12

CHAPTER

The Effects of Flavonoids on the

Immune System
F.J. Prez-Cano, . Franch, T. Prez-Berezo, S. Ramos-Romero, C. Castellote,
M. Castell
Universitat de Barcelona, Barcelona, Spain

ABBREVIATIONS
APC Antigen-presenting cell
BCR B-cell receptor
CCL Chemokine (CC motif) ligand
CCR Chemokine (CC motif) receptor
CD Cluster of differentiation
Con-A Concanavalin-A
DC Dendritic cell
EGCG Epigalocatechin gallate
ERK Extracellular signal-regulated kinases
GALT Gut-associated lymphoid tissue
IFNg Interferon g
Ig Immunoglobulin
IL Interleukin
JNK c-Jun N-terminal kinases
MHC Major histocompatibility complex
NF-kB Nuclear factor kappa light chain enhancer of activated B cells
NK Natural killer
OVA Ovalbumin
PBMC Peripheral blood mononuclear cells
PMA Phorbol myristate acetate
SEB Staphylococcal enterotoxin B
T-bet A transcription factor from the T-box family expressed in T cells
TCR T-cell receptor
Th Helper lymphocyte

1. INTRODUCTION
The immune system has evolved to ensure protection against invading pathogens while
avoiding reactions to innocuous self- and non-self-antigens, in a fashion that promotes
immune tolerance. The function of the immune system depends on many intricate cell
cell interactions that even today are not fully understood. A focus for many nutritionists
Bioactive Food as Dietary Interventions for Arthritis and Related Inflammatory Diseases
http://dx.doi.org/10.1016/B978-0-12-397156-2.00011-9

2013 Elsevier Inc.

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and scientists in the immunonutrition field is the research of food compounds that may
influence the functionality of the immune system.
Flavonoids are products of the secondary metabolism of plants that are regularly
ingested in small quantities in many edible plants. Chemically, they have a polyphenolic
structure showing antioxidant activities. These properties have aroused an increasing
interest in assessing their possible beneficial role in the prevention of various diseases,
as evidenced by the large number of studies focused on the effect of flavonoids on health
over the last decade. Flavonoids can be classified into flavonols, flavones, isoflavones,
flavanones, flavanols, and anthocyanidins. Flavonols such as quercetin, kaempferol,
galangin, morin, rutin, myricetin, isorhamnetin, and isoquercetin can be found in onions,
apples, berries, kale, leeks, broccoli, blueberries, red wine, and tea. Flavones such as
glycosides of luteolin, chrysin, and apigenin are commonly found in fruit skins, parsley,
and celery. Isoflavones such as genistein, daidzein, and glycitein are exclusively present
in leguminous plants, mainly soy and soy products. Flavanones such as naringenin, eriodictyol, and hesperidin are exclusive to citrus fruits. Flavanols include monomers such
as epicatechin, catechin, gallocatechin, epigallocatechin, and epigallocatechin gallate
(EGCG), and also polymers called proanthocyanidins. Proanthocyanidins or condensed
tannins can appear as procyanidins (epicatechin and catechin polymers, mainly found in
cocoa), prodelphinidins (epigallocatechin or gallocatechin polymers), and propelargonidins (epiafzelechin or afzelechin polymers). Anthocyanidins include pelargonidin, cyanidin, and malvidin, whose sources are red wine and berry fruits.
This chapter focuses on the effect of flavonoids on the immune system. Specifically, as
the relationship between flavonoids and innate immune responses, including inflammation, has been extensively discussed in other chapters, the aim of this chapter is to provide
an integrated update on the action of flavonoids on the acquired immune response, including the most recent studies related to this topic and, particularly, those using
preclinical and clinical approaches. First, we include a short summary of the specific
or acquired immune response.

