The Role of Vitamin E in Immunity

1Vitamin E is the collective term for four tocopherols (α-, β-, γ-, and δ-tocopherols) and four tocotrienols (α-, β-, γ-, and δ-tocotrienols) found in food. These forms have antioxidant activities, but cannot be interconverted, and only α-tocopherol meets the human vitamin E requirement. Tocopherols have a chromanol ring and a phytyl tail, while tocotrienols have a chromanol ring and an unsaturated tail. The α-, β-, γ-, and δ- forms differ in the number and position of methyl groups on the chromanol structure. Natural tocopherols have only RRR stereochemistry, but synthetic tocopherols are mixtures of eight stereoisomers (RRR-, RSR-, RRS-, RSS-, SRR-, SSR-, SRS-, SSS-), because there are three asymmetric carbon atoms (2R, 4’R, 8’R) present in the phytyl tail. The structures of tocopherols and tocotrienols are shown in Figure 1.

2The major dietary sources of vitamin E are vegetable oils. Nuts are good sources of vitamin E as well. Soybean, sunflower, corn, walnut, cottonseed, palm, and wheat germ oils contain relatively higher amounts (more than approximately 50 mg vitamin E/100 g oil) of vitamin E than other oils. The proportions of α-, β-, γ-, and δ-tocopherols vary depending on the oil type. Safflower and sunflower oils are high in α-tocopherol, soybean and corn oils contain mainly γ-tocopherol, and cottonseed oil contains similar proportions of α- and γ-tocopherols. Therefore, the types of oils consumed through the diet affect the dietary intake levels of α-tocopherol. Vitamin E supplements are quite popular and contribute considerably to vitamin E intake among some populations. Either natural or synthetic forms of α-tocopherol are used as supplements.

3Despite the relatively higher intake of γ-tocopherol from the diet than α-tocopherol, α-tocopherol is the major form of vitamin E in the circulation because α-tocopherol transfer protein (α-TTP) has the preferential binding affinity for α-tocopherol. α-TTP is involved in the transfer of α-tocopherol to the plasma membrane.

4Vitamin E is a major fat-soluble antioxidant that scavenges peroxyl radicals and terminates the oxidation of polyunsaturated fatty acids (PUFAs). In the presence of vitamin E, peroxyl radicals react with α-tocopherol instead of lipid hydroperoxide, the chain reaction of peroxyl radical production is stopped, and further oxidation of PUFAs in the membrane is prevented. Tocopheroxyl radicals—produced from α-tocopherol and peroxyl radicals—are reduced by vitamin C or glutathione, form tocopherol dimers, undergo further oxidation, or act as prooxidants. The antioxidant activity of vitamin E may be responsible for the regulation of several enzymes involved in signal transduction because the activity of signaling enzymes is regulated by the redox state.

5Vitamin E inhibits protein kinase C (PKC) activity by increasing PKC-α dephosphorylation through the activation of protein phosphatase 2A. The inhibition of PKC by vitamin E has been reported in various cells, and consequently, the inhibition of platelet aggregation; reduced proliferation of monocytes, macrophages, neutrophils, and vascular smooth muscle cells; and decreased superoxide production in neutrophils and macrophages have been observed.

6Vitamin E may directly bind to the enzymes involved in the generation of lipid mediators or to the transport proteins involved in signal transduction. Vitamin E may affect the membrane protein interaction and translocation of the enzymes to the plasma membrane and therefore change the activity of signal transduction enzymes.

7Dietary interventions of vitamin E at supplemental levels have been shown to enhance cell-mediated and humoral immune responses in various species of animals. Increased lymphocyte proliferation, immunoglobulin levels, antibody responses, natural killer (NK) cell activity, and interleukin (IL)-2 production have been reported with vitamin E supplementation (Table 1).

8In humans, many intervention studies have reported increased lymphocyte proliferation in response to mitogenic stimulation, enhanced delayed type hypersensitivity (DTH) response, increased IL-2 production, and decreased IL-6 production with vitamin E supplementation above the recommended levels. However, some studies showed no difference or decreased lymphocyte proliferation responses and decreased chemiluminescence. (Table 2). Differences in dose of vitamin E supplementation used, magnitude of vitamin E level changes with supplementation, age of subjects, and methodology (determination of antibody levels with or without specific vaccination) might have contributed to the different results observed.

