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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 55:B496-B503 (2000)
© 2000 The Gerontological Society of America

Effect of Long-term Dietary Antioxidant Supplementation on Influenza Virus Infection

Sung Nim Hana, Mohsen Meydania, Dayong Wua, Bradley S. Benderb, Donald E. Smitha, José Viñac, Guohua Caod, Ronald L. Priora and Simin Nikbin Meydania,e

a Jean Mayer Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts
b Department of Medicine, University of Florida College of Medicine, and Geriatric Research, Education and Clinical Center, Malcolm Randall Veterans Affairs Medical Center, Gainesville, Florida
c Departmento Fisología, Facultad de Medicina, University of Valencia, Spain
d Nutritional Science Department, University of Connecticut, Storrs
e Department of Pathology, Sackler Graduate School of Biomedical Sciences, Tufts University

Simin Nikbin Meydani, Nutritional Immunology Laboratory, Jean Mayer Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111 E-mail: smeydani{at}hnrc.tufts.edu.

Jay Roberts, PhD


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
This study compared the effect of vitamin E on the course of influenza infection with that of other antioxidants. (In a previous study we showed that short-term vitamin E supplementation significantly decreased pulmonary viral titer in influenza-infected old mice). Eighteen-month-old C57BL/6NCrlBR mice were fed one of the following semisynthetic diets for 6 months: control, vitamin E supplemented, glutathione supplemented, vitamin E and glutathione supplemented, melatonin supplemented, or strawberry extract supplemented. After influenza virus challenge, mice fed vitamin E-supplemented diet had significantly lower pulmonary viral titers compared to those fed the control diet (102.6 vs 104.0, p < .05) and were able to maintain their body weight after infection (1.8 ± 0.9 g weight loss/5 days postinfection in vitamin E group vs 6.8 ± 1.4 g weight loss/5 days postinfection in control group, p < .05). Other antioxidants did not have a significant effect on viral titer or weight loss. There was a significant inverse correlation of weight loss with food intake (r = –.96, p < .01), indicating that the observed weight changes were mainly due to decreased food intake. Pulmonary interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-{alpha} levels increased significantly postinfection. The vitamin E group had lower lung IL-6 and TNF-a levels following infection compared to the control group. In addition, there was a significant positive correlation between weight loss and lung IL-6 (r = .77, p < .01) and TNF-{alpha} (r = .68, p < .01) levels. Because IL-6 and TNF-{alpha} have been shown to contribute to the anorexic effect of infectious agents, the prevention of weight loss by vitamin E might be due to its reduced production of IL-6 and TNF-{alpha} following infection. Thus, among the antioxidants tested, only vitamin E was effective in reducing pulmonary viral titers and preventing an influenza-mediated decrease in food intake and weight loss. Other dietary antioxidant supplementations that reduced one or more measures of oxidative stress (4-hydroxynonenal, malondialdehyde, and hydrogen peroxide) did not have an effect on viral titer, which suggests that, in addition to its antioxidant activity, other mechanisms might be involved in vitamin E's beneficial effect on lowering viral titer and preventing weight loss.

THE incidence of infectious diseases increases with age and is associated with higher morbidity and mortality. As infectious diseases may require hospitalization, they contribute greatly to the medical costs associated with the care of elderly people. Respiratory infections are particularly common (1), and influenza viruses are the most challenging when development of more effective preventive strategies is considered: they change antigenic character at irregular intervals, infect individuals of all ages, produce yearly epidemics associated with excess morbidity and mortality, and predispose the host to bacterial superinfections (2).

Influenza virus predominantly damages epithelial cells of the lung and airways. Although the molecular mechanisms of influenza virus pathogenesis are still largely obscure, possible mechanisms include apoptosis and generation of reactive oxygen species (ROS) (3)(4)(5). Neutrophils and macrophages produce superoxide (O-2) and hydrogen peroxide (H2O2), which normally are involved in the killing of ingested or invading microbes. However, these ROS are toxic to a wide variety of eukaryotic cells and could contribute to the tissue damage seen in lungs after influenza virus infection (3)(6). Generation of ROS by alveolar phagocytic cells increases with influenza virus infection (7). Other than phagocyte-generated ROS, influenza virus itself is involved in the direct generation of ROS. Pahl and Baeuerle (8) reported that expression of influenza virus hemagglutinin (HA) activates nuclear transcription factor (NF)-{kappa}B DNA binding and transactivation in HeLa and 293 cells. Activation was inhibited in the presence of the antioxidant dithiothreitol, suggesting that HA increases the production of ROS within the cell, which may act as second messengers to activate NF-{kappa}B. Other than this direct role in viral pathogenesis, oxidative stress can also have indirect effects on the infectivity of influenza virus by influencing the balance of protease/antiprotease or by influencing the interplay of cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1, or interferons (IFNs) (9).