2. ACQUIRED IMMUNITY: THE TAILORED RESPONSE AGAINST ANTIGEN

Acquired immunity is characterized by antigen specificity and memory. It principally
involves the function of lymphocytes. These cells remain in a resting state waiting to bind
to a specific antigen and thus become activated. Lymphocytes can be T or B cells and have
surface antigen-specific receptors called T-cell receptors (TCRs) and B-cell receptors
(BCRs), respectively. Each lymphocyte is genetically programmed to be capable of
recognizing just one particular antigen.
When an antigen enters the body, it is captured by specialized cells called antigenpresenting cells (APCs), mainly dendritic cells (DCs). DCs are sentinels that have the ability to integrate a wide array of incoming signals and convey them to lymphocytes. After

The Effects of Flavonoids on the Immune System

the capture of the antigen, DCs start a maturation process that involves the enhancement
of the expression of the major histocompatibility complex class II (MHC-II) and accessory molecules, and the production of cytokines and chemokines. After the antigen
processing, the DCs present it to T-helper lymphocytes (Th or CD4 (cluster of differentiation 4) lymphocytes), leading to a molecular DCTh interaction called
immunological synapse (Figure 12.1). This interaction includes the binding of the processed antigen to a specific TCR, as well as the linkage of CD4 on the Th cell with
MHC-II molecules on the APC. The APCTh cell interaction is tightened with costimulatory molecules such as CD54 and CD11a. Moreover, CD3 on Th cells forms
a complex with TCRs playing a role in the transduction of stimuli into the cell. CD3
stimulation is associated with the tyrosine phosphorylation of multiple proteins, resulting
in the activation of various signaling pathways, eventually causing T-cell proliferation
and secretion of cytokines.
Activated Th cells produce interleukin (IL)-2 and proliferate to give a progeny of
activated antigen-specific Th cells. Similarly, B cells can become activated when the
specific antigen binds to their BCR and receive the appropriate cell interaction from
Th cells. Activated B cells will proliferate and differentiate to become antibodyproducing cells or plasma cells, highly specialized in synthesizing antibodies or immunoglobulins (Ig), such as IgG, IgM, IgA, or IgE, which specifically bind to the antigen that
has induced their synthesis.
According to the type of antigen and the environment where the cell activation
occurs, Th cells will become effector Th cells with different profiles (Figure 12.1).
Although there are other recently described types of effector Th cells, Th1 and Th2
are the most abundant. These cells produce different patterns of cytokines resulting in
different responses. Th1 response produces interferon g (IFNg) and IL-2, and enhances
macrophage, cytotoxic T-cell, and natural-killer (NK) cell functions, as well as the synthesis of antibodies capable of activating the complement system. These factors enhance
cell killing by phagocytosis and cytotoxicity, both important actions in the inflammatory
process. Conversely, the activated Th2 cells produce cytokines such as IL-4, IL-5, IL-10,
IL-13, which activate mast cells, eosinophils, and the synthesis of antibodies such as IgE
and IgA, which do not activate the complement system but are involved in the defense
against parasites, allergic reactions, and the mucosal immune response, among others.

3. FLAVONOIDS IN THE IMMUNE SYSTEM

Most studies evaluating the effects of flavonoids on the immune system are performed in
vitro. These studies allow one to approach the molecular mechanisms and targets affected
by flavonoids. However, the in vivo studies can better reflect the effects of these
compounds after absorption and metabolism.

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Antigen presentation
Th lymphocyte
(antigen-specific TCR)

Antigen
MHC-II-antigen - TCR
(immunological synapse)

Dendritic cell
(APC)

Flavonoids

Flavonoids

Activated
Th

IL-2 secretion

Antigen-specific Th cell
proliferation

Flavonoids

Differentiation
Flavonoids

IFN-g

Flavonoids
Effector
Th1

IL-2

Effector
Th2

IL-4

IL-5

IL-13

B
NK
B
Eosinophil

Neutrophil
Macrophage

Mast cell

IgE
IgG
IgA
Inflammatory response
Immune response against
intracellular antigens

Allergic response
Immune response against
extracellular antigens

Figure 12.1 Acquired immune response. Schematic cellular and molecular events from the antigen
entrance until the activation of effector Th1 and Th2 responses. The main targets of flavonoids are
marked.