9The immunostimulatory effect of vitamin E has resulted in enhanced resistance against several pathogens. Animal studies in which infectious disease models were used to test the effects of vitamin E supplementation are listed in Table 3.

10The mechanisms involved with protection against infectious agents were increased macrophage activity and antibody (Ab) production for D. pneumoniae type 1, and higher NK activity and Th1 response for influenza virus.

11In humans, the effects of vitamin E on the natural incidence of infectious diseases have been determined in several studies (Table 4). Many studies provided evidence that the immunostimulatory effects of vitamin E confer improved resistance to infections. However, the magnitudes of the effects were rather small, and in some studies, positive effects were only observed in subgroups of subjects.

12The immunomodulatory mechanisms of α-tocopherol in immune cells are depicted in Figure 2.

13Macrophages, important effector cells in the innate immune response, serve as antigen presenting cells (APC) and regulate NK cells and T cells by producing cytokines, reactive oxygen species (ROS), reactive nitrogen species (RNS), and prostaglandins. Cytokines produced by T cells and other immune cells can shift the macrophages into different populations with distinct physiologies.

14The effects of vitamin E on prostaglandin (PG)E2 production by macrophages from the aged have been suggested as one of the mechanisms by which vitamin E improves the age-associated decrease in the T cell-mediated immune response. In a co-culture experiment in which purified T cells and macrophages from young and old mice were cultured together, T cells from young mice showed suppressed proliferation and IL-2 secretion when cultured with macrophages from old mice. When macrophages from old mice were pre-incubated with 10 µg/mL vitamin E for 4 h, co-cultures of old macrophages and young T cells showed significant improvement in proliferation. Vitamin E pre-incubation of old macrophages improved proliferation and IL-2 production in co-cultures of old macrophages and old T cells. Macrophages from old mice produced significantly higher levels of PGE2, which was due to higher cyclooxygenase (COX) activity. Macrophages from old mice expressed higher levels of inducible COX2 protein and mRNA. These increases in PGE2 synthesis and COX activity were lowered by in vivo vitamin E supplementation. Macrophages isolated from old mice fed a diet containing 500 ppm vitamin E for 30 days produced lower amounts of PGE2 and had lower COX activity than those from old mice fed a control diet containing 30 ppm vitamin E, but the COX2 mRNA levels and protein expression of the control and supplemented groups did not differ. Thus, vitamin E’s effect on COX activity seemed to be through post-translational mechanisms rather than through its effect at transcriptome or translational levels. In a subsequent study, it was shown that vitamin E reduced COX activity in macrophages from old mice by decreasing peroxynitrite production. The inhibition of COX activity by vitamin E in old mice disappeared specifically with the addition of a nitric oxide (NO) donor in the presence of a superoxide to elevate peroxynitrite levels in the macrophage culture. There is a complex interplay between the nitric oxide synthase (NOS) and COX pathways and NO increases COX2 activity, which seems to be due to the NO preventing self-deactivation of COX by the superoxide as NO interacts with the superoxide.

15In vivo supplementation of vitamin E (1500 IU d-α-tocopheryl acetate/day for 16 weeks) in allergic asthmatic patients prevented the suppression of alveolar macrophage nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity after allergen challenge. This study presented the possibility of vitamin E’s protective role in allergies and asthmas through regulation of macrophage NRF2 activity, but, further studies are needed to confirm the findings because of the small number of patients (nine mild non-smoking allergic asthmatics) and the lack of appropriate controls.

16NK activity seems to be related with vitamin E status. The NK activity of a boy with Shwachman syndrome who had a severe vitamin E deficiency was low, but improved after eight weeks of 100 mg/d α-tocopherol supplementation. When α-tocopherol supplementation was stopped, NK activity and CD16+ CD56+ cells decreased. NK activity and CD16+ CD56+ cells were restored upon resuming eight weeks of 100 mg/d α-tocopherol supplementation. In 37 women aged 90–106 years old, NK cell cytotoxicity was positively associated with plasma vitamin E concentration. A two-week supplementation of 750 mg vitamin E in colorectal cancer patients resulted in increased NK activity in six out of seven patients. Vitamin E treatment did not result in changes in perforin expression or IFN-γ production; therefore, mechanisms of improved NK activity by vitamin E could not be determined from the study.

