Modulation of Immune Functions by Foods

Evidence is rapidly accumulating as to the beneficial effects of foods. However, it is not always clear whether the information is based on data evaluated impartially in a scientific fashion. Human research into whether foods modulate immune functions in either intervention studies or randomized controlled trials can be classified into three categories according to the physical state of subjects enrolled for investigation: (i) studies examining the effect of foods in healthy individuals; (ii) studies analyzing the effect of foods on patients with hypersensitivity; and (iii) studies checking the effect of foods on immunocompromized subjects, including patients who had undergone surgical resection of cancer and newborns. The systematization of reported studies has made it reasonable to conclude that foods are able to modulate immune functions manifesting as either innate immunity (phagocytic activity, NK cell activity) or acquired immunity (T cell response, antibody production). Moreover, improvement of immune functions by foods can normalize the physical state of allergic patients or cancer patients, and may reduce the risk of diseases in healthy individuals. Therefore, it is valuable to assess the immune-modulating abilities of foods by measuring at least one parameter of either innate or acquired immunity.


Introduction
Foods contain various substances that can control the physiological functions of the body, and modulating immune responses is one of the most important functions of foods. Immune functions are indispensable for defending the body against attack by pathogens or cancer cells, and thus play a pivotal role in the maintenance of health. However, the immune functions are disturbed by malnutrition, aging, physical and mental stress or undesirable lifestyle. Therefore, the ingestion of foods with immune-modulating activities is considered an efficient way to prevent immune functions from declining and reduce the risk of infection or cancer.
In order to establish a diet capable of preserving immune functions, it is necessary to search and systematize reliable results on the immune-modulating effects of food-derived substances. To this end, we have selected reports that evaluated the immune-modulating abilities of foods in an intervention study or a randomized controlled trial. Thereafter, we classified these studies according to the physical state of their subjects into three categories: (i) studies examining immune parameters of healthy individuals whose immune functions are poorer than expected; (ii) studies analyzing immune parameters of patients with hypersensitivity; and (iii) studies checking immune parameters of immunocompromized subjects, including patients who had undergone surgical resection of cancer and newborns. We found that the measurement of at least one parameter representing either innate or acquired immunity was useful for evaluating the immune-modulating abilities of foods. This review summarizes the immune-modulating characteristics of foods that have been verified in human as well as animal studies. In addition, we briefly describe the pathways by which food-derived substances are absorbed into the body and the mechanisms through which food-derived substances exert their immunemodulating effects.

Intestinal Transport of Foods
Molecules found in foods can be absorbed by multiple routes. Carbohydrates, proteins and lipids are broken down into monosaccharides, amino acids and fatty acids, respectively, by diverse hydrolases secreted in the gut lumen or associated with membranes of intestinal epithelial cells (IECs). These food components are actively transported via specific carrier molecules on the surface membrane of IECs and used as nutrients (Fig. 1). Vitamins and minerals in foods are also absorbed through IECs by passive diffusion or active transport using specific carrier molecules (Fig. 1). The main role of vitamins and minerals is to regulate the various physiological functions of cells. Indigestible macromolecules such as rigid proteins are incorporated into Peyer's patches (PPs) developing throughout the intestine (1). PPs are organized lymphoid tissues that are covered by a particular epithelial layer, the follicle-associated envelope (FAE), and composed of both follicles rich in B cells and an interfollicular area filled with antigen-presenting cells and T cells. Horseradish peroxidase (HRP) given orally to mice was detectable in M cells (microfold cells) within the FAE (2). Moreover, a study using isolated intestinal loops from piglets has revealed that the absorptive rate of HRP was higher in the intestinal segments with PPs than without (3). These results demonstrate that M cells in FAE are the route for efficiently incorporating indigestible proteins (Fig. 1). Macromolecules incorporated into PPs are taken by antigen-presenting cells and induce antigen-specific immune responses. On the other hand, macromolecules with higher molecular size such as carrageenan (88 110 kDa) are hardly absorbed in the intestine.
Small molecules can be transported through IECs by endocytosis (Fig. 1). By contrast, food-derived substances are not usually transported between IECs (paracellular transport), because IECs closely connected by tight junctions do not usually allow food-derived substances to pass through. However, the barrier function of tight junctions is not stiff and breast milk-derived proteins can be transported without degradation between IECs in newborns (4) (Fig. 1). As the immune system in newborns is immature, it is reasonable that newborns incorporate breast milk-derived proteins including lactoferrin and maternal IgG to protect from infection. In contrast, dysfunction of the tight junction due to genetic defect is dangerous. Patients with food allergy have intestines with a

