Dietary Agents in Cancer Prevention: An Immunological Perspective

Abstract

Skin cancer is the most common form of cancer diagnosed in the United States. Exposure to solar ultraviolet (UV) radiations is believed to be the primary cause for skin cancer. Excessive UV radiation can lead to genetic mutations and damage in the skin's cellular DNA that in turn can lead to skin cancer. Lately, chemoprevention by administering naturally occurring non-toxic dietary compounds has proven to be a potential strategy to prevent the occurrence of tumors. Attention has been drawn towards several natural dietary agents such as resveratrol, one of the major components found in grapes, red wines, berries and peanuts, proanthocyanidins from grape seeds, (-)-epigallocatechin-3-gallate (EGCG) from green tea etc. However, the effect these dietary agents have on the immune system and the immunological mechanisms involved there in are still being explored. In this review we shall focus on the role of key chemopreventive agents on various immune cells and discuss their potential as anti-tumor agents with an immunological perspective.

Introduction

Progressive, uncontrollable growth of a single cell that has accumulated genetic, cellular and molecular changes over a period of time results in the development of malignant neoplasms or cancer. Carcinogenesis refers to the process of transformation of a normal cell into a cancerous cell (1). Chemoprevention is aimed at preventing carcinogenesis by use of synthetic and/or naturally occurring compounds primarily by interfering with cellular and molecular targets of signal transduction pathways. Of all the cell and tissue types that are affected by cancer, skin has been the most preferred tissue for study not only because it is the first barrier that protects the body from external agents but also due to various other factors such as ease of accessibility, presence of well determined precursors, ease of drug administration and presence of various mouse models exposing the mechanisms underlying skin carcinogenesis (2). Skin cancer can be broadly divided into melanoma and non-melanoma cancer (which includes basal and squamous cell carcinomas) depending on the cell type. Though both environmental and genetics factors are known to induce skin cancer, solar UV radiation remains the predominant causative agent. Exposure to UV radiation alters the genetic and immunological profile of the cells inducing immunosuppression, DNA damage (including oncogene activation and mutations to tumor suppressor genes such as p53), inflammation, oxidative stress, free radical production and photo aging (3). Moreover, UV radiation is also known to modulate the immune system of the skin by altering the antigen presenting capability, production of immunosuppressive cytokines and modulating contact and delayed type hypersensitivity reactions. In summary UV radiation, causes DNA damage and promotes tumor escape from immune surveillance. Of late, extensive research has highlighted the role of natural dietary agents in controlling skin carcinogenesis and tumor growth in other models (4). While several molecular mechanisms have been shown to be responsible for the effectiveness of these agents in controlling tumor progression (5), a comprehensive immunological evaluation is still pending. Here we review the current literature highlighting the potential immunological mechanisms that may be responsible for the effectiveness of these agents in controlling tumors (skin or otherwise) and autoimmune diseases. However, before discussing the immunomodulatory activities of key dietary chemopreventive agents we herein briefly describe various immune cells that play an important role in mounting an immune response and contribute to tumor development or tumor control.

Role of immune cells in cancer

While significant research elucidating the role of various chemo-preventive agents have been conducted, little effort has been directed in understanding the immunological mechanisms that are affected by these potent agents. The immune response could be divided into innate and adaptive immunity. While the innate immune system comprises of dendritic cells, macrophages, neutrophils, natural killer (NK) cells, granulocytes, basophils, eosinophils and mast cells, the adaptive immune system consists predominantly of T and B cells. In addition to the cellular components various chemokines, cytokines, immunoglobulins and other soluble factors aid in the immune defense mechanism. Simplistically, the interplay between antigen presenting cells (dendritic cells, macrophage) and the effector cells (T cell, B cell) is an important determinant in eliciting an anti-tumor immune response.

Dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells (APC), capable of priming naive T cells, and play a key role in the activation of T-cell-mediated immune responses. Dendritic cells can be tolerogenic or immunogenic based on the microenvironment clues and thereby affect the immune outcome. Immature epidermal dendritic cells especially Langerhans cells (LCs) process and present antigens to CD4⁺ helper and CD8⁺ cytotoxic T cells. It has also been reported that LCs have the ability to cross present epidermal antigens that may permit direct activation of the T cells (6). Chronic exposure to UV may deplete the LCs and hence lead to immune suppression, thereby promoting cancer progression (7). Also, LCs may promote tumor growth indirectly by facilitating UVR induced tolerance (8). Furthermore, human LCs have been found to express indolamine 2,3-dioxygenase (IDO) on stimulation with interferon-γ (IFN-γ), which in turn suppresses T cell immunity. In conjunction, interleukin (IL)-10 and transforming growth factor (TGF)-β found in the tumor microenvironment prevent IFN-γ from down regulating IDO (7).

T cells

T cells are important in regulating the adaptive immune response to a specific antigen. T cells are further categorized based on the cell-surface expression of the co-receptor molecules CD4 and CD8. CD4⁺ T cells are also referred as “T helper ” (Th) cells as they provide help by activating and modulating other immune cells to initiate the body's response to invading microorganisms. CD8⁺ T cells on the other hand are referred as “T cytotoxic” (Tc) cells and are known to destroy/kill cells that have been infected with foreign invading microorganisms. Both CD4⁺ and CD8⁺ T cells are important in autoimmunity, asthma, and allergic responses as well as in tumor immunity. During TCR activation in a particular cytokine milieu, naive CD4⁺ T cells and CD8⁺ T cells may differentiate into one of several lineages of Th or Tc, including Th1/Tc1, Th2/Tc2, Th17/Tc17, and iTreg (induced regulatory T cells, T regulatory cells induced from CD25⁻ cells in vitro), as defined by their pattern of cytokine production and function (Table 1) (9-11). Thus, innate differences between cytokines and signaling requirements for T cell development, networks of transcription factors involved in T cell differentiation, epigenetic regulation of key T cell cytokines, together dictate the type of immune response as well as the survival, and persistence of the lymphocyte subsets – that in turn affects a meaningful immune outcome. Dietary agents can indeed modulate the T cell response and affect the disease state.

Table 1.

Signature cytokines secreted and transcription factors involved in different T cell subsets

No.	T cell subset	Cytokine(s) required for differentiation	Signature Transcription factor (s)	Cytokines secreted	References
1	Th1/Tc1	IL-12	T-bet	IFNγ, TNF-α	(10)
2.	Th2/Tc2	IL-4	GATA3, IRF4	IL-4, IL-5, IL-9, IL-10, IL-13	(10)
3	Th9/Tc9	TGF-β, IL-4	PU1, IRF4	IL-9, IL-10	(11)
4	Th17/TC17	TGF-β, IL-6, IL-21, IL-23	RORγt, RORα, IRF4	IL-9, IL-17, IL-21, TNF-α	(9)
5	Th22/Tc22	IL-6, TNF	AHR	IL-22, TNF	(10)
6	Treg	TGF-β, IL-2	FOXP3	IL-9, IL-10, IL-35, TGF-β	(10)

AHR: Aryl hydrocarbon receptor; IRF: Interferon regulatory factor; ROR: Retinoic acid-related orphan receptor

Apart from the conventional αβ T cells, the skin epithelium consists of a large proportion of epithelial-γδ T cells. The proportion and the composition of these γδ T cells vary between species. These epithelium resident γδ T cells have restricted TCR receptor diversity and are predominantly known to recognize tissue specific stress or damage induced self-ligands that play a role in tissue homeostasis and immune surveillance (12-13). There are reports that mice lacking γδ T cells are highly susceptible to cutaneous malignancies on exposure to carcinogens (14). γδ T cells produce a variety of cytokines like IFN-γ and IL-2 and cytolytic factors such as perforin and granzymes that contribute to anti-tumor response (15-18).

