Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
JID 1997;176 (July) Concise Communications 12. HIV/AIDS Quarterly Epidemiology Report. 2nd Quarter. Seattle, WA: Seattle – King County Department of Health, 1996. 13. Musiani M, Zerbini M, Gentilomi G, Plazzi M, Gallinella G, Venturoli S. Parvovirus B19 clearance from peripheral blood after acute infection. J Infect Dis 1995; 172:1360 – 3. 273 14. Musiani M, Zerbini M, Gentilomi G, et al. Persistent B19 parvovirus infections in haemophilic HIV-1 infected patients. J Med Virol 1995;46:103–8. 15. Patou G, Pillay D, Myint S, Pattison J. Characterization of a nested polymerase chain reaction assay for detection of parvovirus B19. J Clin Microbiol 1993; 31:540 – 6. Vitamin E Supplementation Decreases Lung Virus Titers in Mice Infected with Influenza Michael G. Hayek,* Scott F. Taylor, Bradley S. Bender, Sung Nim Han, Mohsen Meydani, Donald E. Smith, Shahriar Eghtesada, and Simin Nikbin Meydani Nutritional Immunology Laboratory, Vascular Biology Laboratory, and Department of Comparative Biology and Medicine, Jean Mayer Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts; Department of Medicine and Geriatric Research, Education and Clinical Center, Veterans Affairs Medical Center, Gainesville, Florida 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. Infections, particularly those affecting the respiratory system, rank among the leading causes of death in older adults [1]. Like humans, aged mice have more severe disease with an influenza infection [2], and most of our knowledge of the pathogenesis of and the host defenses against influenza was first established in mice and later confirmed in humans [2]. Many factors contribute to recovery from influenza infection. The most important is cellular immunity, in particular antiinfluenza cytotoxic T lymphocyte (CTL) activity. In aged mice, lower CTL activity correlates with prolonged duration of dis- Received 5 August 1996; revised 2 December 1996. 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. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government. Grant support: US Department of Agriculture (Agricultural Research Service, contract 53-K06-01). Reprints or correspondence: Dr. Simin Nikbin Meydani, Nutritional Immunology Laboratory, JM USDA HNRCA at Tufts University, 711 Washington St., Boston, MA 02111. * Present address: The Iams Company, P.O. Box 189, Lewisburg, OH 45338. The Journal of Infectious Diseases 1997;176:273–6 q 1997 by The University of Chicago. All rights reserved. 0022–1899/97/7601–0039$02.00 ease [2]. Other immune functions that appear to aid in recovery from influenza include NK cell activity, high titers of neutralizing serum antibody, and, possibly, mucosal IgA [2]. Influenza infection in mice causes a decrease in lung and liver levels of the antioxidant nutrients [3]. One of the biologic changes associated with aging is a decrease in antioxidant defense status, which results in increased free radical formation and lipid peroxidation. Increased lipid peroxidation, including that of prostaglandin E2 (PGE2) production, has been implicated in the age-associated dysregulation of cytokine production and decrease in lymphocyte proliferation and CTL and NK cell activity [4]. Influenza infection is associated with the release of a variety of cytokines and eicosanoids in mice [5]. Vitamin E supplementation increases delayed-type hypersensitivity skin response, T cell proliferation, and interleukin (IL)-2 production [6, 7] and decreases PGE2 production and plasma lipid peroxide levels. Vitamin E supplementation also prevents antigen-induced decreased NK cell activity in old mice [8]. Therefore, in the present study we determined whether vitamin E supplementation would reduce lung virus titers in young and aged mice infected with influenza virus, and if so, whether this would be due to its effect on CTL, NK cell activity, or antibody titer. Materials and Methods Animals. Specific pathogen–free male young (4 months old) and old (22 months old) C57BL/6NIA mice were obtained from 274 Concise Communications NIA colonies at Charles River Laboratories (Kingston, NY). Mice were housed singly in microisolator cages at a constant temperature (237C) with a 12-h light-dark cycle and fed ad libitum with a semipurified diet supplemented with either 30 (an adequate level of vitamin E) or 500 ppm of dl-a-tocopherol acetate (vitamin E) for 6 weeks [6]. We previously showed that there was no difference in food intake or weight gain between