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

Högner K, Wolff T, Pleschka S, Plog S, Gruber AD, Kalinke U, Walmrath HD, Bodner J, Gattenlöhner S, Lewe-Schlosser P, Matrosovich M, Seeger W, Lohmeyer J, Herold S - PLoS Pathog. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine II, University of Giessen Lung Center, Giessen, Germany.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585175&req=5

ppat-1003188-g006: 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.

Mentions: To address whether epithelial IV-infection was required for macrophage TRAIL-induced apoptosis induction, mock- or A/PR8-infected AECs were co-cultured with infected AM in the presence of anti-TRAIL or control IgG isotype Ab. AEC apoptosis was induced in both mock- and A/PR8-infected co-cultured AEC, however, apoptosis levels in infected AEC, expressing increased levels of DR5 (Fig. 5A and B) exceeded those of mock-infected AEC. Addition of neutralizing anti-TRAIL Ab significantly reduced AEC apoptosis induction in both mock- or A/PR8-infected AEC (Fig. 6A). To address whether the macrophage-specific signalling events mediating IV-induced TRAIL expression indeed promoted AEC apoptosis, we mock- or A/PR8-infected wt, pkr−/−, ifnar−/−, or trail−/− AM for 24 h, co-cultured them with AEC for further 48 h and then analysed the proportion of AnnexinV+ AEC. AEC apoptosis rates were not increased upon co-culture with mock-infected wt, pkr−/−, ifnar−/−, or trail−/− AM, compared to uninfected, mono-cultured AEC (8.3±1.8%, data not shown). However, co-culture with infected wt AM strongly induced apoptosis in AEC, and this was significantly reduced when AEC were co-cultured with infected pkr−/−, ifnar−/−, or trail−/− AM (Fig. 6B). Similar data were obtained when expression of cleaved caspase-3 was analysed by western blot in co-cultured AEC (Fig. 6C). Correspondingly, levels of sTRAIL in supernatants were significantly increased upon infection of wt, but not pkr−/−, ifnar−/−, or trail−/− AM (Fig. 6D). Finally, use of blocking DR5 Ab and of AEC derived from DR5-deficient mice in co-cultures revealed that AM-mediated AEC apoptosis induction was dependent on the epithelial TRAIL receptor DR5 (Fig. 6E). Collectively, these data demonstrate that PKR-IFN-β/IFNAR-dependent macrophage TRAIL- induces apoptotic cell death via DR5 in both non-infected and IV-infected AEC ex vivo.


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

Högner K, Wolff T, Pleschka S, Plog S, Gruber AD, Kalinke U, Walmrath HD, Bodner J, Gattenlöhner S, Lewe-Schlosser P, Matrosovich M, Seeger W, Lohmeyer J, Herold S - PLoS Pathog. (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585175&req=5

ppat-1003188-g006: 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.
Mentions: To address whether epithelial IV-infection was required for macrophage TRAIL-induced apoptosis induction, mock- or A/PR8-infected AECs were co-cultured with infected AM in the presence of anti-TRAIL or control IgG isotype Ab. AEC apoptosis was induced in both mock- and A/PR8-infected co-cultured AEC, however, apoptosis levels in infected AEC, expressing increased levels of DR5 (Fig. 5A and B) exceeded those of mock-infected AEC. Addition of neutralizing anti-TRAIL Ab significantly reduced AEC apoptosis induction in both mock- or A/PR8-infected AEC (Fig. 6A). To address whether the macrophage-specific signalling events mediating IV-induced TRAIL expression indeed promoted AEC apoptosis, we mock- or A/PR8-infected wt, pkr−/−, ifnar−/−, or trail−/− AM for 24 h, co-cultured them with AEC for further 48 h and then analysed the proportion of AnnexinV+ AEC. AEC apoptosis rates were not increased upon co-culture with mock-infected wt, pkr−/−, ifnar−/−, or trail−/− AM, compared to uninfected, mono-cultured AEC (8.3±1.8%, data not shown). However, co-culture with infected wt AM strongly induced apoptosis in AEC, and this was significantly reduced when AEC were co-cultured with infected pkr−/−, ifnar−/−, or trail−/− AM (Fig. 6B). Similar data were obtained when expression of cleaved caspase-3 was analysed by western blot in co-cultured AEC (Fig. 6C). Correspondingly, levels of sTRAIL in supernatants were significantly increased upon infection of wt, but not pkr−/−, ifnar−/−, or trail−/− AM (Fig. 6D). Finally, use of blocking DR5 Ab and of AEC derived from DR5-deficient mice in co-cultures revealed that AM-mediated AEC apoptosis induction was dependent on the epithelial TRAIL receptor DR5 (Fig. 6E). Collectively, these data demonstrate that PKR-IFN-β/IFNAR-dependent macrophage TRAIL- induces apoptotic cell death via DR5 in both non-infected and IV-infected AEC ex vivo.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine II, University of Giessen Lung Center, Giessen, Germany.

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.

Show MeSH
Related in: MedlinePlus