Another subset of T cells that have a significant role in suppressing T cell activation are the CD4⁺CD25⁺ regulatory T (Treg) cells which also express the forkhead box P3 (Foxp3) transcription factor and constitute a major proportion of tumor infiltrating lymphocytes. Steady state levels of Tregs are present in the skin and these Tregs are CD44⁺, CD103^high and express CCR4, CCR5, CCR6 and CCR7 chemokine receptors (19-21). Treg cells could suppress the immune responses by secretion of immunosuppressive cytokines such as IL-10 and TGF-β that could lead to the generation of anergic T cells (22-24). Tregs also indirectly affect T cell stimulation by down regulating expression of costimulatory molecules CD80 and CD86 through cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) and lymphocyte function-associated antigen 1 (LFA-1) and also by stimulating DCs to express IDO (25-26). Further, exposure to UV induced receptor activator of nuclear factor kappa-B ligand (RANKL) activated LCs mediated development of UV induced Tregs. In addition to RANKL, IL-10 and OX40 ligand expressing LCs also induce Tregs (27-28). Certain murine Tregs also express toll-like receptors (TLR) that upon activation could alter the maintenance and expansion of Tregs (29-30). In addition to Tregs, TGF-β along with IL-6 differentiates CD4 T cells to Th17 subsets, which are pro-inflammatory in nature and require IL-23 for their maintenance. The IL23/IL17 pathway may promote induction of tumors and may also inhibit tumor progression by stimulating anti-tumor activity. Hence the interplay of Tregs and Th17 may play an important role in tumor progression.

Myeloid derived suppressor cells

MDSCs are heterogeneous population of immature myeloid cells and myeloid progenitor cells that contribute to the negative regulation of immune responses. MDSCs suppress T cell function by production of arginase, nitric oxide (NO), reactive oxygen species (ROS) and peroxynitrate molecules. Also MDSCs, in the presence of tumor specific T cells, IFN-γ and IL-10 have been shown to induce Tregs (31). However, in the tumor microenvironment MDSCs could differentiate into tumor-associated macrophages (TAMs) and express arginase and inducible nitric oxide synthase (iNOS) and down regulate production of ROS (32).

Mechanistically, optimal activation and clonal expansion of T cell is a result of three specific signals (Fig. 1). While signal 1 (antigen) and signal 2 (co-stimulation) are conveyed to T cells when it interacts with an APC, signal 3 is essentially the cytokine support required for T cell activation, proliferation and maintenance during the course of an immune response. DCs process and present antigens to T cells, with the help of major histocompatibility complex (MHC) that is expressed on the cell surface. Antigen presentation is considered as the first signal in the activation of T cells. Moreover, maturation of DCs also leads to an increase in the expression of co-stimulatory ligands (CD80 and CD86 also known as B7.1 and B7.2) on the surface of DCs. These co-stimulatory ligands are recognized by CD28 and other co-stimulatory receptors that are expressed on the surface of T cells. Co-stimulation is considered as a second signal that is required for proper activation of T cells. Recent studies have indicated that cytokines (IL-12 or IFN-γ) secreted by DCs or other APCs can act as the third signal that is responsible for activation, expansion and optimal generation of effector and memory T cells. Absence of either signal 2 or signal 3 may render these cells tolerant or unresponsive to stimulation (33-34). Moreover, the tumor microenvironment in which the antigen is encountered by DCs also plays an important role in deciding the fate of T cells – i.e. in becoming a tolerant or an effector T cell. Antigen encounter by DCs in an inflammatory microenvironment leads to proper maturation of DCs that are capable of generating a strong immune response. However, tumor microenvironment fails to provide such inflammatory signals which leads to improper activation of DCs. These DCs acquire tolerogenic properties and result in maintenance of T cell tolerance to tumor antigens (35). Additionally, tumors themselves produce immunosuppressive cytokines such as IL-10 and TGF- β that are responsible for further dampening proper DC activation. In addition, the increased number of Treg cells in a tumor microenvironment can also dampen the immune response by secreting suppressive cytokines that can either act directly on effector T cells or DCs (36-37). Importantly, modulating one of the three essential signals (Fig. 1) required for mounting an optimal immune response could lead to susceptibility to diseases. The effect of UV radiation on immune response has been widely studied and implicated in skin cancer progression. We discuss below different immune mechanisms known to be effected by UV radiation and thereafter discuss how each of the immune cells (as in Fig. 1) could be modulated by the dietary agents. Due to lack of literature focused on understanding the modulation of immune mechanisms by dietary agents specifically in skin cancer we have cited studies that highlight immunomodulation by dietary agents in other cancer models. We believe that it is important to understand the paradox that exists in literature when it comes to corroborating the immunological mechanisms to the functionality of these dietary agents in order to design future studies for comprehensive immunological evaluations.

Figure 1. Activation of naive T cells to undergo clonal expansion and develop effector function requires three signals.

(Signal 1) Antigen presentation, (Signal 2) Co-stimulation, and (Signal 3) Cytokine or adjuvant. Further activation of naïve T cell can be restricted by different inhibitory signals from T regulatory cells (Tregs), Tumor associated macrophages (TAMs), Myeloid-derived suppressor cells (MDSCs).

Modulation of immune responses by UV

Absorption of UV-B by the DNA leads to the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) (38). Initiation of CPDs leads to a series of immunosuppressive signals such as impaired secretion of IL-12 caused probably due to poor antigen presentation by UV damaged LCs in the draining lymph nodes (39). Further, UV has been shown to suppress the immune system even post immunization by inducing secretion of cytokines such as IL-10. In addition, exposure to UV post immunization also leads to increase in CD1-restricted CD4⁺ suppressor T cells, which resemble Natural Killer T cells (40). The severity of UV exposure can even vary the magnitude of T cell response. Though both acute and chronic UV exposure can lead to loss of epidermal T cells, acute UV exposure has been shown to result in a proliferative T cell response while chronic UV exposure skewed the response to a Th2 phenotype with concomitant increase in Treg (41). Alongside with cytokines and chemokines, biologically active lipid mediators, such as platelet activating factor (PAF) is also important in generation of immune suppression, where PAF receptor binding plays a critical role the immune-suppressive pathway (42-43). In addition to lipid mediators, certain endogenous messengers such as alarmins are released following cellular damage and activate both innate and adaptive immune responses. IL-33 is one such alarmin that has been found in the epidermal and dermal skin layers on exposure to UVB. Large amounts of IL-33 were produced in UV induced squamous cell carcinomas leading to immune destruction (44). Thus it is noted that the exposure to UV results in the alteration of the immune responses and usage of dietary agents to modulate the UV induced immunosuppression will be an important strategy.

