Autophagy, an important therapeutic target for pulmonary fibrosis diseases

Journal Pre-proofs Review Autophagy, an important therapeutic target for pulmonary fibrosis diseases Hong Zhao, Yiqun Wang, Tingting Qiu, Wei Liu, Pingbo Yao PII: DOI: Reference:

S0009-8981(19)32185-0 https://doi.org/10.1016/j.cca.2019.12.016 CCA 15966

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Clinica Chimica Acta

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15 September 2019 19 December 2019 19 December 2019

Please cite this article as: H. Zhao, Y. Wang, T. Qiu, W. Liu, P. Yao, Autophagy, an important therapeutic target for pulmonary fibrosis diseases, Clinica Chimica Acta (2019), doi: https://doi.org/10.1016/j.cca.2019.12.016

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Autophagy, an important therapeutic target for pulmonary fibrosis diseases Hong Zhao†1, Yiqun Wang†4, Tingting Qiu1, Wei Liu3, Pingbo Yao2 1. Nursing College, University of South China, Hengyang, 42001, China 2. Department of Clinical Technology, Changsha Health Vocational College, Changsha, 410100, China 3. Intensive Care Units of the Affiliated, Nanhua Hospital of University of South China, Hengyang, 421002, China 4. Department of Anaesthesiology, Nanhua Hospital of University of South China, Hengyang, 421002, China

†Hong Zhao and Yiqun Wang contributed equally to this work.

 Corresponding authors: Pingbo Yao* and Wei Liu * Address: Changsha Health Vocational College, Changsha, 410100, China; Intensive Care Units of the Affiliated Nanhua Hospital of University of South China, Hengyang, 421002, China. E-mail: [email protected]; [email protected] 1 / 24

Abstract As an evolutionarily conserved intracellular degradation pathway, autophagy is essential to cellular homeostasis. Several studies have demonstrated that autophagy showed an important effect on some pulmonary fibrosis diseases, including idiopathic pulmonary fibrosis (IPF), cystic fibrosis lung disease, silicosis and smoking-induced pulmonary fibrosis. For example, autophagy mitigates the pathological progression of IPF by regulating the apoptosis of fibroblasts and the senescence of alveolar epithelial cells. In addition, autophagy ameliorates cystic fibrosis lung disease via rescuing transmembrane conductance regulators (CFTRs) to the plasma membrane. Furthermore, autophagy alleviates the silica-induced pulmonary fibrosis by decreasing apoptosis of alveolar epithelial cells in silicosis. However, excessive macrophage autophagy aggravates the pathogenesis of silicosis fibrosis by promoting the proliferation and migration of lung fibroblasts in silicosis. Autophagy is also involved in smokinginduced pulmonary fibrosis, coal workers' pneumoconiosis, ionizing radiationmediated pulmonary fibrosis and heavy metal nanoparticle-mediated pulmonary fibrosis. In this review, the role and signalling mechanisms of autophagy in the progression of pulmonary fibrosis diseases have been systematically analysed. It has provided a new insight into the therapeutic potential associated with autophagy in pulmonary fibrosis diseases. In conclusion, the targeting of autophagy might prove to be a prospective avenue for the therapeutic intervention of pulmonary fibrosis diseases. Key words: Autophagy; idiopathic pulmonary fibrosis (IPF); cystic fibrosis lung disease; silicosis; smoking-induced pulmonary fibrosis;

