(A) Treatment protocol: CD45.1⁺ wt mice were lethally irradiated (6 Gy) and transplanted 1×10⁶ CD45.2⁺ wt, pkr^−/−, ifnar^−/− or trail^−/− BM cells to generate chimeric mice. 12w later, when >90% of AM were of donor (wt, pkr^−/−, ifnar^−/− or trail^−/−) phenotype, chimeric mice were mock- or A/PR8-infected and subjected to analyses at 2 d or 7 d pi. (B, C) depict IV-induced sTRAIL concentrations in BALF (B) and proportions of mTRAIL-expressing AM and ExMac (exudate macrophages) in BALF of chimeric mice at d2 pi (C). (D, E) AEC apoptosis was quantified in mock- or A/PR8-infected chimeric mice at d7 pi by FACS (D, depicted as Annexin V⁺ proportion of CD31⁻CD45⁻EpCam⁺lung cells, left panel; representative FACS plots, right panel) or by western blot using lysates of AEC isolated from mock- or A/PR8-infected chimeric mice and a cleaved caspase-3-specific Ab (E, top panel, western blot of 3 independent experiments; bottom panel, quantification of western blot data by densitometry). (F) Alveolar albumin leakage was analysed in mock- or A/PR8-infected wt and trail^−/− chimeric mice at d7 pi by intravenous injection of FITC-labelled albumin and is depicted as ratio of serum and BALF FITC-fluorescence in arbitrary units (AU). (G) Body weight of wt and trail^−/−chimeric mice was determined post A/PR8 infection (350 pfu/∼30%LD50). Bar graphs show means ± SD of (B, C, D, E, F) 5 animals/group and (G) 8 animals/group. * p<0,05; ** p<0,01; ***p<0,001; n.d.; not determined; BMT, bone marrow transplantation; dpi, days post infection; pi, post infection; sTRAIL, soluble TRAIL; mTRAIL, membrane bound TRAIL; Ab, antibody.

https://doi.org/10.1371/journal.ppat.1005716.g001