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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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Related in: MedlinePlus

Lung I/R injury triggers the inflammation response in lung tissues of minipigs. Different groups of minipigs underwent 1 h left lung ischemia followed by reperfusion for 1 h (IR1h), 3 h (IR3h) or 6 h (IR6h), or only received sham operations (Sham). (a) Heat maps showing hierarchical clustering of upregulated expressed transcripts of inflammation-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) qPCR analysis of Tnf, Il1b, Il12 and Il6 mRNA expression in right (R) or left (L) lung tissues from the sham group or minipigs undergoing lung I/R as indicated (n=3). (c) Immunohistochemical staining of IL-1β and TNF expression in left lung tissues from the sham group or minipigs undergoing lung I/R as indicated. (d) qPCR analysis of Tnf, Il1b, Il12 and Il6 mRNA expression in alveolar macrophages from the sham group or minipigs undergoing lung I/R as indicated (n=3). (e) Immunofluorescence staining of IL-1β, F4/80 (macrophage marker), Ly6G (neutrophil marker) and SP-C (alveolar epithelial cell marker) in left lung tissues from minipigs subjected to lung ischemia followed by reperfusion for 1 h. Original magnification, × 400. Data are representative (c and e), or mean±S.E.M. (b and d), of three individual experiments. *P<0.05, **P<0.01, ***P<0.001
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fig1: Lung I/R injury triggers the inflammation response in lung tissues of minipigs. Different groups of minipigs underwent 1 h left lung ischemia followed by reperfusion for 1 h (IR1h), 3 h (IR3h) or 6 h (IR6h), or only received sham operations (Sham). (a) Heat maps showing hierarchical clustering of upregulated expressed transcripts of inflammation-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) qPCR analysis of Tnf, Il1b, Il12 and Il6 mRNA expression in right (R) or left (L) lung tissues from the sham group or minipigs undergoing lung I/R as indicated (n=3). (c) Immunohistochemical staining of IL-1β and TNF expression in left lung tissues from the sham group or minipigs undergoing lung I/R as indicated. (d) qPCR analysis of Tnf, Il1b, Il12 and Il6 mRNA expression in alveolar macrophages from the sham group or minipigs undergoing lung I/R as indicated (n=3). (e) Immunofluorescence staining of IL-1β, F4/80 (macrophage marker), Ly6G (neutrophil marker) and SP-C (alveolar epithelial cell marker) in left lung tissues from minipigs subjected to lung ischemia followed by reperfusion for 1 h. Original magnification, × 400. Data are representative (c and e), or mean±S.E.M. (b and d), of three individual experiments. *P<0.05, **P<0.01, ***P<0.001

Mentions: We constructed a left lung I/R injury model of minipigs and monitored the inflammation response in lung tissues. Total RNA from crude left or right lung tissues of minipigs in the sham or I/R group was subjected to microarray analysis. The results showed that 54 of a set of 315 inflammation-related genes were significantly upregulated in tissues of both left and right lungs of minipigs undergoing I/R compared with those in the sham group (Figure 1a, Supplementary Figure S1a and Supplementary Table S1). As shown in Figure 1b, the increase in Tnf, Il1b, Il12 and Il6 mRNA expression was detectable as early as 1 h after the commencement of reperfusion, which is more pronounced in left lungs that had been subjected to I/R injury. Similarly, the expression of IL-1β and TNF at the protein level was also upregulated in left lungs of minipigs in the I/R group (Figure 1c and Supplementary Figure S2). Consistent with the important role of macrophages in the initiation and generation of the early inflammatory response to lung I/R injury,12, 16 alveolar macrophages isolated from bronchoalveolar lavage fluid (BALF) of minipigs with I/R injury expressed elevated levels of Tnf, Il1b and Il12 mRNA (Figure 1d). In support, immunofluorescence analysis showed that alveolar macrophages (F4/80+) but not neutrophils (Ly6G+) or alveolar epithelial cells (SP-C+) were the source of IL-1β production in left lung tissues of minipigs with lung ischemia followed by reperfusion for 1 h (Figure 1e and Supplementary Figure S3). These findings, along with the previous results showing that alveolar macrophage depletion protects lungs from I/R-induced injury and significantly reduces cytokine/chemokine production,16, 17, 18 indicate that lung I/R primarily triggers production of inflammatory cytokines by alveolar macrophages, which in turn have