1. Introduction 2 / 24

Autophagy is a conserved catabolic process for recycling cytosolic organelles and proteins via the lysosome-mediated degradation pathway [1]. Presently, there are three main autophagy types in mammals: macroautophagy, microautophagy and chaperonemediated autophagy. In the process of macroautophagy, unwanted organelles and proteins are contained in a double membrane-bound vesicle for the formation of autophagosomes. Subsequently, autophagosomes are delivered to the lysosome for degradation [2]. This process requires the concerted coordination of autophagy-related proteins encoded by autophagy-related genes (Atgs), such as Atg5 or Atg7, two essential genes for autophagy [3]. In the process of microautophagy, cargoes are first engulfed in the lysosome through the invagination of the lysosomal membrane. Then, these engulfed cargoes are degraded in the lysosomal lumen. In the process of chaperone-mediated autophagy, proteins are first recognised by the molecular chaperone Hsc70 via a specific signalling sequence, such as the KFERQ-like pentapeptide motif [4]. Finally, these recognised proteins are selectively degraded by the autophagy pathway. In general, during the process of autophagy, unwanted organelles and proteins are ultimately transported to lysosomal degradation by a variety of pathways. Thus, autophagy is also characterised by a dynamic process to maintain cellular homeostasis in response to stress and various diseases. Autophagy acts as a complicated but crucial moderator in pulmonary diseases, especially in pulmonary fibrosis diseases [5]. Pulmonary fibrosis is the basic pathologic process in a variety of chronic pulmonary diseases. In the process of pulmonary fibrosis, the over-population and the excessive activation of fibroblasts and myofibroblasts can disrupt the normal structure and function of lung tissue. As a result, the distinctive fibroblasts and myofibroblasts are responsible for abnormal lung remodelling through the production of excessive extracellular matrix (ECM) [6]. In addition, the proapoptotic action on lung fibroblasts and myofibroblasts induced by carnosic acid can alleviate the pulmonary fibrosis, thereby causing an important antifibrotic effect [7]. Autophagy also maintains the normal fate of lung fibroblasts and offers an intervention against pulmonary fibrotic diseases. Inhibition of autophagy by the transforming growth factor-beta1 (TGF-β1) has been found to mediate fibroblast to myofibroblast differentiation, leading to induction of pulmonary fibrosis [8]. Moreover, autophagy ameliorates bleomycin-induced pulmonary fibrosis by inhibiting the apoptosis of lung epithelial cells [9]. The block in autophagy has been found to disrupt cellular proteostasis, inducing interstitial lung disease [10]. Furthermore, autophagy ameliorates angiotensin II-induced pulmonary fibrosis by regulating the activation of NLRP3 inflammasomes [11]. However, extensive macroautophagy is activated in amiodarone-induced pulmonary fibrosis. Additionally, the extensive macroautophagy causes the apoptosis of type II alveolar epithelial cells 3 / 24

(AECII) and the profibrotic effect in amiodarone-induced pulmonary fibrosis [12]. This is a contradictory phenomenon. These studies have suggested that the role of autophagy is complicated in bleomycin-induced pulmonary fibrosis and amiodarone-induced pulmonary fibrosis. Physiological autophagy is beneficial for inhibiting the progression of pulmonary fibrosis, but excessive autophagy may mediate the development of pulmonary fibrosis. The type of autophagy and the signalling mechanisms associated with it may be different in various pulmonary fibrosis diseases. Autophagy triggered by TGF-β1 also induces epithelial-mesenchymal transition, leading to the production of pulmonary fibrosis [13]. TGF-β1 also facilitates the secretion of pro-inflammatory mediators interleukin 4 (IL-4) and 13 (IL-13) in alveolar macrophages, which can contribute to the progression of lung fibrogenesis [14]. Inhibition of TGF-β1 decreases the neutrophils, macrophages and lymphocytes in bronchoalveolar lavage fluids, ultimately leading to the relief of bleomycin-induced pulmonary fibrosis [15]. IL-37, an anti-inflammatory cytokine, decreases lung fibroblast proliferation and shows the antifibrotic activity by enhancing autophagy and inhibiting TGF-β1-induced inflammation in pulmonary fibrosis [16]. The role of autophagy in pulmonary fibrosis may be complicated or even opposite in different cell types of lung tissue such as epithelial cells, fibroblasts, and macrophages. Macrophage autophagy is involved in the neutrophil extracellular trap (NET) activation and release (NETosis) by recruited neutrophils and macrophages in lung tissue [17]. NETosis is a process by which neutrophil nuclear DNA decorated with proteins are released into the extracellular matrix. Autophagy is indispensable for NETosis formation. It promotes NETosis formation and ameliorates pseudomonas aeruginosa pneumonia in mice [17]. The acceleration of NETosis reduces proinflammatory cytokine secretion, resulting in the limitation of inflammatory response [18]. Thus, autophagy may relieve pulmonary fibrosis by regulating NETosis formation and inflammatory response. Taken together, this evidence suggests that autophagy plays a complicated but vital role in pulmonary fibrosis diseases. In addition, the role of autophagy in different types of pulmonary fibrosis diseases may be different or even opposite, even in the same pulmonary fibrosis. The importance of autophagy is worthy of systematic analysis in pulmonary fibrosis. Therefore, the role and regulatory mechanisms associated with autophagy in pulmonary fibrosis diseases, such as idiopathic pulmonary fibrosis (IPF), cystic fibrosis lung disease, silica-induced pulmonary fibrosis and other pulmonary fibrosis diseases have been analysed in detail in this review. It is suggested that the targeting of autophagy may be used as a new therapeutic intervention for pulmonary fibrosis diseases. 2. The occurrence of autophagy in pulmonary fibrosis 4 / 24

