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

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

Activation of STAT4 is partially dependent on the direct action of type I IFNs during influenza infection.

<p>NK cells from infected (open histograms) and uninfected (shaded histograms) mice were analyzed for intracellular pSTAT4 following adoptive transfer (A) or co-culture (B). Adoptive transfer was as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051858#pone-0051858-g002" target="_blank">Figure 2</a>. For in vitro infection, CD45.2<b>⁺</b> splenocytes from IFNAR<b>^+/−</b> or IFNAR<b>^−/−</b> mice were combined with CD45.1<b>⁺</b> B6 splenocytes at 1∶1 ratio, then infected with flu. NK cells (NK1.1<b>⁺</b>CD3<b>⁻</b>) from infected (open histograms) and uninfected (shaded histograms) samples were analyzed for intracellular pSTAT4. Values represent the percentages of pSTAT4<b>⁺</b> NK cells. Data are representative of three independent experiments with 2–4 (A) or 1–3 (B) mice per group.</p

STAT1, but not STAT4, is required for granzyme B induction by NK cells in response to flu infection.

<p>CD45.2<b>⁺</b> splenocytes from 129/Sv WT and STAT1^−/− (A) or from BALB/c WT and STAT4^−/− (B) mice were combined with CD45.1<b>⁺</b> B6 splenocytes in vitro, then infected with flu. NK cells (DX5<b>⁺</b>CD3<b>⁻</b>CD19<b>⁻</b>) were analyzed for expression levels of IFN-γ (left panels) and granzyme B (right panels). Bar graphs represent the mean differences in percentage of IFN-γ<b>⁺</b> or granzyme B<b>⁺</b> CD45.2<b>⁺</b> NK cells from infected samples over uninfected controls. Error bars represent the SEM of triplicate samples. Data are representative of four independent experiments. **, <i>P</i><0.001; ***, <i>P</i><0.0001; ns, not significant.</p

Direct action of type I IFNs is critical for activation of NK cells following flu infection.

<p>Splenocytes from IFNAR<b>^+/−</b> or IFNAR<b>^−/−</b> (CD45.2<b>⁺</b>) were transferred into CD45.1<b>⁺</b> B6 WT recipients by i.v. injection prior to infection with flu. Splenic NK cells from indicated mice were analyzed post-infection. (A) Expression levels of IFN-γ (upper panels) and granzyme B (lower panels) were analyzed in NK cells after transfer and infection. Donor and recipient genotypes are indicated, and upper and lower quadrants for each dot plot represent the donor and host NK cells, respectively. Inset values indicate the percentages of IFN-γ<b>⁺</b> or granzyme B<b>⁺</b> NK cells. (B) CD69 expression of NK cells from infected (open histograms) and uninfected (shaded histograms) mice. Percentages of NK cells located within the CD69<b>⁺</b>gate are indicated. (C) IFN-γ and CD107a expression were analyzed after in vitro stimulation of splenocytes with YAC-1 cells. Numbers represent the relative percentages of CD107a<b>⁺</b> and IFN-γ<b>⁺</b> NK cells for donor and host cells. Data are representative of four separate experiments with 2–4 mice per group.</p