around three thousand articles that have
Innate immunity been published during the last decade. Interestingly, less than hundred articles involved humans and rodents as
Adaptive immunity tested models, out of which 20% is of interest to this paper excluding studies done on the nervous and cardiac
MHC molecules
systems, and in pregnant and cancer cases. In this review, information pertaining to the experimental setup of
Cytokines
various studies, namely, the tested model, the study type (in vivo or in vitro), and caffeine dose is covered to
discern the behaviour of major cellular and molecular immune components in light of caffeine exposure.
Although it is hard to extrapolate results done in rodents to humans and to relay conclusions from in vitro to in
vivo studies, most of the collected data favor the suppressive effects of caffeine on the proliferation of stimulated
lymphocytes. Macrophages and natural killer cells also exhibited a reduced activity in the presence of high
caffeine doses compared to increased activity at low doses. Immunosuppression is also supported by reduced
levels of major anti-inflammatory cytokines, IL-2, IL-6, TNF-α. Moreover, certain innate and adaptive immune
receptors, such as TLR1, TLR2, TLR4, and MHC class I-related chain B (MICB) molecules, exhibited decreased
expression levels. Thus, we support the use of caffeine to alleviate various inflammatory conditions and auto-
immune diseases.

1. Introduction
Caffeine is the most generally expanded behavior-affecting sub-
stance in the world (Strong et al., 2000). It acts as a psychoactive drug
that is legally accessible without any regulations, influencing the brain
or state of mind of any individual regardless of ethnicity or age. Caf-
feine is a typical additive in many diets, expanding its presence from
coffee to soda, tea, chocolate, and energy drinks (Ashihara and Crozier,
2001); yet, it is not an essential nutrient, nor fundamental for the
wellbeing (Peacock et al., 2017). It is known to be an alkaloid with
central nervous system and metabolic stimulatory properties. A dosage
of 100 mg/day of caffeine is able to positively affect several cognitive
functions, decrease lethargy and fatigue, and increase vigor. However,
high concentrations of caffeine exceeding 400 mg/day can become
toxic, leading to vomiting, abdominal pain, as well as rhabdomyolysis
(Judelson et al., 2013). To date, most research has been done on the
effect of caffeine on the cardiovascular system (Clarke et al., 2016; Suvi
et al., 2017), respiratory system (Yu et al., 2016), psychological state
(Visram et al., 2016), cancer cells (Wang et al., 2015) and liver fibrosis
(Cachon et al., 2017). The spectrum of research on the effect of caffeine
on the immune system is exponentially growing during the last decade,
so does the confusion inferred from its contradictory results. Since the
last review published on the effect of caffeine on the immune system by
Horrigan et al. (2006), roughly three thousand articles have been added
to the literature database as retrieved while using caffeine and immune
system as keywords. For our search, we used three databases: PubMed,
Science Direct, and Scopus. Search restrictions were based on the lan-
guage (English), year of publication between 2007 and 2018, type of
publication set to journal, and experimental models mainly done on
rodents and humans. Out of the scored hits, only 96 articles met our
selection criteria. Almost eighty percent involved studies on the ner-
vous system and aquatic animal models that were excluded from our
screening process (Fig. 1). The selected reports were collected in an
EndNote library and further checked for duplicates and full screen of
titles and abstracts. Thus, our main objective was to provide an updated
review while incorporating the latest 20% reports matching our search
criteria. Reports were analyzed and information pertaining to the ex-
perimental setup, namely, the tested model (rodents or humans), the
study type (in vivo or in vitro), and caffeine dose was covered to discern
the behavior of different immune components in light of caffeine

⁎
Corresponding author at: Faculty of Natural and Applied Sciences, Department of Sciences, Lebanon.
E-mail address: eghanem@ndu.edu.lb (E. Ghanem).

https://doi.org/10.1016/j.imbio.2018.08.011
Received 6 June 2018; Received in revised form 15 August 2018; Accepted 19 August 2018
0171-2985/ © 2018 Elsevier GmbH. All rights reserved.

Please cite this article as: Al Reef, T., Immunobiology (2018), https://doi.org/10.1016/j.imbio.2018.08.011
T. Al Reef, E. Ghanem
exposure. The platform to discuss the collected information was split
into categories starting with the most general unspecific immune re-
sponses and ending with specific immune indicators. Thus, first we
discuss the non-specific innate key players, mainly C-reactive proteins
(CRP) and toll-like receptors (TLR), followed by the activity of macro-
phages and cytotoxicity of natural killer cells. Moving then to more
specific responses, such as the profile of cytokines and lymphocytes.
Ending our revision with the effect of caffeine on major histocompat-
ibility class (MHC) molecules as specific indicators of adaptive im-
munity. Before shedding light on the allocated categories, we introduce
the general binding properties of caffeine and its physiological func-
tions to potentiate the link between caffeine and the immune system.
