Macrophage-expressed IFN-β contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia

Abstract

Influenza viruses (IV) cause pneumonia in humans with progression to lung failure and fatal outcome. Dysregulated release of cytokines including type I interferons (IFNs) has been attributed a crucial role in immune-mediated pulmonary injury during severe IV infection. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage (AM)-expressed IFN-β significantly contributes to IV-induced alveolar epithelial cell (AEC) injury by autocrine induction of the pro-apoptotic factor TNF-related apoptosis-inducing ligand (TRAIL). Of note, TRAIL was highly upregulated in and released from AM of patients with pandemic H1N1 IV-induced acute lung injury. Elucidating the cell-specific underlying signalling pathways revealed that IV infection induced IFN-β release in AM in a protein kinase R- (PKR-) and NF-κB-dependent way. Bone marrow chimeric mice lacking these signalling mediators in resident and lung-recruited AM and mice subjected to alveolar neutralization of IFN-β and TRAIL displayed reduced alveolar epithelial cell apoptosis and attenuated lung injury during severe IV pneumonia. Together, we demonstrate that macrophage-released type I IFNs, apart from their well-known anti-viral properties, contribute to IV-induced AEC damage and lung injury by autocrine induction of the pro-apoptotic factor TRAIL. Our data suggest that therapeutic targeting of the macrophage IFN-β-TRAIL axis might represent a promising strategy to attenuate IV-induced acute lung injury.

Figure 1. IV-induced IFN-β is mainly macrophage-derived and induces AEC apoptosis ex vivo and in vivo.

(A) C57BL/6 wt mice were infected with 500 pfu A/PR8 and the IFN-α and IFN-β levels (left panel) as well as the amount of infectious virus particles (right panel) were quantified from BALF at indicated time points. (B) AEC apoptosis was quantified in mock or A/PR8 infected mice at d7 pi after intratracheal treatment with IgG isotype Ab, anti IFN-α Ab, anti-IFN-β Ab or both (d5 pi). (C) AEC apoptosis (left panel) and alveolar protein leakage (right panel) were determined at d7 pi after intratracheal treatment with rIFN-β or vehicle (d5 pi). (D) Murine AEC or AM were mock- or A/PR8-infected (live virus or heat-inactivated) ex vivo (MOI = 0.1) and IFN-β release was quantified in supernatants at 24 h pi. (E) Murine AM were ex vivo infected with live or heat-inactivated A/PR8 at the indicated MOI and IFN-β mRNA expression was quantified at the given times and is depicted as fold induction of mock-infected controls. (F) Uninfected murine AEC were mono- or co-cultured with non-infected AM ex vivo in the presence or absence of IFN-β (180 U/ml) for 24 h, and AEC apoptosis rates were quantified. (G) Murine AM were ex vivo infected with live or heat-inactivated A/PR8 at the given MOI and TRAIL mRNA expression was quantified and is depicted as fold induction of mock-infected controls. (H) Murine AEC or AM were ex vivo infected A/PR8 at the indicated MOI and TRAIL mRNA expression was quantified at the given time and is depicted as fold induction of mock-infected controls. Bar graphs represent means ± SD of (A) 4 animals/group, (B, C) 5 animals/group or of 4 (D, E, G) and 3 (F, H) independent experiments. * p<0.05; ** p<0.01; ***p<0.001; ctrl, control; mAEC, murine alveolar epithelial cells; hi, heat inactivated; Ab, antibody, mAM, murine AM; MOI, multiplicity of infection; pi, post infection; n.s., not significant; iso, isotype; rIFN-β, recombinant IFN-β.

Figure 2. The pro-apoptotic factor TRAIL is upregulated in alveolar macrophages of patients with severe IV pneumonia.

