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The Journal of Nutrition
Nutritional Immunology
Zinc Supplementation Increases Zinc Status and
Thymopoiesis in Aged Mice
1,2
Carmen P. Wong,
3
Yang Song,
3
Valerie D. Elias,
3
Kathy R. Magnusson,
4
and Emily Ho
3,5
*
3
Department of Nutrition and Exercise Sciences, Oregon State University, OR 97331; and
4
Department of Biomedical Sciences, and
5
Linus Pauling Institute, Oregon State University, Corvallis, OR 97331
Abstract
The age-related decline in lymphocyte development and function coincides with impaired zinc status in the elderly. Thymic
involution and reduced immune responsiveness are classic hallmarks of both aging and zinc deficiency, resulting in
decreased host defense and an increased susceptibility to infections. Thus, compromised zinc status associated with
aging may be an important contributing factor in reduced thymopoiesis and impaired immune functions. Our goal in this
study was to understand how dietary zinc supplementation affects thymopoiesis in aged mice. We hypothesized that
impaired zinc status associated with aging would mediate the decline in thymic function and output and that restoring
plasma zinc concentrations via zinc supplementation would improve thymopoiesis and thymic functions. In this study,
groups of young (8 wk) and aged (22 mo) mice were fed a zinc-adequate (30 mg/kg zinc) or zinc-supplemented diet (300
mg/kg) for 25 d. Aged mice had impaired zinc status, with zinc supplementation restoring plasma zinc to a concentration
not different from those of young male C57Bl/6 mice. Zinc supplementation in aged mice improved thymopoiesis, as
assessed by increased total thymocyte numbers. In addition, improved thymic output was mediated in part by reducing
the age-related accumulation of immature CD4
2
CD8
2
CD44
+
CD25
2
thymocytes, as well as by decreasing the expression
of stem cell factor, a thymosuppressive cytokine. Taken together, our results showed that in mice, zinc supplementation
can reverse some age-related thymic defects and may be of considerable benefit in improving immune function and overall
health in elderly populations. J. Nutr. 139: 1393–1397, 2009.
Introduction
Age-related decline in immune function encompasses multiple
defects (1). Among them, thymic involution and reduced T-cell
production are one of the most recognized hallmarks of aging.
Whereas the age-dependent changes in thymus function have
been well described, factors important in controlling the process
remain to be fully elucidated. The progressive age-related decline
in thymic function and output coincides with reduced zinc status
and suppressed immune responses in the elderly population.
Thus, the reduced zinc status that occurs with aging may play an
important role in mediating reduced thymopoiesis and contribute
to a progressive decline in immune responsiveness. This culmi-
nates in a higher incidence of infection, cancer, and autoimmune
diseases with increasing age (2–4).
Zinc is a key component for the functions of numerous
proteins and is an essential micronutrient required for numerous
cellular processes. In particular, zinc is necessary for the normal
development and function of the immune system (5). Alterations
in dietary zinc intake, zinc uptake, retention, or secretion can
lead to zinc deficiency and affect zinc-dependent functions. Zinc
deficiency can significantly depress immune response and impair
host defense (6). Zinc homeostasis is critically involved in the
signaling events in immune cells and changes in zinc status affect
multiple immune cell types involved in both innate and adap-
tive immunity (7–9). Severe zinc deficiency dramatically affects
the development of the immune system, resulting in immune
dysfunction. Hallmarks of severe zinc deficiency include thymic
involution, lymphopenia, and accelerated apoptosis in lympho-
cytes. One population particularly at risk for zinc deficiency is
the elderly, who have impaired zinc absorption and reduced
dietary intake (10,11). Elderly patients with reduced zinc status
have higher frequencies of infections (12) and reduced immune
responses to vaccinations (13–15). On the other hand, restoring
normal zinc levels in individuals with low zinc status via zinc
supplementation can improve T-cell–mediated functions and
decrease the incidence of infections in the elderly (16–18).