Chemopreventive agents

Carcinogenesis is a multifunctional process that can be divided into three major stages namely -initiation, promotion and progression. These stages of carcinogenesis manipulate various genes and factors that regulate extracellular and intracellular functions of the cell (45-46). Chemopreventive agents act at multiple steps of various pathways to block carcinogenesis such as tumor incidence, onset and progression. Chemoprevention is primarily attributed to use of naturally occurring agents, which are found in the food we consume and hence not toxic and easily available. These agents by one or multiple pathways are aimed at altering the induction of cellular and molecular factors that cause tumor progression by mechanisms such as induction of apoptosis, inhibition of ROS mediated signaling, inhibition of angiogenesis and modulation of the immune system (Table 2) (47-94). Some of the common photo protective agents include resveratrol, tea polyphenols, caffeine, kamperol, silymarin, curcumin, genistein and delphinidin, flavonoids, piceatannol, pomegranate fruit extract, grape seed proanthocyanidins and kaempherol. Resveratrol, which chemically consists of 3,5,4′-trihydroxystilbene, is a phytoalexin found in grapes, red wines, berries and peanuts. Green tea is characterized by the presence of polyphenols that include presence of catechin/epicatechin and their derivatives such as (-)-epicatechin, (-)-epigallocatechin, (-)-epicate-chin-3-gallate, and EGCG. Of these EGCG has been shown to be the major component of green tea (95-96). Spices are a major source of phytochemicals such as terpenes and phenylproponoids that are present in coriander, cinnamon, saffron, ginger, turmeric, cumin, cloves, vanilla beans etc. Curcumin, which is (1,7-bis (4-hydroxy-3-methoxy-phenyl) hepta-1, 6-diene-3, 5-dione), a diarylhepatonoid, is a major active ingredient of turmeric (97). Cruciferous vegetables, such as broccoli, contain sulphorafane, an isothiocyanate (98). Kaempherol, 3,5,7,4′ tetrahydroxy flavones, is a common dietary flavonoid found in tea, propolis and grape fruit (99-100). Similarly, silymarin is a plant flavonoid extracted from milk thistle. Silymarin is primarily composed of silibinin (<90%) in combination with small amounts of other silibinin stereoisomer's, such as isosilybin, dihydrosilybin, silydianin and silychristin (66, 101). Of particular interest are the actions of some of these chemoprotective agents and their influence on the immune response at various stages of cancer development as discussed below.

Table 2.

Effect of natural dietary agents on tumor and disease state

Agents	Type	Source	Effect on tumors/diseases	References
Apigenin	Flavonoid	Present in common fruits and vegetables such as parsley, onions, oranges, tea, chamomile, wheat sprouts, apple, guava, tomato, broccoli and in some seasonings. Apigenin is present in tea and wine	-topical application of apigenin prior to UV irradiation prevents UV-induced tumorigenesis in mice -inhibits the growth of human cervical carcinoma cells -exhibits anti-proliferative effects on human breast cancer cell lines with different levels of HER2/neu expression. -promotes growth inhibitory activity in HER2/neu-overexpressing breast cancer cells -induces apoptosis in HER2/neu-overexpressing breast cancer cells -inhibits the growth of human cervical carcinoma HeLa cells -induces apoptosis in human leukemia HL-60 cells -inhibits A549 lung cancer cell proliferation and vascular endothelial growth factor (VEGF) transcriptional activation -inhibits the growth of androgen-responsive human prostate carcinoma LNCaP cells	(51, 56, 72-73, 78, 87, 91-92, 94)
Curcumin	Polyphenol	Root of Curcuma longa	-induces apoptosis in human basal cell carcinoma by increasing the expressions of p53, p21Waf1 and Gadd45 proteins -curcumin in human epidermoid carcinoma A431 cells reduces UV induced intracellular oxidative stress -induces apoptosis and reduces cell proliferation in colon cancer in rats of all ages except in middle age -inhibits cancer development in rat stomach initiated by N-methyl-N0-nitro-N-nitrosoguanidine (MNNG). -known as a good anti-angiogenesis agent	(48-49, 53, 58-59, 61, 70)
Delphinidin	Flavonoid	From flowers of Nymphaea caerulea, Eichhornia crassipes, Consolida armenlaca and Petunia reitzii	-suppresses UVB-induced cyclooxygenases-2 expression through inhibition of MAPKK4 and PI-3 kinase -inhibits proliferation and increases apoptosis in breast cancer	(69, 77, 79, 83, 85)
Epigallocatechin-3-gallate (EGCG)	Polyphenol	Green Tea	-affords protection against UVB radiation-induced Tumorigenesis -promotes partial regression of skin papillomas in mice -shows protective effects against experimentally induced lung cancer, fore stomach cancer, colon cancer, breast cancer, and prostate cancer	(57, 65, 67, 81, 89-90, 93)
Genistein	Polyphenols	Soybean	-reduces UV radiation-induced activation of c-fos and c-jun in a dose-dependent manner -reduces UV radiation-induced oxidative and photodynamic DNA damage -exhibits preventive effects against prostate cancer -induces cell death in breast cancer cells	(62, 75, 86, 88)
Proanthocyanidins	A subgroup of flavonoid	Fruits, vegetables, nuts, grape seeds, flowers and bark	-prevents malignant progression of UVB-induced papillomas to carcinomas in mice -inhibits lipid peroxidation, platelet aggregation, capillary permeability and fragility -induces apoptosis in a colon cancer cell line -protects against heart disease	(52, 54, 68, 74, 76, 84)
Resveratrol	Polyphenol	Red grapes, eucalyptus, spruce, blueberries, mulberries, peanuts, red wine, grape skins	-inhibits UVB-induced skin tumor initiation, promotion and progression -inhibits vascular disease, cancer, viral infection and neurodegenerative processes	(50, 55, 60, 64, 71, 80, 82)
Silymarin	Flavonoid	Milk thistle	-has anti-photocarcinogenic activity in laboratory Animals -inhibits UVB-induced skin tumor development in SKH-1 hairless mice in terms of tumor incidence, tumor multiplicity and growth of the tumors. -inhibits endothelial cell growth and survival through induction of apoptosis -inhibits endothelial MMP-2 secretion and expression	(63, 66)

