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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.

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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.
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ppat-1003188-g004: 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.

Mentions: Several pattern recognition receptors and downstream signalling events were previously shown to be involved in type I IFN induction in IV-infected epithelial cells [22], [23]. However, the cell-specific mechanisms of IV recognition and subsequent IFN-β release in primary AM are far less defined. Given our previous results on protein kinase R (PKR) as important inflammatory signal transducer to NF-κB-mediated transcriptional activity in primary alveolar mouse macrophages [24], we questioned whether the IV-induced type I IFN response in AM similarly depended on PKR, known to directly associate with viral RNA in IV-infected epithelial cells [25]. Indeed, ex vivo A/PR8 infection of murine AM resulted in phosphorylation of PKR, most prominent at 2–4 h pi (Fig. 4A). Moreover, the p65 NF-κB subunit was substantially activated at 4–8 h pi in response to A/PR8 infection in wt but not pkr−/− AM (Fig. 4B). Of note, the infection-triggered activation or expression of transcription factors IRF3 and IRF7, respectively, known to induce IV-dependent type I IFN responses in epithelial cells [23], [26], did not differ in A/PR8-infected wt and pkr−/− AM (Fig. 4C). To test whether the IV-induced type I IFN response in AM depended on the PKR-NF-κB axis, we quantified IFN-β release from A/PR8-infected wt vs. pkr−/− AM or in wt AM after NF-κB inhibition. As shown in Fig. 4D, both PKR-deficiency and treatment with a selective IκB kinase (IKK) phosphorylation inhibitor abrogated IFN-β release in A/PR8-infected AM, indicating that IV-induced macrophage IFN-β expression required signal transduction via both PKR and NF-κB. Finally, to address the significance of this signalling pathway for IV-induced TRAIL expression in AM, we stimulated wt or pkr−/− AM with recombinant IFN-β or infected them with A/PR8 in presence or absence of the IKK inhibitor with or without neutralizing anti-IFN-β Ab. As expected, TRAIL gene expression was significantly induced upon IFN-β stimulation in both wt and pkr−/− AM, regardless of NF-κB inhibition (Fig. 4E, left gray graphs). However, when AM were A/PR8-infected, TRAIL gene induction depended on presence of PKR and activation of NF-κB. Macrophage IFN-β blockade in addition to NF-κB inhibition did not further decrease TRAIL expression significantly (Fig. 4E, right black graphs). Altogether, these data demonstrate that IV-infection of AM induces a PKR-NF-κB-dependent autocrine IFN-β signaling loop resulting in expression of the pro-apoptotic ligand TRAIL in IV-infected AM.


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)

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.
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ppat-1003188-g004: 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.
Mentions: Several pattern recognition receptors and downstream signalling events were previously shown to be involved in type I IFN induction in IV-infected epithelial cells [22], [23]. However, the cell-specific mechanisms of IV recognition and subsequent IFN-β release in primary AM are far less defined. Given our previous results on protein kinase R (PKR) as important inflammatory signal transducer to NF-κB-mediated transcriptional activity in primary alveolar mouse macrophages [24], we questioned whether the IV-induced type I IFN response in AM similarly depended on PKR, known to directly associate with viral RNA in IV-infected epithelial cells [25]. Indeed, ex vivo A/PR8 infection of murine AM resulted in phosphorylation of PKR, most prominent at 2–4 h pi (Fig. 4A). Moreover, the p65 NF-κB subunit was substantially activated at 4–8 h pi in response to A/PR8 infection in wt but not pkr−/− AM (Fig. 4B). Of note, the infection-triggered activation or expression of transcription factors IRF3 and IRF7, respectively, known to induce IV-dependent type I IFN responses in epithelial cells [23], [26], did not differ in A/PR8-infected wt and pkr−/− AM (Fig. 4C). To test whether the IV-induced type I IFN response in AM depended on the PKR-NF-κB axis, we quantified IFN-β release from A/PR8-infected wt vs. pkr−/− AM or in wt AM after NF-κB inhibition. As shown in Fig. 4D, both PKR-deficiency and treatment with a selective IκB kinase (IKK) phosphorylation inhibitor abrogated IFN-β release in A/PR8-infected AM, indicating that IV-induced macrophage IFN-β expression required signal transduction via both PKR and NF-κB. Finally, to address the significance of this signalling pathway for IV-induced TRAIL expression in AM, we stimulated wt or pkr−/− AM with recombinant IFN-β or infected them with A/PR8 in presence or absence of the IKK inhibitor with or without neutralizing anti-IFN-β Ab. As expected, TRAIL gene expression was significantly induced upon IFN-β stimulation in both wt and pkr−/− AM, regardless of NF-κB inhibition (Fig. 4E, left gray graphs). However, when AM were A/PR8-infected, TRAIL gene induction depended on presence of PKR and activation of NF-κB. Macrophage IFN-β blockade in addition to NF-κB inhibition did not further decrease TRAIL expression significantly (Fig. 4E, right black graphs). Altogether, these data demonstrate that IV-infection of AM induces a PKR-NF-κB-dependent autocrine IFN-β signaling loop resulting in expression of the pro-apoptotic ligand TRAIL in IV-infected AM.

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