Zinc supplementation has been shown to reverse thymus
involution in aged mice (19,20). In human studies, zinc supple-
mentation improved immune functions in the elderly. However,
the mechanism of how this is accomplished is currently unclear.
In particular, whether improving zinc status through zinc supple-
mentation would improve thymopoiesis and thymic functions in
aged individuals remains to be investigated. We studied the
1
Supported by Oregon Agricultural Experiment Station (OR00735) and the
Environmental Health Science Center at Oregon State University (NIEHS P30
ES00210) and Linus Pauling Institute Pilot Grant Program.
2
Author disclosures: C. P. Wong, Y. Song, V. D. Elias, K. R. Magnusson, and
E. Ho, no conflicts of interest.
* To whom correspondence should be addressed. E-mail: emily.ho@
oregonstate.edu.
0022-3166/08 $8.00 ã2009 American Society for Nutrition.
Manuscript received February 19, 2009. Initial review completed February 26, 2009. Revision accepted May 5, 2009. 1393
First published online May 27, 2009; doi:10.3945/jn.109.106021.
effects of zinc supplementation on thymic development and
function in aged mice. We hypothesized that the impaired zinc
status of the elderly plays an important role in the age-related
decline in thymopoiesis and that zinc supplementation of an
aged population would improve zinc status and have direct
effects on thymic health by reversing age-related defects in the
thymus.
Materials and Methods
Mice, diets, and study design. Young (8 wk) C57Bl/6 male mice were
purchased from Jackson Laboratory. Aged (22 mo) C57Bl/6 male mice
were purchased from the National Institute on Aging. Mice were housed
in stainless steel suspended cages in a temperature- and humidity-
controlled environment and randomly assigned to either a zinc-adequate
(ZA)
6
diet containing 30 mg/kg zinc or a zinc-supplemented (ZS) diet
containing 10 times the zinc concentration (300 mg/kg zinc) of the ZA
diet that was previously shown to be well tolerated (21). Purified diets
were purchased from Research Diets and were custom prepared using an
egg white-based AIN-93G diet with zinc provided as zinc carbonate
(Table 1) (22). Mice were fed the assigned diets for 25 d and consumed
food and water ad libitum. The dietary intakes and body weights of all
mice were monitored throughout the study. At the termination of the
experiments, mice were killed by CO
2
asphyxiation. Blood was collected
for plasma isolation. Thymus from each mouse was collected and made
into single cell suspensions. Cell suspensions were passed through a 70-
mm cell strainer to remove cell debris and prepared for cell counting and
flow cytometry analysis. The animal protocol was approved by the
Oregon State University Institutional Laboratory Animal Care and Use
Committee.
Plasma zinc concentrations. Plasma zinc concentrations were mea-
sured using inductively coupled plasma-optical emission spectroscopy
as previously described, with minor modification (23). Briefly, plasma
samples (100 mL) were added to 1 mL 70% ultrapure nitric acid and
incubated overnight. Incubated samples were diluted with chelex-treated
nanopure water to a final concentration of 7% nitric acid, centrifuged
at 3000 3g; 1 min at 258C, and analyzed using the Prodigy High
Dispersion inductively coupled plasma-optical emission spectroscopy
instrument (Teledyne Leeman Labs) against known standards.
Cell counts and flow cytometry analysis. Thymocyte numbers were
determined using the Z1 Coulter Particle counter (Beckman Coulter).
After counting, one-half of the thymocytes were used for RNA isolation
and the remaining thymocytes were used in flow cytometry. For flow
cytometry analysis, thymocytes were resuspended in flow cytometry
buffer (PBS, 2% fetal bovine serum, 1 mmol/L EDTA). Cells were
incubated with CD4-FITC, CD25-PE, CD44-PerCP-Cy5.5, and CD8-
APC for 30 min on ice in the dark. All antibodies were purchased from
eBioscience. After extensive washing, cells were resuspended in buffer
for flow cytometry acquisition and analysis. A minimum of 300,000
events in the lymphocyte gate were collected. Data were acquired using
FACSCalibur (BD Biosciences). Data analyses were performed using
Summit software (DakoCytomation).
