Respiratory Influenza A Virus Infection Triggers Local and Systemic Natural Killer Cell Activation via Toll-Like Receptor 7

Abstract

The innate immune system senses influenza A virus (IAV) through different pathogen-recognition receptors including Toll-like receptor 7 (TLR7). Downstream of viral recognition natural killer (NK) cells are activated as part of the anti-IAV immune response. Despite the known decisive role of TLR7 for NK cell activation by therapeutic immunostimulatory RNAs, the contribution of TLR7 to the NK cell response following IAV infection has not been addressed. We have analyzed lung cytokine responses as well as the activation, interferon (IFN)-γ production, and cytotoxicity of lung and splenic NK cells following sublethal respiratory IAV infection in wild-type and TLR7ko mice. Early airway IFN-γ levels as well as the induction of lung NK cell CD69 expression and IFN-γ production in response to IAV infection were significantly attenuated in TLR7-deficient hosts. Strikingly, respiratory IAV infection also primed splenic NK cells for IFN-γ production, degranulation, and target cell lysis, all of which were fully dependent on TLR7. At the same time, lung type I IFN levels were significantly reduced in TLR7ko mice early following IAV infection, displaying a potential upstream mechanism of the attenuated NK cell activation observed. Taken together, our data clearly demonstrate a specific role for TLR7 signaling in local and systemic NK cell activation following respiratory IAV infection despite the presence of redundant innate IAV-recognition pathways.

Keywords: Toll-like receptor 7; influenza A virus; innate immunity; natural killer cells; pathogen-recognition receptors; respiratory infection.

Figure 1

The early lung interferon-γ (IFN-γ) response following influenza A virus (IAV) infection is attenuated in toll-like receptor (TLR)7-deficient mice while the lung natural killer (NK) cell frequency is unchanged. Wild-type (WT) (black bars and symbols) and TLR7ko (open bars and symbols) mice were infected with 0.04 LD₅₀ IAV PR8 or treated with PBS and sacrificed at the indicated time points. (A) IFN-γ levels in bronchoalveolar lavage were assessed by enzyme-linked immunosorbent assay. Data show the mean ± SEM of n = 3 uninfected and n ≥ 8 infected mice per strain with samples derived from three independent experiments. (B) IAV nucleoprotein (NP) copies were quantified in whole lung tissue cDNA as a measure for the viral load. Data show individual mice and the mean/group of samples collected from two independent infection experiments. (C) Lymphocytes isolated from the lung tissue were analyzed for the frequency of NK cells (CD3⁻/NK1.1⁺). Frequency ± SEM of NK cells within the lymphocyte population from n ≥ 6 mice per group compiled from at least two independent experiments. (D) Data show the absolute number of NK cells isolated per mouse lung for individual mice and the group mean compiled from at least two independent experiments. (E) Lymphocytes isolated from the lung tissue were analyzed for NK cell (CD3⁻/NK1.1⁺) IFN-γ production by intracellular flow cytometry ex vivo. Data show the frequency ± SEM of IFN-γ-positive NK cells within the NK cell population on day 3 post IAV infection for n ≥ 5 mice per group compiled from two independent experiments. Groups were compared by two-way ANOVA with Bonferroni multiple comparisons test (*p < 0.05, **p < 0.005, ***p < 0.0005).

Figure 2

The induction of natural killer (NK) cell CD69 expression in the lung following respiratory influenza A virus (IAV) infection is delayed in Toll-like receptor (TLR)7-deficient hosts. Wild-type (WT) (black bars) and TLR7ko (white bars) mice were infected with 0.04 LD₅₀ IAV PR8 or treated with PBS and sacrificed at the indicated time points. Lung NK cells (CD3⁻/NK1.1⁺) were analyzed for the expression of CD69 (A) and the co-expression of CD11b and CD27 (B) by flow cytometry. Data show the mean ± SEM of ≥6 mice/group from at last two independent experiments and representative flow-cytometric results for an uninfected and an IAV-infected WT mouse on day 4 post infection. Data were compared by two-way ANOVA with Bonferroni multiple comparisons test (*p < 0.05, ****p < 0.0001).

