The Mechanism Of AEC Autophagy Dysfunction In UFPs-induced Pulmonary Fibrosis

The rapid development of economy in China leads to serious air pollution, such as haze, which draws govermental and public concerns. The haze is mainly caused by particulate matter (PM), especially PM2.5 which is a kind of particle with aerodynamic diameters (AD)< 2.5μm. The respiratory system is considered to be a main route by which PM accesses human body. Accumulating epidemiological evidence indicate that exposure to PM is involved in various pulmonary diseases including chronic obstructive pulmonary disease (COPD), asthma, lung cancer, and so on. Generally, it has been accepted that the toxicity of PM is related with its AD, the smaller the particle size, the more severe health impact. However, studies on the health effects of UFPs are not comprehensive and of in-depth. It should be noted that the complex composition and heterogeneous size distribution of particles collected from real air sample make the study difficult to repeat and analysize. Thus, nano-sized particles with a single component and diameter have usually been used to mimic UFPs for investigations.Pulmonary fibrosis (PF) is an important pathological change during chronic lung diseases. It is characterized by sustained damage of alveolar epithelial cells (AECs), fibroblast proliferation and activation, excessive collagen deposition, finally leading to the decline of lung function. Until now, the mechanisms of its pathogenesis are still unclear while it has been suggested that the PF is a result of multiple mechanisms. The initial hypothesis for development of PF is that chronic inflammation was the underlying cause of PF. Moreover, important cytokines, ROS as well as activation and proliferation of fibroblasts were also thought to play critical roles in PF. Recently, AEC injury was considered to contribute to development of PF. It has been reported that UFPs induced PF after instillation in animal model. But, the mechanisms by which UFPs induced PF were confined to cytokines, ROS and inflammation, whether AECs injury participates in UFP-associated PF is unclear. To explore the contribution of AEC damage in UFPs-induced PF, we used artificial nano-sized particle to mimic UFPs to investigate the molecular mechanism of UFPs-induced AECs damage and its role in subsequent PF.Firstly, the intratracheally instilled mice which mimic UFPs exposure via pulmonary system were used to build PF model. After instillation for 1 month, we tested lung compliance to evaluate pulmonary function. Then, the lung tissues were harvested for further determination. The result showed pulmonary compliance was significantly reduced after UFP treatment; white and spot diffused distribution was observed on the surface of lung. Moreover, the lung was enlarged and the lung/body coefficients were significantly increased in UFP-exposed mice. The Masson’s staining and Sirius red staining were used to determine collagen deposition. The results revealed an increased collagen production in lung tissues of UFP-exposed mice. Meanwhile, UFPs treatment increased hydroxyproline (HYP) level, connective tissue growth factor (CTGF) mRNA level and a-SMA protein level. Taken together, these data demonstrate that the UFP-induced PF model has been successfully established.Our further TEM observation showed that intratracheally instilled UFPs were entered into the type II alveolar epithelial cells, and induced apoptosis in lung tissue. We also found the uptake of UFPs in A549 cells, which triggered apoptosis. Collectively, our data suggest UFPs might target AECs, indicating that AEC injury is implicated in UFP-induced pulmonary fibrosis. Autophagy is a conserved eukaryotic catabolic reaction that sequesters protein aggregates and damaged organelles into autophagosomes for lysosomal degradation. It is important for the cells to maintain cellular homeostasis and respond to environmental stress. Though autophagy is suggested to participate in various progress of PF, no evidence has been found to support its contribution in UFP-induced PF. Thus, we detected microtubule-associated protein 1 light chain 3 (LC3) expression level, as a marker of autophagy, in lung tissues with the immunoblotting analysis and immunohistochemistry. The results showed the LC3-Ⅱ protein was significantly accumulated in lung tissues from UFPs-treated groups, suggesting that autophgy might be involved in UFP-induced PF.To explore the molecular mechanism of autophagy, we exposed A549 cells with UFPs. The results revealed UFPs induced autophagosome accumulation in vitro. To thoroughly understand the underlying mechanisms of autophagosome accumulation, we determined the effects of UFPs on induction of autophagy, the progress of the autophagosome-lysosome fusion, and autolysosomal degradation. Firstly, we determined p-mTOR protein level after UFPs treatment, and found that p-mTOR was not inhibited, suggesting classic mTOR pathway was not involved in UFP-associated autophagy. We also detected the p62 protein level, a substrate that is preferentially degraded by autophagy. Interestingly, UFPs raised rather decreased the p62 protein, indicating that autophagosome accumulation might be contributed to inhibition of autophagic degradation. Generally, the autophagosome was fused with lysosome after induction, and degraded in lysosome. The results showed that autophagic flux inhibition was contributed to the decreased lysosomal degradative capacity, instead of impaired autophagosome-lysosome fusion. In addition, UFPs enlarged lysosome, and inhibited lysosomal acidification and maturation of cathepsin D in AECs. Moreover, reacidification of lysosomes by cAMP enhanced autophagic degradation thus protected A549 cells from apoptosis. Additionally, UFPs upregulated IL-1 and IL-6 mRNA level in A549 cells, and the IL-1 and IL-6 mRNA level were reduced with treatment of cAMP or rapamycin, suggesting that UFP-induced autophagy dysfunction in AECs could also trigger inflammation. Meanwhile, the particles collected in air increased LC3, p62 protein levels in A549 as well, suggesting inhibition of autophagic flux could occurr under real air exposure.Finally, we used the mouse model to confirm our findings of UFP-inhibited autophagic degradation. Our in vivo data revealed that the p62 expression level was increased in lung tissues from UFPs intratracheally instilled mice, indicating UFPs inhibit autophagic degradation in vivo. To enhance in vivo autophagic degradation, we treated the intratracheally instilled mice with rapamycin, and the Sirius red staining showed rapamycin treatment decreased collagen deposition in UFP-treated mice. Furthermore, rapamycin significantly reduced hydroxyproline content and a-SMA protein level. These data demonstrate that enhancement of autophagic degradation could attenuate PF induced by UFPs. Our in vivo results further supported that inhibition of autophagic degradation was implicated in UFP-induced PF.Taken together, we conclude that UFPs inhibit autophagic degradation in AECs by inhibiting lysosomal acidification, and thus contribute to apoptosis and inflammation, and subsequent pulmonary fibrosis.