mice fed 30 or 500 ppm of vitamin E [6]. After the 6-week dietary period, mice were infected with Influenza A/Port Chalmers/1/73 (H3N2) virus according to the method of Bender et al. [9]. Mice were then sacrificed via CO2 asphyxiation 0, 2, 5, or 7 days after infection. Tissue preparation. Noses and lungs were removed and processed as previously described [10]. Livers were perfused with 3 mL of ice-cold sterile PBS, removed, immediately wrapped in foil, and placed in liquid nitrogen. NK cell and CTL activity. Lung cells were enriched for T cells by adding the cell suspensions to nylon wool columns and incubating them at 377C for 1 h, eluting nonadherent cells from the columns, and washing once in supplemented Iscove’s medium. Enriched T cells were resuspended in 10-mL of medium, counted using trypan blue dye exclusion, and adjusted to 106/mL. These cells were subsequently used for either NK cell (2 days after infection) or CTL (7 days after infection) assays. Stein-Streilein et al. [11] reported optimal NK cell activity 2 days after infection. Our preliminary experiment using samples from days 5, 6, 7, 8, 9, and 11 after infection showed optimal CTL activity on day 7 after infection. NK cell activity against Yac-1 target cells was assessed as previously described [8]. The CTL assays were performed as previously described [9]. Target cells (LB27) were sensitized with influenza A/Port Chalmers/1/73 (H3N2) virus (107.5 EID50) or influenza B virus and incubated at 377C, 5% CO2 for 1.25 h with occasional mixing. The final effector-to-target ratio for this assay was 20:1. Virus titers. Virus titers for lung and nose samples were measured as previously described [9, 10]. Serum antibody. Sera were frozen, and a hemagglutination inhibition assay was performed [12]. Statistical analysis. Data were analyzed using the SYSTAT statistical package (SYSTAT, Evanston, IL) by a 2 1 2 factorial two-tailed analysis of variance with individual differences analyzed by single degree of freedom comparison using the Fisher’s least significant difference procedure and are reported as mean { SE. Significance was set at P õ.05. Results Virus titers. Following influenza challenge of healthy young mice, the virus reached a peak pulmonary titer of Ç103 – 104 TCID50 on days 2 – 5 after inoculation and was cleared from the lungs by day 7 (figure 1). As previously reported [2, 9, 10], aging had a significant impact on viral clearance, with significantly higher virus titers in old mice than in young mice on days 2 and 7 after infection (P õ.01). Vitamin E supplementation decreased virus titers of young mice only on day 5 (figure 1) but significantly decreased the virus titers of old mice on JID 1997;176 (July) Figure 1. Pulmonary virus titers of young (4 months) and old (22 months) C57Bl/6NIA mice fed 30 or 500 ppm of vitamin E and given total respiratory tract infection with H3N2 influenza virus. Mice were fed respective diets for 6 weeks, when they were infected intranasally and sacrificed 0, 2, 5, and 7 days after infection. Lungs were excised, tissue was homogenized, and fluid was frozen for virus titer analysis. Means { SE are shown. Nos. of animals per group were as follows: 7, 8, 4, and 6 for day 2; 7, 9, 4, and 6 for day 5; and 6, 5, 5, and 6 for day 7 for young mice receiving 30 ppm, young mice receiving 500 ppm, old mice receiving 30 ppm, and old mice receiving 500 ppm, respectively. Means not sharing same letters above bars on given day are significantly different at P õ.01 for day 2 and õ.05 for days 5 and 7. all days. Even on day 5, vitamin E had a more dramatic effect on viral clearance in aged than in young mice. Titers were 25fold lower in old vs. 15-fold lower in young mice supplemented with 500 ppm of vitamin E than in old and young mice fed the control diet (30 ppm of vitamin E). Vitamin E had no impact on nasal virus titer (data not shown). Immune response. Primary pulmonary CTL activity was measured against influenza H3N2-sensitized LB27 cells. In contrast to results of our previous studies with aged female mice [2], no age-associated decline in pulmonary CTL activity was observed in male C57BL/6NIA mice. In addition, vitamin E supplementation did not affect CTL activity in either young or old animals (50.1 { 8.9, n Å 7; 48.8 { 9.7, n Å 8; 47.1 { 8.9, n Å 4; 42.6 { 8.9, n Å 6 in young mice consuming 30 ppm of vitamin E, young mice consuming 500 ppm of vitamin E, old mice consuming 30 ppm of vitamin E, and old mice consuming 500 ppm of vitamin E, respectively). The