Effects And Mechanism Of Autophagy On LPS-induced Cell Death In Human Alveolar Epithelial Cells

1.Background and objectiveAlveolar epithelial cell damage has been recognized as a prominent feature of acute lung injury/acute respiratory distress syndrome(ALI/ARDS) and is known to contribute to the pathogenesis of this syndrome. The degree of alveolar epithelial injury has been considered to represent an important prognostic marker of ARDS. Lipopolysaccharide(LPS), has been considered one of the important factors leading to ALI.Previous studies suggested that uncontrolled LPS-toll-like receptor 4 signalling response may result in excessive inflammation, which is a primary contributor to diffuse alveolar damage. However, therapeutic strategies aimed at reducing the inflammatory response have shown limited clinical benefit to date. Accordingly, it is necessary to further characterise the functions and mechanisms through which LPS causes alveolar epithelial cell damage.The autophagy has a dual effect on cell survival: cytoprotection and cytotoxicity. Evidence has been presented that autophagy can promote cell survival or cell death depending on the cell type, the specific circumstances and stimuli present, and the duration and strength of the stimulus exposure. Moreover, our preliminary experiment data showed that LPS increased the autophagy in human alveolar epithelial A549 cells. However, it is not well-known whether autophagy plays a role in either the initiation or inhibition of cell death processes when human alveolar epithelial cells are challenged with LPS.Recently, accumulating data have provided evidence that the activation of the three pathway of the unfolded protein response(UPR), upon endoplasmic reticulum(ER) serves an important trigger of autophagy. Specifically, the UPR triggered by the ER stress response to insults plays an important role in the activation of autophagy as well as in the determination of life-or-death cell fates. However, whether LPS-induced autophagic activity is associated with ER stress and the UPR in A549 cells has not been established.In this study, we investigated the effect of LPS-induced autophagy on A549 cell fate outcome and further explored the underlying mechanisms involved.2.Methods(1)To explore the influence of autophagy on LPS-induced A549 cells death.(1) A549 cells were cultured and stimulated with LPS, proteins involved in the induction or progression of the autophagic pathway, including autophagic protein microtubule-associated protein 1 light chain 3(LC3B), Beclin-1 and autophagy related genes(Atg)5, as well as the degradation of p62(an autophagic substrate) were detected by immunoblotting. The occurrence of EGFP-LC3-labeled structures(EGFP-LC3 puncta) was examined by laser confocal scanning microscopy and the morphological indices of autophagy were identified via transmission electron microscopy. In addition, polymyxin B, an inhibitor of LPS was used to verify whether the increased autophagy was due to the presence of LPS.(2) The effect of LPS on the viability of A549 cells was determined using a CCK-8assay. In addition, the effect of 50 μg/ml LPS on the apoptosis of A549 cells was analysed via double-labelled flow cytometry using annexin-V/PI and the necrosis of A549 cells was analysed using the rate of lactate dehydrogenase(LDH) leakag.(3) A specific pharmacological inhibitor of autophagy, 3-methyladenine(3-MA), the autophagy inducer rapamycin(Rapa) and an apoptosis inhibitor Z-VAD were used to examine the effects of autophagy and apoptosis on the LPS-induced death of A549 cells.(4) The expression of Beclin-1 or Atg5, two critical genes involved in the autophagy pathways, was interrupted in A549 cells with short interfering RNA(si RNA), specific to further confirm the functional role of autophagy in LPS-induced cell death in human alveolar epithelial cells.(2)The administration of autophagy inhibitor lowered the alteration of the alveolar capillary barrie in LPS-induced ALI mice.(1) ALI was induced by LPS peritoneal injection in C57BL/6J mice The expression of LC3 B in lung tissues was detected by immunoblotting and immunofluorescence. The morphological indices of autophagy were identified via transmission electron microscopy.(2) Pretreated mice with autophagy inhibitor 3-MA or chloroquine four hours before a 15mg/kg dose of LPS was injected into the mice to establish ALI mouse model then the lung tissues and the bronchoalveolar lavage fluid were collected and lung wet/dry weight ratios and total protein concentrations in the Bronchoalveolar lavage fluid(BALF) were determined.(3)The underlying mechanisms for LPS-induced autophagic cell death were investigated with the model of A549 cells in vitro.(1) A549 cells were cultured and stimulated with LPS, the expression of two ER stress markers, the ER-resident glucose-regulated protein(GRP)78 and GRP94 proteins were detected by immunoblotting. Next, the ER stress inhibitor 4-phenylbutyrate(4-PBA) was used to examine the effects of ER stress on the activation of autophagy in LPS-treated A549 cells.