Figure 2

The induction of natural killer (NK) cell CD69 expression in the lung following respiratory influenza A virus (IAV) infection is delayed in Toll-like receptor (TLR)7-deficient hosts. Wild-type (WT) (black bars) and TLR7ko (white bars) mice were infected with 0.04 LD₅₀ IAV PR8 or treated with PBS and sacrificed at the indicated time points. Lung NK cells (CD3⁻/NK1.1⁺) were analyzed for the expression of CD69 (A) and the co-expression of CD11b and CD27 (B) by flow cytometry. Data show the mean ± SEM of ≥6 mice/group from at last two independent experiments and representative flow-cytometric results for an uninfected and an IAV-infected WT mouse on day 4 post infection. Data were compared by two-way ANOVA with Bonferroni multiple comparisons test (*p < 0.05, ****p < 0.0001).

Figure 3

The expression of CD69 and CD27/CD11b by splenic natural killer (NK) cells in response to respiratory influenza A virus (IAV) infection is significantly altered in toll-like receptor (TLR)7-deficient mice. Wild-type (black bars) and TLR7ko (white bars) mice were infected with 0.04 LD₅₀ IAV PR8 or treated with PBS and sacrificed at the indicated time points. Splenic NK cells were analyzed for the expression of intracellular interferon-γ (IFN-γ) (A) and surface CD69 (B) as well as CD11b/CD27 (C) by flow cytometry ex vivo. (A) Data show the mean ± SEM of 6 mice/group from two independent experiments. (B) Data show the mean ± SEM of ≥6 mice/group from at least two independent experiments. (C) Data show the mean ± SEM of ≥6 mice/group from at least two independent experiments. Data were compared by two-way ANOVA with Bonferroni multiple comparisons test (*p < 0.05, **p < 0.005, ***p < 0.0005).

Figure 4

Respiratory influenza A virus (IAV) infection primes splenic natural killer (NK) cells for in vitro interferon-γ (IFN-γ) expression and target cell-directed cytotoxicity in a toll-like receptor (TLR)7-dependent fashion. Wild-type (WT) (black bars and symbols) and TLR7ko (white bars and symbols) mice were infected with 0.04 LD₅₀ IAV PR8 or treated with PBS and were sacrificed on day 4 post infection. (A) The frequency of IFN-γ-positive NK cells (CD3⁻/NK1.1⁺) was quantified by flow cytometry following in vitro stimulation of splenocytes. Data represent the mean ± SEM of n = 6 mice from two independent experiments. (B) Specific target cell lysis in splenocyte/YAC-1 target cell co-cultures was assessed. Results show results for individual mice and the mean/group from one representative out of two independent experiments. (C) The frequency of CD107a⁺ NK cells (CD3⁻/NK1.1⁺) within splenocytes following co-incubation with YAC-1 target cells was assessed by flow cytometry. Data represent the mean ± SEM of n = 6 mice compiled from two independent experiments. (D) Geometric mean fluorescence intensity (FI) of the CD107a staining of the NK cell population assessed by flow cytometry following co-incubation of splenocytes with YAC-1 target cells. Results are shown for individual mice and the mean/group from one representative out of two independent experiments. Groups were compared by two-way ANOVA with Bonferroni multiple comparisons test (*p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001).

Figure 5

Respiratory influenza A virus (IAV) infection does not lead to significant degranulation of natural killer (NK) cells in the lung or spleen in vivo. Wild-type (WT) (black symbols) and toll-like receptor (TLR)7-deficient (open symbols) mice were infected with 0.04 LD₅₀ IAV PR8 or treated with PBS and sacrificed on day 4 post infection. Lung (A) and splenic (B) NK cells were analyzed for surface CD107a expression by flow cytometry ex vivo. Data show the frequency of CD107a⁺ NK cells for 12 individual mice/groups compiled from four independent experiments. Data were compared by two-way ANOVA with Bonferroni multiple comparisons test.

Figure 6

The early respiratory Il-12p40 and interferon (IFN) I response following influenza A virus (IAV) infection is attenuated in toll-like receptor (TLR)7-deficient mice. Wild-type (WT) (black bars and symbols) and TLR7ko mice (white bars and symbols) were infected with 0.04 LD₅₀ IAV PR8 or treated with PBS and sacrificed at the indicated time points post infection. Il-12p70 (A), Il-12p40 (B), and Il-18 (C) were quantified in bronchoalveolar lavage (BAL). Data are shown for individual mice and indicate the mean/group of samples collected from at least two independent infection experiments. The dotted horizontal lines indicate the detection limit of the respective assay. (D) Bioactive IFN I was quantified in BAL. Data are shown as mean ± SEM of n ≥ 10 mice per group, and samples were collected in at least two independent infection experiments. Groups were compared by two-way ANOVA with Bonferroni multiple comparisons test (*p < 0.05, **p < 0.005).