Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis

Abstract

Introduction: It is not well understood whether the process of autophagy is accelerated or blocked in sepsis, and whether it is beneficial or harmful to the immune defense mechanism over a time course during sepsis. Our aim was to determine both the kinetics and the role of autophagy in sepsis.

Methods: We examined autophagosome and autolysosome formation in a cecal ligation and puncture (CLP) mouse model of sepsis (in C57BL/6N mice and GFP-LC3 transgenic mice), using western blotting, immunofluorescence, and electron microscopy. We also investigated the effect of chloroquine inhibition of autophagy on these processes.

Results: Autophagy, as demonstrated by increased LC3-II/LC3-I ratios, is induced in the liver, heart, and spleen over 24 h after CLP. In the liver, autophagosome formation peaks at 6 h and declines by 24 h. Immunofluorescent localization of GFP-LC3 dots (alone and with lysosome-associated membrane protein type 1 (LAMP1)), as well as electron microscopic examination, demonstrate that both autophagosomes and autolysosomes are increased after CLP, suggesting that intact autophagy mechanisms operate in the liver in this model. Furthermore, inhibition of autophagy process by chloroquine administration immediately after CLP resulted in elevated serum transaminase levels and a significant increase in mortality.

Conclusions: All autophagy-related processes are properly activated in the liver in a mouse model of sepsis; autophagy appears to play a protective role in septic animals.

Figures

Figure 1

Cecal ligation and puncture (CLP) induces autophagy in the liver of CLP model mice. For B-D and F, data are expressed as mean ± SD; data were analyzed for statistical significance using the Mann-Whitney test. (A) Western blotting analysis of microtubule-associated protein light chain 3 (LC3) in the liver. Sepsis was induced by CLP. Liver samples were prepared from sham-operated and CLP mice at each indicated time point post surgery. Results are representative of seven independent experiments. β-tubulin served as standard. (B) The ratio between the levels of LC3-II and LC3-I at each time point. *P <0.05, as compared to sham-operated group (n = 7/group). (C) The ratio of LC3-II/LC3-I expression in various organs at 6 and 24 h after CLP. Values in CLP mice are expressed as fold-change relative to each sham-operated group. MLN, mesenteric lymph node. (D)LC3 mRNA expression in the liver at each time point assessed by real-time PCR. *P <0.05, as compared to sham-operated group (n = 7/group). (E) Confocal images of liver samples obtained from GFP-LC3 transgenic mice. Green-fluorescing GFP-LC3 dots were present in the cytosol indicated by the arrow head. (F) The number of GFP-LC3 dots per cellular confocal image was quantified. Differences in the number of GFP-LC3 dots in CLP mice at 6 h compared with sham-operated mice at 6 h and CLP mice at 24 h were statistically significant (*P <0.05; n = 50 cells /animal; n = 4 animals).

Figure 2

Co-localization of green fluorescent protein (GFP)-Microtubule-associated protein light chain 3 (LC3) dots with lysosome-associated membrane protein type 1 (LAMP1) in the liver after cecal ligation and puncture (CLP). (A) Confocal images of liver samples obtained from GFP-LC3 transgenic mice. Green-fluorescing GFP-LC3 dots were present in the cytosol. LAMP1 was stained using Cy3-conjugated IgG secondary antibodies. Merged images demonstrate co-localization of GFP-LC3 dots and LAMP1. (B) The number of GFP-LC3 or GFP-LC3 + LAMP1 dots per cellular confocal image was quantified at 6 and 24 h after CLP. All data are expressed as mean ± SD. Data were analyzed for statistical significance using the Mann-Whitney test. Differences in the number of GFP-LC3 or GFP-LC3 dots in CLP mice at 6 h compared with CLP mice at 24 h were statistically significant (*P <0.05; n = 50 cells /animal; n = 4 animals). (C) Relative expression of p62 protein in the liver at 6 and 24 h after sham or CLP operation. The amount of p62 protein was normalized to that of β-tubulin by evaluation of band intensity from western blotting. All data were expressed as the mean ± SD. Data were analyzed for statistical significance using the Mann-Whitney test (*P <0.05; n = 5 in each group).

Figure 3

Electron microscopic analysis of the liver. (A) The number of autophagosomes and autolysosomes are compared in CLP and sham animals. All data are expressed as the mean ± SD. Data were analyzed for statistical significance using the Mann-Whitney test. Increase in autolysosomes in the CLP group was statistically significant (*P <0.05; n = 3); mean increases in autophagosomes in CLP compared to sham did not reach statistical significance. NS, not significant. (B) Images of electron microscopy of the liver; a: Liver sample obtained from sham-operated mice. Organelles in the hepatocyte are generally intact and lysosomes do not contain discrete membrane structures, although the inhomogeneous electron-dense material often seen in (hetero)lyosomes most certainly represent end-stage degradation of phospholipid and other cytoplasmic materials (a material at the light microscopic level referred to as lipofuscin); b-e: CLP-operated mice. Double arrow heads identify complex structures bounded by two membranes (autophagosomes); arrow heads identify single membrane-bound lysosomal complexes with degraded organellar content (autolysosomes); e: the double arrow head identifies an autophagosome that clearly contains an injured mitochondrion.

Figure 4

Blockade of the autophagic process by chloroquine. Green fluorescent protein- microtubule-associated protein light chain 3 (GFP-LC3) transgenic mice were administered saline or chloroquine (CQ) (60 mg/kg intraperitoneally) at 1 h after CLP or sham surgery. Samples were obtained from 6 or 24 h after surgery. (A) Confocal images of liver samples obtained from GFP-LC3 transgenic mice with or without CQ treatment. Blockade of autophagic process is indicated by the arrow head. (B) The number of GFP-LC3 or GFP-LC3 + lysosome-associated membrane protein type 1 (LAMP1) dots per cellular confocal image obtained from GFP-LC3 transgenic mice with or without CQ treatment was quantified at 6 and 24 h after cecal ligation and puncture (CLP). All data are expressed as the mean ± SD. Data were analyzed for statistical significance using the Student-Newman-Keuls test. Differences in the number of GFP-LC3 or GFP-LC3 + LAMP1 dots in CLP mice at 24 h compared with CLP mice at 24 h were statistically significant (*P <0.05; n = 50 cells /animal; n = 4 animals).

Figure 5

Inhibition of autophagy enhances cecal ligation and puncture (CLP)-induced liver injury. (A) Histological findings of mouse liver by hematoxylin and eosin staining (original magnification 100×) with a larger view (insets, 400×). Liver tissue was obtained from either sham-operated or CLP mice with or without chloroquine (CQ) treatment 6 h after surgery. (B) Hepatocellular damage as defined by serum aspartate aminotransferase (AST) and arginin aminotransferase (ALT) levels. Samples were obtained from either sham-operated or CLP mice with or without CQ treatment 6 and 24 h after surgery (n = 7/group). Data are shown as mean ± SD. *P <0.05 versus saline treatment (one-way analysis of variance); individual group means were compared using the Student-Newman-Keuls test. (C) Survival after CQ or sterile saline administration (n = 18/group) in sham-operated or CLP mice; 3/18 mice in the CLP + saline group and 12/18 mice in the CLP + CQ group were sacrificed because they were moribund. Comparisons of overall survival were performed using the log-rank test (P = 0.003; CLP + saline versus CLP + CQ).