AM were isolated from BALF of patients with pH1N1-induced ARDS or non-viral (i.e. bacterial pneumonia- or sepsis-associated) ARDS and TRAIL mRNA (A) and mTRAIL expression (B) were quantified by qRT-PCR and FACS, respectively, and compared to AM of control patients who underwent bronchoscopy due to diagnostic reasons but revealed normal cell numbers and differential counts (‘healthy’ control). C depicts sTRAIL levels in BALF of patients with pH1N1-induced ARDS or with confirmed bacterial pneumonia (BP). The graphs show means ± SD. * p<0.05; ** p<0.01; ***p<0.001; n.s., not significant; mTRAIL, membrane bound TRAIL; sTRAIL, soluble TRAIL.

Figure 3. IV-induced macrophage IFN-β upregulates macrophage TRAIL.

(A) Murine AM were treated ex vivo with rIFN-β at the given concentrations and TRAIL mRNA expression was quantified and is depicted as fold induction of unstimulated controls. (B) Murine AM were A/PR8-infected (MOI = 0.1) or treated with 180 U/ml rIFN-β ex vivo in presence of a protease inhibitor cocktail to prevent TRAIL shedding and mTRAIL abundance was analysed by FACS after 24 h. Shown are histograms from a representative experiment (top panel) or mean fluorescence intensities (MFI, bottom panel). (C) Murine wt AM were A/PR8 infected at the given MOI and treated with anti-IFN-β Ab, Jak/STAT inhibitor, or DMSO/isotype IgG Ab as control. Murine ifnar^−/− AM were A/PR8 infected and left untreated. TRAIL mRNA expression was quantified and is depicted as fold induction of mock-infected cells. Bar graphs represent means ± SD of (A) 6; (B) 4 and (C) 5 independent experiments. * p<0.05; ** p<0.01; ***p<0.001; MOI, multiplicity of infection; IgG, IgG isotype control; rIFN-β, recombinant IFN-β; mTRAIL, membrane bound TRAIL; Ab, antibody.

Figure 4. IV-induced IFN-β release and subsequent TRAIL expression depend on activation of PKR and NF-κB in alveolar macrophages.

(A) Murine AM were A/PR8 infected ex vivo and expression of phosphorylated PKR (p-PKR) and total PKR were assessed by western blot. Wt or pkr^{−/ −} AM were A/PR8-infected ex vivo and NF-κB p65 activation was comparatively analysed by TransAM assay (quantifying p65 binding to a consensus-binding site oligo by a colorimetric method) and depicted as fold activation of mock-infected control (B) or phosphorylated IRF-3 in relation to total IRF-3 protein expression or total IRF-7 expression were determined by western blot at the given time points pi and densitometry data is depicted as relative expression (C). (D) Wt vs. pkr^{−/ −} AM or IKK inhibitor- vs. DMSO-treated wt AM were A/PR8-infected ex vivo and IFN-β concentrations in supernatants were analysed 24 h pi. (E) Wt or pkr^{−/ −} AM were either A/PR8-infected or stimulated with 180 U/ml rIFN-β, respectively, and additionally treated with IKK inhibitor vs. DMSO or anti-IFN-β vs. isotype Ab and TRAIL mRNA expression was quantified 16 h pi. (A) shows a representative western blot of 3 independent experiments. Bar graphs represent means ± SD of 3 (B, C) or 4 (D, E) independent experiments. * p<0.05; ** p<0.01; ***p<0.005; ctrl, control; iso, isotype; pi, post infection; Ab, antibody.

Figure 5. DR5 is upregulated in AEC upon IV infection in an IFN-β-independent way.