2. Caffeine binding properties and its general physiological
functions
Caffeine is a drug that belongs to the family of methylxantines,
which are derived from a purine, specifically from guanine. A way for
this substance to target different types of cells is by binding to purine
analogues, namely adenosine receptors (Iglesias et al., 2014) that are
abundant in T and B lymphocytes (Sakowicz-Burkiewicz et al., 2012;
Vincenzi et al., 2013), macrophages (Joos et al., 2017), brain, heart,
vessels and kidneys (Marx et al., 2016). Caffeine is known for its an-
tagonistic activity on adenosine receptors in the brain (Yamaguchi
et al., 2017), empowering the focal sensory system and building an
individual's focus and alertness (Nagata and Urade, 2012). Usually
when adenosine binds to its A1 receptors, there will be an inhibition of
adenylyl cyclase leading to a decrease in cAMP causing a hyperpolar-
ization by increasing K + efflux and therefore inhibiting calcium cur-
rent (Kanyshkova et al., 2009). Physiologically, this can be explained by
a reduction in blood flow, neuronal excitability and neurotransmitter
transmission especially dopamine, therefore promoting a state of rest,
reduced arousal, and suppressed physical alertness (Kim et al., 2015).
In contrast, caffeine antagonizes adenosine effect in the brain and
produces a fight or flight response leading to increased blood flow,
reduced sleep and improved physical activity and alertness (Volkow
et al., 2015). For instance, methylxanthine has major effects on many
brain related diseases such as Schizophrenia, depression, Alzheimer’s
disease, Huntington’s disease, Parkinson’s disease (Ribeiro and
Sebastiao, 2010), and impaired sleepiness (Prather et al., 2016). In
addition to caffeine’s binding ability to A1 receptors in the brain, it has
a high affinity to A2A receptors present on the surface of immune cells
(Ohta and Sitkovsky, 2001; Ongini and Fredholm, 1996); thereby ex-
erting an anti-inflammatory action (Alfaro et al., 2017; Fredholm et al.,
2
Immunobiology xxx (xxxx) xxx–xxx
Fig. 1. Summary of the literature search and screening process
used to retrieve relevant articles published between 2007 and
2018 to evaluate the immonomodulatory effects of caffeine.
The following databases were used PubMed, Scopus, and
Science Direct using caffeine and immune system as keywords.
The initial screening resulted in 2934 articles, out of which
2838 were excluded as our search criteria involved only
human and rodents test models. Last exclusion process filtered
out 79 studies related to the nervous system, microglia re-
sponses, heart rate, metabolism, pregnancy and tumor.
Overall, 17 articles were analyzed and included in our review.
2003). Unlike the inhibitory effect of adenylyl cyclase upon binding of
adenosine to A1 receptors, adenosine binding to A2A receptors en-
hances adenylyl cyclase activity therefore accumulating intracellular
cAMP. To intensify cAMP levels, caffeine acts as a phosphodiesterase
(PDE) inhibitor that will lead to the accumulation of cAMP in the in-
tracellular space and adenosine in the extracellular space (Rohrig et al.,
2017). cAMP activates protein kinase A (PKA), which in turn blocks the
release of pro-inflammatory cytokines suppressing the action of many
immune players, such as macrophages activity and natural killer (NK)
cell cytotoxicity (Fig. 2). A more elaborative description on the beha-
vior of macrophages and NK cells upon caffeine treatment will be
eventually considered in this review. Moreover, caffeine withdrawal or
addiction might not only affect the psychological and immunological
body systems, but also other physiological systems including the car-
diovascular system. For instance, mice treated with low amounts of
caffeine showed weight loss and an increase in physical activity most
likely due to the fight or flight response induced by higher epinephrine
levels in the blood. This demonstrates the protective effect that caffeine
can exhibit on human health by reducing the risk of obesity (Dangol
et al., 2017) and cardiovascular diseases (Di Lorenzo et al., 2017).
However, high levels of caffeine uptake, such as 20 mg/kg spanning a
30 day period in mice, resulted in a significant weight gain that could
be linked to unhealthy symptoms like stress, depression, anxiety, panic
disorders (Ramanaviciene et al., 2004). These findings demonstrate that
caffeine modulate different physiological systems, including the im-
mune system, in a dose dependent manner.
