Fig 1.
Ly49-deficient mice are protected from lethal influenza infection.
(A, B) Groups of sex-matched WT (B6), Ifnar1-/- and B2m-/- mice were infected intranasally with 600 PFU of FM-MA virus. Single-cell suspensions were prepared from uninfected lungs and those infected with the virus for 5 days. Cells were stained with antibodies against H-2Kb or H-2Db and CD326 (EpCAM—epithelial cell marker), and analyzed by flow cytometry. Surface expression of H-2Kb or H-2Db was determined on EpCAM+ lung epithelial cells. The following mAb were used in this experiment: anti-LFA-1, anti-EpCAM, anti-H-2Kb, and anti-H-2Db. One representative image from each group is shown. This experiment was performed three times with similar results. (C, D) Ly49G, I1, O, R, and V expression was detected on lung NK cells of uninfected WT and NKCKD mice using anti-NKp46, anti-TCRβ, and a combination of 4D11, 4E5 and 14B11 mAb. NKG2D, NKp46, CD11b, and CD27 expression was detected on lung NK cells, defined using anti-CD49b (DX5) and anti-TCRβ. The gray line represents staining with an isotype antibody. This experiment was performed three times with similar results. (E) Groups of age and sex-matched WT, Ly49QKO, and NKCKD mice were infected with FM-MA virus (1050 PFU) and monitored for 2 weeks. Data are pooled from two independent experiments (n = 19 in each group). (F) Groups of age and sex-matched NKCKD mice with or without NK depletion by anti-asialoGM1 were infected and monitored as above. A group of uninfected, NK-depleted mice was included as a control (n = 10 in each group). The percentage of surviving mice is shown. *p < 0.05, **p < 0.01 and ***p < 0.001. Statistical analysis was performed with the log rank test.
Fig 2.
Increased numbers of activated, uneducated NK cells correlates with protection from influenza.
Groups of age and sex-matched WT mice were infected as in Fig 1. On day 5 p.i., mice were sacrificed, and single cell suspensions prepared from the lungs. Flow cytometry was performed as above, using cells stained with a fixable viability dye, anti-NKp46, anti-TCRβ, anti-Ly49C/I (5E6), anti-Ly49G (4D11), anti-Ki-67, anti-IFN-γ, and anti-CD107a mAbs. Each symbol represents one mouse. Cell counts are given in absolute number calculated per lung. Horizontal bars represent mean values, *p < 0.05, **p < 0.01, and ***p < 0.001. Statistical analysis was performed using a one-way ANOVA followed by a Bonferroni post-hoc test.
Fig 3.
Lung immunopathology of influenza-infected WT and NKCKD mice.
(A-D) WT and NKCKD mice were inoculated intranasally with 600 PFU of FM-MA virus. Lungs were collected from WT and NKCKD mice 7 days p.i., fixed in neutral-buffered 10% formalin, sectioned, and stained with H&E. Images were acquired at 100x magnification. One representative image from each group is shown. Images were scored by a pathologist blind to the experimental conditions. Regions of tissue damage are indicated as follows: ‘→’ pulmonary edema; ‘*’ diffuse alveolar damage; ‘‡’ lymphocytic and neutrophilic infiltrate; ‘&’ bronchi filled with cellular debris. This experiment was performed three times with similar results. Two to three mice were used for each group per experiment. (E) Lungs from infected (600 PFU) age- and sex-matched WT and NKCKD mice were collected on day 5 p.i., weighed, and virus titer (presented as PFU/g of lung tissue) was assessed in lung homogenates by plaque assay on MDCK cells. Data are pooled from three independent experiments (n = 10 in each group). Each symbol represents a single mouse. Horizontal bars represent mean values. ***p < 0.001. Statistical analysis was performed using Student’s t-test.
Fig 4.
Cytokine and chemokine levels in the lungs of influenza virus-infected mice.
