(A) Representative sections of n = 3 murine lungs d7 after PBS (ctrl) (left panel) or 500 pfu PR8 (IAV) inoculation in vivo, stained sequentially with anti-NKAα1 antibody (red) and DAPI (blue), followed by H&E staining. Sections of IAV-infected mice were taken from highly inflammatory (middle panel) and less inflammatory regions (right panel). Scale bars: 50 μm; arrows mark edematous regions. (B and C) Arterial partial pressure of oxygen (pO₂) (B) and in vivo AFC measurements (C) d7 pi after inoculation of PBS or PR8. (D) Gating strategy showing representative dot plots for live cells (7AAD^–), epithelial cells (EpCAM⁺), and representative histograms of NKAα1⁺ staining or the respective IgG control from murine AEC cultures. SSC, side scatter. (E and F) Densitometric quantification of immunoblots of NKAα1 in relation to β-actin at the indicated time points in total cell lysates of mAEC (E) or hAEC (F) inoculated in vitro with PBS or PR8 for 24 hours. (G and I) Relative MFI of NKAα1 detected by FACS on live mAEC (G) or hAEC (I) treated in vitro with PBS (ctrl) or PR8 (IAV) at MOI 0.1 for 24 or 16 hours, respectively. (H and J) Densitometric analysis of NKAα1 expression in comparison to the housekeeping protein glucose transporter 1 (Glut1) within the cell surface fraction of PR8-infected mAEC at the indicated time points (H) or hAEC 16h pi (I). Values of PBS-treated control conditions were normalized to 1. Representative blots and bar graphs or dot plots show means ±SEM of 7–9 independent experiments for B, C, E, F, and H and 5–6 independent experiments for G, I, and J. Statistical significance was analyzed by unpaired Student’s t test (B, F, G, I, and J) or by 1-way ANOVA and post-hoc Tukey (C, E, and H). *P < 0.05; **P < 0.01; ***P < 0.005.

(A) Flow cytometric analysis of NKAα1 subunit expression on EpCAM⁺ epithelial cells from distal lung homogenate. WT mice were inoculated with PBS (ctrl) or PR8 (IAV) and sacrificed d2 or d7 pi. (B) Relative MFI of NKAα1 detected by FACS on live mAEC inoculated with PBS (ctrl) or PR8 at MOI 0.1 (IAV) and treated with 50 μM salbutamol. (C) Representative Western blot of 3 independent experiments blotting mAEC cell lysates for NKAα1 or GAPDH 72 hours after transfection with adenoviruses carrying NKAα1 or NKAβ1 at MOI 5 and 24 hours after PR8 infection at MOI 0.1. (D and E) WT mice were inoculated with PBS (ctrl) or PR8 (IAV) at d3 after intratracheal infection with adenoviruses overexpressing NKAα1 (Ad-NKAα) and NKAβ1 (Ad-NKAβ) or treated with 10 mg/kg salbutamol i.p. at d3 and d5. Mice were sacrificed at d7 after IAV infection. (D) Flow cytometric analysis of NKAα1 subunit expression on EpCAM⁺ epithelial cells from distal lung homogenate. (E) In vivo measurements of AFC rates over a time interval of 30 minutes. Graphs show single data points plus means ±SEM of 4–10 independent experiments for A, D, and E. Data sets depicting control conditions are identical to (AFC) and (NKAα1 expression) and included for better comparison between experimental conditions. Bar graph (B) represents means ±SEM of 3 independent experiments. Nonbracketed asterisk indicate statistical significance to control conditions. Statistical significance was analyzed by 1-way ANOVA and post-hoc Tukey. **P < 0.01; ***P < 0.005.

(A and B) Densitometric quantification of Western blot of NKAα1 expression compared with β-actin at 24h pi in total cell lysates of mAEC after coculture with AM (A) or BMM (B) without infection (ctrl), infection of only macrophages (AM; BMM), or of both cell types (AEC/AM; AEC/BMM). (C) Densitometric analysis of NKAα1 expression compared with glucose transporter 1 (Glut1) within the cell surface fraction of 24h pi in PR8-infected mAEC cocultured with BMM. (D) Relative MFI of NKAα1 detected by FACS on live mAEC cocultured with BMM. (E) Gating strategy showing representative histograms for viral HA expression and NKAα1 expression on PBS-treated ctrl, IAV-infected HA⁺, or HA^– AEC. F and G depict NKAα1 MFI of HA⁺ vs. HA^– cell populations in mAEC (F) or hAEC (G) 24h pi with PR8 in comparison with PBS-treated cells (ctrl). (H) Flow cytometric analysis of NKAα1 subunit expression on EpCAM⁺ epithelial cells from distal lung homogenate. NKAα1 subunit expression was analyzed in AEC of PBS-treated mice (ctrl) or on the HA⁺ vs. HA^– cell population of AEC isolated from PR8-inoculated WT mice sacrificed at d7 pi. (I) Analysis of NKAα1 MFI of mAEC treated for 2 hours with conditioned media from 16 hours infected (IAV) or PBS-treated (ctrl) cells. For A–D, F, G, and I, values of PBS-treated control conditions were normalized to 1. Representative blots, histograms, and bar graphs showing means ±SEM of n = 8–10 experiments (A–C), n = 6 (D and F), n = 3 (G), n = 6–10 (H), and n = 4 (I). Data set depicting control conditions in H are identical to A and are included for better comparison between experimental conditions. Statistical significance was analyzed by 1-way ANOVA and post-hoc Tukey. *P < 0.05; **P < 0.01; ***P < 0.005.