Currently, the autophagy activity is mainly dissected by some autophagy-related protein expression and immunofluorescence, as well as the number and the location of autophagosomes. The occurrence of autophagy is controlled by at least 30 known autophagy genes (ATG) and some other proteins in pulmonary fibrosis diseases (Table 1). Studies have shown that the autophagic activity was assessed by some autophagyrelated proteins, such as Beclin1, p62 and LC3. Immunohistochemical staining results have demonstrated that the expressions of Belin 1, LC3 and p62 were observed in macrophages and proliferating fibroblasts from the fibrosing areas of paraquat-induced pulmonary fibrosis [19]. The expressions of Beclin 1, LC3 and the numbers of autophagosomes are also decreased by TGF-β via the activation of mTORC1 in lung tissues from fibrosis patients [20]. Thus, autophagy dysfunction is involved in the promotion of fibrogenesis. Importantly, autophagy dysfunction induces fibroblasts to resist the apoptotic system due to an overexpression of the anti-apoptotic protein Bcl-2 in pulmonary fibrotic disease [21]. Impaired apoptosis of fibroblasts and fibroblast accumulation were key features of the pathologic correlate of pulmonary fibrosis [22]. Additionally, LC3B levels and autophagic flux were decreased in human pulmonary fibrosis tissues compared to normal controls [9]. Acceleration of autophagic flux inhibits apoptosis and proliferation of epithelial cells, alleviating bleomycin-induced pulmonary fibrosis. Moreover, impaired autophagic activity and ATG4B deficiency are found in tunicamycin-induced pulmonary fibrosis [23] and bleomycin-induced pulmonary fibrosis [24]. Impaired autophagy exacerbates the lung epithelial apoptosis and the development of pulmonary fibrosis. Impaired autophagy also induces a significantly higher inflammatory response and increases collagen accumulation. Loss of ATG7 and damaged autophagic flux upregulate TGFβ signalling, leading to pulmonary fibrosis via regulation of endothelial-to-mesenchymal transition [25]. Overall, these studies have suggested that autophagy may play a crucial protective role in the regulation of the fibrotic responses. 3. Some signalling pathways related to autophagy are involved in pulmonary fibrosis 3.1 PI3K-Akt-mTOR signalling suppresses autophagy in pulmonary fibrosis The mammalian target of rapamycin (mTOR) is also involved in pulmonary fibrotic diseases [26]. Studies have shown that the overactivation of mTOR caused the insufficient autophagy in alveolar epithelial cells, resulting in the pathogenesis of pulmonary fibrosis [27], and the aberrant Akt/mTOR axis suppresses autophagic activity, inducing the process of pulmonary fibrosis on collagen [28]. Autophagy was also inhibited through the activation of the PI3K/Akt/mTOR pathway in leptin-induced pulmonary fibrosis development [29]. Of note, the PI3K-Akt-mTOR pathway inhibits 5 / 24

