Limits...
Autophagy induced by DAMPs facilitates the inflammation response in lungs undergoing ischemia-reperfusion injury through promoting TRAF6 ubiquitination

View Article: PubMed Central - PubMed

ABSTRACT

Lung ischemia-reperfusion (I/R) injury remains one of the most common complications after various cardiopulmonary surgeries. The inflammation response triggered by the released damage-associated molecular patterns (DAMPs) aggravates lung tissue damage. However, little is known about the role of autophagy in the pathogenesis of lung I/R injury. Here, we report that a variety of inflammation-related and autophagy-associated genes are rapidly upregulated, which facilitate the inflammation response in a minipig lung I/R injury model. Left lung I/R injury triggered inflammatory cytokine production and activated the autophagy flux as evidenced in crude lung tissues and alveolar macrophages. This was associated with the release of DAMPs, such as high mobility group protein B1 (HMGB1) and heat shock protein 60 (HSP60). Indeed, treatment with recombinant HMGB1 or HSP60 induced autophagy in alveolar macrophages, whereas autophagy inhibition by knockdown of ATG7 or BECN1 markedly reduced DAMP-triggered production of inflammatory cytokines including IL-1β, TNF and IL12 in alveolar macrophages. This appeared to be because of decreased activation of MAPK and NF-κB signaling. Furthermore, knockdown of ATG7 or BECN1 inhibited Lys63 (K63)-linked ubiquitination of TNF receptor-associated factor 6 (TRAF6) in DAMP-treated alveolar macrophages. Consistently, treatment with 3-MA inhibited K63-linked ubiquitination of TRAF6 in I/R-injured lung tissues in vivo. Collectively, these results indicate that autophagy triggered by DAMPs during lung I/R injury amplifies the inflammatory response through enhancing K63-linked ubiquitination of TRAF6 and activation of the downstream MAPK and NF-κB signaling.

No MeSH data available.


Related in: MedlinePlus

Lung I/R injury induces autophagy in lung tissues of minipigs. (a) Heat maps showing hierarchical clustering of differentially expressed transcripts of autophagy related genes in right or left lung tissues from the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. (b) Immunoblotting analysis of LC3, BECN1, ATG5 and β-actin (as a loading control) in lysates of right or left lung tissue from the sham group or minipigs subjected to lung I/R as indicated. (c) Immunohistochemical staining of BECN1 and ATG5 in left lung tissue of the sham group or minipigs subjected to lung I/R as indicated. (d) Immunoblotting analysis of LC3, BECN1, ATG7 and β-actin (as a loading control) in lysates of alveolar macrophages from BALF of the sham group or minipigs subjected to lung I/R as indicated. (e) Immunofluorescence analysis of LC3 in alveolar macrophages from BALF of the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. Original magnification, × 630. Quantification of cells with autophagosomes was also shown. (f) Immunoblotting analysis of LC3 and β-actin (as a loading control) in lysates of lung tissues (upper) or alveolar macrophages (lower) from the sham group or minipigs subjected to 1 h left lung ischemia and perfusion with pulmonary protective solution containing E64d (15 μg/ml) and Pepstatin A (15 μg/ml) or DMSO followed by reperfusion for 1 h. The band densitometry was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Values below lanes represent the relative intensities of the corresponding proteins (LC3-II, BECN1, ATG5 and ATG7) to β-actin in the same lane. The relative band intensities of LC3-II/β-actin were calculated from three independent experiments and shown as mean±S.E.M. Data are representative of three individual experiments (b–f). *P<0.05, **P<0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384028&req=5

fig2: Lung I/R injury induces autophagy in lung tissues of minipigs. (a) Heat maps showing hierarchical clustering of differentially expressed transcripts of autophagy related genes in right or left lung tissues from the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. (b) Immunoblotting analysis of LC3, BECN1, ATG5 and β-actin (as a loading control) in lysates of right or left lung tissue from the sham group or minipigs subjected to lung I/R as indicated. (c) Immunohistochemical staining of BECN1 and ATG5 in left lung tissue of the sham group or minipigs subjected to lung I/R as indicated. (d) Immunoblotting analysis of LC3, BECN1, ATG7 and β-actin (as a loading control) in lysates of alveolar macrophages from BALF of the sham group or minipigs subjected to lung I/R as indicated. (e) Immunofluorescence analysis of LC3 in alveolar macrophages from BALF of the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. Original magnification, × 630. Quantification of cells with autophagosomes was also shown. (f) Immunoblotting analysis of LC3 and β-actin (as a loading control) in lysates of lung tissues (upper) or alveolar macrophages (lower) from the sham group or minipigs subjected to 1 h left lung ischemia and perfusion with pulmonary protective solution containing E64d (15 μg/ml) and Pepstatin A (15 μg/ml) or DMSO followed by reperfusion for 1 h. The band densitometry was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Values below lanes represent the relative intensities of the corresponding proteins (LC3-II, BECN1, ATG5 and ATG7) to β-actin in the same lane. The relative band intensities of LC3-II/β-actin were calculated from three independent experiments and shown as mean±S.E.M. Data are representative of three individual experiments (b–f). *P<0.05, **P<0.01