A wide variety of systems contribute to cellular antioxidant defense, including such enzymes as superoxide dismutase, catalase, glutathione peroxidase, and small molecules such as vitamin E, vitamin C, and glutathione. Jacoby and Choi (10) showed that viral infection increases the expression of the antioxidant enzymes manganese superoxide dismutase (MnSOD) and indoleamine 2,3-dioxygenase (IDO), which may be an important defense against oxygen radical-mediated damage. Hennet and colleagues (4) showed that influenza infection resulted in a decrease in the total concentrations of the antioxidants glutathione and vitamin E from lung and liver tissues. This loss of antioxidants may be due to increased production of oxidants.

In a previous study (11), we showed that old mice whose diet was supplemented with 500 ppm vitamin E for 6 weeks had significantly lower lung viral titer than old mice fed control levels (30 ppm) of vitamin E following influenza infection. Vitamin E supplementation preserved the antioxidant status in tissues following influenza virus infection, and a highly significant inverse correlation was observed between viral titers and vitamin E levels. Recently, melatonin, in addition to its hormonal function, has been recognized as having antioxidant capacity in vitro (12). Further, supplementing C57BL/6 mice with melatonin was shown to improve immune function and longevity in middle-aged mice (13). Glutathione, another antioxidant, has also been shown to increase cell-mediated immunity in old mice (14). The phenolic compounds of fruits and vegetables have been shown to have antioxidant capacity. Strawberry extract was shown to have the highest in vitro antioxidant capacity among the 12 fruits tested using the automated oxygen radical absorbance capacity (ORAC) assay (15). Therefore, we tested the efficacy of these antioxidants compared to that of vitamin E on the clinical manifestations (pulmonary viral titers and weight loss) of influenza infection. We hypothesized that if the effect of vitamin E was through its antioxidant function, other antioxidants would show a similar effect.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals and Infection
Pathogen-free male C57BL/6NCrlBR mice (18 months old) bred in our colony were individually housed in filtered cages in an environmentally controlled atmosphere (temperature 23°C; 45% relative humidity) with 15 air changes per hour and a 12/12 hour light-dark cycle. Mice (15 mice/group) were fed ad libitum with one of the following semipurified diets for 6 months: control diet (30 ppm vitamin E), vitamin E-supplemented diet [control + 470 ppm vitamin E ({alpha}-tocopherol acetate, Harlan Teklad, Madison, WI)], glutathione-supplemented diet [control + 0.5% glutathione (Kyowa Hakko Koygo Co., Tokyo, Japan)], vitamin E + glutathione-supplemented diet (control + 470 ppm vitamin E + 0.5% glutathione), melatonin-supplemented diet [control + 11 ppm melatonin (Sigma Chemical Co., St. Louis, MO)], or strawberry extract-supplemented diet (control + 1% strawberry extract). Strawberry extract was prepared from fresh stock of strawberries as described (15). The levels of antioxidants were chosen based on our previous experiments (11)(14) or those reported in the literature (13). Control diet (all ingredients by wt) contained 33.55% corn starch, 33.40% sucrose, 5% cellulose, 3.5% AIN-76A salt mixture, 1% AIN-76A vitamin mixture, 0.3% D-L methionine, 0.25% choline bitartrate (Harlan Teklad), 18% vitamin-free casein (Bioserve, Frenchtown, NJ), and 5% vitamin E-stripped soybean oil (Bioserve). All conditions and handling of the animals were approved by the Animal Care and Use Committee at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University and followed NIH Guidelines for the Care and Use of Laboratory Animals.

After 6-month dietary supplementation, 10 mice from each diet group were euthanized via CO2 asphyxiation and used as preinfection or day 0 group for different measurements. In addition, 5 mice from each diet group were infected with influenza A/Port Chalmers/1/73 (H3N2) virus according to the method of Bender and colleagues (16). Mice were anesthetized with an intraperitoneal injection of 0.4 mg pentobarbital and infected intranasally with 20 µL of H3N2 virus (TCID50 106.75/10 µL). Mice were euthanized via CO2 asphyxiation 5 days after infection. Day 5 postinfection was chosen because a significant inverse correlation between liver vitamin E level and viral titer previously occurred on that day (11). Weight loss due to influenza infection was calculated using the weight measured on the day before viral challenge and the weight measured before animals were euthanized. Food intake was calculated by subtracting the weight of leftover food in each cage (one mouse per cage) from the food provided. Specialized food containers that restrict food spillage (Lab Products, Maywood, NJ) were used to reduce food loss due to digging.

Tissue Preparation
Blood was drawn by cardiac puncture and the plasma separated and stored at -70°C for later analysis of cytokine levels. The lungs were processed as previously described (17), cut into small pieces, and incubated at 37°C for 1 hour in 6 mL of Iscove's medium with 10% fetal calf serum (FCS) and 40 U/mL of collagenase (Sigma). Lung pieces were ground and washed through a 60-mesh stainless steel screen. After centrifugation at 1000 rpm for 5 minutes, supernatant was removed and frozen for virus quantification and IL-6, IL-1ß, and TNF-{alpha} measurements. Lung cells were resuspended and red blood cells were lysed using lysing buffer (Sigma). Lung cells were then counted and cultured for H2O2 production.