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reduced barrier function, and therefore an antigenic load in the gut lumen can be easily incorporated into the body (5). The intestine and liver are important organs in terms of supply of nutrition and self-defense, and these organs are equipped with specialized immune systems. A huge number of IgA-producing cells and intraepithelial T lymphocytes (IELs) with unique physiological functions are colonized in the gut and play a pivotal role in defense against pathogens (6). Moreover, natural killer (NK) T cells are enriched in liver and highly effective in the eradication of tumor cells (7). Hazardous substances (pathogens, toxins and allergens) are recognized as antigens and activate the immune system, but most gut antigenic loads (food-derived molecules and indigenous intestinal bacteria) are harmless and the immune response to these antigens is suppressed in healthy humans (oral tolerance). In contrast, the immune system of patients with inflammatory bowel disease responds excessively to the indigenous intestinal bacteria, causing inflammation in the intestine (8).
It is of great interest that some substances in foods can open tight junctions between IECs. When the capsianoside contained in a sweet pepper was added to the apical side of a monolayer formed by the human IEC line Caco-2, the tight junctions transiently opened, followed by a drop of electric resistance between the apical and basal sides (9). While the tight junction basically acts as a barrier to pathogens or toxic substances in the intestine, the transient opening of tight junctions may be so important that antigens can be captured by dendritic cells in the intestinal lamina propria and immune responses to these antigens are efficiently evoked (10).

Regulation of Immune Functions by Foods
Immune-modulating abilities of foods have been investigated in a number of human studies. We tentatively classified these researches into three categories according to the state of immune system in subjects enrolled for investigation: (i) healthy individuals; (ii) patients with hypersensitivity; and (iii) subjects in immunocompromised state.

Foods Capable of Improving Immune Functions in Healthy Individuals
Immune functions are not stable and usually fluctuate within fixed limits. In addition, various endogenous and exogenous factors can influence immune functions. Corticosteroids suppress a broad range of immune functions efficiently and exhibit anti-inflammatory activity. Malnutrition, aging, stress and undesirable lifestyle are also factors lowering immune functions. The elderly exhibit higher susceptibility to infection than the young, and delayed type hypersensitivity (DTH), antigen-specific antibody production, the proliferative response of T cells and the relative proportion of T cells decline with aging (11)(12)(13)(14)(15)(16). Many kinds of physical and mental stress also disturb immune functions. For instance, a surgical operation exhausts patients and is accompanied by a decline in their DTH, and caregivers of dementia patients show a decrease in NK cell activity, antigen-specific antibody production and T cell proliferation on account of depression (17,18). Moreover, NK cell activity deteriorates under not only mental stress after divorce but also physical stress of heavy exercise (19)(20)(21)(22)(23)(24). It is widely known that systemic malnutrition associated with a deficiency of protein and energy causes a decline in immune functions and results in susceptibility to infection (25)(26)(27)(28). A deficiency in vitamins and minerals induces an attenuation of immune functions including phagocytic activity, NK cell activity, DTH, antigen-specific antibody production, and the proliferative response of T cells (29,30). In addition, NK cell activity and the proliferative response of T cells decline in patients with chronic fatigue syndrome (31).
The deterioration of immune functions possibly causes loss of health. A higher risk of infection is closely linked with low NK cell activity, and increased risk of mortality in the elderly after pathogenic infection is correlated with a decline in DTH (32)(33)(34)(35). Bodily dysfunctions in chronic fatigue syndrome patients are negatively correlated with the proliferative response of T cells (36).
Summarizing the results reported so far, it is reasonable to conclude that the effect of foods on immune functions can be evaluated in healthy subjects by measuring either parameters concerning innate immunity (phagocytic activity and NK cell activity) or parameters concerning acquired immunity (DTH, antigen-specific antibody production, the proliferative response of T cells and T cell number) (Table 1). Therefore, despite fears that immune functions may decline due to malnutrition, aging, stress or undesirable lifestyle, one can remain healthy and reduce the risk of infection or cancer by eating foods capable of enhancing phagocytic activity, NK cell activity, DTH, antigen-specific antibody production, the proliferative response of T cells and/or T cell numbers.

