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

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

Activation of STAT1 in NK cells requires 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 pSTAT1 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 pSTAT1. Values represent the percentages of pSTAT1<b>⁺</b> NK cells. Data are representative of three independent experiments with 2–4 (A) or 1–3 (B) mice per group.</p

Type I IFNs are required for NK cell activation in response to flu infection.

<p>Following i.v. infection with flu, splenic NK cells (NK1.1<b>⁺</b>CD3<b>⁻</b>CD19<b>⁻</b>) from B6 WT, IFNAR<b>^−/−</b>, IL-12R<b>^−/−</b> and IL-18R<b>^−/−</b> mice were analyzed at 9h post-infection. (A) IFN-γ and granzyme B expression are shown. Mouse genotypes are indicated above the dot plots or histograms. Inset values represent the percentages of IFN-γ<b>⁺</b> (upper panels) or granzyme B<b>⁺</b> (lower panels) NK cells. (B) CD69 expression levels on NK cells from uninfected and infected mice are shown. Percentages of NK cells located within the CD69<b>⁺</b>gate are indicated. (C) CD107a expression and IFN-γ production were analyzed in NK cells after incubation with YAC-1 cells. Percentages of NK cells within each quadrant are indicated. Data are representative of at least three 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

Activation mechanisms of natural killer cells during influenza virus infection

During early viral infection, activation of natural killer (NK) cells elicits the effector functions of target cell lysis and cytokine production. However, the cellular and molecular mechanisms leading to NK cell activation during viral infections are incompletely understood. In this study, using a model of acute viral infection, we investigated the mechanisms controlling cytotoxic activity and cytokine production in response to influenza (flu) virus. Analysis of cytokine receptor deficient mice demonstrated that type I interferons (IFNs), but not IL-12 or IL-18, were critical for the NK cell expression of both IFN-γ and granzyme B in response to flu infection. Further, adoptive transfer experiments revealed that NK cell activation was mediated by type I IFNs acting directly on NK cells. Analysis of signal transduction molecules showed that during flu infection, STAT1 activation in NK cells was completely dependent on direct type I IFN signaling, whereas STAT4 activation was only partially dependent. In addition, granzyme B induction in NK cells was mediated by signaling primarily through STAT1, but not STAT4, while IFN-γ production was mediated by signaling through STAT4, but not STAT1. Therefore, our findings demonstrate the importance of direct action of type I IFNs on NK cells to mount effective NK cell responses in the context of flu infection and delineate NK cell signaling pathways responsible for controlling cytotoxic activity and cytokine production