17NO appears to be involved in the impairment of NK cell function. Co-culture of NK cells and myeloid-derived suppressor cells (MDSCs) showed that NK cell cytotoxicity and IFN-γ were impaired by MDSCs and that the inhibition of inducible nitric oxide synthase (iNOS) rescued the impairment by MDSCs. Exposure of NK cells to NO by treatment with an NO producer caused the nitration of tyrosine residues on CD16+ NK cells. These results suggested that MDSCs impair NK cell function via the production of NO and the nitration of protein tyrosine residues. Vitamin E might exert its effects on NK cell function by modulating NO levels.

18Dendritic cells (DCs) are effective antigen-presenting cells that recognize pathogens and present pathogen-derived antigens to T cells. The interaction of DCs with pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) elicits the activation and maturation of DCs. The increased expression of surface major histocompatibility complex (MHC) molecules and co-stimulatory molecules and the increased production of cytokines occur with the activation of DCs, which allows the effective induction of the T cell response. DCs are also involved in tolerance and autoimmunity. DCs might promote tolerance by the generation of Treg cells and/or by the induction of T cell unresponsiveness. DCs might be involved in the pathogenesis of autoimmune disease by promoting the priming or differentiation of self-reactive T cells. Therefore, understanding the regulation of DCs by vitamin E will provide insight into the mechanisms of vitamin E’s immune response modulation and implications of vitamin E in immunological diseases.

19Several studies have shown that vitamin E could regulate the maturation and functions of DCs. Tan et al. investigated the effects of α-tocopherol and vitamin C, alone or in combination, on the phenotype and functions of human DCs generated from peripheral blood mononuclear cells (PBMCs). During the differentiation of human PBMCs into DCs, various concentrations of α-tocopherol were treated in culture starting from day 2, cells were stimulated on day 5, and then the surface phenotype was determined on day 6. The expression of human leukocyte antigen(HLA)-DR, CD40 CD80, and CD86 appeared to be increased with lower concentrations of α-tocopherol (<0.05 mM), but the combination of vitamin E and C prevented DC activation, as the upregulation of surface markers was not observed. DCs treated with 0.5 mM vitamin E and 10 mM vitamin C showed lower levels of intracellular ROS and inhibition of the nuclear factor (NF)-κB, PKC, and p38 mitogen-activated protein kinase (MAPK) pathways. When bone marrow-derived dendritic cells (BMDCs) from Balb/c mice were treated with 500 µM of α-tocopherol for 2 h, upregulation of phosphorylated inhibitor of κB (IκB) by lipopolysaccharide (LPS)-stimulation was suppressed. Vitamin E treatment for 24 h resulted in a reduced number of CD11+CD86+ cells and ROS-positive cells, lower production of IL-12p70 and TNF-α, and decreased transwell migration of BMDCs. These effects of vitamin E on BMDCs were partly dependent on Klotho expression. Vitamin E treatment on BMDCs resulted in higher Klotho transcript and protein levels, and silencing of Klotho by transfection of Klotho siRNA abolished the inhibitory effects of vitamin E on IL-12p70 production, number of ROS-positive cells, and DC migration. Klotho is a membrane protein that has been shown to mediate calcium transport into the cells; regulate intracellular signaling pathways such as p53/p21, cyclin adenosine monophosphate (cAMP), PKC, and Wnt; and inhibit the NF-κB pathway. Therefore, the upregulation of Klotho by vitamin E could be one of the mechanisms by which vitamin E modulates NF-κB mediated DC function and maturation. However, the level of α-tocopherol used for in vitro treatment (500 µM) was high and, therefore, further research is needed to elucidate the physiological relevance of vitamin E treatment on the expression of Klotho and its involvement in the modulation of DC function.