autophagy and promotes lung fibroblast proliferation [30]. Moreover, autophagy inhibited by the PI3K-Akt-mTOR pathway is mainly attributed to the depleting thymocyte differentiation antigen-1 (Thy-1) and upregulating integrin β3 (Itgb3) in lung fibroblasts [31]. Importantly, the pre-treatment with rapamycin mitigates the bleomycin-mediated pulmonary fibrosis [27]. Collectively, these results have supported that the suppression of mTOR may be used for treatment of pulmonary fibrosis diseases via enhancing autophagy. 3.2 Endoplasmic reticulum (ER) stress and autophagy regulate pulmonary fibrosis Several studies have confirmed that ER stress promotes the pathogenesis of pulmonary fibrosis [32-34]. In addition, ER stress activates the unfolded protein response (UPR) and impairs proteostasis in alveolar epithelial cells in pulmonary fibrosis disease [34]. ER stress is also known to mediate the occurrence of autophagy [35]. Furthermore, impaired autophagy also induces ER stress and further exacerbates development of tunicamycin-induced pulmonary fibrosis [23]. However, in silicosis, the activation of ER stress and autophagy can simultaneously augment the proliferation and migration of fibroblasts, promoting the pathological process of pulmonary fibroblasts [36]. This evidence has revealed that the role of ER stress and autophagy in pulmonary fibrosis diseases may be complex and worth of further exploration. 3.3 Elongation factor-2 kinase (eEF2K) and p38 MAPK activates autophagy in pulmonary fibroblasts eEF2K, a member of the calmodulin kinase family, not only regulates autophagy under nutrient deprivation and growth factor inhibition [37], but also acts as a various of downstream signalling pathways, such as the AMPK, ERK1/2, and p38 MAPK pathways [38]. In addition, the eEF2K and downstream of p38 MAPK regulate autophagy and apoptosis in human lung fibroblasts [39]. Furthermore, eEF2K-activated autophagy can improve the differentiation of lung fibroblasts into myofibroblasts, thus causing an antifibrotic effect. Additionally, Janus kinase 2 (JAK2), a receptorassociated tyrosine kinase, plays an important role in growth factor signalling. JAK2 and the signalling transducer and the activator of transcription 3 (STAT3) suppress autophagy, resulting in the fibroblast to myofibroblast transition [40]. Moreover, multityrosine kinase inhibitor ameliorates pulmonary fibrosis progression and symptoms [41]. Based on the information presented above, it has been suggested that numerous kinases are involved in regulating autophagy in pulmonary fibrosis. 3.4 The p65 and Keap1/Nrf2 signalling pathway regulates autophagy in pulmonary fibrosis The upregulation of nuclear factor p65, a subunit of nuclear factor (NF)-κB, is found in lipopolysaccharides (LPS)- and TGF-β1-induced lung fibrosis [42]. The level of p65 and autophagy are inhibited in high-dose paraquat-induced pulmonary fibrosis. 6 / 24