Measurements
Determination of lung virus titer..-- Lung virus titer was determined by inoculation of lung supernatant into Madin Darby canine kidney cells in round-bottom 96-well plates (16). The TCID50 was calculated using the method of Reed and Muench (18).

Determination of hydrogen peroxide (H2O2) production..-- H2O2 was measured after lung cells (106 cells) were stimulated with 1 mg/mL (final concentration) of zymosan (Sigma) for 1 hour at 37°C. Production of H2O2 was measured by H2O2-dependent oxidation of homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid, HVA) to a highly fluorescent dimer (2,2'-dihydroxy-3,3'-dimethoxydiphenyl-5,5'-diacetic acid), which is mediated by horseradish peroxidase (19). A linear relationship between fluorescence ({lambda}ex = 312 nm and {lambda}em = 420 nm) and amount of H2O2 was found in the range of 0.1–10 nmoles.

Determination of liver lipid peroxidation..-- Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were measured as markers of lipid peroxidation in the liver using LPO-586 kit (R&D Systems, Minneapolis, MN).

Vitamin E, glutathione, and melatonin assessment..-- Status of vitamin E in control, vitamin E, and vitamin E + glutathione (GSH) groups was assessed by measuring {alpha}-tocopherol content of plasma by high-performance liquid chromatography using electrochemical detector (20). Status of glutathione was assessed by measuring GSH level of liver by using glutathione S-transferase method (21). Because there was a limitation in availability of lung samples, we measured liver content of GSH and plasma content of {alpha}-tocopherol. Previous reports have shown that liver content of GSH correlates with GSH level in lung following influenza infection (4). Furthermore, using similar dietary treatments, we have shown that there is a good correlation between plasma and lung levels of {alpha}-tocopherol (22). Melatonin-level in plasma of control and melatonin-supplemented groups was measured by radioimmunoassay using a commercially available kit (Elias, Osceola, WI).

IL-6, IL-1ß, and TNF-{alpha} determination..-- IL-6, IL-1ß, and TNF-{alpha} were measured by ELISA according to the manufacturer's instruction using rat anti-mouse IL-6 or TNF-{alpha} monoclonal antibodies (PharMingen, San Diego, CA) or rat anti-mouse IL-1ß polyclonal antibody (Endogen, Cambridge, MA) and biotinylated rat anti-mouse IL-6, TNF-{alpha}, or IL-1ß antibodies.

Statistical Analysis
Data were analyzed by analysis of variance (ANOVA) for overall effects of diet followed by Fisher's least significant difference post hoc test for individual comparisons using the SYSTAT 1992 statistical package (SYSTAT, Evanston, IL). Student's paired t test was used to test for differences in body weights before and after infection. Kruskal-Wallis nonparametric test was used for data that were not normally distributed. Pearson correlation was used to determine association. Data are reported as mean ± SEM. Significance was set at p < .05. Sample size was calculated to achieve 30% difference in influenza titer with {alpha} = .05 and 80% power using mean and standard deviation from our previous study (11).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Lung Viral Titer and Prevented Weight Loss Following Influenza Virus Infection
As shown in Table 1 , animals fed vitamin E or vitamin E + GSH had significantly higher plasma vitamin E levels compared to those fed the control diet. Animals fed the melatonin diet showed a significant increase in plasma melatonin levels compared to those fed the control diet. Animals fed glutathione-supplemented diet did not have significantly different liver glutathione levels compared to those fed the control diet. This may be due to the fact that the glutathione level was measured from uninfected animals. Most normal cells have a large excess of glutathione. However, decreased tissue concentration of glutathione has been reported in several disease states including Wilson's disease, HIV infection, and influenza virus infection (4)(23). Increased splenocyte-SH content occurred in mice fed 1% GSH only after 68 hours of stimulation with ConA, but no effect of supplementation was seen in the absence of the mitogen (14). Alternatively, the 1% GSH level added to the diet might not have been effective in changing the tightly regulated GSH level in presence of adequate protein levels (24)(25). Animals fed vitamin E + GSH supplemented diet, however, did tend to have higher liver GSH levels compared to those fed the control diet ( p = .09). This might be due to vitamin E's protective effect against the oxidation of glutathione.


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  		Table 1. Effect of Long-term Antioxidant Supplementation on Plasma {alpha}-Tocopherol and Melatonin and Liver Glutathione (GSH) Concentrations

 
There was a significant effect of diets on lung viral titer (ANOVA, p = .026). Lung influenza viral titers were significantly lower ( p = .017) in the vitamin E-supplemented group compared to the control group (Fig. 1). There was no significant difference between viral titers in animals fed the other antioxidant-supplemented diets and those fed the control diet.



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  		Figure 1. Pulmonary viral titers of 24-month-old C57BL/6NCrlBR mice fed different diets for 6 months before infection with H3N2 influenza virus. Mice were euthanized 5 days postinfection. Values are means ± SEM, n = 5. ANOVA result indicates a significant effect of diet ( p = .026). *Significantly different from mice fed the control diet at p = .017 by Fisher's least significant test. VitE = vitamin E, GSH = glutathione.