20In vivo supplementation of α-tocopherol at 150, 250, and 500 mg/kg diet in allergic female mice reduced the lung CD11b+ DCs and mRNA levels of IL-4, IL-33, thymic stromal lymphopoietin (TSLP), eotaxin 1 (CCL11), and eotaxin 2 (CCL24) in allergen challenged pups. Furthermore, when BMDCs from 10-day-old neonates born to a control female were treated with 80 µM α-tocopherol for 24 h, the number of CD45+ CD11b+ CD11+ DCs and the number of CD45+ CD11b+ CD11c+ Ly6c− MHCII− DCs were reduced. Maternal supplementation with α-tocopherol was effective in decreasing allergic responses in offspring from allergic mothers by affecting the development of subsets of DCs that are critical for allergic responses. On the other hand, γ-tocopherol supplementation exerted an opposite response in the same model. In vivo supplementation of γ-tocopherol at 250 mg/kg diet in allergic female mice resulted in a higher number of lung eosinophils, a higher number of lung CD11c+ CD11b+ DCs, and higher levels of lung lavage CCL11 in the offspring.

21Modulation of the immune response by vitamin E has been observed in animal and human studies, and DCs play a critical role in bridging innate and adaptive immune systems and initiating adaptive immune responses. Despite the importance of DCs’ role in adaptive immune responses and in diseases such as autoimmune diseases, few studies have investigated the DC-specific effect of vitamin E.

22The effects of vitamin E on immune cells have been studied the most with T cells. The dysregulation of immune function occurs with aging and the most significant changes are observed in T cells. Age-associated changes in T cells include, but are not limited to, (1) defects in T cell receptor (TCR) signal transduction such as a decrease in linker for the activation of T cells (LAT) phosphorylation by zeta chain of T cell receptor associated protein kinase 70 (ZAP-70), (2) decreased intracellular influx of calcium following stimulation, (3) diminished synapse formation, (4) diminished activation of the mitogen activated protein kinase (MAP kinase) pathway, (5) decreased nuclear factor of activated T-cells (NFAT) binding activity, and (6) a shift of the T cell population toward memory T cells. As a result, diminished production of IL-2 and reduced proliferative capacity of naive T cells are observed and impaired T cell functions contribute to increased susceptibility to infectious diseases and poor response to immunization.

23Vitamin E has been shown to increase the cell division and IL-2 producing capacity of naïve T cells, increase the percentage of T cells capable of forming an effective immune synapse, and reverse the age-associated defect in the phosphorylation of LAT in T cells from old animals.

24In vitro pre-incubation with 46 µM vitamin E for 4 h increased proliferation and IL-2 production in T cells purified from old mice stimulated with anti-CD3 and anti-CD28. Increased IL-2 production was due to both an increase in the number of activation-induced IL-2+ cells and an increase in the level of IL-2 accumulated per cell. Vitamin E specifically increased the naive T cells’ ability to progress through the cell division cycle in old mice. The gene expression profile of T cells isolated from young and old mice fed a diet supplemented with 500 ppm vitamin E for four weeks provided evidence that vitamin E influences cell cycle-related molecules at the gene expression level. Higher expression of cell cycle-related genes Ccnb2, Cdc2, and Cdc6 was observed in stimulated T cells from old mice fed the vitamin E-supplemented diet compared with those fed the control diet, which was not observed in young mice. Cyclin B2, encoded by Ccnb2, binds to cyclin-dependent kinase 1 (also known as Cdc2) and regulates the events during both the G2/M transition and progression through mitosis. Cdc6 is a key regulator in the early steps of DNA replication, as the binding of Cdc6 to chromatin is a necessary and universal step in the acquisition of replication competences. These alterations in the expression of cell cycle-related genes observed with vitamin E might contribute to vitamin E improving the proliferative ability of old T cells.