3. General effects of caffeine on non-specific immune players
In vivo studies on normal mice showed that caffeine levels up to
20 mg/kg increase the counts of isolated polymorphonuclear cells
(PMNs) as well as their activity to secrete lysozyme in a dose dependent
manner. For instance, compared with untreated mice, blood count
analysis collected from mice heart after being exposed to 20 mg/kg
caffeine, showed almost 2.5 times increase in both monocytes and
neutrophils’ counts, and a 3.5 times increase in eosinophils. On the
same note, serum withdrawn from mice administered a dose of 20 mg/
kg caffeine showed 1.6 times increase in lysosomal activity compared
with untreated mice, while increasing caffeine dose to 40 mg/kg re-
sulted in a 1.8 times activity increase. Thus, caffeine enhanced the
immunological activity of certain PMNs. Exceptionally, basophils’
counts did not alter upon caffeine treatment (Ramanaviciene et al.,
2004). On the other hand, at high doses of caffeine (40–200 mg/kg) a
negative correlation was assessed relative to the overall count and
T. Al Reef, E. Ghanem
activity of innate leukocytes including eosinophils, macrophages, nat-
ural killer cells, and neutrophils. Caffeine is speculated to increase
PMNs’ levels, by either acting directly on the bone marrow or through
the central nervous system by inducing epinephrine release by the
adrenal gland (Ramanaviciene et al., 2004). A recent in vitro study by
Abbasi et al., 2018 observed the vitality of neutrophils isolated from
cardiac blood of rats after a co-culture with caffeine-treated-LPS
primed-mesenchymal stem cells. An increase in the survival of co-cul-
ture neutrophils was observed upon treatment with 0.2 mg/mL caffeine
as assessed by MTT and neutral red uptake assays compared to a re-
duced survival in the absence of caffeine. Thus, caffeine promotes the
cell longevity of co-cultured neutrophils (Abbasi et al., 2018). In ad-
dition to the increment in the innate immune players, caffeine induced
an increase in the release of C-reactive protein (CRP) levels in human
hepatoma cell lines, Hep3B and NPLC/PRF/5. CRP is an acute phase
protein that is released from hepatocytes as an indicator of inflamma-
tion (Ganapathi et al., 1990). CRP increase was assessed by im-
munoprecipitation of radiolabeled CRP from the cell media, mRNA
quantification, and by western blot assays. A linear response was ob-
served for CRP in response to caffeine doses between 0.5 and 2 mM.
Thus, a dose-dependent effect of caffeine is exerted on CRP release.
Interestingly, a synergistic increase in CRP levels was observed upon
simultaneous treatment of cells with caffeine and certain cytokines,
such as IL-6 and IL-1α. It remains intriguing to unravel the actual role of
caffeine in the signal transduction pathway leading to a cytokine-de-
pendent increase in CRP levels. Another indirect effect of caffeine on
CRP levels could be via the activation of the complement cascade
system (Thompson et al., 1999). This finding aligned with other studies
in the literature (Jacobs et al., 2014; Yamashita et al., 2012) are op-
posed by a null association of CRP and serum amyloid-A (SAA) with
coffee consumption in humans (Loftfield et al., 2015) and low serum
levels of CRP exposed to daily consumption of caffeine (15 mg/kg) for
21 days in Wistar rats (Owoyele et al., 2015). Several factors might
underline this discrepancy in the reported results and hinders our in-
sight on whether caffeine prompts a pro- or anti- inflammatory effect on
CRP. Major factors might include the type of tested model, in vitro or in
vivo experimental applications, dose of administered caffeine, and the
combinatorial effect of caffeine with other mediators, such as cytokines.
Minor factors might include the mode of coffee preparation (filtered
3
Immunobiology xxx (xxxx) xxx–xxx
Fig. 2. A schematic model representing the
major modulatory effects of caffeine on various
immune parameters. Once caffeine binds to its
A2A receptor, adenylyl cyclase is activated
converting ATP to cyclic adenosine monopho-
sphate (cAMP). This action induces an in-
tracellular signaling cascade mediated by
cAMP upregulation via phosphodiesterase
(PDE) inhibition. Thus, the extracellular
binding of caffeine is amplified intracellularly
by the second messenger, cAMP. High con-
centrations of cAMP activate protein kinase A
(PKA), which in turn blocks the release of pro-
inflammatory cytokines. A suppressed in-
flammation tunes down the activity of several
immune players, such as macrophages, natural
killer (NK) cell cytotoxicity, T and B cell pro-
liferation, and antibody production. In addi-
tion, it modulates the expression levels of toll
like receptors (TLRs) and major histocompat-
ibility class I molecules (MHC I).
versus unfiltered) and other masked effects, such as cigarette smoking,
obesity or overweight, and type of diet (Mediterranean- style versus
Western) (Clrs et al., 2017). Furthermore, serum caffeine levels could
be biased by factors as suggested by Horrigan et al., such as acute versus
chronic exposure and peak concentration of caffeine at the time of
experimental practice (Horrigan et al., 2006).
Another influence of caffeine on non-specific immune players in-
cludes its effect on toll-like receptors (TLRs). TLRs recognize pathogens
specific associated patterns and play a crucial role in initiating the in-
nate inflammatory signaling cascade. TLRs expression induced by caf-
feine is mainly examined in neonates. In an in vivo study by Tunc et al.,
baby rats were administered breastmilk with 20 mg/kg/day of caffeine
starting from postnatal day 1 to day 10. Caffeine induced an increase in
the levels of TLR9 in newborn lungs as compared to untreated breast-
milk, while exhibiting insignificant changes in TLR2 and TLR4 ex-
pression levels (Tunc et al., 2013). In another study done on LPS-acti-
vated cord blood monocytes (CBM) taken from infants, TLR gene
expression level (qRT PCR) was altered by caffeine in a dose-dependent
manner. Caffeine doses at 0.01 and 0.02 mg/mL downregulated the
expression of TLR1 and TLR2 by almost 75%. However, TLR1 or TLR2
mRNA levels did not change at higher doses of caffeine (0.04 mg/mL).