Foods Capable of Improving Clinical Symptoms in Patients With Hypersensitivity
Immune reactions are usually evoked in response to externally derived hazardous antigens. However, in patients with hypersensitivity represented by immediate type allergy, immune reaction to non-toxic antigens and sometimes to the body's own molecules is induced. The causes of hypersensitivity are mainly genetic, but environmental factors, including air pollution, dietary components and residential conditions, also play an important role. As clinical condition and immune parameters change concomitantly in allergic patients, it is possible to observe the effects of foods by measuring the immune parameters associated with allergic reactions.
Generation of pro-inflammatory cytokines and chemokines and expression of cell adhesion molecules are involved in the progression of allergic diseases including atopic dermatitis, pollinosis and allergic rhinitis. Levels of pro-inflammatory cytokines and chemokines increase and the expression of cell adhesion molecules is enhanced in allergic patients (65)(66)(67)(68)(69)(70). Furthermore, eosinophils as well as mast cells secrete chemical mediators and worsen the clinical symptoms in the inflammatory areas (71,72). In order to establish an objective assessment of the clinical state of allergic patients, a skin test, the antigen-induced response and the SCORAD score have all been utilized (73,74).
Based on findings reported to date, we conclude that the following immune parameters can be used to evaluate the effects of foods on the clinical symptoms of allergic patients: (i) parameters to directly assess clinical symptoms in allergic patients: skin test (75,82), skin-induced response (76) (Table 1).
An allergic reaction is a sequential immune response involving the processing and presentation of the allergen, activation of allergen-specific T and B cells, production of IgE against the allergen, and activation of mast cells and eosinophils triggered by the allergen. Therefore, food-derived materials could prevent allergy by counteracting at least one step in the cascade of allergic reactions. It has been reported that a variety of foods contain substances able to prevent an allergic reaction (100)(101)(102).

Foods Capable of Improving Immune Functions in Subjects in an Immunocompromised State
Cancer patients are usually immunosuppressed and at high risk of infection due to a reduction of immune functions. Therefore, foods capable of enhancing the immune responses of cancer patients with disturbed immune functions are valuable.
Invading pathogenic bacteria or viruses are captured and killed by phagocytes such as neutrophils and macrophages, and NK cells recognize and lyse infected cells. Activated NK cells and T cells produce huge amounts of IFN-␥, which further augments the anti-bacterial activity of macrophages (103)(104)(105)(106).
Pathogens that have escaped capture by phagocytes or NK cells are incorporated and processed by professional antigenpresenting cells, which stimulate T cell clones expressing

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Modulation of immune functions by foods antigen receptors specific for pathogens. Activated antigen-specific T cells secrete various arrays of cytokines necessary for antibody production, and pathogen-specific antibodies play an important role in the exclusion of pathogens invading the airway, intestine and urinary tract (107)(108)(109). IgA secreted in the intestinal mucosa can neutralize toxins produced by pathogens and prevents diarrhea (110), and IgG circulating in sera is principally for defense against infection in the upper respiratory tract (107). The incidence of infection increases and the aggravation of infectious diseases occurs when innate and acquired immune functions decline or are insufficient. Patients with undetectable levels of NK cell activity suffer frequent viral infections and the transfer of NK cells into suckling mice can render the recipient mice resistant to infection for murine cytomegalovirus (111,112). Patients with Gaucher disease, who were highly susceptible to serious bacterial infections, had macrophages with impaired anti-bacterial activity and the rate of infection among marrow transplant recipients 100-365 days after transplantation was negatively correlated with the total number of B cells and monocytes (113,114). On the other hand, several reports have shown that the improvement of depressed immune functions by ingesting foods reduced infection rates and mitigated the severity of infectious disease (43,(115)(116)(117)(118). When assessing the anti-infectious capabilities of foods, phagocytic activity, NK cell activity, T cell number, production of antigen-specific antibodies and total IgG level can be regarded as useful parameters.
NK cells exhibit cytotoxic activity against not only infected cells but also cancer cells (119)(120)(121). IFN-␥ produced by activated NK cells suppresses the proliferation of cancer cells and activates cytotoxic T cells and macrophages (122,123). While NK cells kill cancer cells in an antigen non-specific manner, cytotoxic T cells recognize specific antigens of cancer cells for killing. Moreover, macrophages secrete molecules toxic to cancer cells and induce the apoptosis of cancer cells (124)(125)(126).
The proliferation and metastasis of cancer cells accelerate when immune functions are disturbed. It has been found that cancer patients have lower NK cell activity than healthy controls and persons with lower NK cell activity are subject to higher rates of cancer incidence, metastasis and aggravation of cancer (127)(128)(129)(130)(131). The macrophages infiltrating solid tumor have less phagocytic activity (132). On the other hand, when cancer patients ingest foods capable of improving immune functions, the prognosis becomes much better (133)(134)(135)(136)(137). Based on the reports of clinical trials with cancer patients, phagocytic activity, NK cell number, T cell number, DTH and IFN-␥ production are all useful immune parameters for assessing the effect of foods on prognosis after surgical operation for cancer (Table 1). Moreover, it has been reported that NK cell activity deteriorates in AIDS patients (138), and branched chain amino acids, probiotics and vitamin A improves virus-triggered diseases (139)(140)(141).
Newborns exhibit immature immune functions and are vulnerable to pathogenic infection. Supplementation of vitamins in malnourished children and ingestion of probiotics in newborns enhance immune functions and prevent viral infection (118,142).

