PICK1 Deficiency Induces Autophagy Dysfunction via Lysosomal Impairment and Amplifies Sepsis-Induced Acute Lung Injury

Abstract

Sepsis is a systemic inflammatory reaction caused by infection. Multiple organ failure ultimately leads to high morbidity and mortality. Unfortunately, therapies against these responses have been unsuccessful due to the insufficient underlying pathophysiological evidence. Protein interacting with C-kinase 1 (PICK1) has received considerable attention because of its important physiological functions in many tissues. However, its role in sepsis-induced acute lung injury (ALI) is unclear. In this study, we used cecal ligation and puncture (CLP) to establish a septic model and found that decreased microtubule-associated protein-1light chain 3 (LC3)-II/LC3-I in PICK1^-/- septic mice was caused by autophagy dysfunction. Consistently, the transmission electron microscopy (TEM) of bone marrow-derived macrophages (BMDMs) from PICK1^-/- mice showed the accumulation of autophagosomes as well. However, more serious damage was caused by PICK1 deficiency indicating that the disrupted autophagic flux was harmful to sepsis-induced ALI. We also observed that it was the impaired lysosomal function that mediated autophagic flux blockade, and the autophagy progress was relevant to PI3K-Akt-mTOR pathway. These findings will aid in the potential development of PICK1 with novel evidence of autophagy in sepsis treatment and prevention.

Figure 1

PICK1 participates in autophagic progression in sepsis-induced ALI. (a, b) TNF-α and IL-1β concentrations were measured in BALF. (c, d) Lung injury was evaluated by lung wet : dry weight ratio, and permeability was assessed by detecting changes in total BALF protein concentration. (e) Lungs were harvested at 4 h, 8 h, and 24 h post-CLP, and Western blot was performed to analyze PICK1, LC3-II/LC3-I, and p62 content. Representative Western blot images and the statistical density of PICK1 are shown in the graphs. (f, g) PICK1 and LC3 mRNA levels measured in the lungs by quantitative PCR. Results represent the mean ± SEM (n = 8/group). ^∗P < 0.05 vs. sham-operated groups; ^#P < 0.05 vs. 4 h post-CLP; ^†P < 0.05 vs. 8 h post-CLP.

Figure 2

PICK1 deficiency disrupted the autophagic flux. Lungs were harvested 24 h post-CLP. (a, b) Representative Western blot images and statistical analysis of LC3-II/LC3-I ratio and p62 levels in PICK1^−/− mice. (c) Representative TEM images of autophagic structures in BMDMs. Autophagosomes are indicated by arrowheads and autolysosomes are shown by arrows (magnification, 10,500x). (d) Statistical analysis of autophagosomes and autolysosomes. Results represent the mean ± SEM of independent experiments of animals (n = 6) and cells (n = 3). ^∗P < 0.05 vs. sham-operated group; ^#P < 0.05 vs. CLP mice in the WT group.

Figure 3

PICK1 deficiency leads to more serious damage. (a, b) Changes in TNF-α and IL-1β concentrations in BALF between groups. (c) Representative photomicrographs of lung sections stained with H&E at 24 h post-CLP in WT and PICK1^−/− mice (magnification, 200x). (d) Lung wet : dry weight ratio in WT and PICK1^−/− mice. Results represent the mean ± SEM (n = 6/group). ^∗P < 0.05 vs. sham-operated group; ^#P < 0.05 vs. CLP mice in the WT group; ^†P < 0.05 vs. injection of chloroquine after CLP in the WT group.

Figure 4

Lysosome is impaired in PICK1^−/− mice. (a, b) Lungs were harvested 24 h post-CLP. Representative Western blot images and statistical analysis of LAMP1 and cathepsin B in PICK1^−/− mice. (c, d) Immunofluorescence and statistical analysis of LAMP1 in PICK1^−/− mice post-CLP (magnification, 200x). Results represent the mean ± SEM (n = 6/group). ^∗P < 0.05 vs. sham-operated group in WT mice; ^#P < 0.05 vs. sham-operated group in PICK1^−/− mice.

Figure 5

PICK1 participates in autophagy through dephosphorylation of Akt and mTOR. (a) Representative Western blot images and statistical analysis of p-Akt/t-Akt and p-mTOR/t-mTOR levels in PICK1^−/− mice. Results represent the mean ± SEM of independent experiments in mice (n = 4). ^∗P < 0.05 vs. sham-operated group, ^#P < 0.05 vs. CLP group.

Figure 6

Proposed model for PICK1 deficiency-related autophagy in sepsis-induced acute lung injury. In PICK1^−/− mice, impaired lysosomal structure decreases the function of lysosomes and influences the formation of autophagosomes with it. Accumulation of autophagosomes eventually result in autophagy dysfunction.