25Marko et al. showed that pre-incubation of CD4+ T cells isolated from old T cells with 46 µM vitamin E for 4 h increased the percentage of CD4+ T cells displaying effective immune synapses. Redistribution of Zap70, LAT, Vav, and phospholipase Cγ (PLCγ) into immune synapse increased significantly with vitamin E treatment. This change was confirmed with in vivo supplementation of vitamin E. In old mice fed a diet containing 500 ppm vitamin E for eight weeks, LAT and Vav showed significantly higher redistribution into the T cell/APC contact area when purified CD4+ T cells were stimulated with murine CD3ε hybridoma. In a subsequent study, it was shown that vitamin E could reverse the age-associated defect in the phosphorylation of LAT on tyrosine 191. The phosphorylation of LAT is required for the recruitment of adaptor and effector proteins. Therefore, it plays a pivotal role in the assembly of microcluster structures in the initiation of T cell activation signals. This evidence suggests that vitamin E can modulate the early stages of T cell activation.

26Vitamin E seems to modulate Th1 and Th2 responses. The polarization of CD4 T cells to T helper (Th)1 or Th2 cells has implications for the protection against different pathogens (intracellular vs. extracellular pathogens) and the development of different types of chronic diseases (inflammatory vs. allergic diseases). PBMCs isolated from allergic donors treated with vitamin E (12.5–50 µM) showed dose-dependent decreases in IL-4 production. IL-4 mRNA levels in activated PBMCs were downregulated by 25 µM vitamin E treatment. Jurkat T cells treated with 50 µM vitamin E exhibited downregulation of IL-4 promoter activity, which might be related to vitamin E blocking the interaction of transcription factors with PRE-1 and P1. In vivo supplementation of vitamin E enhancing the Th1 response has been observed in mice infected with influenza virus and in colorectal cancer patients. In colorectal cancer patients, two weeks of supplementation with 750 mg vitamin E led to an increased frequency of IL-2 producing CD4+ T cells and increased IFN-γ production. In old mice infected with influenza virus, 500 ppm vitamin E supplementation for eight weeks prior to infection lowered the viral titer in the lung, and this protective effect of vitamin E was associated with the enhancement of Th1 response. IFN-γ production levels correlated negatively with viral titer, and old mice fed a vitamin E-supplemented diet produced significantly higher levels of IFN-γ and IL-2. The gene expression profile of T cells isolated from young and old mice fed a diet supplemented with 500 ppm vitamin E for four weeks provided evidence that vitamin E influences the Th1/Th2 balance at the gene expression level. The increase in IL-4 expression following stimulation was lower in T cells from old mice fed the vitamin E-supplemented diet compared with those fed the control diet, and the ratio of IFN-γ and IL-4 expression levels was significantly higher in the vitamin E group than in the control group.

27Vitamin E can affect activation-induced cell death in T cells. In vitro treatment of primary human T cells with 25 µM vitamin E suppressed CD95L expression and activation-induced cell death. The reduction of CD95L mRNA levels and the proportion of CD95L-positive cells were related to the suppression of NF-κB and AP-1 binding to the CD95L promoter target site by vitamin E. On the other hand, α-tocopheryl succinate was shown to trigger apoptosis in Jurkat cells with caspase-activation involved.

28Vitamin E supplementation has been reported to enhance humoral responses. Higher antibody responses have been observed in animals and humans. However, it is hard to differentiate whether vitamin E’s direct effect on B cells or indirect effect through T cells contributes to higher antibody responses.

29Vitamin E has been shown to enhance immune responses in animal and human models and to confer protection against several infectious diseases. Suggested mechanisms involved with these changes are (1) the reduction of PGE2 production by the inhibition of COX2 activity mediated through decreasing NO production, (2) the improvement of effective immune synapse formation in naive T cells and the initiation of T cell activation signals, and (3) the modulation of Th1/Th2 balance. Higher NK activity and changes in dendritic function such as lower IL-12 production and migration were observed with vitamin E, but underlying mechanisms need to be further elucidated

30Several considerations are warranted for the advancement in our understanding of vitamin E’s role in immunity. For in vitro studies to support implications for the regulation of immunological diseases, the physiological relevance of vitamin E levels used for treatment should be considered. Different forms of vitamin E exert differential effects on immune cells. Cell-specific effects of vitamin E provide valuable evidence regarding the immunomodulatory mechanisms of vitamin E, but the interplay between immune cells should not be ignored, because interactions between immune cells are critical in the regulation of immune function.