Mechanisms by which Foods Influence Immune Functions
Food-derived substances incorporated into the body via various routes modulate immune functions. Taking into consideration that malnutrition or calorie restriction cause reduced activity in immune functions, nutritional condition is indispensable for the development of the immune system (143). Moreover, food-derived substances exhibit a special role in influencing immune functions.
The way that food-derived substances modulate immune functions is either indirect or direct. Comparative analyses of conventional and germ-free animals revealed that indigenous intestinal microflora play a pivotal role in the development of host immune systems. Ingestion of probiotics stabilizes the intestinal microflora, and normalization of the intestinal microflora by probiotics could lead to modulation of the host immune system (144). In addition, probiotics such as lactic acid bacteria are recognized by specific receptors on the surface of phagocytic cells. Additionally, vitamins, minerals or fatty acids affect cellular functions by preserving the cell membrane or regulating gene expression after being incorporated into lymphocytes (Fig. 3). One group of foods represented by lactic acid bacteria stimulates innate immunity (phagocytic activity, NK cell activity), while other foods, including vitamins and minerals, activate acquired immunity (T cell response, antibody production). However, as innate immunity and acquired immunity are closely linked, both groups of foods may regulate both immune systems. It has been reported that various nutrients found in foods exhibit anti-infectious functions (145). We would like to briefly It has been clarified that agonists of TLRs transmit signals to DNA by activating intracellular adaptor molecules such as MyD88. In contrast, vitamins and minerals prevent the oxidation of cell membranes and affect gene expression. Furthermore, fatty acids modulate immune functions by stabilizing the cell membrane and regulate the transcription of genes. describe the pathways by which food-derived substances exert their immune-modulating abilities.
Probiotics ingested may be partially digested in the gut and incorporated into M cells present in FAE, and then captured by dendritic cells or macrophages in the interfollicular area of PPs. These professional phagocytic cells hold various receptors on their surface capable of binding common structures of microbes, the pathogen-associated molecular patterns (PAMPs). Among the receptors for PAMPs, molecular structure and functions of TLRs (Toll-like receptors) have been recently unveiled. Ten TLR families (TLR1-TLR10) have been identified and ligands recognized by some TLRs have been determined. TLR2 recognizes peptidoglycans and lipopeptides as TLR4 does lipoteichoic acids and lipopolysaccharides. Moreover, the CpG oligonucleotides universally detected in bacterial DNA are recognized by TLR9. The signaling response to stimuli recognized by TLRs is mainly mediated by an intracellular adaptor molecule, MyD88 (myeloid differentiation factor 88). Thereafter, the nuclear transport of NF-B (nuclear factor-B) is stimulated and de novo synthesis of cytokines is induced (146). It has been proposed that stimuli through TLR2 activate both JNK (c-Jun N-terminal kinase) and ERK (extracellular signal regulated kinase) and induce production of IL-10, while stimuli through TLR4 activate JNK and induce production of IL-12 (147).
Immune-modulating effects of amino acids such as glutamine and arginine have been evaluated. Ingestion of glutamine improved nitrogen retention and lowered incidence of bacteremia in patients with trauma, and enteral supplementation of glutamine-enriched diet enhanced the recovery of immune functions and reduced the length of hospital stay after surgical operation in cancer patients (148,149). Glutamine is a nutrient for immune cells and acts as precursor for glutathione, which circumvents oxidant stress and improves cell-mediated immunity. Arginine is a substrate for synthesis of nitric oxide and improves helper T-cell numbers. Peri-operative feeding of arginine and n-3 polyunsaturated fatty acids (PUFAs) restored DTH and decreased infection rates in colorectal cancer patients (150).
Nucleotides are rich in foods containing nucleic acid/ nucleoprotein and supplementation of nucleotides is important for growth of infants. Addition of nucleotides increased the proportion of TCR␥␦-bearing IELs through stimulating IL-7 production by IECs in mice (151), and ingestion of formula supplemented with nucleotides augmented NK cell activity and IL-2 production in human infants (152). It remains to be elucidated whether immune cells may utilize ingested nucleotides as substrates for synthesis of nucleic acids.
Vitamins and minerals exhibit important immune-modulating functions by entering cells and regulating gene expression. Vitamin A affects the differentiation of epithelial cells and inhibits IFN-␥ production by T cells at the transcriptional level, which results in stimulation of antibody-mediated immune responses (153). Vitamin C prevents the production of reactive oxygen intermediates and reduces DNA damage in immune cells. Moreover, vitamin C inhibits the transcription of NF-B, and down-regulates the production of pro-inflammatory cytokines (154). Vitamin E is also an anti-oxidant and exerts an anti-inflammatory effect. Vitamin E stabilizes the membrane of immune cells and enhances the binding of antigen-presenting cells and T cells (155).
Minerals prevent the oxidation of lipids in the cell membrane, which can reduce oxidative stress affecting immune cells. For instance, selenium is indispensable to the function of reducing enzymes such as glutathione peroxidase and thioredoxin reductase, and is needed to stimulate cell-mediated immune functions (156). Furthermore, zinc may be required for the translocation and binding of NF-B to DNA (157).
Long-chain PUFAs in foods can modulate immune functions. Dietary n-3 PUFAs alter the lipid composition of the cell membrane and regulate the function of immune cells. Antigenpresenting cells from mice and humans fed n-3 PUFAs exhibited the capacity to suppress excessive activation of T cells (158,159). As a result, n-3 PUFAs can act as antiinflammatory agents.
Major food-derived substances and their immune-modulating functions are summarized in Table 2.