While TLR4 gene expression was unchanged in CBM exposed to
0.01 mg/mL of caffeine, it was up-regulated by 2.6-fold in the presence
of 0.04 mg/mL of caffeine (Chavez-Valdez et al., 2016; Tunc et al.,
2013). This is inconsistent with another ex vivo study done on rats
administered 1 mg/mL caffeine from green tea extracts, resulting in
decrease of total TLR-4 mRNA levels of isolated lymphocytes (Molina
et al., 2015). Thus, the reduced amounts of TLR1 and TLR2 in humans
and TLR4 in rats favor an anti-inflammatory microenvironment pro-
duced by caffeine at specific doses.
3.1. Caffeine modulates macrophage activity
Within the spectrum of innate immunity, macrophages activity
isolated from whole human blood of young adults ranging from 20 to
30 years was tested for a three-day period in the presence of different
doses of caffeine. Only during the first day, macrophages’ activity to
phagocytose increased when treated with a caffeine dose of 0.15 mg/
mL. This is most likely due to the saturation of adenosine receptors,
T. Al Reef, E. Ghanem
thereby enhancing inflammation regardless of its PDE inhibitory action.
However, as of the second day, regardless of the dose and treatment
duration, macrophages’ activity significantly decreased, but the addi-
tion of PKA inhibitor reversed this effect (Steck et al., 2015). This was
confirmed also by Li & Funahashi in 2010, where phagocytosis of sperm
cells by isolated peripheral cow blood PMNs decreased with increasing
doses of caffeine (Li and Funahashi, 2010). This most likely indicates
that the phagocytic inhibition by caffeine is through the cAMP/PKA
pathway (Ferreira et al., 2014). Once PKA is activated by caffeine, the
role of pro-inflammatory cytokines to activate macrophages (Kyaw
et al., 2017) is silenced (Quiroz et al., 2006; Sugimachi et al., 2016). On
the other hand, the viability of alveolar macrophages isolated from rat
lungs exhibited a dose dependent variation up to a caffeine dose of ∼
4 mg/mL and cAMP levels of ∼ 0.03 mM. Above such levels, the via-
bility drastically dropped (Jafari and Rabbani, 2004). These observa-
tions support the finding that high cAMP level precedes apoptosis
(Kesavardhana and Kanneganti, 2017; Ortiz et al., 2001). Similarly,
treating PKA induced human mononuclear phagocytes with a dose of
caffeine (0.02 mg/mL) for 22 h or a dose of (0.19419 mg/mL) for 72 h
has been shown to augment the anti-inflammatory effect of mesench-
ymal stem cells (MSCs) on macrophages (Steck et al., 2015) and in that
way decreasing macrophage phagocytosis and IL-10 secretion levels
upon incubation with caffeine-pulsed MSCs isolated from bone marrow
of rats (Shushtari and Abtahi Froushani, 2017; Steck et al., 2015). In
short, the collected data reinforces the anti-inflammatory role of caf-
feine on macrophages and is in line with the reduced release of nitric
oxide (NO) from induced murine macrophages (RAW264.7 cell line) in
the presence of caffeine doses up to 0.23 mg/mL (Hwang et al., 2016;
Samieirad et al., 2017).
3.2. Caffeine and Natural killer (NK) cell cytotoxicity
Known for their vital innate cytotoxic role in defeating pathogens,
NK cell activity seems to be modulated by the dose of caffeine exposure
as tested by Kantamala et al., 1990. The NK cytotoxic assay was as-
sessed both in vivo upon treatment of rats for 120 consecutive days with
different doses of caffeine (6, 12, 18 mg/kg/day) and in vitro upon a 4-
hour caffeine treatment (0.01, 0.02 and 0.04 mg caffeine/mL) of iso-
lated splenic NK and mononuclear cells against YAC-1 lymphoma cell
line. Interestingly, in vivo groups of rats administered low doses of
caffeine (6 mg/kg/day) resulted in a significant decreased activity of
NK cells.
However, upon high caffeine doses (18 mg/kg/day) the effect was
reversed and NK cell cytotoxicity was not detected. This reversed pat-
tern of NK cell activity appearing after high doses might be a result of
the saturation of adenosine receptors (Bortoluzzi et al., 2016), which
become unresponsive to the PDE inhibitory effect of caffeine (Steck
et al., 2015). In the same study, in vitro caffeine assessment showed
insignificant interference of caffeine with NK cell cytolysis. Such a
difference in NK cell pattern in view of caffeine exposure could be due
to the chronic effect of rat exposure to caffeine resulting in an increase
in Ca2+ intracellular levels, thereby affecting NK cell activity
(Kantamala et al., 1990).
