Sung Nim Han

Vitamin E and immune response in the aged: molecular mechanisms and clinical implications

Summary:  Nutritional status has been indicated as a contributing factor to age‐related dysregulation of the immune response. Vitamin E, a lipid‐soluble antioxidant vitamin, is important for normal function of the immune cells. The elderly are at a greater risk for vitamin E intake that is lower than recommended levels. Vitamin E supplementation above currently recommended levels has been shown to improve immune functions in the aged including delayed‐type hypersensitivity skin response and antibody production in response to vaccination, which was shown to be mediated through increased production of interleukin (IL)‐2, leading to enhanced proliferation of T cells, and through reduced production of prostaglandin E2, a T‐cell suppressive factor, as a result of a decreased peroxynitrite formation. Vitamin E increased both cell‐dividing and IL‐producing capacities of naïve T cells, but not memory T cells. The vitamin E‐induced enhancement of immune functions in the aged was associated with significant improvement in resistance to influenza infection in aged mice and a reduced risk of acquiring upper respiratory infections in nursing home residents. Further studies are needed to determine the signaling mechanisms involved in the upregulation of naïve T‐cell function by vitamin E as well as the specific mechanisms involved in reduction of risk for upper respiratory infections.

Macrophage prostaglandin production contributes to the age-associated decrease in T cell function which is reversed by the dietary antioxidant vitamin E

The aging process is associated with a decline in T cell-mediated immunity, including decreased interleukin (IL)-2 production and mitogen-induced T cell proliferation. Because macrophages (M phi) from old mice have higher production of prostaglandin (PG) E2 than young mice, and PGE2 has been shown to suppress T cell-mediated function, we hypothesized that increased production of PGE2 would contribute to decreased T cell function with aging and that decrease in PGE2 production by dietary antioxidants would enhance T cell-mediated function. Experiments were conducted in which combinations of purified M phi and T cells (> 95% pure) from young or old C57BL/6N1A mice were cultured together. Co-cultures containing T cells and M phi from old mice had reduced ConA-stimulated proliferation and IL-2 secretion than those consisting of T cells and M phi from young mice. Addition of M phi from old mice suppressed proliferation and IL-2 secretion by T cells from young mice. Likewise, T cells from old mice secreted more IL-2 when cultured with M phi from young mice compared to those cultured with M phi from old mice. Addition of PGE2, at concentrations produced by old M phi, decreased proliferation and IL-2 production by young but not old T cells. Neither addition of H2O2 at physiological levels, nor catalase changed the response of cultures from young or old mice. However, addition of indomethacin and the antioxidant nutrient vitamin E, both of which decreased PGE2 production, improved T cell proliferation and IL-2 production. These experiments demonstrate that increased production of PGE2 by M phi contributes to the age-associated decline in T cell function. Vitamin E improves T cell responsiveness in old mice mostly by reducing M phi PGE2 production, although a direct effect of vitamin E on T cells was also observed.

Age-associated increase in PGE2 synthesis and COX activity in murine macrophages is reversed by vitamin E