The Effects of Flavonoids on the Immune System

3.1 In Vitro Studies of Flavonoids in the Immune System

As detailed above, the first events following the entrance of antigen into the body are its
uptake by APCs and its presentation to Th (CD4) lymphocytes. At that moment, the
cellcell interaction between APCs and Th cells is crucial for triggering the acquired
immune response. Two studies have reported that both quercetin (flavonol) and EGCG
(flavanol) were able to inhibit the expression of MHC class II and co-stimulatory molecules, such as CD11c, CD40, CD80, CD83, and CD86, in activated DCs (Huang et al.,
2010; Yoneyama et al., 2008). Quercetin and EGCG also downregulated the antigen
loading in DCs. Moreover, quercetin reduced the DC migration induced by the
CCL21 (chemokine (CC motif) ligand 21). The mechanism induced by quercetin
was the disruption of the extracellular signal-regulated kinases (ERK), c-Jun N-terminal
kinases (JNK), Akt, and nuclear factor kappa light chain enhancer of activated B cell (NFkB) pathways involved in DC activation (Huang et al., 2010).
The following phase in the acquired immune response includes the activation of
specific Th lymphocytes. In consequence, Th cells synthesize and secrete high amounts
of IL-2, which induces the specific Th-cell proliferation. Several flavonoids have demonstrated their ability to control lymphocyte proliferation and modulate IL-2 secretion
(Table 12.1). Isoflavones such as genistein decreased proliferation and the secretion of
IL-2 in stimulated human peripheral blood mononuclear cells (PBMCs) and T cells
(Gredel et al., 2008). Flavonols such as quercetin also reduced IL-2 synthesis in activated
Table 12.1 In Vitro Effect of Flavonoids on the Cytokine Production of Lymphocytes
Cytokine Effect
Flavonoid
Target

IL-2

EGCG

Epicatechin
Catechin
Procyanidin
B2
Epicatechin
Cocoa extract

#
#

Genistein

Quercetin

Kaempferol

Reference

Concanavalin-A (Con-A)- and/or

Staphylococcal enterotoxin B (SEB)stimulated human PBMC; murine
splenocytes or mesenteric lymph node
cells
Jurkat T cells

Watson
et al. (2005)

Phorbol myristate acetate (PMA)stimulated EL-4.BU.OU6

lymphocytic murine cell line
Con-A-stimulated human PBMC

Ramiro
et al. (2005)

Activated T-bet-deficient and T-bet

transgenic/deficient mouse Th cells
Con-A-stimulated human
lymphocytes

Mackenzie
et al. (2004)

Gredel
et al. (2008)
Yu et al.
(2008)
Miles et al.
(2005)
Continued

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F.J. Prez-Cano et al.

Table 12.1 In Vitro Effect of Flavonoids on the Cytokine Production of Lymphocytescont'd

Cytokine Effect
Flavonoid
Target
Reference

mRNA

EGCG

Jurkat T cells

Wu et al.
(2009)

Cocoa extract
(flavanols)
EGCG

Phytohemagglutinin (PHA)stimulated human PBMC

Con-A- and/or SEB-stimulated
human PBMC; murine splenocytes or
mesenteric lymph node cells
Primary Th cells from immunized
animals and EL4 T-cell line

Jenny et al.
(2009)
Watson
et al., 2005

Con-A-stimulated human
lymphocytes
Con-A-stimulated human PBMC

Miles et al.
(2005)
Gredel
et al. (2008)
Yu et al.
(2008)

#
#

Park et al.
(2005)

Formononetin
Daidzein
Equol
Kaempferol

Genistein

Quercetin

#mRNA

EGCG

PMA-stimulated EL-4.BU.OU6
lymphocytic murine cell line
Primary Th cells from immunized
animals and EL4 T-cell line

