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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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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.
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ppat-1003188-g007: 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.

Mentions: To investigate whether IFN-β induced pro-apoptotic AM-AEC cross-talk operates also during in vivo IV infection, A/PR8-infected wt mice were intratracheally treated with IFN-β at d5 pi. As shown in Fig. 7A, sTRAIL concentrations in BALF at d7 pi were significantly increased compared to vehicle-treated or mock-infected mice. IFN-β-induced (both endogenous and exogenously applied IFN-β) AEC apoptosis and alveolar protein leakage were attenuated upon intratracheal treatment with an anti-TRAIL Ab (Fig. 7B, C). To address the role of PKR activation and subsequent type I IFN release in macrophage TRAIL-induced epithelial cell damage, we next created bone marrow chimeric mice of wt recipient phenotype which were transplanted wt, pkr−/−, ifnar−/−, or trail−/− BM cells. Chimeric mice displayed >90% of circulating and tissue-resident macrophages of donor phenotype at 12w post BMT (Fig. S5), and were subjected to A/PR8-infection as outlined in Fig. 8A. Quantification of sTRAIL in BALF revealed induction in A/PR8-infected wt BM transplanted mice, whereas sTRAIL concentrations were significantly reduced in infected chimeric mice with pkr−/−, ifnar−/− or trail−/− AM (Fig. 8B). Likewise, the proportions of mTRAIL expressing alveolar resident (AM) and recruited exudate (ExMac) macrophages ranged at ∼15% in wt BM transplanted mice, and were widely reduced when resident or exudate macrophages were PKR-, or IFNAR-deficient (Fig. 8C, flow cytometric gating strategies for AM and ExMac depicted in Fig. S6). Notably, mTRAIL was not significantly expressed on macrophages of mock-infected mice or on further myeloid lung-recruited leukocyte populations in BALF (neutrophils; data not shown) or lung homogenates (CD11b+ dendritic cells, CD103+ dendritic cells; data not shown) in the different groups of chimeric mice after A/PR8 infection. Correspondingly, the fractions of apoptotic AEC, increased in A/PR8-infected compared to mock-infected wt BM transplanted mice, were reduced in mice transplanted pkr−/−, ifnar−/− or trail−/− BM, as demonstrated by flow cytometric quantification of AnnexinV+ AEC or levels of cleaved caspase-3 in AEC isolated from mock- or A/PR8-infected chimeric mice (Fig. 8D, E, respectively). Minor differences in between the AnnexinV- and cleaved caspase 3-based AEC apoptosis quantification might result from the analyses of different signalling events within the apoptotic cascade. Of note, viral clearance was not affected in mice lacking leukocyte TRAIL or IFNAR, however, PKR expression in leukocytes was required for effective antiviral host defense as demonstrated by quantification of A/PR8 titers in BALF at d7 pi (Fig. S7). Finally, macrophage TRAIL deficiency was associated with attenuated IV-induced lung injury and reduced morbidity as shown by alveolar albumin leakage at d7 pi (Fig. 8F) and body weight loss in the time course pi (Fig. 8G). Together, these data reveal that TRAIL expression is induced in a PKR- and type I IFN-dependent way in AM upon A/PR8 infection in vivo which significantly contributes to IV-induced lung injury.


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)

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.
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ppat-1003188-g007: 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.
Mentions: To investigate whether IFN-β induced pro-apoptotic AM-AEC cross-talk operates also during in vivo IV infection, A/PR8-infected wt mice were intratracheally treated with IFN-β at d5 pi. As shown in Fig. 7A, sTRAIL concentrations in BALF at d7 pi were significantly increased compared to vehicle-treated or mock-infected mice. IFN-β-induced (both endogenous and exogenously applied IFN-β) AEC apoptosis and alveolar protein leakage were attenuated upon intratracheal treatment with an anti-TRAIL Ab (Fig. 7B, C). To address the role of PKR activation and subsequent type I IFN release in macrophage TRAIL-induced epithelial cell damage, we next created bone marrow chimeric mice of wt recipient phenotype which were transplanted wt, pkr−/−, ifnar−/−, or trail−/− BM cells. Chimeric mice displayed >90% of circulating and tissue-resident macrophages of donor phenotype at 12w post BMT (Fig. S5), and were subjected to A/PR8-infection as outlined in Fig. 8A. Quantification of sTRAIL in BALF revealed induction in A/PR8-infected wt BM transplanted mice, whereas sTRAIL concentrations were significantly reduced in infected chimeric mice with pkr−/−, ifnar−/− or trail−/− AM (Fig. 8B). Likewise, the proportions of mTRAIL expressing alveolar resident (AM) and recruited exudate (ExMac) macrophages ranged at ∼15% in wt BM transplanted mice, and were widely reduced when resident or exudate macrophages were PKR-, or IFNAR-deficient (Fig. 8C, flow cytometric gating strategies for AM and ExMac depicted in Fig. S6). Notably, mTRAIL was not significantly expressed on macrophages of mock-infected mice or on further myeloid lung-recruited leukocyte populations in BALF (neutrophils; data not shown) or lung homogenates (CD11b+ dendritic cells, CD103+ dendritic cells; data not shown) in the different groups of chimeric mice after A/PR8 infection. Correspondingly, the fractions of apoptotic AEC, increased in A/PR8-infected compared to mock-infected wt BM transplanted mice, were reduced in mice transplanted pkr−/−, ifnar−/− or trail−/− BM, as demonstrated by flow cytometric quantification of AnnexinV+ AEC or levels of cleaved caspase-3 in AEC isolated from mock- or A/PR8-infected chimeric mice (Fig. 8D, E, respectively). Minor differences in between the AnnexinV- and cleaved caspase 3-based AEC apoptosis quantification might result from the analyses of different signalling events within the apoptotic cascade. Of note, viral clearance was not affected in mice lacking leukocyte TRAIL or IFNAR, however, PKR expression in leukocytes was required for effective antiviral host defense as demonstrated by quantification of A/PR8 titers in BALF at d7 pi (Fig. S7). Finally, macrophage TRAIL deficiency was associated with attenuated IV-induced lung injury and reduced morbidity as shown by alveolar albumin leakage at d7 pi (Fig. 8F) and body weight loss in the time course pi (Fig. 8G). Together, these data reveal that TRAIL expression is induced in a PKR- and type I IFN-dependent way in AM upon A/PR8 infection in vivo which significantly contributes to IV-induced lung injury.

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