 
There was no significant difference in baseline weight between different dietary groups (Table 2 ). Weight loss after influenza virus infection in the vitamin E-supplemented group (1.84 ± 0.91 g/5 days postinfection) was significantly less ( p = .016) than that of the group fed the control diet (6.78 ± 1.39 g/5 days postinfection). There was no significant difference in weight loss between mice fed the control diet and that of mice fed the other diets. Mice fed the control diet, GSH-supplemented diet, and melatonin-supplemented diet showed significant weight loss following influenza infection ( p = .008, .008, and .004, respectively) compared to their weight before infection. Vitamin E + GSH-, and strawberry extract-supplemented groups tended to lose weight after infection ( p = .054 and .065, respectively), whereas mice in the vitamin E group did not exhibit a significant weight loss compared to their weight before infection ( p = .114). The weight loss appears to be mainly due to decreased food intake, as there was a highly significant inverse correlation (r = -.96, p < .001; Fig. 2) between weight loss and food intake. Furthermore, the vitamin E-supplemented group had significantly higher food intake during 5 days postinfection compared to other groups ( p < .05; Table 2 ).


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  		Table 2. Effect of Dietary Antioxidant Supplementation on Weight Loss and Food Intake Following Influenza Virus Infection

 


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  		Figure 2. Correlation between weight loss and food intake during 5 days post influenza virus infection. Significant inverse correlation (r = -.96, p < .001, n = 29) was observed by Pearson correlation.

 
Malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE) in Liver
There was a significant effect of antioxidant supplementation on the levels of lipid peroxidation markers in the liver ( p = .001 for MDA, p = .004 for 4-HNE). Mice fed vitamin E- or vitamin E + GSH-supplemented diet had significantly lower levels of MDA and 4-HNE levels in the liver compared to mice fed the control diet. Mice fed the strawberry extract-supplemented diet had significantly lower 4-HNE but not MDA levels than mice fed the control diet (Table 3 ). GSH or melatonin supplementation did not have significant effects on liver 4-HNE and MDA levels.


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  		Table 3. Effect of Dietary Antioxidant Supplementation on Liver 4-Hydroxynonenal (4-HNE) and Malondialdehyde (MDA) Levels

 
Hydrogen Peroxide Production
ANOVA results indicate that H2O2 production by lung cells significantly increased with influenza infection (2.86 ± 0.22 nmoles/mL in Day 0 vs 3.79 ± 0.12 nmoles/mL in Day 5, p = .001). There was no significant overall effect of dietary antioxidant supplementation on H2O2 production before or following infection. However, H2O2 production on Day 0 tended to be lower in vitamin E (2.45 ± 0.5 nmoles/mL, n = 7, p = .076) and melatonin (2.32 ± 0.45 nmoles/mL, n = 5, p = .092) groups compared to the control group (3.63 ± 0.35 nmoles/mL, n = 7). Levels of H2O2 production by GSH (2.71 ± 0.35 nmoles/mL), vitamin E+GSH (2.94 ± 1.02 nmoles/mL), and strawberry (2.84 ± 0.74 nmoles/mL) groups on Day 0 were not significantly different from the control group.

Plasma IL-6 Level
Plasma IL-6 level increased significantly 5 days after influenza infection in all diet groups ( p < .005; Fig. 3). There was, however, no significant effect of dietary antioxidant supplementation on plasma IL-6 levels before or after influenza infection. Furthermore, there was no significant correlation between plasma IL-6 levels and weight loss or food intake.



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  		Figure 3. Plasma IL-6 levels of 24-month-old C57BL/6NCrlBR mice fed different diets for 6 months prior to infection with H3N2 influenza virus. Day 0 (hatched bar) represents results from noninfected mice, and Day 5 (solid bar) represents results from mice 5 days postinfection. Values are means ± SEM, n = 5–7. **Significantly different from Day 0 at p < .05, Kruskal-Wallis nonparametric test. p values are .024, .047, .014, .016, .009, and .014 for control, VitE (vitamin E), GSH (glutathione), VitE + GSH, melatonin, and strawberry groups, respectively.

 
Lung IL-1ß, IL-6, and TNF-{alpha} Levels
ANOVA results indicate that there was a significant effect of influenza infection on IL-1ß levels in lung homogenate ( p = .001). There was no significant effect of dietary antioxidant supplementation on lung IL-1ß levels before or after influenza infection.

As shown in Fig. 4, IL-6 levels in lung homogenate increased significantly with infection ( p < .001). Overall, there was no effect of dietary antioxidant supplementation on IL-6 levels and no significant interaction between infection and diet. However, as only the vitamin E group showed lower viral titer, confirming our previous observations (11), and no significant weight loss following infection, IL-6 levels in each group were compared to that of the control group using a post-hoc test. IL-6 levels following infection were significantly lower in the vitamin E group (1519.5 ± 496.2 pg/mL) compared to the control group (3372.5 ± 419.2 pg/mL, p = .009). Also, a significant positive correlation (r = .77, p < .001) was observed between IL-6 levels and weight loss (Fig. 5).



