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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.

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Autophagy is involved in TRAF6 ubiquitination triggered by lung I/R injury. (a–d) Alveolar macrophages were transfected with the control siRNA, Atg7 siRNA (a and b) or Becn1 siRNA (c and d). Sixty hours later, cells were treated with rpHMGB1 (1 μg/ml) (a and c) or rpHSP60 (1 μg/ml) (b and d) for 30 min or left untreated. (e) Minipigs underwent lung ischemia and perfusion with pulmonary protective solution containing 3-MA (5 mM) or DMSO followed by reperfusion for 30 min or received sham operations (Sham). Whole-cell lysates of alveolar macrophages (a–d) or lysates of left lung tissues (e) were subjected to immunoprecipitation with anti-TRAF6 antibody followed by immunoblotting with anti-K63 ubiquitin and anti-TRAF6 antibodies, or immunoblotted with anti-TRAF6 and β-actin antibodies. S: sham group. Data are representative of three individual precipitation experiments
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fig6: Autophagy is involved in TRAF6 ubiquitination triggered by lung I/R injury. (a–d) Alveolar macrophages were transfected with the control siRNA, Atg7 siRNA (a and b) or Becn1 siRNA (c and d). Sixty hours later, cells were treated with rpHMGB1 (1 μg/ml) (a and c) or rpHSP60 (1 μg/ml) (b and d) for 30 min or left untreated. (e) Minipigs underwent lung ischemia and perfusion with pulmonary protective solution containing 3-MA (5 mM) or DMSO followed by reperfusion for 30 min or received sham operations (Sham). Whole-cell lysates of alveolar macrophages (a–d) or lysates of left lung tissues (e) were subjected to immunoprecipitation with anti-TRAF6 antibody followed by immunoblotting with anti-K63 ubiquitin and anti-TRAF6 antibodies, or immunoblotted with anti-TRAF6 and β-actin antibodies. S: sham group. Data are representative of three individual precipitation experiments

Mentions: Since HMGB1 and HSP60 is recognized by TLR4, which subsequently induces Lys63 (K63)-linked ubiquitination of TRAF6 leading to activation of downstream MAPK and NFKB signaling and production of inflammatory cytokines,23, 24 we examined whether TRAF6 ubiquitination participates in autophagy-mediated activation of MAPK and NF-κB signaling induced by DAMPs in lungs undergoing I/R injury. Indeed, knockdown of ATG7 or BECN1 markedly reduced K63-linked ubiquitination of TRAF6 triggered by rpHMGB1 or rpHSP60 in alveolar macrophages (Figures 6a–d). In addition, the pulmonary protective solution with addition of the autophagy inhibitor 3-MA substantially attenuated K63-linked ubiquitination of TRAF6 in left lung tissues triggered by I/R injury (Figure 6e). The data from 3-MA treatment suggest that autophagy is involved in K63-linked ubiquitination of TRAF6 in lung tissues triggered by I/R, which has an important role in I/R injury-induced activation of MAPK and NF-κB signaling and the inflammatory response.


Autophagy induced by DAMPs facilitates the inflammation response in lungs undergoing ischemia-reperfusion injury through promoting TRAF6 ubiquitination
Autophagy is involved in TRAF6 ubiquitination triggered by lung I/R injury. (a–d) Alveolar macrophages were transfected with the control siRNA, Atg7 siRNA (a and b) or Becn1 siRNA (c and d). Sixty hours later, cells were treated with rpHMGB1 (1 μg/ml) (a and c) or rpHSP60 (1 μg/ml) (b and d) for 30 min or left untreated. (e) Minipigs underwent lung ischemia and perfusion with pulmonary protective solution containing 3-MA (5 mM) or DMSO followed by reperfusion for 30 min or received sham operations (Sham). Whole-cell lysates of alveolar macrophages (a–d) or lysates of left lung tissues (e) were subjected to immunoprecipitation with anti-TRAF6 antibody followed by immunoblotting with anti-K63 ubiquitin and anti-TRAF6 antibodies, or immunoblotted with anti-TRAF6 and β-actin antibodies. S: sham group. Data are representative of three individual precipitation experiments
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fig6: Autophagy is involved in TRAF6 ubiquitination triggered by lung I/R injury. (a–d) Alveolar macrophages were transfected with the control siRNA, Atg7 siRNA (a and b) or Becn1 siRNA (c and d). Sixty hours later, cells were treated with rpHMGB1 (1 μg/ml) (a and c) or rpHSP60 (1 μg/ml) (b and d) for 30 min or left untreated. (e) Minipigs underwent lung ischemia and perfusion with pulmonary protective solution containing 3-MA (5 mM) or DMSO followed by reperfusion for 30 min or received sham operations (Sham). Whole-cell lysates of alveolar macrophages (a–d) or lysates of left lung tissues (e) were subjected to immunoprecipitation with anti-TRAF6 antibody followed by immunoblotting with anti-K63 ubiquitin and anti-TRAF6 antibodies, or immunoblotted with anti-TRAF6 and β-actin antibodies. S: sham group. Data are representative of three individual precipitation experiments
Mentions: Since HMGB1 and HSP60 is recognized by TLR4, which subsequently induces Lys63 (K63)-linked ubiquitination of TRAF6 leading to activation of downstream MAPK and NFKB signaling and production of inflammatory cytokines,23, 24 we examined whether TRAF6 ubiquitination participates in autophagy-mediated activation of MAPK and NF-κB signaling induced by DAMPs in lungs undergoing I/R injury. Indeed, knockdown of ATG7 or BECN1 markedly reduced K63-linked ubiquitination of TRAF6 triggered by rpHMGB1 or rpHSP60 in alveolar macrophages (Figures 6a–d). In addition, the pulmonary protective solution with addition of the autophagy inhibitor 3-MA substantially attenuated K63-linked ubiquitination of TRAF6 in left lung tissues triggered by I/R injury (Figure 6e). The data from 3-MA treatment suggest that autophagy is involved in K63-linked ubiquitination of TRAF6 in lung tissues triggered by I/R, which has an important role in I/R injury-induced activation of MAPK and NF-κB signaling and the inflammatory response.

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