an important role in the pathogenesis of acute 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 triggers the inflammation response in lung tissues of minipigs. Different groups of minipigs underwent 1 h left lung ischemia followed by reperfusion for 1 h (IR1h), 3 h (IR3h) or 6 h (IR6h), or only received sham operations (Sham). (a) Heat maps showing hierarchical clustering of upregulated expressed transcripts of inflammation-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) qPCR analysis of Tnf, Il1b, Il12 and Il6 mRNA expression in right (R) or left (L) lung tissues from the sham group or minipigs undergoing lung I/R as indicated (n=3). (c) Immunohistochemical staining of IL-1β and TNF expression in left lung tissues from the sham group or minipigs undergoing lung I/R as indicated. (d) qPCR analysis of Tnf, Il1b, Il12 and Il6 mRNA expression in alveolar macrophages from the sham group or minipigs undergoing lung I/R as indicated (n=3). (e) Immunofluorescence staining of IL-1β, F4/80 (macrophage marker), Ly6G (neutrophil marker) and SP-C (alveolar epithelial cell marker) in left lung tissues from minipigs subjected to lung ischemia followed by reperfusion for 1 h. Original magnification, × 400. Data are representative (c and e), or mean±S.E.M. (b and d), of three individual experiments. *P<0.05, **P<0.01, ***P<0.001
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fig1: Lung I/R injury triggers the inflammation response in lung tissues of minipigs. Different groups of minipigs underwent 1 h left lung ischemia followed by reperfusion for 1 h (IR1h), 3 h (IR3h) or 6 h (IR6h), or only received sham operations (Sham). (a) Heat maps showing hierarchical clustering of upregulated expressed transcripts of inflammation-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) qPCR analysis of Tnf, Il1b, Il12 and Il6 mRNA expression in right (R) or left (L) lung tissues from the sham group or minipigs undergoing lung I/R as indicated (n=3). (c) Immunohistochemical staining of IL-1β and TNF expression in left lung tissues from the sham group or minipigs undergoing lung I/R as indicated. (d) qPCR analysis of Tnf, Il1b, Il12 and Il6 mRNA expression in alveolar macrophages from the sham group or minipigs undergoing lung I/R as indicated (n=3). (e) Immunofluorescence staining of IL-1β, F4/80 (macrophage marker), Ly6G (neutrophil marker) and SP-C (alveolar epithelial cell marker) in left lung tissues from minipigs subjected to lung ischemia followed by reperfusion for 1 h. Original magnification, × 400. Data are representative (c and e), or mean±S.E.M. (b and d), of three individual experiments. *P<0.05, **P<0.01, ***P<0.001
Mentions: We constructed a left lung I/R injury model of minipigs and monitored the inflammation response in lung tissues. Total RNA from crude left or right lung tissues of minipigs in the sham or I/R group was subjected to microarray analysis. The results showed that 54 of a set of 315 inflammation-related genes were significantly upregulated in tissues of both left and right lungs of minipigs undergoing I/R compared with those in the sham group (Figure 1a, Supplementary Figure S1a and Supplementary Table S1). As shown in Figure 1b, the increase in Tnf, Il1b, Il12 and Il6 mRNA expression was detectable as early as 1 h after the commencement of reperfusion, which is more pronounced in left lungs that had been subjected to I/R injury. Similarly, the expression of IL-1β and TNF at the protein level was also upregulated in left lungs of minipigs in the I/R group (Figure 1c and Supplementary Figure S2). Consistent with the important role of macrophages in the initiation and generation of the early inflammatory response to lung I/R injury,12, 16 alveolar macrophages isolated from bronchoalveolar lavage fluid (BALF) of minipigs with I/R injury expressed elevated levels of Tnf, Il1b and Il12 mRNA (Figure 1d). In support, immunofluorescence analysis showed that alveolar macrophages (F4/80+) but not neutrophils (Ly6G+) or alveolar epithelial cells (SP-C+) were the source of IL-1β production in left lung tissues of minipigs with lung ischemia followed by reperfusion for 1 h (Figure 1e and Supplementary Figure S3). These findings, along with the previous results showing that alveolar macrophage depletion protects lungs from I/R-induced injury and significantly reduces cytokine/chemokine production,16, 17, 18 indicate that lung I/R primarily triggers production of inflammatory cytokines by alveolar macrophages, which in turn have an important role in the pathogenesis of acute 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