Curcumin

Curcumin (diferulomethane), a yellow pigment extracted from turmeric, is widely used to inhibit tumor progression. Since it can either promote or suppress the immune system, how curcumin affects the immune system in tumor-bearing bodies is not yet clear. A recent study attributed the beneficial effect of curcumin in treatment of concanavalin A (Con A) induced hepatitis to be partly mediated by inhibiting the expression levels of TLR2, TLR4 and TLR9 in the liver (102). This study showed that mice receiving curcumin (200mg/kg body weight) by gavage prior to the intravenous administration of ConA significantly reduced liver necrosis and reduced intrahepatic expression of genes encoding pro-inflammatory molecules such as TNF-α and IFN-γ as compared to the vehicle controls. Curcumin treatment also augmented anti-inflammatory cytokine IL-10 secretion. Curcumin has also been shown to be a potent inhibitor of activation-induced T-lymphocyte proliferation. A recent study dissecting the molecular basis of its immunosuppressive effect showed that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4⁺ T-lymphocytes, as well as IL-2 and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28 (103). Curcumin acted downstream of PKC activation and intracellular Ca²⁺ release to inhibit inhibitor of κB (IκB) phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. Curcumin has also been shown to inhibit IL-2-induced phosphorylation of Signal transducer and activator of transcription (STAT)5A and janus kinase (JAK)3, but not JAK1, indicating inhibition of critical proximal events in IL-2R signaling. This study concluded that curcumin inhibits IL-2 signaling by reducing the available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling. However, contrary to the inhibitory action of curcumin, pretreatment of CD4⁺CD25⁺ regulatory/suppressive T-cells with curcumin resulted in an inhibition in the suppressor function along with a decrease in Foxp3. Another study demonstrated that tumor-bearing mice treated consecutively once a day with low-dose curcumin for ten days resulted in retarded tumor growth and prolonged survival of the mice, which might be attributed to T cell-mediated adaptive immune response (104). The in vitro molecular analysis also showed that a high-dose of curcumin decreases T cells whereas a low-dose increases T cells derived from 3LL tumor-bearing mice, especially CD8⁺ T cells. Accordingly, these increased CD8⁺ T cells exhibited enhancement of IFN-γ secretion, proliferation and cytotoxicity specifically against 3LL tumor cells, which may result in the success of antitumor immunity. In support of the anti-tumor property of curcumin another study has shown that curcumin reverses T cell mediated adaptive immune dysfunctions in tumor bearing hosts (105). While addressing the role of curcumin in preventing tumor-induced dysfunction of T cell-based immune responses, these authors observed loss of both effector and memory T-cell populations, downregulation of type-1 and upregulation of type-2 immune responses and decreased proliferation of effector T cells in the presence of tumors. Curcumin, in turn, prevented this loss of T cells, expanded central memory T cell (T_CM)/effector memory T cell (T_EM) populations, reversed the type-2 immune bias and attenuated the tumor-induced inhibition of T-cell proliferation in tumor-bearing hosts. Further they show that tumor burden upregulated Treg cell populations and stimulated the production of immunosuppressive cytokines TGF- β and IL-10 in these cells. Curcumin, however, inhibited the suppressive activity of Treg cells by downregulating the production of TGF- β and IL-10 in these cells. More importantly, curcumin treatment enhanced the ability of effector T cells to kill cancer cells. Overall, these data suggest for unique properties of curcumin that can be exploited for successful attenuation of tumor-induced suppression of cell-mediated immune responses. Apart from the role of curcumin in downregulating Th1 cytokine secretion, its role in inhibiting Th17 response and disease progression in experimental autoimmune encephalomyelitis (EAE) model has also been documented (106). This study showed that treatment of Lewis rats with curcumin significantly reduced the clinical severity of EAE, and had a dramatic reduction in the number of inflammatory cells infiltration in the spinal cord. The proliferation of the myelin basic protein (MBP)-reactive T cells was also reduced in a curcumin dose-dependent manner. Furthermore, the mRNA expression of the cytokine profiles revealed a dramatic decrease in IL-17, TGF- β, IL-6, IL-21, STAT3, and retinoic acid-related orphan receptor (ROR)γt expression in curcumin-treated groups.

As far as the role of curcumin in modulating APCs is concerned, it has been lately shown by several studies that curcumin arrests maturation of DCs and induces a tolerogenic phenotype that subsequently promotes functional FoxP3⁺ Tregs in vitro and in vivo. Rogers et al. (107), compared the cell surface maturation markers and functional capacity of human monocytederived DC generated in the presence or absence of 25μM curcumin, and matured using lipopolysaccharide (1 μg/ml). Their data shows that DCs generated in presence of curcumin had minimal CD83 expression (<2%), down-regulated levels of CD80 and CD86 (50% and 30%, respectively) and reduction (10%) in both MHC class II and CD40 expression compared to those DCs that were differentiated in absence of curcumin. A decrease in RelB and IL-12 mRNA, decrease in allo-stimulatory capacity by up to 60% (P < 0·001) and a decrease in intracellular interferon (IFN-γ) expression in the responding T cell population by 50% (P < 0·05) was also observed in DCs cultured in presence of curcumin. This study also showed an increase in generation of CD4⁺CD25^highCD127^low FoxP3⁺ Tregs that exerted suppressive functions on naïve syngeneic T cells, although the effect was not antigen-specific. This property of curcumin to induce a tolerogenic DC phenotype and regulatory T cell population has been successfully exploited to treat autoimmune disease such as colitis (108). Based on this observation it was concluded that curcumin modulated BM-derived DC to express IL-10. Co-culture of CD4+ T cells with curcumin-treated DC that produced IL-10, TGF- β and retinoic acid resulted in differentiation of naïve CD4⁺ T cells into Treg resembling Treg in the intestine, including both CD4⁺CD25⁺ Foxp3⁺ Treg and IL-10-producing Tr1 cells that inhibited antigen-specific T-cell activation in vitro and also inhibited colitis due to antigen-specific pathogenic T cells in vivo.