fact that the CTLs exhibited very low lysis of influenza B – sensitized LB27 cells (11% – 16%) demonstrated that we were measuring specific antiinfluenza A CTL activity. JID 1997;176 (July) Concise Communications The NK cell activity was significantly lower (P õ .01) in old animals fed 30 ppm of vitamin E (6.34 { 6.03, n Å 7). Vitamin E supplementation had no significant effect on NK activity in young animals (nonsignificant decrease; 14.51 { 4.92, n Å 8 in young mice fed 500 ppm of vitamin E vs. 28.25 { 5.4, n Å 7 in young mice fed 30 ppm of vitamin E); however, it tended to enhance NK cell activity in old mice, such that NK cell activity in the old animals fed 500 ppm of vitamin E (18.18 { 5.3, n Å 6) was not significantly different from that of young mice fed 30 ppm of vitamin E (28.25 { 5.4, n Å 7). Serum antiinfluenza hemagglutinin (HA) inhibition titers were measured 2, 5, and 7 days after infection. Aged animals had significantly lower serum titers (P õ .001, data not shown) than young mice. Vitamin E – supplemented animals tended to have higher serum titers than control mice (difference not statistically significant, data not shown). Discussion The most important finding of this study is that old mice supplemented with 500 ppm of vitamin E have significantly lower virus titers following influenza challenge than old animals fed normal dietary levels of vitamin E (30 ppm). This effect of vitamin E does not appear to be mediated through enhanced CTL activity, T cells that are important in viral clearance. On the other hand, preservation of NK cell activity and antioxidant status may contribute to the observed effect. To our knowledge, this is the first time that a higher than adequate intake of a nutrient has demonstrated a beneficial effect on influenza in animals. The only other dietary intervention that has demonstrated any protective effect against influenza infection in old rodents is caloric restriction (40% reduction), an intervention that is unlikely to be practical for most humans [13]. The biologic effect of food restriction in the aged is due, at least in part, to its reduction of oxidative stress [14]. In this experiment, vitamin E was more effective in reducing virus titers in old mice that show higher oxidative stress than in young mice. The mechanism for the effect of vitamin E on reducing influenza virus titers could not be determined from our experiments. We originally hypothesized that the effect of vitamin E would be mediated through enhanced CTL activity, but no such effect was observed. Nevertheless, this does not rule out the possibility that vitamin E might influence pulmonary CTL activity in vivo. We eliminated macrophages from the CTL preparation, and thus, the main source of prostaglandins, which have been shown to inhibit CTL activity. We previously showed that cyclooxygenase and 5-lipoxygenase products increase with age and that supplementation with vitamin E decreases their production [6, 7]. Therefore, it is feasible that elimination of macrophages in our CTL culture system eliminated vitamin E – induced differences in CTL activity. In this experiment, only one effector-to-target ratio was used in the CTL assay. It 275 is possible that vitamin E could be effective in enhancing CTL activity at lower effector-to-target ratios. Vitamin E supplementation prevented an age-associated decrease in NK cell activity of old mice, which suggests that the effect of vitamin E in old mice may be mediated through the preservation of NK cell activity. NK cell activity decreases in old mice, and NK cells may play a role in influenza infection [11]. Influenza virus infection has been shown to increase production of cytokines, including IL-1, IL-4, IL-6, interferon-g, and tumor necrosis factor, which may contribute to the pathogenesis of the disease [3]. Treatment with antioxidant enzymes was shown to decrease pathogenicity of influenza virus [15]. Nuclear transcription factor kB (NF-kB) activation is needed for expression of mRNA for several of these cytokines. A recent report indicated that influenza virus HA activation of NF-kB might be involved in the influenza-induced increase in cytokine production. However, HA-induced activation of NF-kB was inhibited by the antioxidant dithiothreitol [16]. Other studies have shown that vitamin E supplementation of old subjects inhibited IL-6 production and prevented exercise-induced increases in IL-1 and tumor necrosis factor production by peripheral blood mononuclear cells [17]. Thus, the