CONCLUDING REMARKS
We have reviewed and systematized studies reporting the effects of food-derived materials on immune functions in intervention studies or randomized controlled trials in order to clarify whether the immune-modulating activities of foods have been evaluated in a scientific manner. This search has revealed the following points: (i) many foods or food-derived materials improve or enhance immune functions in a wide range of human subjects; and (ii) foods with immune-modulating activities affect either innate or acquired immunity. Phagocytic activity

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and NK cell activity are representative parameters of innate immunity, and phagocytes and NK cells rapidly kill pathogenic bacteria, viruses and cancer cells in an antigen-independent manner. In contrast, DTH, antigen-specific antibody production and the proliferative response of T cells are major parameters reflecting acquired immunity, which is responsible for the antigen-specific exclusion of pathogenic bacteria, viruses and cancer cells. Many kinds of foods can improve parameters exhibiting either innate or acquired immunity.
Ingestion of foods does not always change many immune parameters. Therefore, it is useful to define immune parameters affected by foods. Vitamins, minerals, amino acids, proteins, carbohydrates or lipids, for example, enhance parameters of acquired immunity. In contrast, probiotics, including lactic acid bacteria, mainly augment parameters of innate immunity. These findings support that food-derived materials act on different immune cells or distinct molecules of the cells and improve at least one parameter of either innate or acquired immunity. In other words, these results mean that one can evaluate the immune-modulating abilities of foods by analyzing parameters of either innate or acquired immunity.
The components in foods that improve immune functions and the mechanisms by which foods exert immune-modulating effects are still far from fully understood. To confirm the scientific basis of the immune-modulating activities of foods, there is a need to keep on systematizing newly obtained scientific data on foods.