(2) The constituents involved in these three pathways of UPR signalling were analysed by immunoblotting to assess whether all three branches of the UPR are activated upon LPS-induced stress in A549 cells. Furthermore, the expression of double-stranded RNA-activated protein kinase-like ER kinase(PERK) or activating transcription factor(ATF)4 was interrupted with si RNAs to confirm the role of PERK branche of the UPR in LPS-induced autophagy and cell death in human alveolar epithelial cells.3.Results(1)Treating cells with LPS resulted in an increase in total LC3 B expression and in the dose- and time-dependent accumulation of its active form, LC3B-II. Consistent with the observed increase in LC3 B expression, Beclin-1 and Atg5 were upregulated after LPS treatment. These events were also accompanied by the degradation of p62, an autophagic substrate. Pre-incubation of LPS with polymyxin B decreased the formation of LC3B-II protein. LPS treatment induced the formation of EGFP-LC3 puncta in EGFP- LC3-transfected A549 cells. The quantification of these results revealed that the number of LC3 puncta per cross-sectioned cell increased after the LPS treatment relative to the control treatment. The accumulation of double-membrane autophagosomes was identified in the A549 cells treated with LPS, while this ultrastructural feature was rarely observed in the control cells.(2)Treatment of the cells with LPS at concentrations of 50, 100, and 150 μg/ml resulted in significant decreases in cell viability at 16 h, whereas the treatment of the cells with LPS at 0.1, 1, 10, and 25 μg/ml did not affect cell viability. A time-course analysis indicated that 50 μg/ml LPS decreased cell viability at 16 h post-treatment but that viability at 2, 4, and 8 h was not affected. Therefore, 50 μg/ml seemed to be the minimal cytotoxic dose of LPS for A549 cells. Pretreatment of LPS with polymyxin B increased the viability of A549 cells.(3)Treatment with 50 μg/ml LPS for 16 h did not significantly induce the apoptosis of A549 cells and affect the rate of LDH leakage compared to the control.(4)The increase in the level of LC3B-II protein was reversed by pretreatment with 3-MA, whereas the formation of LC3B-II was significantly upregulated when LPS-stimulated A549 cells were pretreated with Rapa. Furthermore, the decrease in the viability of A549 cells exposed to 50 μg/ml LPS for 16 h was efficiently abrogated by pretreatment with 3-MA. In contrast, Rapa led to a further decrease in the viability of A549 cells treated with LPS. Additionally, pretreatment with the apoptosis inhibitor Z-VAD failed to prevent cell death in A549 cells that had been treated with LPS at 50 μg/ml for 16 h(P=0.17792).(5)The knockdown of Beclin-1 or Atg5 treated with si RNAs decreased the expression level of LC3B-II protein and the number of LC3 puncta in the A549 cells treated with LPS. Moreover, the decrease in cell viability after exposure to LPS was efficiently abrogated in A549 cells undergoing Beclin-1 or Atg5 knockdown.(6)Compared with control group, the lung injury score, the total cell numbers and neutrophil percentage in the BALF, the concentrations of proinflammatory cytokines, IL-1β, TNF-α and MIP-2, the total BALF protein concentration, the lung wet/dry weight ratio were remarkably increased in the LPS treated group.(7)The Western blot and immunofluorescence results have demonstrated that LPS administration can significantly increase the expression of LC3 B in lung tissues. The accumulation of double-membrane autophagosomes was identified in the in lung tissues of mice treated with LPS, while this ultrastructural feature was rarely observed in the control group.(8)Compared with control group, the lung injury score, the total BALF protein concentration and the lung wet/dry weight ratio were significantly decreased in 3-MA or chloroquine(CLQ) pretreated group. Pretreatment of CLQ led to the decrease of concentrations of proinflammatory cytokines(IL-1β, TNF-α, MIP-2) in BALF.(9)The protein expression levels of both GRP78 and GRP94 were upregulated after LPS treatment in a dose- and time-dependent manner. In addition, in A549 cells exposed to 50 μg/ml LPS for 16 h, the upregulation of GRP78 and GRP94 was reduced by pretreating the cells with the ER stress inhibitor 4-PBA, which was accompanied by a decrease in the level of LC3B-II.(10)LPS treatment resulted in the accumulation of phosphorylated PERK(p-PERK) and constituents of the PERK cascade, phosphorylated eukaryotic initiation factor-2(p-e IF2α), nuclear ATF4 and growth arrest DNA damage gene 34(GADD34). However, there were hardly any changes in the splicing of X box binding protein 1(XBP1) or in the nuclear cleaved ATF6 in LPS-treated A549 cells compared with the control cells.(11)The transfection of the cells with an si RNA targeted against PERK or ATF4 inhibited the expression of the proteins. In addition, the knockdown of PERK or ATF4 decreased the formation of LC3B-II and the punctate accumulation of LC3, as well as partially increased the viability of LPS-treated A549 cells.4.ConclusionIn conclusion, our present findings demonstrate that alveolar epithelial cells can undergo autophagic cell death following a challenge with a minimal cytotoxic dose of LPS and that this process depends upon the activation of the PERK pathway of the UPR upon ER stress. These molecular events might represent another important mechanism contributing to the alveolar epithelial cell damage induced by LPS, which likely results in the pathogenesis of ALI. These findings may provide potential clues for exploiting possible therapeutic drugs for the management of alveolar epithelial cell death during ALI.