Groups of age and sex-matched WT and NKCKD mice were infected intranasally with 600 PFU of FM-MA virus. Lungs were collected on day 5 p.i., and lung tissue homogenates were prepared. The levels of (A) IFN-γ, (B) TNF-α, (C) IL-17, (D) MCP-1, (E) MCP-3, and (F) MIP-1β in the lung tissue homogenates were measured by flow cytometry using FlowCytomix Multiplex kits. Data are pooled from three independent experiments. (G, H) NK cells were isolated from infected lungs, stained, and analyzed by flow cytometry as above. Cells were stained with a fixable viability dye, anti-NKp46, anti-TCRβ, and anti-IFN-γ (G) or anti-TNF-α (H). Horizontal bars represent mean values. Each symbol represents data from a single mouse. *p < 0.05, **p < 0.01 and ***p < 0.001. Statistical analysis was performed using Student’s t-test.
Fig 5.
Lymphocyte recruitment to the lungs following influenza virus infection.
WT and NKCKD mice were infected intranasally with 600 PFU of FM-MA virus. Single-cell suspensions were prepared from uninfected lungs and those infected with the virus for 5 or 7 days. The following mAb were used in this experiment: anti-CD8, anti-CD3, anti-CD4, and anti-NKp46 mAb; as well, Kb-NS2114−121 tetramers and I-Ab–NP311-325 tetramers were used. Both the frequency (%) and absolute number (#) of cell populations is reported. (A, B) NS2114-121 CD8+ specific T cells were gated out of CD3+ CD8+ T cells. (C, D) NP311-325 CD4+specific T cells were gated out of CD3+ CD4+ T cells. (E, F) NK cells were detected as CD3- NKp46+ cells. Data are pooled from two independent experiments (n = 6 in each group). Horizontal bars represent mean values. Each symbol represents data from a single mouse. *p < 0.05 and **p < 0.01. Statistical analysis was performed using Student’s t-test.
Fig 6.
Involvement of Ly49 inhibitory receptors in the pathogenesis of influenza virus.
(A) Groups of influenza-infected age and sex-matched WT, NKCKD, and littermate NKCKD-Ly49Itg mice were infected with 600 PFU of FM-MA virus, then monitored as before. Data are pooled from two independent experiments (n = 16 in each group). (B) Groups of influenza-infected age and sex-matched WT (B6) and B2m-/- mice were monitored as in Fig 1. One group each of WT or B2m-/- mice was treated with 200 μg anti-NK1.1 mAb per mouse i.p. two days prior to influenza virus infection, on the day of infection (600 PFU), and every two days after until day 10 p.i. Data are pooled from two independent experiments (n = 10 in each group). (C) Groups of age and sex-matched WT (B6) mice were treated or not with 200 μg of 5E6 F(ab')2 mAbs per mouse i.p. two days prior to influenza virus infection (600 PFU), on the day of infection, and every two days post-influenza virus infection until day 10 p.i. Data are pooled from two independent experiments (n = 16 in each group). *p < 0.05, **p < 0.01 and ***p < 0.001. Statistical analysis was performed with the log rank test.
Fig 7.
IFN-γ and perforin are required for protection against influenza virus infection.
(A) Survival of infected NKCKD (n = 18) and NKCKD-Prf-/- (n = 12) mice (600 PFU) was monitored as before. Data are pooled from two independent experiments. (B) Groups of influenza virus-infected and uninfected NKCKD mice (600 PFU) were treated or not with 200 μg anti-IFN-γ mAb per mouse i.p. two days prior to influenza virus infection, on the day of infection, and every two days post-influenza virus infection until day 10 p.i. Data are pooled from two independent experiments, with the following groups: infected NKCKD+anti-IFN-γ (n = 18), infected NKCKD (n = 10), and uninfected NKCKD+anti-IFN-γ (n = 6). *p < 0.05, **p < 0.01 and ***p < 0.001. Statistical analysis was performed with the log rank test. (C) Lungs from infected (600 PFU) age- and sex-matched WT and NKCKD mice were collected on day 5 p.i., weighed, and virus titer (presented as PFU/g of lung tissue) was assessed in lung homogenates by plaque assay on MDCK cells. (D) Schematic representation of NK cell education/licensing and pathogen recognition by NK cells that express self-MHC-I-specific inhibitory Ly49 (licensed) and those that do not (unlicensed).