RNA isolation, cDNA synthesis, and real-time quantitative PCR.
Total RNA from thymocytes was isolated using Trizol reagent
(Invitrogen). One microgram of total RNA was reverse transcribed
into cDNA using SuperScript III First-Strand Synthesis SuperMix for
quantitative real-time PCR (Invitrogen). Real-time PCR was performed
using primers specific for mouse stem cell factor (SCF) (forward:
59-CAACTGCTCCTATTTAATCCTC-39, reverse: 59-TGTATTACCA-
TATCTCGTAGCC-39), interleukin-7 (IL-7) (forward: 59-CTAACAG-
TATCACAAGGCACAC-39, reverse: 59-TCAACCTCTCCAAGTATAT-
GAACC-39), or 18S ribosomal RNA (forward: 59-CCGCAGCTAG-
GAATAATGGAAT-39, reverse: 59-CGAACCTCCGACTTTCGTTCT-
39). Real-time PCR were performed using DyNAmo HS SYBR Green
qPCR kit (New England Biolabs). Gene copies were determined using
the standard curve method. A standard curve was generated from serial
dilutions of purified plasmid DNA that encoded for each gene of interest.
Data represent the copy number of the gene of interest normalized to the
copy number of the 18S ribosomal RNA housekeeping gene in individual
mice.
Statistical analysis. Data are reported as means 6SEM. The main
effects of age and dietary zinc and their interaction were analyzed using
2-way ANOVA, followed by the Bonferroni post hoc test when the
interaction was significant. Where necessary, data were log transformed
to correct for unequal variances prior to statistical analyses. Non-
transformed data are shown in tables and figures. All analyses were
performed using GraphPad Prism version 4.01. Significance was defined
as P,0.05.
Results
Plasma zinc concentration. Both dietary zinc and age affected
zinc status as assessed by the plasma zinc concentration (Fig.
1A). It was lower in aged mice than in young mice and was
greater in ZS mice than ZA mice. The greater plasma zinc in the
ZS mice was not due to differences in food intake or body weight
gain; these variables did not differ between ZS and ZA mice of
either age (data not shown).
Total and double negative thymocyte numbers. Dietary
zinc, age, and their interaction affected the number of thymo-
cytes (Fig. 1B). As expected, aged mice had fewer thymocytes
than young mice. Zinc supplementation did not alter thymocyte
numbers in young mice but resulted in a significant 52% increase
in thymocytes in aged mice.
We evaluated whether zinc status affected thymocyte devel-
opment in young and aged mice. Thymocyte maturation is
divided into 4 main differentiation stages based primarily on the
TABLE 1 Diet composition
1
ZA diet ZS diet
g/kg
Egg white, spray dried 203 203
L-Histidine 0 0.04
Corn starch 494.5 494.5
Maltodextrin 10 35 35
Sucrose 100 100
Cellulose 50 50
Soybean oil 70 70
t-Butylhydroquinone 0.014 0.014
Mineral mix S19409 (no added Ca, P, K, or Zn)
2
77
Potassium phosphate, monobasic 6.86 6.86
Calcium carbonate 8 8
Potassium citrate 2.48 2.48
Calcium phosphate 6 6
Vitamin mix V10037
2
10 10
Biotin, 1% 0.4 0.4
Choline bitartrate 2.5 2.5
Zinc carbonate (52.1% zinc) 0.058 0.58
1
Final zinc concentration is 30 mg/kg zinc in the ZA diet and 300 mg/kg zinc in the ZS
diet.
2
Mineral and vitamin mix as previously described (22).
6
Abbreviations used: DN, double negative; DP, double positive; IL-7,
interleukin-7; SP, single positive; SCF, stem cell factor; ZA, zinc adequate; ZS,
zinc supplemented.
