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C57BL/6 Mice Are Altered by Caloric
NK Cell Maturation and Function in
Elizabeth M. Gardner
Jonathan F. Clinthorne, Eleni Beli, David M. Duriancik and
2013; 190:712-722; Prepublished online 14
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The Journal of Immunology
NK Cell Maturation and Function in C57BL/6 Mice Are
Altered by Caloric Restriction
Jonathan F. Clinthorne, Eleni Beli, David M. Duriancik, and Elizabeth M. Gardner
NK cells are a heterogenous population of innate lymphocytes with diverse functional attributes critical for early protection from
viral infections. We have previously reported a decrease in influenza-induced NK cell cytotoxicity in 6-mo-old C57BL/6 calorically
restricted (CR) mice. In the current study, we extend our findings on the influence of CR on NK cell phenotype and function in the
absence ofinfection. We demonstrate that reduced mature NK cell subsets result in increased frequencies of CD127+NK cells in CR
mice, skewing the function of the total NK cell pool. NK cells from CR mice produced TNF-a and GM-CSF at a higher level,
whereas IFN-g production was impaired following IL-2 plus IL-12 or anti-NK1.1 stimulation. NK cells from CR mice were highly
responsive to stimulation with YAC-1 cells such that CD272CD11b+NK cells from CR mice produced granzyme B and degranu-
lated at a higher frequency than CD272CD11b+NK cells from ad libitum fed mice. CR has been shown to be a potent dietary
intervention, yet the mechanisms by which the CR increases life span have yet to be fully understood. To our knowledge, these
findings are the first in-depth analysis of the effects of caloric intake on NK cell phenotype and function and provide important
implications regarding potential ways in which CR alters NK cell function prior to infection or cancer.
nology, 2013, 190: 712–722.
with increased incidence of disease, CR has been found to decrease
the severity of autoimmune disease, and decrease the incidence of
cardiac, kidney, or nervous system dysfunction (1–4). Other ben-
efits of CR include decreased triglycerides and blood pressure,
lower central adiposity, improved insulin sensitivity, and delayed
age-related immunosenescence (5, 6). It has been established that,
in laboratory conditions, CR reduces the incidence of spontaneous
tumors and cancers in aged rodents, and slows the age-related
decline in T cell proliferation, cytokine production, and CTL ac-
tivity that is often observed during aging (7, 8). Lifelong CR of
mice preserves thymopoiesis in the face of aging, and has been
shown to enhance influenza-specific Abs and splenic lymphocyte
proliferation after vaccination of mice with influenza (8, 9). These
beneficial changes to the adaptive immune system have been well
characterized; however, it has also been found that CR influences
innate immune function (10, 11). Several decades ago, Weindruch
et al. (12) reported that CR resulted in decreased splenic NK cell
cytotoxicity compared with aged matched controls, although this
could be ameliorated by polyinosinic:polycytidylic acid. More
recently, we have shown CR results in increased susceptibility to
primary influenza infection and decreased influenza-induced NK
The Journal of Immu-
aloric restriction (CR) is a dietary intervention that has
been shown to extend the life span of laboratory animals
(1). Whereas excess energy intake has been associated
cell cytotoxicity in young and aged mice (13, 14). This was ac-
companied by the observation that NK cell numbers and frequency
are decreased in the spleen of young CR mice (14). Overall, these
findings have raised concerns about the effects of CR on innate
immunity, and may predispose CR individuals to suffer more se-
vere primary infections (10, 15). However, at this time, few studies
have focused on understanding the effects of CR on innate immune
cell development and function.
NK cells are responsible for recognizing virally infected cells, as
well as transformed cells, including neoplasms and tumor cells
(16–18). Development of NK cells takes place mainly in the bone
marrow (BM), and signals from stromal cells and cytokines result
in the microenvironment required for NK cell generation (19, 20).
NK cell commitment takes place through upregulation of the
shared IL-2/IL-15R b-chain (CD122), followed by acquisition of
the NK cell marker NK1.1 in B6 mice (19, 21). Interactions with
stromal cells within the BM regulate gene expression leading to
programmed expression of surface molecules, including integrins,
cytokine receptors, and a family of NK cell receptors (22–25). NK
cell maturation is classified by using both surface phenotype and
functional capacity. Phenotypic maturation takes place in stepwise
fashion; expression of the integrin CD49b (DX5) is used to define
early mature NK cells (26). Following acquisition of DX5, NK
cells in the BM upregulate CD11b and CD43, which correlates
strongly with the capability of a NK cell to produce large amounts
of IFN-g (19). Once mature, NK cells seed various lymphoid and
nonlymphoid peripheral tissues, with the majority of NK cells
expressing high levels of DX5, CD11b, and CD43 (19, 27).
tissues, DX5+NK cells continue to adapt to their environment; the
downregulation of CD27 and TRAIL and upregulation of killer cell
lectin-like receptor G1 (KLRG1) are associated with peripheral NK
cell maturation (28, 29). The application of the marker CD27 has
allowed the DX5+NK cell pool to be further divided into subsets in
mice in which there is a linear progression from CD27+CD11b2
early mature NK cells to CD27+CD11b+(double-positive [DP]) NK
phenotypic changes further reflect changes to NK cell function, as
Department of Food Science and Human Nutrition, Michigan State University, East
Lansing, MI 48824
Received for publication July 3, 2012. Accepted for publication November 13, 2012.
This work was supported by National Institutes of Health Grant R01AG034949-01A1
Address correspondence and reprint requests to Dr. Elizabeth M. Gardner, Depart-
ment of Food Science and Human Nutrition, Michigan State University, 234C GM
Trout Building, East Lansing, MI 48824. E-mail address: firstname.lastname@example.org
Abbreviations used in this article: AL, ad libitum; BM, bone marrow; CR, caloric
restriction; DN, double-negative; DP, double-positive; Eomes, eomesodermin;
KLRG1, killer cell lectin-like receptor G1; LN, lymph node; MRI, magnetic reso-
nance imaging; mTOR, mammalian target of rapamycin; NIA, National Institute on
Aging; PEM, protein energy malnutrition.
by guest on October 17, 2015
in a laboratory setting (10, 13, 15, 39, 70–72). Infection via these
pathogens results in substantial weight loss, which could be det-
rimental to CR mice because of limited energy reserves (7).
However, it is also plausible that CR initiated before adulthood
results in immunological changes such as those presented in this
work, that increase susceptibility to specific pathogens, limiting
the usefulness of this intervention in humans. However, future
studies are required to determine whether this is specific for
respiratory viruses as we have shown, or whether other viral
infections such as HSV-1, mousepox, and murine CMV pose
a greater threat to CR mice as well (16, 73). Our study utilizing
dietary manipulation in a mouse model led us to wonder whether
CR has a similar effect on NK cells in humans. Indeed, the NIA
has begun a series of human trials to determine the efficacy of CR
in humans; this study, known as the Comprehensive Assessment of
the Long-Term Effects of Reducing Intake of Energy (CALERIE),
should allow for further study of the influence of CR on immune
function in humans (74). Furthermore, because CR is designed as
a dietary intervention to delay aging, it will be interesting to de-
termine whether any age-related changes in NK cell phenotype
that occur in mice are ameliorated or exacerbated by CR in
humans and mice (36, 73).
We thank Dr. Jeannine Scott for insightful discussion regarding the manu-
script. We also thank Brooke Roman for technical assistance.
The authors have no financial conflicts of interest.
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722 CR ALTERS NK CELL PHENOTYPE AND FUNCTION
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