(A and B) IFNα (A) and TRAIL (B) concentrations of AEC mono- and coculture supernatant 24h pi quantified by ELISA. mAEC were monocultured and inoculated in vitro with PBS (ctrl) or PR8 (AEC) or cocultured with BMM without infection (ctrl), with infection of only macrophages (BMM), or with infection of both cell types (AEC/BMM). (C) TRAIL mRNA expression quantified by qPCR in BMM isolated from WT or Ifnar^–/– mice noninfected (ctrl) or PR8-infected (IAV) at 16h pi. (D–G) NKAα1 relative MFI on/of mAEC treated with 25U/ml mouse rIFNα or/and 100 pg/ml mouse rTRAIL for 16 hours (D); mAEC derived from Ifnar^–/– or Trail^–/– mice inoculated with PBS (ctrl) or PR8 (IAV) 24h pi (E); mAEC derived from Ifnar^–/– mice inoculated for 2 hours with conditioned media from PBS (ctrl) or PR8 (IAV) WT AEC infected for 16 hours (F); and mAEC derived from WT, Ifnar^–/–, or Dr5^–/– mice cocultured with BMM from WT or Trail^–/– mice and infection of none (ctrl) or both cell types (AEC/BMM) in vitro (G). Value of control conditions were normalized to 1. (H and I) Vectorial water transport of hAEC monoculture 6 hours after inoculation with PBS (ctrl), PR8 (IAV), rIFNα (150 pg/ml), and/or rTRAIL (100 pg/ml) or in coculture with human AM without additional treatment (–), in presence of neutralizing antibodies against human IFNα (0.5 μg/ml) and human TRAIL (0.1 μg/ml) (anti-IFN + anti-TRAIL) or the respective IgG controls (IgG). hAEC maintained barrier integrity in all assay conditions (). Bar graphs represent means ±SEM of 4–6 independent experiments (A, B, and E–I), 3 independent experiments (C), and 6–8 independent experiments (D). Statistical significance was analyzed by Student’s t test (F) or 1-way ANOVA and post-hoc Tukey (A–E and G–I). *P < 0.05; **P < 0.01; ***P < 0.005.

(A–D) Representative Western blots of n = 3–4 independent experiments of AMPK (62 kDa) and the AMPK substrate pACC (280 kDa) mAEC treated with PBS (ctrl) or PR8 (IAV) (A) for 24 hours. (B) Cocultures of murine AEC and BMM were left uninfected (ctrl); only macrophages were infected (BMM) or both cell types were infected (AEC/BMM). (C) mAEC were treated with conditioned media (CM) of 16 hours PR8-infected (IAV) or PBS-treated (ctrl) mAEC for 2 hours. (D) mAEC treated with the AICAR (1 mM), rTRAIL (100 g/ml) , or rIFNα (25 U/ml) for 16 hours. (E–G) Relative MFI of NKAα1 on mAEC inoculated with PBS (ctrl) or PR8 (IAV) for 24 hours without additional treatment or in presence of 1 mM AICAR or 20 μM Compound C (E); on mAEC cocultured with BMM without infection (ctrl), with infection of only macrophages (BMM), or with infection of both cell types (AEC/BMM) without additional treatment or in presence of 20 μM Compound C (F); on mAEC inoculated with PBS (ctrl) or PR8 (IAV) and incubated for 24 hours without additional treatment or in presence 0,1 μM TAK1 inhibitor (5Z)-7-Oxozeanol or the 0,5 μM CaMKKβ inhibitor STO-609 or 10 μM of the Ca²⁺ chelator BAPTA-AM (G). (H) Representative Western blot of n = 3–4 independent experiments of AMPK and pACC from 24 hours PR8-infected mAEC without additional treatment or in the presence of 20 μM Compound C, 0,1 μM (5Z)-7-Oxozeanol, or the 0,5 μM STO-609 and schematic depiction of the used inhibitors. (I) Vectorial water transport of confluent hAEC culture 6 hours after inoculation with PBS (ctrl) or PR8 (IAV) without additional treatment (–), in the presence of 25 μM Ouabain, 10 μM Compound C, or 0.5 μM STO-609. Bar graphs represent means ±SEM of 4–6 independent experiments. Values of PBS-treated control conditions were normalized to 1. Statistical significance was analyzed by 1-way ANOVA and post-hoc Tukey. *P < 0.05; **P < 0.01; ***P < 0.005.