Ramiro
et al. (2005)
Park et al.
(2005)

Epicatechin
Cocoa extract
Formononetin
Daidzein
Equol
Genistein

Con-A stimulated human PBMC

EGCG

Kaempferol

Con-A- and/or SEB-stimulated

human PBMC; murine splenocytes or
mesenteric lymph node cells
Con-A-stimulated human
lymphocytes

Gredel
et al. (2008)
Watson
et al. (2005)

IL-5

mRNA

EGCG

Jurkat T cells

IL-13

mRNA

EGCG

Jurkat T cells

IFNg

IL-4

Activated T-bet-deficient and

T-bet-transgenic/deficient mouse
Th cells
Jurkat T cells

Wu et al.
(2009)

Miles et al.
(2005)
Wu et al.
(2009)
Wu et al.
(2009)

Th cells, and this was accompanied by a lower expression of the chain a of IL-2 receptor
(IL-2Ra or CD25) (Yu et al., 2008). In the same way, when the flavanol EGCG was
added to cultures of human PBMC, murine splenocytes or mesenteric lymph node cells
abolished the immune cell proliferation and IL-2 production by mechanisms independent of NF-kB and AP-1 (activator protein 1) DNA-binding activities (Watson et al.,

The Effects of Flavonoids on the Immune System

2005). The immunological synapse may be another possible target for flavonoid action.
In this sense, Kawai et al. (2003) demonstrated that EGCG bound directly to the
cell-surface CD4 molecule, without modifying the surface expression of CD3, CD54,
and CD11a. In agreement with those results, flavonoids from the same EGCG family,
such as epicatechin, catechin, and the dimeric procyanidin B2, have been reported to
inhibit IL-2 synthesis in Jurkat T cells (Mackenzie et al., 2004). In this case, the inhibition
was attributed to the entrance of these flavanols into the lymphocyte nuclei, inhibiting
NF-kB activation at both the early stages of the activation cascade (regulation of oxidant
levels, IkB kinase (IKK) activation, and subsequent IkBa phosphorylation) and at the
later stages by preventing the binding of active NF-kB to kB sites (Mackenzie et al.,
2004). In a similar vein, Ramiro et al. (2005) reported the inhibition of IL-2 secretion
and the downregulation of CD25 by epicatechin and a cocoa extract on stimulated EL-4.
BU.OU6 lymphocytic cells.
After activation and proliferation of specific Th cells, the acquired immune response
proceeds with the differentiation of Th lymphocytes to effector Th1 or Th2 cells that
secrete their typical cytokine profile (see above). IFNg is the main product of Th1 cells,
and IL-4 is the representative cytokine of Th2 cells. Consequently, increases or decreases
in the synthesis of these cytokines could reflect Th1- or Th2-promoting or -inhibiting
activities. Several flavonoids added to lymphocytes in culture have shown the ability to
modify IFNg and IL-4 production (Table 12.1).
The flavonol quercetin reduced IFNg protein and mRNA expression in TCRstimulated Th cells through the modulation of a transcription factor from the T-box family expressed in T-cell (T-bet) expression (Yu et al., 2008). Similarly, Miles et al. (2005)
reported the inhibition of IFNg, but no effect on IL-2 and IL-4 secretion, by the flavonol
kaempferol in stimulated human lymphocytes.
Park et al. (2005) investigated the effect of the isoflavones formononetin, daidzein,
and equol on the production of IFNg and IL-4 in primary Th cells from immunized mice
and the EL4 T-cell line. The isoflavones also decreased IFNg synthesis but enhanced IL-4
production, both at transcriptional level. However, other studies revealed that the
isoflavone genistein diminished the secretion of both IFNg and IL-4 in stimulated human
PBMC (Gredel et al., 2008).
Human PBMCs or murine lymph node cells treated with the flavanol EGCG
significantly reduced IFNg production without modifying IL-4 synthesis (Watson
et al., 2005). Likewise, a cocoa extract rich in flavanols suppressed the production of
IFNg in stimulated human PBMCs (Jenny et al., 2009). On the other hand, epicatechin
and a cocoa extract were able to increase IL-4 production by stimulated EL4.BU.OU6
murine cells (Ramiro et al., 2005). Similarly, Wu et al. (2009) showed that EGCG
significantly upregulated the mRNA expression of Th1/Th2 cytokines including
IL-2, IFNg, IL-5, and IL-13 in Jurkat T cells. The mRNA upregulation of IL-2
and IL-5 was predominantly affected by ERK signaling, whereas IL-13 gene expression, the most responsive to the EGCG treatment, was dependent on neither ERK

