Download Vitamin E Supplementation Decreases Lung Virus Titers in Mice

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.