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  		Figure 4. IL-6 levels in lung homogenate of 24-month-old C57BL/6NCrlBR mice fed different diets for 6 months prior to infection with H3N2 influenza virus. Day 0 (hatched bar) represents results from noninfected mice, and Day 5 (solid bar) represents results from mice 5 days postinfection. Values are means ± SEM, n = 5–7. Overall ANOVA indicates a significant effect of infection ( p < .001) on lung IL-6. Overall, there was no significant effect of diet on lung IL-6. *Significantly different from Day 5 control ( p = .009).

 


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  		Figure 5. Correlation between weight loss and lung IL-6 level. A significant positive correlation (r = .77, p < .001, n = 29) was observed by Pearson correlation.

 
There was a significant effect of infection on TNF-{alpha} levels (ANOVA, p = .005; Fig. 6). There was no main effect of dietary antioxidant supplementation and no significant interaction between infection and diet. However, because TNF-{alpha} has been shown to have significant effects on appetite and body weight, and only the vitamin E group showed no significant weight loss and significantly higher food intake, TNF-{alpha} levels in each group were individually compared to that of the control group using a follow-up test. TNF-{alpha} levels following infection were significantly lower in the vitamin E group (1.66 ± 0.12 U/mL, p = .033) and melatonin group (1.62 ± 0.34 U/mL, p = .030) compared to the control group (3.65 ± 0.46 U/mL). A significant positive correlation (r = .68, p < .001) between TNF-{alpha} levels and weight loss was observed (Fig. 7).



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  		Figure 6. TNF-{alpha} levels in lung homogenate of 24-month-old C57BL/6NCrlBR mice fed different diets for 6 months prior to infection with H3N2 influenza virus. Day 0 (hatched bar) represents results from noninfected mice, and Day 5 (solid bar) represents results from mice 5 days postinfection. Values are means ± SEM, n = 5–7. Overall ANOVA indicates a significant effect of infection (p = .005) on lung TNF-{alpha}. Overall, there was no significant effect of diet on lung TNF-{alpha}. *Significantly different from Day 5 control ( p values are .033 for vitamin E and .030 for melatonin groups).

 


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  		Figure 7. Correlation between weight loss and lung TNF-{alpha} level. A significant positive correlation (r = .68, p < .001, n = 29) was observed by Pearson correlation.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Influenza virus is a significant human pathogen. The clinical expression of infection with influenza virus is variable and greatly influenced by the age, physiological state, and immunological experience of the host. Morbidity and mortality during influenza epidemics are particularly high among elderly people (26). The increased susceptibility of elders to influenza virus infection can be attributed to several factors, including age-associated decline of cell-mediated immune and humoral immune responses and increased oxidative stress with aging. Oxidative stress has been shown to play a role in both pathogenesis and infectivity of influenza virus (4)(9)(27).

Vitamin E, a potent biological antioxidant, has been shown to enhance cell-mediated and humoral immune responses in aged mice and humans (28)(29). We previously demonstrated that vitamin E supplementation in old mice resulted in a significant reduction of lung influenza virus titer after influenza infection (11). In the current study, we tested the effect of long-term supplementation with vitamin E and other dietary and nondietary antioxidants on the course of influenza infection. Of the antioxidants tested, only vitamin E supplementation was effective in decreasing lung viral titer and preventing the weight loss and decreased food intake typically observed with influenza virus infection. GSH, which has been shown to play an important role in the detoxification of ROS and xenobiotics and in regulating immune function (23)(30), had no effect on lung viral titer. Melatonin, which acts as an intracellular scavenger of hydroxyl and peroxyl free radicals (12)(31) and enhances immune response (32), tended to reduce H2O2 production by lung cells, but did not have a significant effect on liver MDA and 4-HNE levels or on lung influenza viral titer. This difference might be due to the differences in the dose (11 mg/kg diet vs 5–10 mg/kg b.w.) or administration route of melatonin [oral vs i.p. injection; (31)(32)]. Strawberry extract has been shown to have high antioxidant activity in vitro, as measured by the ORAC assay (15). Animals fed strawberry extract-supplemented diet had significantly lower levels of liver 4-HNE compared to those fed the control diet, yet supplementation with strawberry extract did not affect lung influenza viral titer.

This lack of effect by other antioxidants on lung viral titer might represent the difference in biological availability of these compounds compared to vitamin E. On the other hand, this lack of an effect suggests that factors other than antioxidant activity might have contributed to the protective effect of vitamin E against influenza infection. This is supported by the observation that viral titers of the animals fed vitamin E + GSH were similar to those of the control, whereas animals fed vitamin E alone had significantly lower viral titer than those fed the control diet despite a similar effect of the two treatments on 4-HNE and MDA levels in the liver. Although hepatic GSH levels tended to be slightly higher in animals fed vitamin E + GSH than in animals fed GSH alone, this higher GSH status did not render any detectable beneficial effects against infection. Rather, GSH abolished vitamin E's beneficial effect. This indicates that there is an interaction between GSH and vitamin E.