Mentions: Next, we investigated whether lung I/R injury activates autophagy in lung tissues. Microarray analysis showed that 25 of 70 autophagy related genes were differentially expressed between lungs undergoing I/R injury and those in the sham group (Figure 2a, Supplementary Figures S1b–d and Supplementary Table S2). Induction of autophagy was evidenced by conversion of microtubule-associated protein 1 light chain 3 α/β (MAP1LC3A/B, LC3A/B)-I into LC3-II and the increase in the protein and mRNA expression of Beclin 1 (BECN1) and autophagy related 5 (ATG5) in crude lung tissues from minipigs subjected to I/R (Figures 2b, c and Supplementary Figure S4). Consistently, conversion of LC3, upregulation of BECN1 and autophagy related 7 (ATG7) as well as increased LC3 puncta were also observed in alveolar macrophages isolated from BALF of lungs of both sides from minipigs with I/R (Figures 2d and e). Activation of the autophagy flux was further confirmed using the lysosomal degradation inhibitors E64d and Pepstatin A in vivo (Figure 2f). A pulmonary protective solution with or without the addition of E64d and Pepstatin A was continuously perfused into left lungs of minipigs in the sham or ischemia group for 1 h. As shown in Figure 2f, more pronounced accumulation of LC3-II was observed in crude lung tissues as well as isolated alveolar macrophages of minipigs with ischemia treated with the protective solution in the presence of E64d and Pepstatin A (Figure 2f). Of note, autophagy was not observed in alveolar epithelial cells of minipigs with lung I/R injury (Supplementary Figure S5). Taken together, these results indicate that the autophagy is induced in lungs, and in particular, in alveolar macrophages at relatively early stages of lung I/R injury.


Autophagy induced by DAMPs facilitates the inflammation response in lungs undergoing ischemia-reperfusion injury through promoting TRAF6 ubiquitination
Lung I/R injury induces autophagy in lung tissues of minipigs. (a) Heat maps showing hierarchical clustering of differentially expressed transcripts of autophagy related genes in right or left lung tissues from the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. (b) Immunoblotting analysis of LC3, BECN1, ATG5 and β-actin (as a loading control) in lysates of right or left lung tissue from the sham group or minipigs subjected to lung I/R as indicated. (c) Immunohistochemical staining of BECN1 and ATG5 in left lung tissue of the sham group or minipigs subjected to lung I/R as indicated. (d) Immunoblotting analysis of LC3, BECN1, ATG7 and β-actin (as a loading control) in lysates of alveolar macrophages from BALF of the sham group or minipigs subjected to lung I/R as indicated. (e) Immunofluorescence analysis of LC3 in alveolar macrophages from BALF of the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. Original magnification, × 630. Quantification of cells with autophagosomes was also shown. (f) Immunoblotting analysis of LC3 and β-actin (as a loading control) in lysates of lung tissues (upper) or alveolar macrophages (lower) from the sham group or minipigs subjected to 1 h left lung ischemia and perfusion with pulmonary protective solution containing E64d (15 μg/ml) and Pepstatin A (15 μg/ml) or DMSO followed by reperfusion for 1 h. The band densitometry was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Values below lanes represent the relative intensities of the corresponding proteins (LC3-II, BECN1, ATG5 and ATG7) to β-actin in the same lane. The relative band intensities of LC3-II/β-actin were calculated from three independent experiments and shown as mean±S.E.M. Data are representative of three individual experiments (b–f). *P<0.05, **P<0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384028&req=5