We previously showed that increased macrophage and PGE2 production with age is due to enhanced cyclooxygenase (COX) activity and COX-2 expression. This study determined the effect of vitamin E supplementation on macrophage PGE2 synthesis in young and old mice and its underlying mechanism. Mice were fed 30 or 500 parts per million vitamin E for 30 days. Lipopolysaccharide (LPS)-stimulated macrophages from old mice produced significantly more PGE2 than those from young mice. Vitamin E supplementation reversed the increased PGE2 production in old mice but had no effect on macrophage PGE2production in young mice. In both LPS-stimulated and unstimulated macrophages, COX activity was significantly higher in old than in young mice at all intervals. Vitamin E supplementation completely reversed the increased COX activity in old mice to levels comparable to those of young mice but had no effect on macrophage COX activity of young mice or on COX-1 and COX-2 protein or COX-2 mRNA expression in young or old mice. Thus vitamin E reverses the age-associated increase in macrophage PGE2 production and COX activity. Vitamin E exerts its effect posttranslationally, by inhibiting COX activity.We previously showed that increased macrophage and PGE2 production with age is due to enhanced cyclooxygenase (COX) activity and COX-2 expression. This study determined the effect of vitamin E supplementation on macrophage PGE2 synthesis in young and old mice and its underlying mechanism. Mice were fed 30 or 500 parts per million vitamin E for 30 days. Lipopolysaccharide (LPS)-stimulated macrophages from old mice produced significantly more PGE2 than those from young mice. Vitamin E supplementation reversed the increased PGE2 production in old mice but had no effect on macrophage PGE2 production in young mice. In both LPS-stimulated and unstimulated macrophages, COX activity was significantly higher in old than in young mice at all intervals. Vitamin E supplementation completely reversed the increased COX activity in old mice to levels comparable to those of young mice but had no effect on macrophage COX activity of young mice or on COX-1 and COX-2 protein or COX-2 mRNA expression in young or old mice. Thus vitamin E reverses the age-associated increase in macrophage PGE2 production and COX activity. Vitamin E exerts its effect posttranslationally, by inhibiting COX activity.

The Effect of Long-term Dietary Supplementation with Antioxidantsa

ABSTRACT: The impact of diet and specific food groups on aging and age‐associated degenerative diseases has been widely recognized in recent years. The modern concept of the free radical theory of aging takes as its basis a shift in the antioxidant/prooxidant balance that leads to increased oxidative stress, dysregulation of cellular function, and aging. In the context of this theory, antioxidants can influence the primary “intrinsic” aging process as well as several secondary age‐associated pathological processes. For the latter, several epidemiological and clinical studies have revealed potential roles for dietary antioxidants in the age‐associated decline of immune function and the reduction of risk of morbidity and mortality from cancer and heart disease. We reported that long‐term supplementation with vitamin E enhances immune function in aged animals and elderly subjects. We have also found that the beneficial effect of vitamin E in the reduction of risk of atherosclerosis is, in part, associated with molecular modulation of the interaction of immune and endothelial cells. Even though the effects of dietary antioxidants on aging have been mostly observed in relation to age‐associated diseases, the effects cannot be totally separated from those related to the intrinsic aging process. For modulation of the aging process by antioxidants, earlier reports have indicated that antioxidant feeding increased the median life span of mice to some extent. To further delineate the effect of dietary antioxidants on aging and longevity, middle‐aged (18 mo) C57BL\6NIA male mice were fed ad libitum semisynthetic AIN‐76 diets supplemented with different antioxidants (vitamin E, glutathione, melatonin, and strawberry extract). We found that dietary antioxidants had no effect on the pathological outcome or on mean and maximum life span of the mice, which was observed despite the reduced level of lipid peroxidation products, 4‐hydroxynonenol, in the liver of animals supplemented with vitamin E and strawberry extract (1.34 ± 0.4 and 1.6 ± 0.5 nmol/g, respectively) compared to animals fed the control diet (2.35 ± 1.4 nmol/g). However, vitamin E‐supplemented mice had significantly lower lung viral levels following influenza infection, a viral challenge associated with oxidative stress. These and other observations indicate that, at present, the effects of dietary antioxidants are mainly demonstrated in connection with age‐associated diseases in which oxidative stress appears to be intimately involved. Further studies are needed to determine the effect of antioxidant supplementation on longevity in the context of moderate caloric restriction.