(A and B) WT mice were inoculated with PBS (ctrl), Ad-Null, or Ad–DN AMPK for d7 prior to infection with PBS or IAV (Udorn) at d2 pi. (A) Flow cytometric analysis of NKAα1 subunit expression on AEC. (B) In vivo measurements of AFC rates over a time interval of 30 minutes. (C and D) WT mice were inoculated with PBS (ctrl) or PR8 (IAV) and treated with 20 mg/kg Compound C i.p. at d5 pi. Mice were sacrificed at d7 pi. (C) Flow cytometric analysis of NKAα1 subunit expression on AEC. (D) In vivo measurements of AFC rates over a time interval of 30 minutes. Graphs show means ±SEM of n = 4–5 (A), 4–9 (B), 4–9 (C), and 4–6 (D) independent experiments. Data set depicting control conditions in C and D are identical to (AFC) and (NKAα1 expression) and were included for better comparison between experimental conditions. Nonbracketed asterisk indicate statistical significance to control conditions. Statistical significance was analyzed by 1-way ANOVA and post-hoc Tukey. **P < 0.01; ***P < 0.005.

(A and B) Flow cytometric analysis of NKAα1 subunit expression on EpCAM⁺ epithelial cells from distal lung homogenate. (C and D) In vivo measurements of AFC rates over a time interval of 30 minutes. (A and C) WT, Ifnar^–/–, or Trail^–/– mice were inoculated with PBS (ctrl) or PR8 (IAV) and sacrificed d7 pi. (B and D) Ccr2^–/– mice were inoculated with PR8 (IAV) and sacrificed d7 pi without further intervention (Ccr2^–/–) or after intratracheal transfer of flow-sorted alveolar BMM from d7 infected WT (WT to Ccr2^–/–) or Trail^–/– (Trail^–/– to Ccr2^–/–) mice at d3 pi. Viral loads were not significantly altered between experimental groups (data not shown). Graphs show single data points plus means ±SEM of 5–10 independent experiments for A and B and 4–6 independent experiments for C and D. Data sets depicting control conditions are similar to (AFC) and (NKAα1 expression). Nonbracketed asterisk indicate statistical significance to control conditions. Statistical significance was analyzed by unpaired Student’s t test (B) or by 1-way ANOVA and post-hoc Tukey. ***P < 0.005.

WT mice were inoculated with 500 pfu PR8 (IAV) and treated with i.p. administration of 150 μg IgG control antibody or anti-TRAIL antibody at d3 and d5 pi together with a BSA vehicle control or anti-IFNα antibody (10,000 IU) intratracheally at d3 pi (IgG + BSA or anti-TRAIL + anti-IFNα, respectively) and sacrificed at d7 pi. (A) Flow cytometric analysis of NKAα1 subunit expression on EpCAM⁺ epithelial cells from distal lung homogenate. (B) In vivo measurements of AFC rates over a time interval of 30 minutes. (C) Daily body weight measurements are given as a percentage of values prior to infection. (D) Representative sections of n = 3 murine lungs stained by H&E (Scale bar: 50 μm; arrows mark edematous regions). Graphs in A and B show single data points plus means ±SEM of 5 independent experiments. Bar graphs represent means ±SEM of 5 mice per group. Data set depicting control conditions in A and B are identical to (NKAα1 expression) and were included for better comparison of between experimental conditions. Nonbracketed asterisk indicate statistical significance to control conditions. Statistical significance was analyzed by 1-way ANOVA and post-hoc Tukey. ***P < 0.005.

IAV infection results in AEC release of IFNα and induction of IFNβ-dependent release of TRAIL in AMs. Ligation to their receptors, IFNAR and particularly DR5, results in activation of the stress kinase AMPK. Activation of AMPK initiates degradation and reduction of Na,K-ATPase abundance on the cell plasma membrane and impacts on fluid reabsorption, leading to the persistence of lung edema after IAV infection.