Another approach to test for the ability of NK cell to lyse target cells
is to assess NK cell surface expression of CD69 which in turn induces the
release of TGF-β (Esplugues et al., 2003). Such approach was carried by
Fletcher and Bishop (2011), on male participants exposed to a single
dose capsule ingestion of caffeine (6 mg/kg) or 3 repeated caffeine
doses of 2 mg/kg each. Venous blood and saliva samples were assessed
for CD69 cell surface expression on antigen stimulated NK (CD3−
CD56+) gated cells. Interestingly, the mean fluorescence intensity of
CD69 was neither altered by the caffeine ingested dose nor by the time
of specimen withdrawal (Dulson and Bishop, 2016; Fletcher and
Bishop, 2011). In contrast, a high dose of caffeine (> 6 mg/mL) in-
creased the number of antigen-stimulated CD3− CD56+ cells expres-
sing CD69 at 1 h following caffeine ingestion (Dulson and Bishop, 2016;
4
Immunobiology xxx (xxxx) xxx–xxx
Fletcher and Bishop, 2012). Thus, we deduce that moderate and acute
levels of caffeine uptake would not interfere with the cytotoxicity ef-
ficiency of NK cells in humans, unlike a reduced activity in rats.
However, high doses and prolonged exposure to caffeine most likely
promote NK cell activity in humans, while exerting no effect in rats.
Future studies are needed to test whether caffeine-induced-NK cells
upregulate the expression of NKG2D receptors to better recognize
genomic stressed or infected cells. This might highlight the role of
caffeine in targeting cancer cell death. Another line of research could be
linked to the hyperactivity of caffeine-induced-NK cells and its potential
link in the development of certain autoimmune diseases.
3.3. Caffeine attenuates pro-inflammatory cytokine profile in the blood
A reliable method to examine the behavior of different immune cells
in the presence of caffeine is to track their cytokine profile. Despite the
well-known effect of concanavalin A (ConA) as a T cell mitogen (Krauss
et al., 1999) and a cytokine stimulator (Buyukozturk et al., 2005;
Karagouni and Kourounakis, 1991), Ritter et al., 2005 showed that in
vitro stimulation of isolated mouse splenocytes with caffeine (0.73 mg/
mL) and ConA (5 mg/mL) significantly reduced the expression of IL-2,
while completely blocked the release of TNF-α and IFN-γ. Upon the
dose increase of caffeine from 0.73 mg/mL to 1.9 mg/mL, IL-2 levels
were completely blocked (Ritter et al., 2005). On the other hand, sti-
mulating mice splenocytes with freeze dried instant coffee powder
0.1 mg/ml or arabinogalactan protein for 24 h showed significant in-
crease in IL-2 levels (Capek et al., 2014). Moreover, isolated mesenteric
lymphocytes from rats administered 1 mg/mL caffeine from green tea
extracts exhibited decreased expression levels of IL-2, IL-6, TNF-α, but
no change was detected in INF-γ levels (Molina et al., 2015). Similar to
the behavior of isolated cells from mice and rats, isolated human per-
ipheral blood mononuclear cells stimulated with mitogens and treated
with caffeine showed no significant effect on INF-γ levels as detected by
ELISA (Gostner et al., 2015). Thus, with the exception of Capek et al.
study, caffeine most likely exerts anti-inflammatory effects depending
on the dose and source of caffeine used.
However, many other factors can bias our analysis. For instance,
switching from in vitro isolated cell model, to in vitro cultured cell lines
or in vivo models might alter the overall reading of caffeine mode of
action.
On the same note, mRNA levels of TNF-α and IL-6 from lipopoly-
saccharide (LPS)-treated murine macrophage-like cell line, RAW 264.7
cells, showed a negative correlation with the presence of coffee ex-
tracts. Upon increasing the roasting levels of coffee (0.5 to 2 mg/mL)
lower mRNA levels of these cytokines were measured (Jung et al.,
2017). Such anti-inflammatory effect of caffeine aligns with the re-
duced expression of IL-6, IL-3, and IL-12 by LPS-treated RAW264.7 in
the presence of caffeine doses up to 0.23 mg/ml (Hwang et al., 2016;
Samieirad et al., 2017). Thus, in vitro studies whether done on isolated
immune cells or using cultured cell lines, support an im-
munosuppressive role of caffeine on major anti-inflammatory cyto-
kines.
