NK cell maturation and function in C57BL/6 mice are altered by caloric restriction - PubMed (original) (raw)

NK cell maturation and function in C57BL/6 mice are altered by caloric restriction

Jonathan F Clinthorne et al. J Immunol. 2013.

Abstract

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 of infection. 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-α and GM-CSF at a higher level, whereas IFN-γ 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 CD27(-)CD11b(+) NK cells from CR mice produced granzyme B and degranulated at a higher frequency than CD27(-)CD11b(+) 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.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1

Food intake and physiological parameters altered by CR. (A) Food intake and body weight were recorded daily for 7 d, and averages for AL and CR mice are shown. (B) Body composition was assessed by MRI on the day animals were sacrificed, and body fat percentage was calculated as the portion of fat mass relative to total mass. (C) Circulating levels of corticosterone, albumin, and leptin were determined in serum from AL and CR mice by ELISA on the day of sacrifice. (D) Serum glucose, triglycerides, and cholesterol from AL and CR mice were measured on day of sacrifice by colorimetric assays. Data are means ± SEM. *Indicates significance, p < 0.05 (n = 8–10 mice/group).

FIGURE 2

Tissue weight and distribution of NK cells in CR mice. (A) The percentage of NK1.1+ CD3− cells of total lymphocytes was determined in various tissues known to contain NK cells and was found to be significantly reduced in the lungs, blood, and spleen (SPL) of CR mice. (B) Wet tissue weights from AL and CR mice were taken immediately following sacrifice of AL and CR mice. NK cell numbers from BM (C), spleen (D), and lungs (E) of CR and AL mice expressed as the number of NK cells per femur or per mg tissue. The absolute number of NK cells was calculated based on the frequency of NK cells of total cells analyzed by flow cytometry and divided by the wet tissue weight. Experiments were repeated twice. Data are means ± SEM. *Indicates significance, p < 0.05 (n = 5 mice/group/experiment).

FIGURE 3

Characterization of surface phenotype of splenic NK cells in CR mice. Histograms are representative and contain either percentage of NK cells within the positive gate for the indicated cell surface Ag or the median fluorescence intensity of the indicated marker (when no gate is shown). (A) Expression of surface markers associated with NK cell maturation on splenic NK cells gated NK1.1+ CD3− from 6-mo-old AL and CR mice. (B) Expression of NK cell receptors and activation markers on splenic NK cells from CR and AL mice. (C) Ly49 repertoire on both CD11b+ (top) and CD11b− (bottom) NK cells. Data are mean ± SEM. Experiments were repeated twice. *Indicates significance, p < 0.05 (n = 5 mice/group/experiment).

FIGURE 4

A greater fraction of NK cells from CR mice expresses CD127. (A) CD127 expression on spleen (SPL), LN, and BM NK cells from AL and CR mice. (B) The absolute number of CD127+ NK cells (NK1.1+ CD3−) in the spleen, LN, and BM of AL and CR mice. (C) Surface phenotype of splenic and BM CD127+ NK cells from gates indicated in (A). Filled gray histogram represents CD127+ NK cells from AL; solid line represents CD127+ NK cells from CR; and dotted line represents splenic CD127− NK cells from AL mice. (D) Gating strategy for identification of thymic NK cells that are identified as NK1.1+ CD3− (top) and CD127+ (bottom). (E) Frequency of NK cells in the thymus represented both as frequency of thymocytes (top) and number of NK cells per mg thymus collected (bottom). (F) Absolute counts of various cell populations identified in the thymus of AL and CR are shown. Flow plots are representative and contain the percentage of NK cells positive for the indicated gates. Experiments were repeated twice. Data are mean ± SEM. *Indicates significance, p < 0.05 (n = 5 mice/group/experiment).

FIGURE 5

Functional characterization of CD127+ NK cells from CR mice. (A) Analysis of cytokine production by NK cells (NK1.1+ CD3−) from AL and CR mice in nonstimulated (NS) controls (top), cells isolated from spleen (middle), and LN (bottom) stimulated with IL-2 (1000 U/ml), IL-12 (10 ng/ml), and PMA (50 ng/ml) plus ionomycin (1 μg/ml). (B) Summary of the frequency of cytokine-producing NK cells in the spleen (top) and LN (bottom) of AL and CR mice. (C) Splenic lymphocytes were gated NK1.1+ CD3−CD127+ and analyzed for cytokine production and cytotoxicity. Histograms of TNF-α and GM-CSF production following IL-2 plus IL-12 stimulation (top) and granzyme B and CD107a (bottom) staining following stimulation with YAC-1 cells (10:1 E:T ratio) in CD127+ NK cells from CR and AL mice. Filled gray histogram represents AL; solid line represents CR; and dotted line represents cells with no stimulation (NS) from CR mice. Flow plots and histograms are representative and contain the percentage of NK cells positive for the indicated gates. Experiments were repeated twice. Data are mean ± SEM. *Indicates significance, p < 0.05 (n = 5 mice/group/experiment).

FIGURE 6

Altered distribution of NK cell subsets in the BM and spleen of CR mice. (A) Distribution of NK cell (NK1.1+ CD3−) subsets based on expression of CD27 and CD11b in the BM (top) and spleens (bottom) of AL and CR mice. (B) Summary of the frequency of NK cells in each subset both as a percentage of NK cells (top) and as a percentage of total cells recovered (bottom). NK cell subsets were defined as CD27−CD11b− (DN), CD27+CD11b−, CD27+CD11b+ (DP), and CD27−CD11b+. (C) Gating strategy for transcription factor analysis (left) and summary of transcription factor expression in splenic NK cells from AL and CR mice (right). (D) Expression of KLRG1 and CD43 on splenic CD27−CD11b+ NK cells from AL and CR mice. Flow plots are representative and contain the percentage of NK cells positive for the indicated gates. Experiments were repeated twice. Data are mean ± SEM. *Indicates significance, p < 0.05 (n = 4–5 mice/group/experiment).

FIGURE 7

Function of NK cells from CR mice is altered after interrogation with various stimuli. (A) IFN-γ production by BM and splenic NK cells (NK1.1+ CD3−) stimulated with IL-2 (1000 U/ml) plus IL-12 (50 ng/ml) (top) and the frequency of splenic NK cell subsets producing IFN-γ from AL and CR mice (bottom). (B–D) Splenic NK cells from AL and CR mice were stimulated with (B) anti-NK1.1 (25 μg/ml), (C) anti-NKp46 (15 μg/ml), and (D) YAC-1 cells (10:1 E:T ratio), and DX5+ CD3− cells were analyzed for production of IFN-γ, granzyme B, and surface CD107a. (E) Histograms representing granzyme B and CD107a staining in NK cell subsets following stimulation of splenic NK cells with YAC-1 cells. NK cells from AL and CR mice were gated DX5+ CD3−, and the indicated NK cell subset was analyzed for granzyme B or CD107a expression. Filled gray histogram represents AL; solid line represents CR; and dotted line represents cells from CR mice that received no stimulation (NS). Flow plots and histograms are representative and contain the percentage of NK cells positive for the indicated gates. Experiments were repeated twice. Data are mean ± SEM. *Indicates significance, p < 0.05 (n = 5 mice/group/experiment).

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NK cell maturation and function in C57BL/6 mice are altered by caloric restriction - PubMed (original) (raw)