Additionally, the overexpression of p65 reverses paraquat-induced autophagy inhibition and pulmonary fibrosis [43]. Notably, p65 induces the release of nuclear factor (erythroid-derived-2)-like 2 (Nrf2) via binding itself to its inhibitor Kelch-like ECH-associated protein 1 (Keap1) [44]. Moreover, the release of Nrf2 eliminates reactive oxygen species (ROS) levels and protects against oxidative stress by binding to antioxidant response elements (AREs) in the nucleus and promoting the transcription of antioxidant genes [45]. Knockdown of Nrf2 reverses the protective role of p65 in pulmonary fibrosis [43], and Nrf2 knockout reduces autophagic gene expression [46], while system-level feedback of NRF2 can regulate autophagy induction according to the level of oxidative stress response [47]. Additionally, the accumulation of p62, as a marker of autophagy, shows the impairment of autophagy in the cytoplasm. Recent studies have confirmed that p62 levels were increased in the fibrotic area [19]. p62 also binds competitively to Keap1, leading to the release of Nrf2 [48]. Likewise, Nrf2 promotes autophagy by activating p62 downstream target genes in a positive feedback loop [46]. Autophagy, as an antioxidant response, is activated by a Keap1–Nrf2–p62 feedback loop to ameliorate the detrimental effects of excessive oxidative stress. In short, both p65 and p62 regulate autophagy by the Keap1/Nrf2 signalling pathway in pulmonary fibrosis. 3.5 Golgi and vimentin regulate autophagy in pulmonary fibrosis The Golgi provides the double-membraned phagophore for the assembly of autophagosomes, while Golgi proteins have been recognized as central players of autophagy formation [49]. A study has shown that the downregulated expression of cisGolgi protein GOLGA2 induced autophagy with Golgi disruption [50]. Autophagy induced by GOLGA2 loss leads to fibrosis along with the reduction of lamellar bodies, which is a component of the surfactant in the lung [51]. These findings demonstrate that the relationship between Golgi and autophagy is close in pulmonary fibrosis. In addition, vimentin regulates autophagy and pulmonary fibrosis. A study has indicated that vimentin intermediate filaments (VimIFs) can contribute to the invasive property of pulmonary fibroblasts by inducing a deficient cellular autophagy [52]. In future, more signalling pathways of autophagy need to be elucidated in pulmonary fibrosis. Overall, these signalling pathways are complicated and connected in different cell types of lung tissue (Figure 1). In lung fibroblasts, both PI3K-Akt-mTOR signalling and VimIFs can suppress autophagy and promote lung fibroblast proliferation, leading to the pulmonary fibrosis pathogenesis. JAK2/STAT3 signalling pathways can suppress autophagy and promote the fibroblast to myofibroblast transition, resulting in pulmonary fibrosis progression. The GOLGA2 also suppresses autophagy and reduces alveolar macrophage survival, thereby exacerbating the progression of pulmonary 7 / 24

