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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 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.
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ppat-1003188-g001: 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.

Mentions: To evaluate the role of type I IFN in IV-induced AEC damage and lung injury, we first quantified type I IFN levels in bronchoalveolar lavage fluid (BALF) during the time course of A/PR8 infection in wildtype (wt) mice. As shown in Fig. 1A (left), IFN-α and -β were both released into the air spaces in response to A/PR8 infection. Corresponding A/PR8 titers in BALF are provided (Fig. 1A, right). To test whether endogenous type I IFNs contributed to the severe lung injury observed in IV-infected mice we analysed AEC injury after alveolar deposition of a neutralizing anti-IFN antibody (Ab). As demonstrated in Fig. 1B, AEC apoptosis was significantly reduced to similar levels when either IFN-α or IFN-β or both were blocked at d5 pi. In turn, alveolar injury at d7 pi was further increased when recombinant IFN-β was applied to the airways of A/PR8-infected mice at d5 pi, as shown in Fig. 1C (left; AEC apoptosis quantified by AnnexinV binding to AEC; right, alveolar protein leakage). Of note, lung viral loads at d7 pi remained unchanged regardless of whether endogenous IFN-β was blocked or recombinant IFN-β was applied intratracheally at d5 pi (data not shown). It is well established that AEC and AM are primary targets for different IV strains in the alveolar compartment of the lung [20]. To determine the predominant source of IV-induced alveolar IFN-β, we next infected primary murine AEC and AM ex vivo with A/PR8 and quantified peak IFN-β levels in the culture supernatants. Of note, both AEC and AM were infected by A/PR8 at different MOIs ex vivo as determined by immunolabelling of viral nucleoprotein (Fig. S1A). In contrast to AEC, A/PR8 infection of AM was found to be abortive as we could not detect infectious virions in the supernatants of AM cultures. As demonstrated in Fig. 1D, murine AM nevertheless released significantly higher IFN-β levels within 24 h compared to murine AEC in response to infection with live, but not heat-inactivated A/PR8, and this was similarly found when we compared human AM and AEC (Fig. S1B). IFN-β expression robustly occurred in ex vivo IV-infected murine AM in a dose- and IV strain-dependent manner (Fig. 1E, S2A), and likewise in human AM (Fig. S2B). To further dissect the mechanisms by which macrophage IFN-β increased AEC apoptosis, we treated non-infected AEC with IFN-β ex vivo and determined AEC apoptosis induction. Interestingly, recombinant IFN-β did not increase apoptotic damage in infected mono-cultured AEC, however, AEC apoptosis was highly increased upon IFN-β treatment when AEC were co-cultured with AM (Fig. 1F). Together, these data indicate that macrophage IFN-β induces apoptotic AEC damage upon IV infection and suggest a second, IFN-β-inducible pro-apoptotic macrophage mediator to be involved.


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585175&req=5

ppat-1003188-g001: 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.
Mentions: To evaluate the role of type I IFN in IV-induced AEC damage and lung injury, we first quantified type I IFN levels in bronchoalveolar lavage fluid (BALF) during the time course of A/PR8 infection in wildtype (wt) mice. As shown in Fig. 1A (left), IFN-α and -β were both released into the air spaces in response to A/PR8 infection. Corresponding A/PR8 titers in BALF are provided (Fig. 1A, right). To test whether endogenous type I IFNs contributed to the severe lung injury observed in IV-infected mice we analysed AEC injury after alveolar deposition of a neutralizing anti-IFN antibody (Ab). As demonstrated in Fig. 1B, AEC apoptosis was significantly reduced to similar levels when either IFN-α or IFN-β or both were blocked at d5 pi. In turn, alveolar injury at d7 pi was further increased when recombinant IFN-β was applied to the airways of A/PR8-infected mice at d5 pi, as shown in Fig. 1C (left; AEC apoptosis quantified by AnnexinV binding to AEC; right, alveolar protein leakage). Of note, lung viral loads at d7 pi remained unchanged regardless of whether endogenous IFN-β was blocked or recombinant IFN-β was applied intratracheally at d5 pi (data not shown). It is well established that AEC and AM are primary targets for different IV strains in the alveolar compartment of the lung [20]. To determine the predominant source of IV-induced alveolar IFN-β, we next infected primary murine AEC and AM ex vivo with A/PR8 and quantified peak IFN-β levels in the culture supernatants. Of note, both AEC and AM were infected by A/PR8 at different MOIs ex vivo as determined by immunolabelling of viral nucleoprotein (Fig. S1A). In contrast to AEC, A/PR8 infection of AM was found to be abortive as we could not detect infectious virions in the supernatants of AM cultures. As demonstrated in Fig. 1D, murine AM nevertheless released significantly higher IFN-β levels within 24 h compared to murine AEC in response to infection with live, but not heat-inactivated A/PR8, and this was similarly found when we compared human AM and AEC (Fig. S1B). IFN-β expression robustly occurred in ex vivo IV-infected murine AM in a dose- and IV strain-dependent manner (Fig. 1E, S2A), and likewise in human AM (Fig. S2B). To further dissect the mechanisms by which macrophage IFN-β increased AEC apoptosis, we treated non-infected AEC with IFN-β ex vivo and determined AEC apoptosis induction. Interestingly, recombinant IFN-β did not increase apoptotic damage in infected mono-cultured AEC, however, AEC apoptosis was highly increased upon IFN-β treatment when AEC were co-cultured with AM (Fig. 1F). Together, these data indicate that macrophage IFN-β induces apoptotic AEC damage upon IV infection and suggest a second, IFN-β-inducible pro-apoptotic macrophage mediator to be involved.

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