Green tea EGCG

Green tea is one of the most popular beverages consumed worldwide. Recently many studies have showed promising results in skin photo protection upon using green tea. Several studies have addressed whether dietary agents could prevent solar radiation-induced oxidative damages to DNA and LCs that lead to immune suppression. A recent study investigated the effect of EGCG, a component of green tea catechin with the strongest biological activity, on human monocyte-derived DCs (MODCs) and showed that, EGCG induces immunosuppressive phenotype on human MODCs, both by induction of apoptosis and suppression of cell surface molecules and antigen presentation (109). Furthermore, the cell surface expression of CD83, CD80, CD1 1c, and MHC class II, which are molecules essential for antigen presentation by DCs, were downregulated by EGCG. EGCG also suppressed the endocytotic ability of immature DCs. Most importantly, mature DCs treated with EGCG inhibited stimulatory activity toward allogeneic T cells while secreting high amounts of immunosuppressive cytokine IL-10. Another study has addressed the molecular mechanisms by which EGCG antagonized LPS-induced DC maturation and implicated that the EGCG-pretreated DC inhibited LPS-induced mitogen-activated protein kinase (MAPKs), such as extracellular signal-regulated protein kinase (ERK)1/2, p38, c-Jun N-terminal kinases (JNK), and nuclear factor (NF)-κB p65 nuclear translocation (110). Similarly, Camouse et al. (111), showed that skin samples analyzed from volunteers or skin explants treated with white tea or green tea after UV irradiation offered equal protection against detrimental effects of UV on cutaneous immunity. Another study by Katiyar & Mukhtar (112) tried to define the molecular mechanism as to how EGCG prevents UV-B-induced immunosuppression in mice. They showed that topical application of EGCG (3 mg/mouse/3 cm² of skin area) to C3H/HeN mice prior to a single dose exposure to UV-B (90 mJ/cm²) radiation inhibited UV-B-induced infiltration of leukocytes, specifically the CD11b⁺ cell type, and myeloperoxidase activity, a marker of tissue infiltration of leukocytes. EGCG treatment was also found to prevent UV-B-induced depletion in the number of antigen-presenting cells when immunohistochemically detected such as class II MHC⁺ Ia⁺ cells. Pretreatment with EGCG decreased the UV-B-induced oxidative stress that is involved in regulating photocarcinogenesis. Thus, EGCG possesses immunosuppressive properties and its ability to engage the immune cells in controlling tumor progression is intriguing and thereby important in current research. An earlier study has shown that a multimodality treatment strategy, such as combining immunotherapy with a tumor-killing cancer drug, such as EGCG, may be an effective anticancer strategy (113). The combination of EGCG and DNA vaccination led to an enhanced tumor-specific T-cell immune response and enhanced antitumor effects, resulting in a higher cure rate than either immunotherapy or EGCG alone. In addition, long-term antitumor protection in cured mice was observed when DNA vaccination was combined with oral EGCG treatment and tumor-free animals rejected a challenge of E7-expressing tumors, such as TC-1 and B16E7. However, E7-negative B16 tumors progressed seven weeks post-combined treatment, indicative of antigen-specific immune response. The increased T cell response in this case could be attributed to the ability of EGCG to induce apoptosis of tumor cells resulting in increased availability of tumor antigens by the surrounding DCs. In addition, another study has shown that while treatment of the DC with EGCG inhibited IL-12 production in a dose-dependent manner, it also enhanced production of TNF-a by DC cultures stimulated with either soluble bacterial muramyldipeptide (MDP) product or infected with the intracellular bacterium Legionella pneumophila (Lp) (114). EGCG has also been shown to protect against many harmful effects of solar ultraviolet radiation. Meeran et. al. (115), have shown that topical treatment of EGCG inhibits UVB-induced immunosuppression in mice through the induction of IL-12, an immunoregularoty cytokine. The application of EGCG resulted in reduction of UVB-induced skin tumor development compared to non-EGCG treated wild-type mice. Moreover, the treatment of IL-12 KO mice with EGCG did not inhibit tumor development in these mice (115). Furthermore, studies of the DNA repair mechanisms suggested that the repair of UV-induced cyclobutane pyrimidine dimmers (CPDs) by EGCG was mediated through stimulation of IL-12 upon topical application of the EGCG. While EGCG did not repair UV-induced CPDs in the skin of IL-12 knockout mice, it repaired it in the skin of the wild-type mouse- thus confirming the role of IL-12 in the repair of DNA damage by this polyphenol (115). Other studies have shown that topical treatment of murine skin with green tea polyphenols (GTP) also inhibited UVB-induced DNA damage (116). Another study focused on the development of a hydrophilic cream that formulated with EGCG that proved to be an exceptionally better photo protective against UV-induced skin tumors in an animal model (117). Moreover, to expand this study, Mantena et. al. (118), reports that topical application of EGCG in a hydrophilic cream inhibits UVB induced markers of angiogenesis and induces activation of cytotoxic T lymphocytes (CD8⁺ T cells) in skin tumors on SKH-1 hairless mice. Their data also suggested that ECGC-directed stimulation or increased recruitment of CD8⁺ T cells in the tumor microenvironment may be responsible for the death of tumor cells, which may contribute to the inhibition of UV-induced tumor growth or regression of developing tumors in an in vivo system. Taken together, these results show that EGCG catechin has a marked effect in modulating production of the immunoregulatory cytokines by stimulated DCs, which are important for eliciting an innate immune response.

The direct effect of GTPs on T cells has also been evaluated. Bayer et al. (119), evaluated the effects of GTPs on in vitro and in vivo T cell immunity. Based on their results the authors suggest that GTPs inhibited IFN-y secretion by cultured monoclonal T cells and by allo-reactive T cells in mixed lymphocyte reactions. Oral administration of GTPs significantly prolonged minor antigen-disparate skin graft survival and decreased the frequency of donor-reactive IFN-γ producing T cells in recipient secondary lymphoid organs compared to controls. These authors attributed the suppressive activity of GTPs to its direct action on T cells rather than on the APC since oral GTPs did not alter dendritic cell phenotype and its trafficking to lymph nodes or affect metalloproteinase activity in the graft. Owing to its action on T cells, these results suggest that oral intake of green tea could act as an adjunctive therapy for prevention of transplant rejection in humans. Thus, the above data suggest that green tea and its biologically active ingredients possess potent immunomodulatory activity that can either skew DC or T cell response.

Proanthocyanidins

Grape seed proanthocyanidin extract (GSPE), which is the antioxidant derived from grape seeds, has been reported to possess a variety of potent immunomodulatory properties. A recent study has shown that the inhibition of UVB-induced immunosuppression by dietary grape seed proanthocyanidins (GSP) is associated with the induction of IL-12 in mice (120). Furthermore, these authors documented the importance of immunogenic cytokine IL-12 and demonstarted that GSPs do not inhibit UVB-induced immunosuppression in IL-12p40 knockout (IL-12 KO) mice and that treatment of these mice with recombinant IL-12 restores the inhibitory effect. Additionally, CD8⁺ T cells from GSP-treated, UVB-exposed mice secreted higher levels (5- to 8-fold) of Th1 cytokines than CD8⁺ T cells from UVB-irradiated mice not treated with GSPs. CD4⁺ T cells from GSP-treated, UVB-exposed mice secreted significantly lower levels (80%-100%) of Th2 cytokines than CD4⁺ T cells from UVB-exposed mice not treated with GSPs. These data suggest that GSPs inhibit UVB-induced immunosuppression by stimulating CD8⁺ effector T cells and diminishing regulatory CD4⁺ T cells. Another study has reported that GSPs differentially regulate FoxP3⁺ regulatory T cells and IL-17⁺ pathogenic T cells in autoimmune arthritis (121). These authors report that GSPE decreased the frequency of IL-17⁺CD4⁺Th17 cells and increased induction of CD4⁺CD25⁺Foxp3⁺Treg cells. In vivo, GSPE effectively attenuated clinical symptoms of established collagen-induced arthritis in mice with concomitant suppression of IL-17 production and enhancement of Foxp3 expression (type II collagen-reactive Treg cells) in CD4⁺ T cells of joints and splenocytes. The presence of GSPE decreased the levels of cytokines IL-21, IL-22, IL-26 and IL-17 production by human CD4⁺ T cells in a STAT3-dependent manner.

Similarly, orally administered apple procyanidins (ACT) has also been shown to protect against atopic dermatitis and experimental inflammatory bowel disease (122). Using experimental models of colitis induced by dextran sulfate sodium (DSS) or oxazolone this study demonstrated that oral administration of ACT before DSS treatment attenuated the DSS-induced mortality rate and decreased body weight loss. ACT also prevented the body weight loss associated with oxazolone-induced colitis. An evaluation for effect of ACT on intraepithelial lymphocytes (IEL), showed that oral administration of ACT leads to increased proportions of TCR γδ and TCR αβ-CD8αα T cells in IEL and suppressed IFN-γ synthesis in stimulated IEL. In addition, ACT inhibited PMA-induced secretion of IL-8 in intestinal epithelial cells. The combined anti-inflammatory and immunomodulatory effects of ACT on intestinal epithelial cells and IEL suggest that it may be an effective oral preventive agent for inflammatory bowel diseases.