fibrosis. ER stress not only augments the proliferation and migration of fibroblasts but also impairs proteostasis in alveolar epithelial cells, which promotes the pathogenesis of pulmonary fibrosis. However, the eEF2K and p38 MAPK signalling pathways activate autophagy and ameliorate lung fibroblast-to-myofibroblast differentiation, leading to an anti-fibrotic effect in pulmonary fibrosis. p65 and Nrf2 also produce protective effects in pulmonary fibrosis by mitigating alveolar epithelial cell apoptosis. 4. The role of autophagy in different pulmonary fibrosis diseases 4.1 Autophagy mitigates the IPF pathogenesis IPF is characterised by alveolar epithelial cell injury, fibroblast-to-myofibroblast differentiation, collagen deposition and epithelial-to-mesenchymal transition [53, 54]. The aberrant alveolar epithelial cells induce the migration, proliferation and activation of fibroblasts. Studies have found that autophagy was inhibited in IPF, and the expression of autophagic Beclin 1 was decreased in fibroblasts from IPF [55]. Notably, the dysregulation of autophagy promotes fibrogenesis by regulating the proliferation and apoptosis of fibroblasts in IPF [56] (Figure 2). Inhibition of autophagy increases the production of collagen and the epithelial-to-mesenchymal transition [57]. Furthermore, fibroblasts resist type I collagen matrix-induced cell death due to low autophagic activity in IPF [58]. In fibroblasts of IPF on collagen, the PTEN-Akt axis reduces the expression of FoxO3a. FoxO3a, as a member of the forkhead family of transcription factors, activates LC3B by binding to the promoter region of LC3B. The aberrantly low FoxO3a expression suppresses LC3B transcription and causes abnormally low autophagy in IPF fibroblasts on collagen. Additionally, the inhibition of autophagy increases the anti-apoptotic protein Bcl-2 expression in fibroblasts [55]. Autophagy was found to be decreased through PI3K/AKT/mTOR activation [59], and insufficient autophagy makes fibroblasts and myofibroblasts resistant to apoptosis in IPF. Autophagy is also suppressed by inhibiting eEF2K and p38 MAPK signalling in IPF [39], and autophagy suppression induces fibroblast-to-myofibroblast differentiation by increasing the proliferation and differentiation of fibroblasts and decreasing apoptosis of fibroblasts in IPF. Additionally, the JAK2/STAT3 signalling pathway damages autophagy, then resulting in the epithelial-to-mesenchymal and fibroblast-to-myofibroblast differentiation [40]. Thus, autophagy represented a protective feature by regulating fibroblast function in IPF. However, the excessive autophagy and the unfolded protein response (UPR) promoted pulmonary fibrosis through the excessive biosynthesis of collagen and fibronectin in lung fibroblasts of IPF [60]. Therefore, more studies are still necessary to clarify the role of autophagy in fibroblasts of IPF. The dysregulation of autophagy also promotes fibrogenesis by regulating the survival and senescence of alveolar epithelial cells in IPF. The injury and the 8 / 24

dysfunction of alveolar epithelial cells can exacerbate the pathogenesis of IPF [61]. Studies have demonstrated that the impairment of autophagy and the induction of ER stress increased the apoptosis of alveolar epithelial cells and exacerbated the development of pulmonary fibrosis [23]. In contrast, increase of autophagy relieves proteostatic stress and promotes lung epithelial cell survival. Moreover, alveolar epithelial cell senescence and aging are involved in the insufficient autophagy-mediated IPF [62]. Insufficient autophagy has been found to accelerate the pathogenesis of IPF by promoting the senescence of epithelial cells and the differentiation of myofibroblasts in pulmonary fibroblasts [63, 64]. Clearing senescent cells alleviates IPF through the suppression of fibrosis factor expression [65]. Autophagy is attenuated by IL-17A in alveolar epithelial cells [57], and increasing autophagy promotes the autophagic degradation of collagen and autophagy-associated cell death, thereby alleviating the progression of pulmonary fibrosis. Thus, this evidence suggested that cellular senescence may decrease autophagy and promote the progression of IPF. Deficient mitophagy mediates the pathological process of IPF. Mitophagy is known as a selective autophagic process in which damaged or dysfunctional mitochondria are degraded by the lysosome pathway to maintain cellular environment homeostasis [66]. Thus, mitophagy controls the quality of mitochondria via degrading damaged or dysfunctional mitochondria. Deficient mitophagy increases the ROS accumulation and causes the mitochondria dysfunction. Increasing studies have confirmed that mitophagy plays a crucial role in the pathogenesis of IPF [67]. The dysfunction of mitochondria increases ROS accumulation and affects the pathogenesis of IPF [68, 69]. Accumulating mitophagy-related proteins and gene expression are upregulated in IPF [70]. Additionally, the PINK1, phosphorylated Drp1 and LC3B proteins are increased via the application of TGF-β1 in lung epithelial cells [71]. Downregulation of parkin and PINK1 by ER stress induces mitophagy deficiency and the profibrotic factor expression in lung epithelial cell death [72]. Importantly, persistent ER stress increases mitochondrial damage through the downregulation of PINK1 [72]. However, there is some evidence that ER stress upregulates the mRNA and the protein levels of parkin [73]. This pitfall requires further studies to demonstrate the relationship between ER stress and mitophagy. Deficient mitophagy increases susceptibility to bleomycin-induced fibrosis, while mitochondria ROS scavenging ameliorates this phenotype. The knockdown of Parkin also promotes pulmonary fibrosis by enhancing myofibroblast differentiation and proliferation [74]. PINK1 expression was reduced with advancing age, and an age-related decline in mitophagy elevates TGF-β1-mediated fibroblast-to-myofibroblast differentiation in IPF [8]. These studies have implied that deficiency of mitophagy may increase myofibroblast differentiation, mediating the pathological process of IPF. Nevertheless, more 9 / 24