H2O2 production in lung increased following influenza infection in this study. Buffinton and colleagues (27) also reported increased H2O2 in the lungs of mice suffering from influenza. Increased production of oxidants can decrease viral clearance by suppressing the immune response. In addition, increased oxidative stress can enhance viral titer by inactivating the antiprotease that affects the activation of virus by proteolytic cleavage (4)(9). In this study, there was no difference in H2O2 production following influenza infection (Day 5) between different dietary groups. On Day 0, vitamin E and melatonin groups tended to have lower H2O2 production compared to the control group. However, only the vitamin E group had significantly lower viral titer. Taken together, these results indicate that vitamin E's effect on lowering viral titer is not solely due to its antioxidant property.

Viral infection results in acute phase responses including lethargy, fever, and anorexia (33). In our study, significant weight loss due to anorexia was observed in all experimental groups except the vitamin E-supplemented group over the 5 days postinfection. The vitamin E-supplemented group consumed about twice as much food and showed about one third of the weight loss of the other groups. The mechanisms by which the host triggers the acute phase responses are not known. Cytokines such as IL-1, IL-6, TNF-{alpha}, and IFN, produced upon viral infection, are capable of causing both fever and anorexia (33)(34). Serum IL-6 levels were shown to be higher in aged than in young mice and to increase following influenza infection (34)(35). Thivierge and Rola-Pleszczynski (36) showed that prostaglandin (PG)E2 can increase the production of IL-6 by macrophages, and vitamin E has been shown to decrease PGE2 and IL-6 production (29)(37). There was no overall effect of dietary antioxidant supplementation on lung IL-6 levels, but mice in the vitamin E group showed significantly lower lung IL-6 levels than those in the control group on Day 5, postinfection. In addition, a significant positive correlation was observed between lung IL-6 levels and weight loss following influenza infection. However, we did not observe a significant effect of antioxidant supplementation on plasma IL-6 levels in this study. In addition, no significant correlation between weight loss and plasma IL-6 levels was observed. Thus, the beneficial effect of vitamin E on weight loss could be explained by the difference in lung IL-6 levels following infection but not by a change in plasma IL-6 levels.

Vitamin E, added in vitro, has been shown to inhibit endotoxin-induced production of TNF-{alpha} by alveolar macrophages and Kupffer cells (38)(39). Dietary supplementation of vitamin E in mice infected with LP-BM5 retrovirus has been shown to decrease the virus-induced elevation of TNF-{alpha} production by lipopolysaccharide (LPS)-stimulated splenocytes (40). In our study, lung TNF-{alpha} levels increased with infection. The vitamin E group had lower levels of pulmonary TNF-{alpha} than the control group after infection although there was no overall effect of dietary antioxidant supplementation on lung TNF-{alpha} levels. In addition, a significant positive correlation was observed between lung TNF-{alpha} levels and weight loss. TNF-{alpha} was not detectable from most of the plasma samples (18/26) in this study (data not shown). Thus, the effect of vitamin E on weight loss might have been mediated through a reduction in TNF-{alpha} and IL-6 production. There was a significant positive correlation between viral titer and lung IL-6 levels (r = .54, p = .004) and a less significant positive correlation between viral titer and lung TNF-{alpha} (r = .33, p = .09).

TNF-{alpha} has a dual role in influenza infection. While it has been shown to have an antiviral property (41), its higher production following infection contributes to pathogenesis of influenza including loss of appetite and weight loss (33)(42). Thus, while the reduction of TNF-{alpha} by vitamin E can contribute to its effect on weight loss, it cannot explain vitamin E-induced reduction of viral titer. Similarly, while reduction of IL-6 by vitamin E supplementation contributes to maintenance of weight, it cannot explain its effect on viral titer, as IL-6 is not known to have direct antiviral effect.

Another possible mechanism of vitamin E's effect on food intake could be suppression of cyclooxygenase products, particularly PGE2. Macrophages and spleens from old mice have significantly higher production of cyclooxygenase products (43)(44). We have previously shown that vitamin E can inhibit cyclooxygenase activity and decrease PGE2 production using similar dietary treatments (29)(45). Shimomura and colleagues (46) showed that suppressed food intake by peripheral administration of IL-1 was partially restored to control levels by ibuprofen, a selective cyclooxygenase inhibitor. Future studies are planned to determine the contribution of IL-6, TNF-{alpha}, and PGE2 in vitamin E-induced prevention of weight loss following influenza infection.

In conclusion, we have demonstrated that long-term vitamin E supplementation is effective in lowering viral titer and preventing decreased food intake and weight loss following influenza infection in aged mice. This beneficial effect was not observed with the other antioxidants tested. Vitamin E's effect on prevention of weight loss following influenza infection appears to be due to reduction in lung IL-6 and TNF-{alpha} levels. While the mechanism of vitamin E's protective effect on viral titer needs to be delineated, these results suggest that mechanisms other than antioxidant protection are likely to be involved.


   Acknowledgments
 
This research was based upon work supported by the U.S. Department of Agriculture, under Agreement 58-1950-9-001. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.