Similar to the beneficial effects of dietary agents discussed above (Table 3), a number of other natural compounds that could not be covered in this review due to the space limitations exhibit potent immunomodulatory properties, e.g. simvastatin has also been recently demonstrated in various experimental models of inflammation (123). This study investigated the potential anti-inflammatory and immunomodulatory mechanisms of these compounds in a murine model of hyper-acute Th1-type ileitis following peroral infection with Toxoplasma gondii and showed that oral treatment ameliorates acute small intestinal inflammation by down-regulating IL-23p19, IFN-γ, TNF-α, I L-6, Monocyte chemotactic protein (MCP)-1 and increasing the number of immunosuppressive Treg cells. In addition, Hushmendy et al. (124) showed that in addition to curcumin, sulforaphane, quercetin and berry extract also significantly inhibited T-cell proliferation. They showed that IL-2 production was also reduced by these agents, implicating a role for this important T-cell cytokine in proliferation suppression. Thus, a large number of the dietary compounds have active biological moieties that could be exploited to treat various diseases and a better understanding of the immunological checkpoints that can be modulated by these agents will indeed pave the way for their future translational use.

Table 3.

Immunomodulation by natural dietary agents

Agents	Immunomodulatory activity	References
Curcumin	-induces hepatitis to be partly mediated by inhibiting the expression levels of TLR2, TLR4 and TLR9 in the liver -augments anti-inflammatory cytokine interleukin 10 (IL-10) -inhibits activation-induced T-lymphocyte proliferation -inhibits interleukin-2 (IL-2) secretion and CD25 upregulation upon T cell activation -high-dose curcumin decreases T cells whereas low-dose increases T cells derived from 3LL tumor-bearing mice, especially CD8+ T cells -reverses T cell mediated adaptive immune dysfunctions in tumor bearing hosts -enhances the ability of effector T cells to kill cancer cells -inhibits Th17 response and disease progression in EAE model -arrests maturation of DC and induces a tolerogenic phenotype -promotes functional FoxP3(+) T(regs) in vitro and in vivo -down regulates expression of CD40, CD80, CD83, CD86, MHC class II	(102-106, 108, 114)
Greentea(-) epigallocatechin-3-gallate (EGCG)	-induces immunosuppressive phenotype on human monocyte derived DC -downregulates CD83, CD80, CD11c, and MHC class II -suppresses the endocytotic ability of immature DCs -mature DCs treated with EGCG inhibits stimulatory activity toward allogeneic T cells while secreting high amounts of immunosuppressive cytokine IL-10 -EGCG-pretreated DC inhibits LPS-induces MAPKs, such as ERK1/2, p38, JNK, and NF-κB p65 translocation -inhibits UV-B-induced infiltration of leukocytes -prevents UV-B-induced depletion in the number of antigen-presenting cells -decreases the UV-B-induced oxidative stress -enhances production of TNF-α -inhibits UVB-induced immunosuppression in mice through the induction of IL 12 -induces repair in UV-induced CPDs through stimulation of IL-12 -inhibits IFN-γ secretion by T cells	(109-110, 113, 115)
Proanthocyanidins	-inhibits UVB-induced immunosuppression by induction of IL-12 in mice -inhibits UVB-induced immunosuppression by stimulating CD8(+) effector T cells and diminishing regulatory CD4(+) T cells -regulates FoxP3+ regulatory T cells and IL-17+ pathogenic T cells in autoimmune arthritis -decreases the frequency of IL-17(+)CD4(+)Th17 cells -increases induction of CD4(+)CD25(+)Foxp3(+) Treg cells -decreases the levels of cytokines IL-21, IL-22, IL-26 and IL-17 production	(120) (121)
Procyanidins	-protects against atopic dermatitis and bowel disease by increasing Tregs -prevents body weight loss associated with oxazolone-induced colitis -increases proportions of TCR γδ and TCRαβ-CD8αα T cells in IEL -suppresses IFN-γ synthesis in stimulated IEL -inhibits IL-8 in intestinal epithelial cells	(122)
Resveratrol	-down regulates CD40, CD80, CD86, MHC class II molecules -induces development of tolerogenic DC phenotype -inhibits the expression of CD80, CD86, MHC classes I and II -suppresses the ability of BM-DC to produce IL-12 p70 -enhances TNF-α, IL-12 and IL-1β production after LPS activation -down-regulates the expression of CD28 and increase IL-10 secreting ability -affects T cells and MDSCs in chronic colitis model -resveratrol treated IL-10(-/-) mice induces immunosuppressive CD1 1b(+) Gr-1(+) MDSCs in the colon -down regulates mucosal and systemic CXCR3(+) expressing effector T cells as well as inflammatory cytokines in the colon -increase in regulatory T cell population in EAE model -inhibits suppressive activity of FoxP3 expressing Treg cells among CD4+CD25+ population -low dose resveratrol enhanced cell-mediate immune response by promoting Th1 cytokine production and influencing macrophage function -low dose resveratrol activates antigen presenting cell induced IL-12 and IFN-γ production	(127-129, 131-135)
Silymarin	-suppresses CD4+ T cells activation and proliferation -decreases Th1 cytokines IL-2 and IFN-γ -inhibits p65/NF-κB phosphorylation -blocks κB (NF-kB) which is known to be responsible for IL-2 transcriptional activation -decreases CD4+ and CD8+ T-cell populations -suppresses T-lymphocyte function	(136-138)
Silibinin	-impairs induction of Th1 response, and a normal cell-mediated immune response -upregulates Th2 cytokine secretion -suppresses expression of CD80, CD86, MHC class I, and MHC class II on DC -impairs LPS-induced IL-12 secretion by DCs	(139-140)

Resveratrol

Resveratrol is a polyphenol found in nature and is known as 3, 5, 4′-trihydroxy-Trans-stilbene. Resveratrol has been identified in more than seventy plant species including grapes, peanuts, fruits, red wine and mulberries. Among these, grape is the most popular good source of resveratrol. It has been proven that resveratrol inhibits diverse cellular events associated with tumor initiation, promotion and progression of skin cancer and cancer of other organs (125). It has also been shown to protect human skin from damage induced due to repeated exposure to UV radiation (126). Studies evaluating the role of resveratrol on monocyte derived DC differentiation and maturation have shown that the expression of costimulatory molecules CD40, CD80 and CD86, and MHC class II molecules were down-regulated in presence of resveratrol (127). This study has also shown that resveratrol induced development of tolerogenic DC phenotype as exhibited by reduced interleukin (IL)-12p70 secretion after activation, and an increased ability to produce IL-10. Resveratrol treated DCs were also poor stimulators of allogeneic T cells and exhibited reduced ability to induce CD4⁺ T-cell migration. Another study has shown the effect of resveratrol on the phenotypic and functional maturation of bone marrow (BM)-derived dendritic cells (DC). This study also showed that resveratrol significantly inhibited the expression of costimulatory molecules (CD80 and CD86), and MHC class I and II significantly, in a dose-dependent manner on DC. Resveratrol also significantly suppressed the ability of BM-DC to produce IL-12 p40/p70 in response to lipopolysaccharides (LPS) stimulation. However, contrary to these immunosuppressive properties of resveratrol on DCs, another study has shown that resveratrol enhanced tumor necrosis factor (TNF)-α, IL-12 and IL-β production after LPS activation of phorbol myristate acetate (PMA) differentiated THP-1 human macrophages (128). This study also showed that expression of CD86 was enhanced on macrophages cultured with resveratrol alone and stimulated with LPS. Although, when macrophages were primed with IFN-γ, resveratrol suppressed the expression of human leukocyte antigen (HLA)-ABC, HLA-DR, CD80, CD86 and inhibited production of TNF-α, IL-12, IL-6 and IL-1 β induced by LPS. The differential impact of resveratrol on macrophages due to IFN-y is intriguing. While generation of tolerogenic APC indicates the immunosuppressive properties of resveratrol and its therapeutic efficacy in controlling chronic immune and/or inflammatory diseases, its widely documented role in controlling tumor growth needs comprehensive immunological evaluation.