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nor JNK signaling. IFNg gene expression was partially mitigated by both inhibitors of
ERK and JNK pathways.
In summary, most of the in vitro studies of the effect of flavonoids on lymphocytes
point to an inhibitory effect of the acquired immune activation, including very early
and late phases of the immune response, as in the case of the interaction between
DCs and Th cells, IL-2 secretion, Th cell proliferation, and Th1 or Th2 effector
responses. However, a skewed effect of flavonoids favoring or inhibiting Th1 or Th2
responses is not clearly established in vitro.

3.2 The Effect of Flavonoid Intake on the Functionality of the

Immune System
In vivo studies of flavonoids include both those performed with a specific flavonoid and
those carried out with extracts or foods rich in certain flavonoids. Most of the preclinical
studies focused on the acquired immune response were performed in experimental
models based on an antigen sensitization (mainly ovalbumin, OVA) followed by a challenge through different routes, searching to provoke a harmful immune response. As
reported in the following section, different types of flavonoids show the potential to
suppress these damaging responses.
3.2.1 Preclinical studies with single flavonoids administered orally
Yano et al. (2007) evaluated the immune response of OVA-sensitized mice, which were
fed diets containing the flavones chrysin and apigenin. Interestingly, total IgE concentration in serum decreased with these diets, whereas concentrations of IgG, IgM, and
IgA were not affected. IL-2, IL-4, IL-10, and IL-13 mRNA expression in splenocytes
were also downregulated by the flavonoid diets. In a similar manner, mice sensitized with
picryl chloride were treated with the flavone naringenin, and this resulted in the inhibition of the proliferation of activated hapten-specific T cells and polyclonal-activated T
cells, and a reduction in CD69, IL-2, and IFNg mRNA expression in activated T cells
(Fang et al., 2010).
Kogiso et al. (2006) established the effect of the isoflavone genistein in OVAimmunized mice. The administration of genistein decreased OVA-specific proliferative
responses and IFNg production. The concentration of OVA-specific IgG1 was also reduced while OVA-specific IgG2a and IgG2b and IL-4 production tended to be decreased
in genistein-treated mice. Similarly, Nazir et al. (2009) established the effect of two isoflavones, 5,7-dihydroxy-6,40 -dimethoxyisoflavone (irisolidone) and 5,40 -dihydroxy-6,7methylenedioxyisoflavone (irilone), isolated from Iris germanica (Iridaceae) and administered
to mice. Both isoflavones produced different effects on the secretion of Th1 cytokines:
The first isoflavone increased IL-2 and IFNg secretion, whereas the second isoflavone induced their decrease. However, both isoflavones downmodulated the secretion of Th2
cytokines (IL-4 and IL-5). The methylated products of both isoflavones showed a drastically