1394 Wong et al.
expression of CD4 and CD8. The earliest immature thymocytes
are double negative (DN) thymocytes (CD4
2
CD8
2
), which tran-
sition to immature double positive (DP) (CD4
+
CD8
+
) thymo-
cytes, and finally differentiate into mature single positive (SP)
CD4
+
CD8
2
and CD8
+
CD4
2
thymocytes that exit the thymus
and enter the bloodstream (Fig. 2A). Dietary zinc and age, but
not their interaction, specifically affected DN thymocytes; there
were more in aged mice than in young mice (Table 2). Zinc
supplementation resulted in a lower frequency of DN thymo-
cytes compared with ZA mice. DP and SP thymocytes were not
affected by dietary zinc in aged or young mice.
DN1 thymocyte subsets. The development of DN thymocytes
can be further divided into 4 distinct maturation steps based on
the expression of CD44 and CD25, namely DN1 (CD44
+
CD25
2
),
DN2 (CD44
+
CD25
+
), DN3 (CD44
2
CD25
+
), and DN4
(CD44
2
CD25
2
) (24). During aging, there is a specific blockade
in thymocyte development that prevents DN1-DN2 transition,
resulting in the accumulation of DN1 thymocytes (Fig. 2A)
(25,26). Dietary zinc and age, but not their interaction, specif-
ically affected DN1 thymocytes (Fig. 2B). As expected, aged
mice had a higher frequency of the DN1 subset than young mice.
Zinc supplementation reduced the proportion of DN1 thymo-
cytes compared with ZA mice.
Thymic cytokines. Dysregulation of various thymic cytokines,
including SCF and IL-7, have been associated with thymic
atrophy and the suppression of thymopoiesis during aging (27).
Dietary zinc, age, and their interaction affected thymic SCF
expression (Fig. 1C). Specifically, SCF expression was greater in
the thymus of aged mice than in young mice. Zinc supplemen-
tation in young mice did not alter SFC expression but signifi-
cantly reduced SCF expression in aged mice. Neither age nor
zinc supplementation affected thymic IL-7 expression (data not
shown).
Discussion
There is a grow ing body of evidence tha t suggests that the impair ed
zinc status associated with aging may directly be involved in age-
related immunological decline. Thus, it has been postulated
that zinc supplementation to restore zinc levels in aged individ-
uals may have beneficial effects on immune health. This study
FIGURE 1 Plasma zinc concentration (A), thymocyte number (B),
and thymic SCF expression (C) in young and aged male C57Bl/6 mice
fed ZA or ZS diets for 25 d. Values are means 6SEM, n=8.
*Different from corresponding ZA, P,0.001. NS, Nonsignificant, P.
0.05.
FIGURE 2 Flow cytometry analysis
of DN (CD4
2
CD8
2
), DP (CD4
+
CD8
+
),
and SP (CD4
+
CD8
2
and CD8
+
CD4
2
)
thymocyte cell populations from
young and aged male C57Bl/6 mice
fed ZA or ZS diets for 25 d. DN cell
population was further analyzed to
determine the frequency of DN1
(CD44
+
CD25
2
), DN2 (CD44
+
CD25
+
),
DN3 (CD44
2
CD25
+
), and DN4
(CD44
2
CD25
2
). Representative flow
cytometry data (A) and mean DN1
frequency (B) are shown. Values are
means 6SEM, n= 8. NS, Nonsignif-
icant, P.0.05.
TABLE 2 CD4/CD8 thymocyte distribution in young and aged
male C57Bl/6 mice fed ZA or ZS diets for 25 d
1
Young Aged ANOVA
ZA ZS ZA ZS Diet Age Interaction
%P-values
DN 2.8 60.7 2.1 60.2 8.2 62.5 2.8 60.2 0.042 0.004 0.183
DP 88.9 60.5 88.0 60.5 81.2 63.6 88.2 60.7 0.162 0.098 0.081
CD4 SP 6.6 60.7 8.0 60.3 8.2 60.8 7.0 60.4 0.908 0.605 0.075
CD8 SP 1.6 60.1 2.0 60.2 2.3 60.3 1.9 60.1 0.968 0.163 0.061
1
Values are means 6SEM, n=8.