Vitamin E supplementation increases T helper 1 cytokine production in old mice infected with influenza virus

Compared with young mice, old mice infected with influenza virus have significantly higher pulmonary viral titres, although these can be reduced significantly with dietary vitamin E supplementation. T helper 1 (Th1) cytokines, especially interferon‐γ (IFN‐γ), play an important role in defending against influenza infection. However, there is an age‐associated loss of Th1 cytokine production. Prostaglandin E2 (PGE2) production, which increases with age, can modulate the T helper cell function by suppressing Th1 cytokine production. To investigate the mechanism of vitamin E supplementation on reduction of influenza severity in old mice, we studied the cytokine production by splenocytes, and PGE2 production by macrophages (Mφ), in young and old C57BL mice fed semipurified diets containing 30 (control) or 500 parts per million (ppm) (supplemented) vitamin E for 8 weeks, and then infected with influenza A/PC/1/73 (H3N2). Old mice fed the control diet had significantly higher viral titres than young mice; old mice fed the vitamin E‐supplemented diet had significantly lower pulmonary viral titres than those fed the control diet (P = 0·02 and 0·001 for overall age and diet effect, respectively). Following influenza infection, interleukin (IL)‐2 and IFN‐γ production was significantly lower in old mice than in young mice. Vitamin E supplementation increased production of IL‐2 and IFN‐γ in old mice; higher IFN‐γ production was associated with lower pulmonary viral titre. Old mice fed the control diet showed significantly higher lipopolysaccharide (LPS)‐stimulated Mφ PGE2 production than old mice fed the vitamin E diet or young mice fed either diet. There was no significant age difference in IL‐6, IL‐1β, or tumour necrosis factor‐α (TNF‐α) production by splenocytes. Young mice fed the vitamin E‐supplemented diet had significantly lower IL‐1β (day 7) and TNF‐α production (day 5) compared with those fed the control diet. Old mice fed the vitamin E‐supplemented diet had significantly lower TNF‐α production (day 2) than those fed the control diet. Our results indicate that the vitamin E‐induced decrease in influenza viral titre is mediated through enhancement of Th1 cytokines, which may be the result of reduced PGE2 production caused by vitamin E.

Disease incidence and longevity are unaltered by dietary antioxidant supplementation initiated during middle age in C57BL/6 mice

The ability of augmented antioxidant consumption to alter disease incidence, lesion burden and/or longevity was studied in adult male C57BL/6 mice. Mice were fed modified AIN76 diet or modified AIN76 supplemented with vitamin E, glutathione (GSH), vitamin E and GSH, melatonin or strawberry extract starting at 18 months of age. All the mice in this study were heavier than reference populations of male C57BL/6 mice fed NIH-07 or NIH-31, which were maintained without a mid-life change in diet. Fatty liver, focal kidney atrophy and proteinacious casts in the renal tubules were observed more frequently in this study population than in the reference populations. Lesion burden and incidence of specific lesions observed amongst the various groups in this study did not differ. There were no differences observed for longevity of any of the study groups. The longevity observed in this study was similar to that previously reported for male C57BL/6 mice. Thus, diet supplementation with antioxidants initiated during middle age did not appear to affect age-associated lesions patterns, lesion burden or longevity for ad libitum fed male C57BL/6 mice.

Vitamin E supplementation decreases lung virus titers in mice infected with influenza

Effects of vitamin E (E) supplementation on influenza infection were examined in young and old C57BL/6NIA mice fed 30 or 500 ppm of E for 6 weeks and subsequently infected with influenza A/Port Chalmers/1/73 (H3N2). Old mice fed 30 ppm of E had significantly higher lung virus titers on days 2 and 7 after infection than young mice fed 30 ppm of E. Titers on all 3 days were significantly lower in old mice fed 500 ppm of E than in those fed 30 ppm. Significant effects of E on lung virus titers in young mice were observed on only day 5, but E caused more reduction of virus titers in old than in young mice (25-fold vs. 15-fold). An age-associated decline in NK cell activity was restored by 500 ppm of E in old but not young mice. Pulmonary cytotoxic T lymphocyte activity on day 7 was not affected by age or E. These experiments demonstrate that high doses of E significantly enhance influenza viral clearance in aged mice but only modestly affect young mice.