The Effects of Flavonoids on the Immune System

decreased activity, revealing the importance of free phenolic groups on their immunomodulating activities. In contrast with these results, Sakai et al. (2010) investigated the
immunomodulatory effects of the isoflavones daidzein and equol in mice by evaluating
the OVA-specific T-cell and B-cell responses. Mice showed a significantly higher level
of OVA-specific IgE than control mice when treated with equol, but not with daidzein.
Moreover, IFNg and IL-4 production were not modified in mice receiving equol, but
IL-13 production was significantly higher than that in control mice.
As well as from the preclinical studies in healthy sensitized animals, models of induced
hypersensitivity were used to establish the possible effects of flavonoids. A murine model of
airway hyperreactivity can be induced by OVA sensitization and posterior challenge with
OVA inhalation. In this context, OVA-sensitized mice that received a daily inhalation of
OVA from day 19 to day 23 were administered daily with the flavone luteolin during the
sensitization or after challenge (from day 26) (Das et al., 2003). The flavonoid reduced
OVA-specific IgE concentration in serum and increased IFNg and decreased IL-4 and
IL-5 secretions in the bronchoalveolar lavage fluid, which significantly attenuated
OVA-induced airway bronchoconstriction and bronchial hyperreactivity (Das et al.,
2003). Similarly, the flavanone naringenin was administered during allergic airway inflammation in mice. Serum total IgE and OVA-specific IgG1, IgG2a, and IgM were not modified, but IL-4, IL-5, and IL-13 from stimulated splenocytes decreased with naringenin
administration. Moreover, there was an attenuation of airway hyperreactivity and eosinophilic infiltration in the bronchioalveolar lavage fluid in flavonoid-treated mice (Iwamura
et al., 2010). Similar promising effects were reported for the flavone apigenin. Yano et al.
(2009) examined the effect of dietary apigenin on a mice model of atopic dermatitis. The
apigenin diet decreased serum IgG1 and IgE concentrations, ameliorated the development
of skin lesions, and reduced IFNg secretion (but not IL-4) in spleen cells.
3.2.2 Preclinical studies using food extracts rich in flavonoids
Regarding the effects of diets with high flavonoid content, we have demonstrated that
a cocoa-enriched diet containing flavanols such as epicatechin and procyanidins is capable of modifying the composition and functionality of several lymphoid tissues, including the gut-associated lymphoid tissue (GALT) in young healthy rats. In
particular, a continuous cocoa intake by young rats reduced the proportion of Th cells
in the spleen, Peyers patches (PP), and mesenteric lymph nodes, although neither the
proliferative response nor IL-2 secretion in these tissues was modified by the diet
(Ramiro-Puig et al., 2007, 2008). Rats fed a cocoa diet showed lower serum IgG,
IgM, and IgA concentrations (Ramiro-Puig et al., 2007) and a reduced ex vivo IL-4
secretion by splenocytes than reference animals (Ramiro-Puig et al., 2007, 2008).
In addition, the effect of a cocoa diet on an OVA-specific immune response in rats
was established (Perez-Berezo et al., 2009). The diet attenuated OVA-specific IgG1
(the main subclass associated with the Th2 immune response in rats), IgG2a, IgG2c,