Zinc status and thymopoiesis in aged mice 1395
demonstrated that zinc status is impaired in aged mice and could
be restored to levels comparable to young mice by dietary zinc
supplementation. Moreover, our results indicated that zinc
supplementation partially reversed the thymic defects associ-
ated with aging. In particular, zinc supplementation increased
total thymocyte numbers in aged mice, in part by reversing the
age-related accumulation of DN1 thymocytes and reducing the
age-related elevated expression of the thymosuppressive cyto-
kine, SCF. These data were consistent with our hypothesis that
zinc status is an important variable in determining overall
thymic health during the aging process. To our knowledge, this is
the first report to directly examine the effects of zinc supplemen-
tation on thymocyte development and function during aging.
Overall, our data suggest that impaired zinc status associated
with the aging process may play an important role in the age-
related decline in thymopoiesis.
Immunosenescence, the age-related progressive decline in
lymphocyte development and function, coincides with impaired
zinc status in the elderly. Involution of the thymus and reduced
immune responsiveness are classic hallmarks shared by aging
and zinc deficiency. In particular, severe zinc deficiency results in
a decline of CD4
+
CD8
+
pre-T-cells in the thymus (5). Disruption
of the hypothalamus-pituitary-adrenal-axis leading to enhanced
circulating corticosterone concentrations has been postulated to
play an important role in zinc-deficiency related dysfunction
(28). Elevated corticosterone levels are also apparent in aging
and may also contribute to immunosenescence (29). Whether
alteration in corticosterone levels with age is directly related to
alterations in zinc status is currently unknown. Decreases in zinc
uptake and absorption increase the risk of zinc deficiency in the
elderly (10,11). Murine models have been used to study the
various effects of aging, including age-associated decline in zinc
status (19,30). In agreement with others, our data demonstrated
that aged C57Bl/6 mice, despite being fed a ZA diet, had a lower
plasma zinc concentration than young mice (Fig. 1A). This
suggested that aged mice, similar to humans, had impaired zinc
uptake/absorption. In addition, the elderly are more prone to
consuming inadequate levels of zinc in their diets. From the
NHANES III data, it is estimated that 12% of the U.S.
population does not consume the current Average Estimated
Requirement for zinc, but this number escalates to 35–45% in
people over the age of 60 y (31). Because a high proportion of
the elderly population (.60 y) consumes a low-zinc diet, there is
likely a synergy between low dietary zinc intake and the age-
related decline in zinc status that leads to immune dysfunction.
Our studies demonstrate that zinc supplementation of aged mice
restored plasma zinc to levels comparable to ZA young mice and
partially restored age-related alterations in thymopoiesis; thus, it
is likely that compromised zinc status only partially accounts for
the age-related decline in immune function. Understanding these
interactions is an important area of future research with
important public health implications and highlights the potential
need for higher zinc requirements in an elderly population.
The differentiation, selection, and output of mature naı
¨ve T-
cells take place in the thymus. The earliest immature thymocytes
are bone marrow-derived precursors that are negative for both
CD4 and CD8 (DN thymocytes). DN thymocytes are further
divided in 4 distinct subsets (DN1–4) based on the expression
of CD44 and CD25 (24). The frequency of immature DN1
thymocytes has been shown to be significantly increased in
aged mice, suggesting an age-dependent defect or blockade in
DN1-DN2 transition during thymocyte maturation (25,26). As
a consequence, a reduced number of thymocytes are available
for maturation and the output of naı
¨ve T-cells that exit the
thymus is reduced. Although zinc supplementation did not fully
restore thymocyte numbers to those of young mice, it neverthe-
less indicated that improving zinc status in aged mice could
enhance thymopoiesis by partially reversing involution of the
thymus (Fig. 1B). Our data further suggested that the increase in
thymocyte numbers was mediated in part by reducing the
accumulation of DN1 thymocytes and relieving the age-specific
DN1-DN2 block during thymopoiesis (Table 1; Fig. 2).