In terms of in vivo studies, two reports examined the modulatory
effect of caffeine on certain cytokines. The first study was done in mice
administered up to 1-week caffeine doses (up to 16 mg/mL). There was
no significant effect on the plasma levels of IL-6, IL-12, and IL-10, and
reduced effect on IL-2 and IL-4 (Pohanka, 2015). Similarly, the second
study involved LPS-activated cord blood monocytes taken from infants
with serum caffeine doses at 0.01 and 0.02 mg/mL. Results showed a
significant drop in the levels of secreted TNF-α and IL-10, while at
concentrations greater than 0.02 mg/mL only decreased IL-10 secretion
and gene expression was detected (Chavez-Valdez et al., 2016).
It is worth noting that a suppressive effect of caffeine on the release
of pro-inflammatory mediators, such as TNF‑α, IFN‑γ and IL-6 and it is
insignificant effect on the anti-inflammatory cytokine IL-10, is most
likely exerted by high levels of cyclic adenosine monophosphate
T. Al Reef, E. Ghanem
(cAMP) (Kotova et al., 1994). As described in Fig. 2, once caffeine binds
to its A2A receptor, cAMP is upregulated via phosphodiesterase (PDE)
inhibition.
On the other hand, high cAMP levels drive a resistant state (anergic)
of responder T cells to T reg suppression; eventually, higher counts of
responder T cells may contribute to an anti-tumor immune response
(Mosenden and Tasken, 2011). However, cAMP elevating substances
may lead to autoimmune diseases resulting from self-reacting effector T
cells (Aricha et al., 2006; Delgado et al., 2002).
3.4. Caffeine modulates lymphocyte profile
Given that IL-2, IFN-γ, and TNF-α are released by activated lym-
phocytes in addition to their release by other immune players (Borish
and Steinke, 2003), we speculate that lower expression levels of these
cytokines in a caffeine-induced microenvironment- as described in the
previous section- imply reduced activity of lymphocyte behavior.
In vitro studies done on mitogen-stimulated murine splenic T cells,
murine T cell clones, and isolated human peripheral lymphocytes
showed an inhibitory effect of caffeine on lymphocyte proliferation and
a decrease in antibody production (Kantamala et al., 1990; Rosenthal
et al., 1992b). The drop in antibody levels is in line with an in vivo
murine study by Pohanka (2015) where caffeine doses (up to 16 mg/
mL) caused a significant decrease in plasma levels of antibodies against
keyhole limpet hemocyanin (KLH) (Pohanka, 2015). In vitro caffeine
treatment of mitogen-stimulated T and B rat lymphocytes suppressed
cellular proliferation in a dose-dependent manner. Interestingly, the
same group reported an increase in T cell proliferation upon in vivo
chronic treatment of rats with high doses of caffeine up to 18 mg/day
(Kantamala et al., 1990). Thus, switching between in vitro and in vivo
studies showed discrepancies in T cell proliferation, but not in antibody
production.
To further understand T cell behavior, data was collected from in
vivo human study whereby venous blood sample from male participants
exposed to 6 mg/mL caffeine was assessed for lymphocyte counts 1 h
prior to a bout of intensive exercise. Total lymphocyte counts in the
blood were enhanced in a resting state compared to placebo (Bassini-
Cameron et al., 2007; Bishop et al., 2005). Interestingly, both subsets of
T lymphocytes, CD4+ and CD8+, exhibited upregulated expression of
CD69 on their cell surface. This might indicate a selective migration of
T cells into the blood sensitizing the rising levels of plasma epinephrine.
CD8+ quantity was accentuated compared to that of CD4+ cells. This is
most likely explained by the fact that CD8+ cells express a more no-
teworthy thickness of β-receptors, also known as adrenergic receptors,
that respond to epinephrine after caffeine treatment (Bishop et al.,
2005; Spielmann et al., 2014). On the other hand, an opposing result
showed no alteration in CD4+ and CD8+ counts after caffeine uptake (2
to 6 mg/mL) in humans compared to placebo (Fletcher and Bishop,
2012).
Similarly, in a recent study, Dulson and Bishop, 2016 demonstrated
that both low and high doses of caffeine had no significant effect on the
count and expression levels of CD69 on antigen-stimulated CD69+ T
cells withdrawn from human blood (Dulson and Bishop, 2016). Im-
munocompromised studies linked to tumors were excluded from this
study. However, one study done on individuals infected with HIV, mi-
micking immunocompromised conditions, revealed significantly re-
duced counts of peripheral blood CD4+ upon a dose of caffeine ex-
posure of 338 mg per day (Ramamoorthy et al., 2017).
Taken together, the contrasting findings in lymphocytes’ behavior
between different studies could be attributed to the experimental pro-
cedure- whether it is applied in vivo or in vitro, the tested model-
whether rodents or humans, and the time of sample withdrawal since
lymphocytes exhibit a circadian rhythm. Time of specimen withdrawal
is usually not mentioned in the experimental settings, which might as
well attribute to bias during the data read-out. Definitely, in vivo studies
done in humans are most reliable as caffeine metabolism and plasma
5
Immunobiology xxx (xxxx) xxx–xxx
half-life might vary dramatically between human and rodents. Thus, in
vivo studies support an enhancement or unremarkable effect on T cell
proliferation compared to suppressive effects reported from in vitro
studies.