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and IgM concentrations but led to higher amounts of anti-OVA, IgG2b (subclass
linked to the Th1 response). This effect was accompanied by decreased IL-4 and increased IFNg secretions (Perez-Berezo et al., 2009). Similarly, a cocoa diet was able to
attenuate the specific antibody response in a model of chronic inflammation (RamosRomero et al., 2011). Moreover, in the GALT, a cocoa-enriched diet decreased IgA
secretion into the gut, and this was accompanied by a reduction in the gene expression
of several molecules involved in IgA-secreting cell activation (IL-6, CD40), gut homing (RARa (retinoic acid receptor a), CCR9 (chemokine (CC motif) receptor-9),
CCL28), and IgA synthesis (transforming growth factor b (TGFb)) (Perez-Berezo
et al., 2011a, b). Likewise, cocoa-fed animals showed a modified toll-like receptors
(TLR) expression pattern in gut tissues, which may reflect a change in the crosstalk
between microbiota and body cells induced by a cocoa diet (Perez-Berezo et al.,
2011a,b). Nevertheless, the effect of cocoa-enriched diets could also be attributed
to compounds other than flavonoids present in cocoa, such as fiber.
On the other hand, Cruz et al. (2008) reported the immunosuppressive action of
an aqueous extract of Kalanchoe pinnata (Crassulaceae) containing quercetin 3-O-a-Larabinopyranosyl (1!2) a-L-rhamnopyranoside, quercitrin, and kaempferol 3-O-aL-arabinopyranosyl (1!2) a-L-rhamnopyranoside. Mice treated daily with this extract
during OVA-sensitization were protected against death when challenged with OVA
and showed a reduced production of OVA-specific IgE antibodies and impaired production of the IL-5 and IL-10 cytokines. Similarly, Akiyama et al. (2005) demonstrated that the intake of apple-condensed tannins inhibited the development of
oral OVA sensitization in mice. This diet produced a decreased serum OVA-specific
IgE and IgG1 synthesis in sensitized mice, which was accompanied by a higher proportion of gut intraepithelial gd T cells and inhibited the reduction of body temperature or the increase of serum histamine concentration associated with antigen
stimulation. Similarly, Medeiros et al. (2008) evaluated the properties of plant extract
collected by Apis mellifera bee, rich in myricetin, tricetin, quercetin, and luteolin, on a
murine model of OVA-induced allergy. The extract produced inhibition of OVAspecific IgE and IgG1 concentrations and, in addition, decreased leukocyte migration
to the bronchoalveolar lavage, thereby protecting against the anaphylactic shock
reaction induced by OVA. Zuercher et al. (2010) evaluated the effect of a polyphenol-enriched apple extract on a similar model. In this case, mice fed with the apple
extract did not show modified OVA-specific IgE, IgG1, or IgG2b concentrations,
and the apple extract also inhibited the release of mast cell protease and reduced symptoms of allergy upon challenge.
In conclusion, most of these studies suggested that diets containing flavonoids, regardless of their class, produce a certain immunosuppressant effect that might be desirable in
IgE allergic reactions. Moreover, it may be useful to explore this effect on oral tolerance
and antibody-mediated diseases such as autoimmune pathologies.

The Effects of Flavonoids on the Immune System

3.2.3 Clinical studies using food or extracts rich in flavonoids

Although there are many in vitro and experimental preclinical studies on the modulatory
effect of flavonoids on acquired immune responses, there are currently a limited number
of trials evaluating this effect in humans. There are evident and ethical obstacles to
evaluating the functionality of the immune system in people. Therefore, the blood compartment, as a noninvasive approach, is widely used to evaluate lymphocyte function by
means of cytokine secretion. However, such products are scarce or undetectable in serum
in most conditions. For this reason, in order to study the Th1/Th2 effector immune
responses modulation by flavonoids, an ex vivo approach after a dietary intervention is
usually designed.
Chen et al. (2005) reported the effects of purple sweet potato leaves, which were rich
in carotenoids and flavonoids, on a randomized crossover study involving 16 healthy
nonsmoking adults. In contrast to most preclinical studies, this diet increased the
proliferative response of PBMCs and enhanced the secretion of IL-2 and IL-4. Karlsen
et al. (2007) assessed the effect of anthocyanins isolated from bilberries and blackcurrants
(Medox capsules for 3 weeks) on a parallel-designed, placebo-controlled clinical trial.
The Medox group showed decreased serum IL-4 and IL-13 concentrations (Th2
cytokines) and also a reduction in pro-inflammatory chemokines such as IL-8, regulated
upon activation, normal T-cell expressed, and secreted (RANTES), and IFNa. Interestingly, Ryan-Borchers et al. (2006) evaluated the effects of soy isoflavones, both in
soymilk and in a supplement form, on the immune system of postmenopausal women.
This 16-week double-blind, placebo-controlled trial included three groups: (1) control,
706 mL cow milk/day plus a placebo supplement; (2) soymilk, 71.6 mg isoflavones
derived from 706 mL soymilk/day plus a placebo supplement; and (3) supplement,
70 mg isoflavones in a supplement plus 706 mL cow milk/day. Isoflavone intervention
in postmenopausal women resulted in a higher proportion of the B-cell population but
did not significantly influence plasma concentrations of IFNg and IL-2.
In conclusion, further preclinical studies and clinical trials should be performed in
order to better delimit the effects of flavonoids on acquired immune response and to
investigate the mechanisms involved in this process.