To date, the precise mechanism of age-induced thymic atrophy
remains unclear. However, it likely involves changes in the
thymic microenvironment, including the dysregulation of thy-
mic cytokines, the loss of thymic epithelium, and/or changes in
T-cell progenitors (1). Dysregulation of thymotrophic as well as
thymosuppressive cytokines have been proposed to contribute to
age-associated thymic involution (27). In particular, elevated
expression of thymosuppressive cytokines such as SCF has been
observed in aged thymus and administration of SCF into young
mice induced acute thymic atrophy in vivo. Although the precise
mechanism by which SCF affects thymopoiesis remains to be
elucidated, we showed that zinc supplementation in aged mice
was associated with a significantly reduced expression of SCF
in the thymus (Fig. 1C). Interestingly, dietary supplementation
of zinc similarly decreased expression of SCF in the small
intestine in vivo (32). In addition to the restoration of normal
expression of SCF in aged mice, zinc supplementation likely
affects thymopoiesis via additional mechanisms. For example,
dietary zinc may modulate the expression of genes involved in
zinc homeostasis. Our preliminary survey of zinc transporters
in the thymus revealed age-specific differences in zinc trans-
porter expression (data not shown). In addition, Moore et al.
(33) reported that the expressions of a number of genes were
modulated in the thymus of zinc-deficient and zinc-supplemented
young mice. Interestingly, genes differentially expressed with
age, including various thymic cytokines (27) and zinc trans-
porters (C. P. Wong and E. Ho, unpublished data), were not
among the list of differentially expressed genes identified in this
murine model of severe zinc deficiency. However, in this model,
p56
lck
, a lymphocyte-specific tyrosine kinase important in the
selection and maturation of thymocytes, was upregulated with
zinc deficiency. This could be an important target for examina-
tion in future studies. Other studies have also found increased
p56
lck
with zinc deficiency but without effects on T-cell matura-
tion (34). It is possible that alterations found with age-related
decline in zinc status are not fully comparable to young growing
mice fed a severely zinc-deficient diet (,1 mg/kg zinc). Both the
severity of zinc deficiency and the physiological response to
zinc status may differ. At the same time, the effects of zinc
supplementation likely have differential effects in young and
aged mice. This is evidenced in our findings that the effect of zinc
supplementation on SCF expression was exclusively observed in
aged mice. Further study is needed to characterize the response
of the immune system to nutritional status in the growing young,
adult, and aged animals. The interactions among nutrition, age,
maturity/decline of the immune system are important to address
to be able to make appropriate nutrient intake recommendations
across the lifespan. Future studies will focus on providing an in-
depth characterization of the differential expression of genes in
the thymus with age and examine how zinc status influences the
expression of other thymic cytokines as well as genes involved
in zinc homeostasis, such as metallothionein and zinc trans-
porters. We will further explore how zinc status and age influence
thymopoiesis. Specifically, it will be of great interest to study
how diet and age interact and affect cell proliferation and/or
apoptosis during thymocyte development.
1396 Wong et al.
In summary, we showed that zinc supplementation improved
impaired zinc status and thymopoiesis in aged mice. Reversing
age-related thymic involution and enhancing thymic develop-
ment and function would be of considerable benefit to improve
overall immune function in aged individuals. In particular, the
elderly are at increased risk of complications and death from
infections such as influenza. Improving thymic development and
function in the elderly should lead to an enhancement in immune
responsiveness and translate to a reduction in complications and
mortality from infections. Our study showed that zinc supple-
mentation can serve as a therapeutic agent in improving thymic
health and provided the foundation for future studies to further
investigate the mechanisms as well as therapeutic effects of zinc
supplementation in reversing thymic involution and in improv-
ing immune response in the elderly.
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