Resveratrol has also been shown to act directly on T cells and down-regulate the expression of CD28 and increase IL-10 cytokine secretion (129). Down-regulation of the protein kinase C (PKC)-θ expression in peripheral blood lymphocytes has also been postulated as a part of the immunosuppressive mechanism of resveratrol (130). In addition, the direct effect of resveratrol on T cells and MDSCs in chronic colitis model has recently been suggested (131). Based on this study, the administration of resveratrol into IL-10^-/- mice induces immunosuppressive CD11b⁺Gr-1⁺ MDSCs in the colon, which correlates with reversal of established chronic colitis. The down regulation of mucosal and systemic CXCR3⁺ expressing effector T cells as well as inflammatory cytokines in the colon was also observed in these mice model. The induction of immunosuppressive CD11b⁺Gr-1⁺ cells by resveratrol during colitis is unique, and suggests an as-yet-unidentified mode of anti-inflammatory action of this plant polyphenol. The effect of reseveratrol in the development of Th17 cells has also been recently illustrated (132). Another study showed a decreased severity of experimental autoimmune encephalomyelitis (EAE) after resveratrol administration and implicated a role for increased IL-17/IL-10 secreting cells, along with repressed macrophagic IL-6 and IL-12/23 p40 expression. It should also be noted that the recent literature suggests both an increase and a decrease in the CD4⁺CD25⁺ Treg cell population in presence of resveratrol. While Petro et al. (132), mentions about an increase in Treg population in EAE model, another study showed a decrease in this suppressive T cell subset (133). Using the EG7 tumor-bearing C57BL/6 mice these authors showed that FoxP3 expressing cells among CD4⁺CD25⁺ population were significantly reduced after resveratrol treatment. Single intraperitoneal administration of 4-mg/kg resveratrol suppressed the CD4⁺CD25⁺ cell population among CD4⁺ cells and downregulated secretion of TGF-P, an immunosuppressive cytokine, as measured from the splenocytes derived from the tumor-bearing mice. Furthermore, resveratrol enhanced IFN-y expression in CD8⁺ T cells both ex vivo and in vivo, leading to immune stimulation. Taken together, these results suggested that resveratrol has a suppressive effect on CD4⁺CD25⁺ cell population and makes peritumoral microenvironment unfavorable to tumor development. The reason for this discrepancy in quantitative and qualitative differences in immune response due to resveratrol could be due to the differences in dose or route of administration that were used in various studies. Low dose resveratrol treatment has been shown to enhance cell-mediated immune response by promoting Th1 cytokine production and influencing on macrophage function (134). This study showed that low dose resveratrol (0.75-6 micromol/L) promoted lymphocyte proliferation and IL-2 production induced by ConA. In addition, Staphylococcus aureus Cowan activated APC induced IL-12 and IFN-γ production were also enhanced by resveratrol, while IL-10 production was inhibited. Thus, taken together resveratrol can potentially acts as an adjuvant in vaccination-based cancer therapies.

Finally, the effect of resveratrol on reversing the pro-inflammatory cytokine profile and oxidative damage in aged mice has also been shown (135). This study showed that resveratrol reversed surface phenotypes of old mice similar to that of young mice by maintaining the CD4⁺ and CD8⁺ population in splenocytes as well as reducing CD8⁺CD44⁺ cells in the aged. Age-dependent increase in 8-Oxo-2′-deoxyguanosine (8OHdG), an oxidative DNA damage marker was ameliorated by reseveratrol. The above data lead us to believe that resveratrol does impact various arms of the immune system and could modulate the immune response effectively and control tumor progression and autoimmunity in a dose dependent manner.

Silymarin

Silymarin, a polyphenolic flavonoid isolated from Silybum marianum, exhibits anti-carcinogenic, anti-inflammatory and cytoprotective effects. A recent study showed that Silymarin suppresses CD4⁺ T cell activation and proliferation (136). This study also showed a decrease in Th1 cytokines IL-2 and IFN-γ secretion from C57BL6 splenocytes that were pretreated with 50 μM of Silymarin. Moreover, silymarin inhibited p65/NF-κB phosphorylation and blocked the NF-κB that is known to be responsible for transcriptional activation of IL-2. However, another study has shown the biphasic effect of silymarin (137). This study evaluated the effect of silymarin on the proliferation and cell cycle progression of Jurkat cells, a human peripheral blood leukemia T cell line. Cells were incubated with various concentrations of silymarin for 24-72 h and examined for cell growth and proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DNA 5-bromo 2′-deoxyuridine (BrdU) colorimetric assays. The results revealed that silymarin increased proliferation of Jurkat cells at 50-400 μM concentrations with 24 h exposure as confirmed by both MTT and BrdU assays. However, Jurkat incubation with silymarin at higher concentrations of 400 μM for 48 h and 200-400 μM for 72 h caused inhibition of DNA synthesis, cell cycle arrest at the G2/M phase and significant cell death. Similar biphasic observation was highlighted by Johnson et. al. (138), where male BABL/c mice (6/group) treated intraperitoneally once daily for five days with 0, 10, 50 or 250 mg/kg of silymarin. While concomitant decrease in CD4⁺ and CD8⁺ T-cell populations were observed but only the CD4⁺ population in mice treated with 10 mg/kg of silymarin was significantly different from control mice. Functional examination of secondary lymphoid cells revealed that phytohemagglutinin-induced T-lymphocyte proliferation was increased in the lowest dose group only. B-lymphocyte blastogenesis induced by LPS was increased following exposure to 10 and 50 mg/kg of silymarin. Similarly, expression of TNF-α, iNOS, IL-1 β and IL-6 mRNA were increased in a dose-dependent manner. The expression of IL-2 and IL-4 were reduced in mice treated with 10 and 50 mg/kg of silymarin although only the 10-mg/kg groups were significantly different from the control mice. The results indicate that in vivo parenteral exposure to silymarin results in suppression of T-lymphocyte function at low doses and stimulation of inflammatory processes at higher doses. Further mechanistic studies investigating the effects of silymarin on the immune system are warranted.