The authors thank Dr. Alison A. Beharka and Robert Cottey for technical assistance and Joanne Meegan for preparation of the manuscript.

Received September 23, 1998

Accepted March 3, 2000


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ruben FL, Dearwater SR, Norden CW, et al. 1995. Clinical infections in the noninstitutionalized geriatric age group: methods utilized and incidence of infections. Am J Epidemiol. 141:145-157. [Abstract/Free Full Text]
  2. Leigh MW, Carson JL, Denny FW, Jr 1991. Pathogenesis of respiratory infections due to influenza virus: implications for developing countries. Rev Infect Dis. 13: (Suppl 6) S501-S508.
  3. Maeda H, Akaike T, 1991. Oxygen free radicals as pathogenic molecules in viral diseases. Proc Soc Exp Biol Med. 198:721-727. [Medline]
  4. Hennet T, Peterhans E, Stocker R, 1992. Alterations in antioxidant defenses in lung and liver of mice infected with influenza A virus. J Gen Virol. 73:39-46. [Abstract/Free Full Text]
  5. Hinshaw VS, Olsen CW, Dybdahl-Sissoko N, Evans D, 1994. Apoptosis: a mechanism of cell killing by influenza A and B viruses. J Gen Virol. 68:3667-3673.
  6. Babior B, 1984. Oxidants from phagocytes: agents of defense and destruction. Blood. 64:959-966. [Free Full Text]
  7. Oda T, Akaike T, Hamamoto T, Suzuki F, Hirano T, Maeda H, 1989. Oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymer-conjugated SOD. Science. 244:974-976. [Abstract/Free Full Text]
  8. Pahl HL, Baeuerle PA, 1995. Expression of influenza virus hemagglutinin activates transcription factor NF-{kappa}B. J Virol. 69:1480-1484. [Abstract]
  9. Peterhans E, 1994. Oxidants and antioxidants in viral diseases: metabolic regulation and autotoxicity. Frei B, , ed.Natural Antioxidants in Human Health and Disease 489-514. Academic Press, San Diego, CA.
  10. Jacoby DB, Choi AMK, 1994. Influenza virus induces expression of antioxidant genes in human epithelial cells. Free Radic Biol Med. 16:821-824. [Medline]
  11. Hayek MG, Taylor SF, Bender BS, et al. 1997. Vitamin E supplementation decreases lung virus titers in mice infected with influenza. J Infect Dis. 176:273-276. [Medline]
  12. Reiter RJ, Pablos MI, Agapito TT, Guerrero JM, 1996. Melatonin in the context of the free radical theory of aging. Ann NY Acad Sci. 786:362-378. [Medline]
  13. Pierpaoli W, Regelson W, 1994. Pineal control of aging: effect of melatonin and pineal grafting on aging mice. Proc Natl Acad Sci USA. 91:787-791. [Abstract/Free Full Text]
  14. Furukawa T, Meydani SN, Blumberg JB, 1987. Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice. Mech Ageing Dev. 38:107-117. [Medline]
  15. Wang H, Cao G, Prior RL, 1996. Total antioxidant capacity of fruits. J Agric Food Chem. 44:701-705.
  16. Bender BS, Croghan T, Zhang L, Small PA, Jr 1992. Transgenic mice lacking class I major histocompatibility complex-restricted T cells have delayed viral clearance and increased mortality after influenza virus challenge. J Exp Med. 175:1143-1145. [Abstract/Free Full Text]
  17. Bender BS, Taylor SF, Zander DS, Cottey R, 1995. Pulmonary immune response of young and aged mice after influenza challenge. J Lab Clin Med. 126:169-177. [Medline]
  18. Reed LJ, Muench H, 1937. A simple method of estimating fifty per cent endpoints. Am J Hygiene. 27:493-497.
  19. Ruch W, Cooper PH, Baggioloni M, 1983. Assay of H2O2 production by macrophages and neutrophils with homovanillic acid and horse-radish peroxidase. J Immunol Methods. 63:347-357. [Medline]
  20. Bieri JG, Tolliver TJ, Catignani GL, 1979. Simultaneous determination of {alpha}-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography. Am J Clin Nutr. 32:2143-2149. [Abstract/Free Full Text]
  21. Brigelius R, Muckel C, Akerboom TP, Sies H, 1983. Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide. Biochem Pharmacol. 32:2529-2534. [Medline]
  22. Meydani SN, Shapiro AC, Meydani M, Macauley JB, Blumberg JB, 1987. Effect of age and dietary fat (fish, corn and coconut oils) on tocopherol status of C57BL/6Nia mice. Lipids. 22:345-350. [Medline]
  23. Taylor CG, Nagy LE, Bray TM, 1996. Nutritional and hormonal regulation of glutathione homeostasis. Curr Top Cell Regul. 34:189-208. [Medline]
  24. Bray TM, Taylor CG, 1993. Tissue glutathione, nutrition, and oxidative stress. Can J Physiol Pharmacol. 71:746-751. [Medline]