4. CONCLUDING REMARKS
Flavonoids comprise a myriad of vegetal products that are included in diets containing
fruits and vegetables, and also wine, tea, and cocoa. Although structurally different, most
of the flavonoids assessed in in vitro and preclinical studies showed similar activities in
terms of lymphocyte function and acquired immune response. The effects induced by
flavonoids are referred to as an attenuation of the immune functionality, a fact that implies
their putative beneficial role on immune hypersensitivity status. However, the few trials

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performed to date do not seem to support this hypothesis. In any case, more preclinical
models and protocols closer to human responses should be conducted in order to better
establish the immune consequences of these dietary compounds.

GLOSSARY
Acquired immune response Immunity mediated by lymphocytes and characterized by antigenspecificity and memory.
Antibody Also known as immunoglobulin, this is a glycoprotein of the g-globulins family produced by B
lineage cells in order to neutralize foreign molecules from bacteria, viruses, and parasites and provide
immune defense.
Antigen Any foreign (pathogen or not) or own molecule capable of being specifically recognized by an
antibody or T-cell receptor.
Antigen-presenting cells Cells that present a processed antigen through the MHC class II molecules to
the T-cell receptor of CD4 T cells. This event is essential for initiating the acquired immune response.
Chemokine Family of structurally related cytokines which selectively direct movements to certain body
environment (chemotaxis) and activation of leukocytes.
Cytokine Soluble and low-molecular-weight proteins that stimulate or inhibit the differentiation,
proliferation, or function of immune cells. The family of cytokine includes, among others,
interleukins (IL) such as IL-2, IL-4, etc.

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Akiyama, H., Sato, Y., Watanabe, T., et al., 2005. Dietary unripe apple polyphenol inhibits the development
of food allergies in murine models. FEBS Letters 579, 44854491.
Chen, C.M., Li, S.C., Lin, Y.L., et al., 2005. Consumption of purple sweet potato leaves modulates human
immune response: T-lymphocyte functions, lytic activity of natural killer cell and antibody production.
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FURTHER READING
Comalada, M., Xaus, J., Galvez, J., 2011. Flavonoids and immunomodulation. In: Bioactive Foods and
Chronic Disease States. Elsevier, Amsterdam.
Gomes, A., Fernandes, E., Lima, J.L.F.C., Mira, L., Corvo, M.L., 2008. Molecular mechanisms of antiinflammatory activity mediated by flavonoids. Current Medicinal Chemistry 15, 15861605.
Gonzalez-Gallego, J., Garca-Mediavilla, M.V., Sanchez-Campos, S., Tunon, M.J., 2010. Fruit polyphenols, immunity and inflammation. British Journal of Nutrition 104, S15S27.
Hamer, M., 2007. The beneficial effects of tea on immune function and inflammation: a review of evidence
from in vitro, animal, and human research. Nutrition Research 27, 373379.
Kawai, M., Hirano, T., Higa, S., et al., 2007. Flavonoids and related compounds as anti-allergic substances.
Allergology International 56, 113123.
Middleton, E., Kandaswami, C., Theoharides, T.C., 2000. The effects of plant flavonoids on mammalian
cells: implications for inflammation, heart disease, and cancer. Pharmacological Reviews 52, 673751.
Ramiro-Puig, E., Castell, M., 2009. Cocoa: antioxidant and immunomodulator. British Journal of Nutrition
101, 931940.
Sakai, T., Kogiso, M., 2008. Soy isoflavones and immunity. The Journal of Medical Investigation
55, 167173.