Silibinin, the primary active compound in silymarin, has also been shown to affect the APC arm of the immune response. It has been demonstrated to exert anti-carcinogenic effects and hepato-protective effects. However, until recently the effects of silibinin on the maturation and immunostimulatory activities exhibited by DCs have not been reported. A recent study showed that Silibinin polarizes Th1/Th2 immune responses through the inhibition of immunostimulatory function of dendritic cells (139). In this study, Silibinin was shown to significantly suppress the expression of CD80, CD86, MHC class I, and MHC class II in the DCs, and was also associated with reduced IL-12 secretion upon LPS-induced stimulation of DCs. Silibinin-treated DCs proved highly efficient with regard to antigen capture via mannose receptor-mediated endocytosis. Silibinin also inhibited LPS-induced activation of MAPKs and the nuclear translocation of NF-κB p65 subunit. Additionally, silibinin-treated DCs exhibited an impaired induction of Th1 response, and a normal cell-mediated immune response. The immunosuppressive effect of silibinin was also exploited in EAE model (140) where Th1 cytokine secretion was inhibited and Th2 cytokine was upregulated non-antigen specifically in a dose-dependent manner. These findings highlight the immunopharmacological functions of silibinin, and may prove useful in the development of therapeutic adjuvants for acute and chronic DC-associated disease.

Future Directions

The primary discussion in this review has centered around the effect of dietary agents on the DCs, T cell subsets and the cytokine modulation - for the reason that these cells have been the focus of the immunological parameters studied thus far. However, apart from these key targets other immune cells like B cells, NK cells, NKT cells, neutrophils, basophils, mast cells, and macrophages also play an important role in shaping of the immune response and regulating disease states (141-144). Moreover, there also exists heterogeneity in T cells (as discussed in Table 1), DCs (immunogenic vs. tolerogenic; conventional vs. plasmcytoid) (37), macrophages (M1 vs. M2) (145) and other immune cells that could be functionally modulated by the dietary agents. Figure 2 shows the effect of the dietary agents discussed above on various arms of immune response. Modulating the immune response by targeting the above mentioned key immune cells that control pro- or anti-inflammatory micro-environment which in turn promotes tumor progression or autoimmune diseases is an area of intense research (146). A number of groups have evaluated how these agents affect the downstream signaling kinetics such as NF-κB, MAPK pathways and p53 pathways. Though there exists data in the literature that throws light independently on how the immune responses are modulated by UV and to a certain extent by chemopreventive agents, there is not much work elucidating the role of immune network that is responsible for arresting progression of skin cancer on exposure to UV radiation by use of chemopreventive agents. Also, while a number of studies have shown the potential of using dietary compounds in treatment of autoimmune diseases and tumor development, the active biological moiety in these complex formulations is still being investigated. In addition, the efficacy of these compounds is often limited by their lack of solubility in aqueous solvents and poor oral bioavailability that needs to be addressed for a sustained objective outcome. In a recent approach curcumin was encapsulated with 97.5% efficiency in biodegradable nanoparticulate formulation based on poly (lactide-co-glycolide) (PLGA) and a stabilizer polyethylene glycol (PEG)-5000 (147). Using Dynamic laser light scattering and transmission electron microscopy the authors indicated a particle diameter of 80.9 nm and that curcumin-loaded PLGA nanoparticles formulation exhibited enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability due to longer half-life in vivo over curcumin. Other strategies such as gold nanoparticles generated and stabilized by water soluble curcumin-polymer conjugate have also been reported (148). Using flow cytometry and confocal microscopy this study showed significant cellular uptake and internalization of the particles by cells that exhibited more cytotoxicity compared to free curcumin. Another study has developed a sustained-release solid dispersion mechanism by employing water-insoluble carrier cellulose acetate for solubility enhancement, release control, and oral bioavailability improvement of curcumin (149). This study showed that solubility/dissolution of curcumin was enhanced in the formulations in comparison with pure drug and sustained-release profiles of curcumin from the solid dispersions were ideally controlled in vitro up to 12 h. The above-mentioned strategies are significant technological developments that would help in taking these compounds to the clinic in near future. Recently it has also been demonstrated that piperine significantly improves the in vivo bioavailability of resveratrol (150). This group employed a standardized LC/MS assay to determine the effect of piperine co-administration with resveratrol on serum levels of resveratrol and resveratrol-3-O-β-D-glucuronide in C57BL/6 mice. Analysis of serum levels for resveratrol and resveratrol-3-O-β-D-glucuronide from mice that were administered resveratrol (100 mg/kg; oral gavage) or resveratrol (100 mg/kg; oral gavage) and piperine (10 mg/kg; oral gavage) showed that the degree of exposure (i.e. AUC) to resveratrol was enhanced to 229% and the maximum serum concentration (C_max) was increased to 1544% with the addition of piperine – thus showing that piperine enhanced the pharmacokinetic parameters of resveratrol via inhibiting its glucuronidation resulting in its slow elimination and increased bioavailability. The other strategy being employed widely is to identify the biologically active moieties in the natural dietary agents and to synthesize bioactive peptides that are more stable and perform the same biological function. A recent report showed that novel curcumin analogs GO-Y030 and GO-Y078 were 7 to 12-fold more potent growth suppressors for myeloma cells, and 6- to 15-fold stronger inhibitors of NF-κB, PI3K/AKT, JAK/STAT3, and Interferon regulatory factor (IRF) 4 pathways than curcumin. GO-Y78 also 14-fold more potently inhibited IL-6 production (151). While further comprehensive evaluation is needed to understand the mechanism of improved bioavailability of resveratrol (and other dietary agents) via its combination with piperine (or other similar compounds) or for evaluating the efficacy of nanoparticles or synthetic peptides, these above mentioned strategies provide a platform for future endeavors directed to overcome the poor in vivo bioavailability of dietary chemopreventive agents in order for their use in translational setting to benefit humans.

Figure 2. Effect of dietary agent on various arms of immune response.

Various dietary components are known to affect signal 1-3 resulting in modulation of immune response.

Biographies

Ya Ying Zheng graduated from College of Charleston with a Bachelor of Science in Biochemistry and a Bachelor of Arts in Chemistry in 2011. She aims to seek a higher degree through MD/PhD program and continue with immunology research.

Bharathi Viswanathan received her PhD in 2011 from National Institute of Immunology, New Delhi, India for her studies on the regulation of macrophage apoptosis. Her research interest lies in understanding the host pathogen interactions with a particular focus on the mechanisms governing cell death and cell signaling during innate immune response.

Pravin Kesarwani obtained his PhD degree in life sciences in 2010. His doctoral study focused on identification of genetic variants associated with inflammatory pathway, which may modulate susceptibility to prostate carcinoma and benign prostate hyperplasia. His current research involves understanding the role of reactive oxygen species in melanoma epitope specific T cell activation, apoptosis, signaling and differentiation.

Shikhar Mehrotra obtained his PhD in Immunology at Sanjay Gandhi Post Graduate Institute of Medical Sciences in India and continued with his post-doctoral training until 2006 at University of Connecticut Health center at Farmington. After moving to MUSC his lab is focused on understanding the pathways regulating life vs. death in anti-tumor specific T cells.