(A) Primary murine or human AEC were mock- or A/PR8-infected ex vivo and DR5 mRNA expression was quantified 48 h pi. (B) Murine AEC were mock- or A/PR8-infected ex vivo and surface DR5 abundance in infected (NP⁺) vs. non-infected (NP⁻) AEC from the same culture was analysed by FACS 48 h pi (representative histogram provided in the left panel) and is depicted as fold MFI (mean fluorescence intensity) of mock-infected cultures (right panel). (C) Murine AEC were stimulated with rIFN-β or vehicle-treated (ctrl) and DR5 mRNA expression was quantified after 6 h and 12 h and is depicted as ΔCT values. (D) DR5 surface expression on AEC from A/PR8-infected wt and ifnar^−/− mice was analysed by FACS. Bar graphs represent means ± SD of 4 (A) and 5 (B, C) independent experiments. Bar graphs in D represent means ± SD of animals/group. * p<0,05; ** p<0,01; ***p<0,001; MOI, multiplicity of infection; hAEC, human AEC; mAEC, murine AEC; NP, nucleoprotein; iso, isotype Ab; rIFN-β, recombinant IFN-β.

Figure 6. Macrophage TRAIL induces AEC apoptosis in a PKR- and type I IFN-dependent way ex vivo.

(A) 48 h co-cultured murine AEC (mock- or A/PR8-infected) and AM (A/PR8-infected) were treated with anti-TRAIL or isotype Ab and AEC apoptosis was quantified by flow cytometry (Annexin V staining). (B, C) Mock- or A/PR8-infected wt, pkr ^−/−, ifnar^{− /−} or trail^−/− AM (MOI = 0.1) were co-cultured with AEC for 48 h until AEC apoptosis was determined by FACS (representative FACS plots provided in bottom panel) or western blot using an anti-cleaved caspase-3 Ab and lysates of staurosporin-treated AEC as positive control. (D) Wt or gene-deficient AM were mock- or A/PR8-infected (MOI = 0.1) ex vivo and TRAIL concentrations were determined at 48 h pi. (E) Mock or A/PR8 infected wt macrophages were co-cultured with wt AEC and a neutralizing anti-DR5 antibody or the isotype control were added to the medium. Additionally, wt mock or A/PR8 infected macrophages were co-cultured with dr5 ^−/− AEC for 48 h and until AEC apoptosis was determined by FACS. (C) shows a representative western blot of 3 independent experiments. Bar graphs show means ± SD of (A) 3, (B, D) 5 and (E) 3 independent experiments. * p<0,05; ** p<0,01; ***p<0,001; AM, alveolar macrophages; AEC, alveolar epithelial cells; hi, heat inactivated; ns, not significant; iso, isotype; sTRAIL, soluble TRAIL.

Figure 7. Blockade of IFN-β dependently induced TRAIL attenuates epithelial injury upon IV infection in vivo.

(A) A/PR8 infected wt mice (500 pfu) were intratracheally treated with rIFN-β (10.000 IU) or vehicle (0.1% BSA in PBS) at d5 pi and sTRAIL was quantified from BALF. (B, C) A/PR8 infected wt mice (500 pfu) were intratracheally treated with rIFN-β or vehicle (0.1% BSA in PBS) and anti-TRAIL Ab or isotype Ab intraperitoneally and AEC apoptosis (B) and alveolar protein leakage (C) were determined at d7 pi. Bar graphs show means ± SD of (A) 6 animals/group and (B, C) 8 animals/group. * p<0,05; ** p<0,01; ***p<0,001. Iso, isotype; sTRAIL, soluble TRAIL; Ab, antibody; pi, post infection.

Figure 8. Blockade of autocrine myeloid IFN-β signalling impairs macrophage TRAIL expression and attenuates epithelial injury upon A/PR8 infection in vivo.

(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.

Figure 9. Proposed model of IFN-β mediated pro-apoptotic AM-AEC cross-talk in IV-induced lung injury.

IFN-β is released from IV-infected AM in a PKR- and NF-κB-dependent way and induces expression and release of macrophage TRAIL via autocrine IFNAR activation. Macrophage TRAIL induces AEC apoptosis via its receptor DR5 which is constitutively expressed on non-infected and upregulated on IV-infected AEC. Through this signalling cascade, IFN-β significantly contributes to AEC damage and lung injury during severe IV pneumonia. IV, influenza virus.