  25. Bauman PF, Smith TK, Bray TM, 1988a. The effect of dietary protein and sulfur amino acids on hepatic glutathione concentration and glutathione-dependent enzyme activities in the rat. Can J Physiol Pharmacol. 66:1048-1052. [Medline]
  26. Kilbourne ED, 1987. Influenza in man. Kilbourne E D, , ed.Influenza 157-218. Plenum Medical Book Company, New York.
  27. Buffinton GD, Christen S, Peterhans E, Stocker R, 1992. Oxidative stress in lungs of mice infected with influenza A virus. Free Radic Res Commun. 16:99-110. [Medline]
  28. Meydani SN, Barklund MP, Liu S, et al. 1990. Vitamin E supplementation enhances cell-mediated immunity in healthy elderly subjects. Am J Clin Nutr. 52:557-563. [Abstract/Free Full Text]
  29. Meydani SN, Meydani M, Verdon CP, Shapiro AA, Blumberg JB, Hayes KC, 1986. Vitamin E supplementation suppresses prostaglandin E2 synthesis and enhances the immune response of aged mice. Mech Ageing Dev. 34:191-201. [Medline]
  30. Droge W, Schulze-Osthoff K, Mihm S, et al. 1994. Functions of glutathione and glutathione disulfide in immunology and immunopathology. FASEB J. 8:1131-1138. [Abstract]
  31. Melchiorri D, Reiter RJ, Sewerynek E, Hara M, Chen L, Nistico G, 1996. Paraquat toxicity and oxidative damage. Reduction by melatonin. Biochem Pharmacol. 51:1095-1099. [Medline]
  32. Maestroni GJM, Conti A, Pierpaoli W, 1987. The pineal gland and the circadian, opiatergic, immunoregulatory role of melatonin. Ann NY Acad Sci. 496:67-77. [Medline]
  33. Conn CA, McClellan JL, Massab HF, Smitka CW, Majde JA, Kluger MJ, 1995. Cytokines and the acute phase response to influenza virus in mice. Am J Physiol. 268:R78-R84. [Abstract/Free Full Text]
  34. Hennet T, Ziltener HJ, Frei K, Peterhans E, 1992. A kinetic study of immune mediators in the lungs of mice infected with influenza A virus. J Immunol. 149:932-939. [Abstract]
  35. Daynes RA, Araneo BA, Ershler WB, Maloney C, Li G-Z, Ryu S-Y, 1993. Altered regulation of IL-6 production with normal aging. Possible linkage to the age-associated decline in dehydroepiandrosterone and its sulfated derivative. J Immunol. 150:5219-5230. [Abstract]
  36. Thivierge M, Rola-Pleszczynski M, 1994. Involvement of both cyclooxygenase and lipoxygenase pathways in platelet-activating factor-induced interleukin-6 production by alveolar macrophage. Ann NY Acad Sci. 725:213-222. [Medline]
  37. Cannon JG, Meydani SN, Fielding RA, et al. 1991. Acute phase response in exercise. II. Associations between vitamin E, cytokines, and muscle proteolysis. Am J Physiol. 260:R1235-R1240. [Abstract/Free Full Text]
  38. Mendez C, Garcia I, Maier RV, 1995. Antioxidants attenuate endotoxin-induced activation of alveolar macrophages. Surgery. 118:412-420. [Medline]
  39. Fox ES, Brower JS, Bellezzo JM, Leingang KA, 1997. N-Acetylcysteine and {alpha}-tocopherol reverse the inflammatory response in activated rat kupffer cells. J Immunol. 158:5418-5423. [Abstract]
  40. Wang Y, Huang DS, Liang B, Watson RR, 1994. Nutritional status and immune responses in mice with murine AIDS are normalized by vitamin E supplementation. J Nutr. 124:2024-2032.
  41. Van Campen H, 1994. Influenza A virus replication is inhibited by tumor necrosis factor-alpha in vitro. Arch Virol. 136:439-446. [Medline]
  42. Peper RL, Van Campen H, 1995. Tumor necrosis factor as a mediator of inflammation in influenza A viral pneumonia. Microb Pathog 19:175-183. [Medline]
  43. Hayek MG, Meydani SN, Meydani M, Blumberg JB, 1994. Age differences in eicosanoid production of mouse splenocytes: effects on mitogen-induced T-cell proliferation. J Gerontol Biol Sci. 49:B197-B207.
  44. Beharka AA, Wu D, Han SN, Meydani SN, 1997. Macrophage prostaglandin production contributes to the age-associated decrease in T cell function which is reversed by the dietary antioxidant vitamin E. Mech Ageing Dev. 93:59-77. [Medline]
  45. Wu D, Han SN, Meydani SN, 1996. Vitamin E supplementation inhibits macrophage cyclooxygenase activity of old mice. FASEB J. 10:A191
  46. Shimomura Y, Inukai T, Kuwabara S, et al. 1992. Both cyclooxygenase and lipoxygenase inhibitor partially restore the anorexia by interleukin-1ß. Life Sci. 51:1419-1426. [Medline]



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