beneficial effect of vitamin E might be mediated through modulation of cytokines involved in the pathogenesis of influenza virus. Clearly, antioxidant status is important for protection against influenza. This may explain the greater protective effect of vitamin E in old rather than young mice. Since one of the biologic changes associated with aging is increased accumulation of free radicals, leading to increased oxidative stress, old animals exposed to virally induced oxidative stress may require higher levels of antioxidant nutrients to control viral replication to the same level as in young animals. This is supported by our preliminary observation (Han et al., unpublished data) that lungs from influenza-infected mice have significantly higher H2O2 production and lower vitamin E levels than noninfected mice and that lungs from old animals have higher zymosanstimulated H2O2 production. Furthermore, vitamin E supplementation decreased H2O2 production in old animals. In conclusion, our results show that supplementation with 500 ppm of vitamin E decreases lung influenza virus titers on days 2, 5, and 7 after infection in old mice. This observation could be of great clinical relevance to the elderly, who have higher morbidity and mortality due to influenza. Further research to delineate the mechanism of the effect of vitamin E and determine the effectiveness of vitamin E supplementation in older humans is warranted. Acknowledgments We thank Timothy S. McElreavy for preparation of this manuscript and Robert Cottey for technical assistance. References 1. Yoshikawa TT. Impact of aging on host to infectious disease. In: Geriatric clinical pharmacology. Wood WG, Strong R, eds. Raven Press, 1987: 107 – 13. 276 Concise Communications 2. Bender BS, Small PA. Influenza: pathogenesis and host defense. Semin Respir Infect 1992; 7:38 – 45. 3. Hennet T, Peterhans E, Stocker R. Alterations in antioxidant defenses in lung and liver of mice infected with influenza A virus. J Gen Virol 1992; 73:39 – 46. 4. Meydani SN, Hayek M. Vitamin E and immune response. In: Chandra RK, ed. Proceedings of the International Congress on Nutrition and Immunity. St. Johns, Canada: ARTS Biomedical Publishers and Distributors, 1992:105 – 28. 5. Hennet T, Ziltener HT, Frei K, Peterhans E. A kinetic study of immune mediators in the lungs of mice infected with influenza A virus. J Immunol 1992; 149:932 – 9. 6. Meydani SN, Meydani M, Verdon CP, Shapiro AC, Blumberg JB, Hayes KC. Vitamin E supplementation suppresses prostaglandin E2 synthesis and enhances the immune response of aged mice. Mech Ageing Dev 1986; 34:191 – 201. 7. Meydani SN, Barklund MP, Liu S, et al. Vitamin E supplementation enhances cell-mediated immunity in healthy elderly subjects. Am J Clin Nutr 1990; 52:557 – 63. 8. Meydani SN, Stocking LM, Shapiro AC, Meydani M, Blumberg JB. Fish oiland tocopherol-induced changes in ex vivo synthesis of spleen and lung leukotriene B4 (LTB4) in mice. Ann NY Acad Sci 1988;524:395–7. 9. Bender BS, Johnson MP, Small PA. Influenza in senescent mice: impaired cytotoxic T-lymphocyte activity is correlated with prolonged infection. Immunology 1991; 72:514 – 9. JID 1997;176 (July) 10. Bender BS, Small PA. Heterotypic immune mice lose protection against influenza virus infection with senescence. J Infect Dis 1993; 168:873 – 80. 11. Stein-Streilein J, Guffee J. In vivo treatment of mice and hamsters with antibodies to asialo GM1 increases morbidity and mortality to pulmonary influenza infection. J Immunol 1986; 136:1435 – 41. 12. Salk JE. Simplified procedure for titrating hemagglutinating capacity of influenza virus and the corresponding antibody. J Immunol 1944; 49: 87 – 98. 13. Effros RB, Walford RL, Weindruch R, Mitcheltree C. Influences of dietary restriction on immunity to influenza in aged mice. J Gerontol 1991; 46: B142 – 7. 14. Shigenaga MK, Ames BN. Oxidants and mitochondrial decay in aging. In: Frei B, ed. Natural antioxidants in human health and disease. New York, NY: Academic Press, 1994:84 – 8. 15. Akaike T, Ando M, Oda T, et al. Dependence on O2 generation by xanthine oxidase of pathogenesis of influenza virus infection in mice. J Clin Invest 1990; 85:739 – 45. 16. Pahl HL, Baeuerle PA. Expression of influenza virus hemagglutinin activates transcription factor NF- kB. J Virol 1995; 69: 1480 – 4. 17. Cannon JG, Meydani SN, Fielding RA, et al. Acute phase response in exercise. II. Associations between vitamin E, cytokines, and muscle proteolysis. Am J Physiol 1991; 260:R1235 – 40.