Antioxidants, Cytokines, and Influenza Infection in Aged Mice and Elderly Humans

The age-associated dysregulation of the immune response contributes to higher incidences of infectious diseases in the aged. Of note, there is dysregulation of cytokines, including a change in T helper (Th) 1/Th2 cytokine balance and an increase in production of proinflammatory cytokines. Synthesis of many cytokines is influenced by changes in the cellular oxidant/antioxidant balance. Because vitamin E supplementation reduces oxidative stress and improves the immune response in the aged, a series of experiments was conducted to determine the effect of supplementation with vitamin E and other antioxidants on resistance to influenza infection in aged mice and the role of cytokines in vitamin E-induced increase in resistance to influenza infection. The results of these studies plus findings by other investigators on the effects of age and antioxidants on production of cytokines in human and animal models are reviewed.

Age and vitamin E-induced changes in gene expression profiles of T cells.

T cells are vulnerable to age-associated changes. Vitamin E has been shown to improve T cell functions in the old. We studied gene expression profiles of T cells to better understand the underlying mechanisms of age and vitamin E-induced changes in T cell function. Young and old C57BL mice were fed diets containing 30 (control) or 500 (supplemented) ppm of vitamin E for 4 wks. Gene expression profiles of T cells were assessed using microarray analysis with/without anti-CD3/anti-CD28 stimulation. Genes associated with cytokines/chemokines, transcriptional regulation, signal transduction, cell cycle, and apoptosis were significantly up-regulated upon stimulation. Higher SOCS3 and lower growth factor independent 1 (Gfi-1) expression in old T cells may contribute to age-associated decline in proliferation. Higher Gadd45 and lower Bcl2 expression may contribute to increased apoptosis in old T cells. Vitamin E supplementation resulted in higher expression of genes involved in cell cycle regulation (Ccnb2, Cdc2, Cdc6) in old T cells. Vitamin E supplementation resulted in higher up-regulation of IL-2 expression in young and old T cells and lower up-regulation of IL-4 expression in old T cells following stimulation. These findings suggest that aging has significant effects on the expression of genes associated with signal transduction, transcriptional regulation, and apoptosis pathways in T cells, and vitamin E has a significant impact on the expression of genes associated with cell cycle and Th1/Th2 balance in old T cells. Further studies are needed to determine whether these changes are due to the effects of aging at a single-cell level or to the shift in the ratio of naïve:memory T cells with age.

Black soybean anthocyanins inhibit adipocyte differentiation in 3T3-L1 cells

Anthocyanins are naturally occurring polyphenolic pigments in plants that have been shown to decrease weight gain and insulin resistance in mice-fed high-fat diets. We investigated the effects of anthocyanins on cell growth, differentiation, and lipolysis in 3T3-L1 cells to test our hypothesis that anthocyanins could reduce adipose tissue mass by acting directly on adipocytes. Anthocyanin extracts from black soybeans were used and composed of 3 of the following major anthocyanins: cyanidine-3-O-glucoside (68.3%), delphinidin-3-O-glucoside (25.2%), and petunidin-3-O-glucoside (6.5%). Treatment with 12.5 and 50 μg/mL of black soybean anthocyanins exhibited inhibitory effects on the proliferation of both preconfluent preadipocytes (P < .01) and maturing postconfluent adipocytes (P < .01). In fully differentiated adipocytes, the number of viable cells was reduced by black soybean anthocyanins (P < .01). Treatment with 50 μg/mL of black soybean anthocyanins slightly increased epinephrine-induced lipolysis but decreased the basal lipolysis of fully differentiated adipocytes (P < .05). Black soybean anthocyanins also reduced lipid accumulation and suppressed the expression of the peroxisome proliferator-activated receptor γ, a major transcription factor for the adipogenic gene (P < .01). These results suggest that black soybean anthocyanins inhibit adipocyte differentiation and basal lipolysis, which may contribute to their antiobesity and antidiabetic properties.