3.5. MHC expression and caffeine
To date the effect of caffeine on the expression levels of major
histocompatibility molecules (MHC) class I and II molecules have not
been well elucidated. Only two studies have addressed the in vitro be-
havior of MHC class I on murine T- cell lymphoma as reported by
Rosenthal et al. in 1992 and 1993 (Rosenthal et al., 1992a) (Rosenthal
and Blank, 1993). In addition, one study assessed MHC class I-related
chain B (MICB) molecules (Tang et al., 2008), and no single hit was
recorded for MHC class II molecules. Given that MHC molecules play an
important role in antigen presentation during adaptive immunity and
they possess a strong association with certain autoimmune and tumor
diseases (Arase, 2016; Sollid et al., 2014; Winchester and Dwyer, 1991);
it becomes quite intriguing to examine their behavior in light of caf-
feine treatment.
Rosenthal and Blank (1993) demonstrated an enhanced cell surface
expression of murine MHC class I alleles, H-2Kk and H-2Dk, on two T
cell lymphoma lines in the presence of various concentrations of caf-
feine (0.1 to 0.4 mg/mL) in a dose dependent manner reaching highest
levels after 2 or 3 days of incubation (Rosenthal and Blank, 1993).
Furthermore, pre-treatment of murine leukemia T-cell lymphoma line
(Kgv) cells with 0.3 mg/mL of caffeine for 3 days, significantly in-
creased H-2Dk, but not H-2Kk expression especially when combined
with high doses of IFN-γ. A synergistic effect occurred upon simulta-
neous incubation of cells with both caffeine and IFNγ, most likely by
acting at distinct promoter regions and activating different signal
transduction pathways. This assumption could be linked to the presence
of cAMP-responsive elements in the promoter regions of major histo-
compatibility complex class I (MHC I) genes (Israel et al., 1989). Thus,
caffeine most likely exerts its modulatory effects at the transcriptional
levels in a cAMP dependent mechanism, mainly by regulating the
promoter regions of MHC class I genes. This paves the way for future
studies to better understand the transcription and post-transcriptional
contribution of caffeine on the cell surface expression of MHC class I
and class II alleles in human cell lines including professional antigen
presenting cells, notably dendritic cells (DC), macrophages, and B cells.
Interestingly, unlike the boosted effect of caffeine on MHC class I levels,
MICB showed reduced cell surface expression levels in human hepato-
cytes, HepG2, and human embryonic kidney cells, HEK293 T, treated
with 0.39 mg/mL caffeine. Caffeine was shown to partially inhibit DNA
damage and DNA demethylation, thereby protecting the cells from NK
cytotoxicity through NKG2D receptor binding to MICB ligands (Tang
et al., 2008).
4. Concluding remarks and future perspectives
The profound impact of caffeine as an immunomodulator mainly
depends on its daily intake dose. Based on the literature, scientists fail
to agree on a specific dose that is universally considered moderate.
Moreover, doses administered to animal models do not necessarily re-
flect the same dose-effect on humans due to its rate of metabolism and
half-life time in the plasma. To conclude, the binding properties of
caffeine, especially on its adenosine receptor, A2A, induce a well-ac-
cepted intracellular signaling pathway that is dose dependent as illu-
strated in Fig. 2. On one hand, caffeine acts non-specifically by en-
hancing the protective immune response and increasing the counts of
macrophages, neutrophils, eosinophils and other immune players. On
the other hand, specific action has been noted on the adaptive immune
cells by increasing their blood counts, but reduced their proliferation
and cytokine production. This could be due to the fact that immune
cells are sensitive to epinephrine present in the blood, which induces
T. Al Reef, E. Ghanem Immunobiology xxx (xxxx) xxx–xxx

Table 1
Summary of the in vivo and in vitro studies assessing the effects of caffeine on various cellular and molecular immune components as retrieved from articles published
between 2007 and 2018.
Cellular components Organism Caffeine Conc./ Dose Time Study type Major outcome Reference

Neutrophils isolated from cardiac blood Rats 0.1 to 1 mM 48 h In vitro ↑ vitality of neutrophils Abbasi et al.