fig2: Lung I/R injury induces autophagy in lung tissues of minipigs. (a) Heat maps showing hierarchical clustering of differentially expressed transcripts of autophagy related genes in right or left lung tissues from the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. (b) Immunoblotting analysis of LC3, BECN1, ATG5 and β-actin (as a loading control) in lysates of right or left lung tissue from the sham group or minipigs subjected to lung I/R as indicated. (c) Immunohistochemical staining of BECN1 and ATG5 in left lung tissue of the sham group or minipigs subjected to lung I/R as indicated. (d) Immunoblotting analysis of LC3, BECN1, ATG7 and β-actin (as a loading control) in lysates of alveolar macrophages from BALF of the sham group or minipigs subjected to lung I/R as indicated. (e) Immunofluorescence analysis of LC3 in alveolar macrophages from BALF of the sham group or minipigs subjected to lung ischemia followed by reperfusion for 1 h. Original magnification, × 630. Quantification of cells with autophagosomes was also shown. (f) Immunoblotting analysis of LC3 and β-actin (as a loading control) in lysates of lung tissues (upper) or alveolar macrophages (lower) from the sham group or minipigs subjected to 1 h left lung ischemia and perfusion with pulmonary protective solution containing E64d (15 μg/ml) and Pepstatin A (15 μg/ml) or DMSO followed by reperfusion for 1 h. The band densitometry was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Values below lanes represent the relative intensities of the corresponding proteins (LC3-II, BECN1, ATG5 and ATG7) to β-actin in the same lane. The relative band intensities of LC3-II/β-actin were calculated from three independent experiments and shown as mean±S.E.M. Data are representative of three individual experiments (b–f). *P<0.05, **P<0.01
Mentions: Next, we investigated whether lung I/R injury activates autophagy in lung tissues. Microarray analysis showed that 25 of 70 autophagy related genes were differentially expressed between lungs undergoing I/R injury and those in the sham group (Figure 2a, Supplementary Figures S1b–d and Supplementary Table S2). Induction of autophagy was evidenced by conversion of microtubule-associated protein 1 light chain 3 α/β (MAP1LC3A/B, LC3A/B)-I into LC3-II and the increase in the protein and mRNA expression of Beclin 1 (BECN1) and autophagy related 5 (ATG5) in crude lung tissues from minipigs subjected to I/R (Figures 2b, c and Supplementary Figure S4). Consistently, conversion of LC3, upregulation of BECN1 and autophagy related 7 (ATG7) as well as increased LC3 puncta were also observed in alveolar macrophages isolated from BALF of lungs of both sides from minipigs with I/R (Figures 2d and e). Activation of the autophagy flux was further confirmed using the lysosomal degradation inhibitors E64d and Pepstatin A in vivo (Figure 2f). A pulmonary protective solution with or without the addition of E64d and Pepstatin A was continuously perfused into left lungs of minipigs in the sham or ischemia group for 1 h. As shown in Figure 2f, more pronounced accumulation of LC3-II was observed in crude lung tissues as well as isolated alveolar macrophages of minipigs with ischemia treated with the protective solution in the presence of E64d and Pepstatin A (Figure 2f). Of note, autophagy was not observed in alveolar epithelial cells of minipigs with lung I/R injury (Supplementary Figure S5). Taken together, these results indicate that the autophagy is induced in lungs, and in particular, in alveolar macrophages at relatively early stages of lung I/R injury.

View Article: PubMed Central - PubMed

ABSTRACT

Lung ischemia-reperfusion (I/R) injury remains one of the most common complications after various cardiopulmonary surgeries. The inflammation response triggered by the released damage-associated molecular patterns (DAMPs) aggravates lung tissue damage. However, little is known about the role of autophagy in the pathogenesis of lung I/R injury. Here, we report that a variety of inflammation-related and autophagy-associated genes are rapidly upregulated, which facilitate the inflammation response in a minipig lung I/R injury model. Left lung I/R injury triggered inflammatory cytokine production and activated the autophagy flux as evidenced in crude lung tissues and alveolar macrophages. This was associated with the release of DAMPs, such as high mobility group protein B1 (HMGB1) and heat shock protein 60 (HSP60). Indeed, treatment with recombinant HMGB1 or HSP60 induced autophagy in alveolar macrophages, whereas autophagy inhibition by knockdown of ATG7 or BECN1 markedly reduced DAMP-triggered production of inflammatory cytokines including IL-1&beta;, TNF and IL12 in alveolar macrophages. This appeared to be because of decreased activation of MAPK and NF-&kappa;B signaling. Furthermore, knockdown of ATG7 or BECN1 inhibited Lys63 (K63)-linked ubiquitination of TNF receptor-associated factor 6 (TRAF6) in DAMP-treated alveolar macrophages. Consistently, treatment with 3-MA inhibited K63-linked ubiquitination of TRAF6 in I/R-injured lung tissues in vivo. Collectively, these results indicate that autophagy triggered by DAMPs during lung I/R injury amplifies the inflammatory response through enhancing K63-linked ubiquitination of TRAF6 and activation of the downstream MAPK and NF-&kappa;B signaling.

No MeSH data available.


Related in: MedlinePlus