(0.02 to 0.2 mg/mL) (2018)
HIV-infected peripheral blood Human 338 mg/day In vivo ↓ CD4+ T cells Ramamoorthy
et al. (2017)
PKA-induced isolated mononuclear Human 0.1 mM 22 h In vitro ↓ phagocytosis Steck et al. (2015)
phagocytes
Lymphocytes from venous blood Human 2-6 mg/kg of body mass 1 to 3.5 h In vivo No effect CD4+ and CD8+ levels ↑ Fletcher and
CD3- CD56+ cells Bishop (2012)

Molecular components Organism Caffeine Conc./ Dose Time Study type Major outcome Reference

Toll-like receptors from newborn lungs Rats 20 mg/kg/day in breast 10 days In vivo ↑ TLR9 levels. Tunc et al. (2013)
milk No effect TLR-2 and TLR-4
Cytokine production by mesenteric Rats 1 mg/ml caffeine in In vitro ↓ IL-2, IL-6, ROS Molina et al.
lymphocytes Green Tea ↓ TNF-α (2015)
↓ in mRNA level of TRL4
No effect on IFN-γ
Cytokines from activated splenocytes Mice 0.1 mg/ml of freeze 24 h In vitro ↑ IL-2 & free radicals Capek et al.
dried instant coffee (2014)
powder or
arabinogalactan protein
Antibody and cytokine production from Mice 1 to 16 mg/kg caffeine 1, 2, 7 days In vivo ↓ antibody production, IL-2, and IL-4. Pohanka (2015)
plasma No effect on IL-6, IL-10, IL-12
Cytokine production by LPS induced Mice 0.1 to 1.2 mM 24 h In vitro ↓ Nitric Oxide (NO), Samieirad et al.
macrophage- Cell lines ↓ IL-6, IL-12, IL-3, COX-2 (2017)
like cell line
(RAW264.7)
Cytokine production by LPS induced Mice 0.1 to 1.2 mM In vitro ↓ NO, COX-2 Hwang et al.
macrophage- Cell lines (up to 0.23 mg/mL) ↓ IL-6, IL-3, IL-12 (2016)
like cell line
(RAW264.7)
Cytokine production by Mitogen- Human 10% of hot brewed 30 mins In vitro Hot brewed coffee, but not caffeine Gostner et al.
stimulated isolated PBMCs coffee or caffeine in showed a significant decrease in IFN-γ (2015)
medium (v/v)
Cytokine production by LPS-induced Human 0 to 0.2 mM In vitro ↓ TNF-α and IL-10 Chavez-Valdez
cord blood monocytes (up to 0.04 mg/mL) et al. (2016)
Cytokine production from plasma and Human 20 mg/kg 24 h - In vivo No effect on IL-1β, IL-2, IL-7, INF-γ, and (Chavez Valdez
tracheal aspirates 1week GM-CSF in plasma et al., 2011)
↑ whereas all measured cytokines were
significantly detected in tracheal
aspirates
Cytokine production by peripheral blood Human Caffeine from infused 1 5 or 24 h In vitro ↑ IFN- γ, ↓ IL-6 (Neyestani et al.,
mononuclear cells black tea bag extract No effect on TNF- α and IL-1 β 2009)
(BTE) ∼
31 mg/dl
MHC class I-related chain B molecules Human cell 2 mM (0.4 mg/mL) 1h In vitro ↓ DNA induced demethylation and Tang et al. (2008)
(MICB) lines damage
↓ MICB expression

their migration from the lymphatic tissues to the periphery. Never-
theless, their larger quantity might pertain to an anergic state, in-
competent of becoming effective.
Last but not least, one should note that caffeine content in a single
coffee serving can considerably vary depending on the type of coffee
beans and its mode of preparation. Moreover, caffeine constitutes one
out of many other coffee substances, such as kahweol and
Arabinogalactan-protein (AGP). Thus, caffeine can act either antag-
onistically or synergistically with other substances changing the overall
effect of purified caffeine. For instance, AGP showed an increase in the
pro-inflammatory response by significantly enhancing the TNF-α pro-
duction in mice splenocytes (Nosalova et al., 2011). Thus, it is still
unclear whether the anti-inflammatory impact of caffeine once blended
with other substances remains valid. In addition, most of the caffeine
studies are either done in vitro or on animal models. Therefore, it is
challenging to translate this modulation to humans knowing that in-
terspecies have different distribution and activation processes related to
caffeine receptors. The increase in adenosine levels has been proven to
have a protective effect by inducing inflammation and preventing tissue
damage by increasing the counts of immune players and promoting
tumor cell death in the targeted sites (Merighi et al., 2002). However
when present in very high doses, it exacerbates the inflammatory pro-
cesses especially by inhibiting T cell activation (Clayton et al., 2011).
For instance, when it accumulates in tumor microenvironment, it is
favoring cancer proliferation and metastasis by switching the functions
of these cells from immune defense to cancer growth (Antonioli et al.,
2013). We believe that the optimum dose of caffeine to exert a mod-
erate and effective function as an adenosine antagonist is still subjected
to further studies. Operative doses might carry potential therapeutic
applications in a vast array of inflammatory diseases and as anti-tumor
agents.
To conclude, the overall trend of caffeine is to downregulate major
cellular and molecular immune components regardless of the employed
dose concentration, experimental settings, study type (in vitro or in vivo)
and the tested model (Table 1).

6
T. Al Reef, E. Ghanem
Disclosure statement
No potential conflict of interest was reported by the authors.
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