morphological evidence of mitophagy deficiency are essential in the pathological process of IPF. The downregulation of PINK1 expression is insufficient to identify the mitophagy deficiency. Targeting autophagy is an important therapeutic strategy for ameliorating the pathological process of IPF. Studies have investigated that rapamycin treatment induced an antifibrotic effect by increasing autophagy on bleomycin-induced IPF [20]. In the process of bleomycin-induced IPF, TGF-β inhibits autophagy via the activation of mTORC1 in fibroblasts, while rapamycin increases autophagy by inhibiting mTORC1. Additionally, rapamycin also decreases the expression of á-smooth muscle actin and fibronectin by fibroblasts in bleomycin-induced IPF. Tubastatin treatment also reduces the expression of type-1 collagen in IPF and ameliorates bleomycin-induced IPF through the upregulation of autophagy [75]. TGF-β1 attenuates the expression of LC3B-II, which is a marker of autophagosome formation in bleomycin-induced IPF. Tubastatin upregulates autophagy via inhibiting the TGFβ-PI3K-Akt pathway. Berberine enhances autophagy by inhibiting mTOR activation. Berberine-induced autophagy protects against the pathological process of IPF by reducing some fibrotic markers, such as α-smooth muscle actin (α-SMA), fibronectin and collagens I and III. Berberine also protects against pulmonary fibrosis via inhibiting the Smad and nonSmad signalling cascades [76]. 4.2 Autophagy ameliorates cystic fibrosis lung disease Cystic fibrosis is a fatal genetic disorder in which lung tissue and the digestive system are most affected [77]. The chronic pulmonary infections and inflammatory responses are crucial pathogenic factors in the pathological process of cystic fibrosis lung disease [78]. Autophagy is triggered through the activation of the EIF2AK4-ATF4 pathway in lung tissues of pseudomonas aeruginosa-mediated cystic fibrosis [79]. And activated autophagy induces a protective effect on the progression of cystic fibrosis [56]. However, autophagy is downregulated due to the mutations of the transmembrane conductance regulator gene (CFTR) [80]. The mutation of CFTR gene mediates the CFTR protein dysfunction via misfolded protein response, resulting in the dysregulation of the ubiquitin-proteasome system (UPS) and autophagy. The CFTR dysfunction also decreases bacterial killing by reduced macrophage autophagy in cystic fibrosis, while the dysfunctional autophagy exacerbates the pathology of cystic fibrosis of the lungs by heightening inflammatory responses [81]. It can be stated that increasing autophagy ameliorates the cystic fibrosis lung disease. Autophagy may be a novel treatment strategy for cystic fibrosis. Strong evidence have demonstrated that rapamycin decreased bacterial burden and drastically reduced inflammation by inducing autophagy in the lung tissue of cystic fibrosis [82]. G4-CYS, a novel cystamine-core dendrimer formulation, alleviates the pseudomonas aeruginosa 10 / 24

infection-induced cystic fibrosis due to augmenting autophagy [83]. G4-CYS and its control, DAB-core dendrimer (G4-DAB), corrects impaired-autophagy and rescues CFTR protein to the plasma membrane. Cysteamine improves macrophage autophagy and increases macrophage-mediated bacterial clearance, resulting in an effective adjunct to mitigate the pathological process of CF [84]. AR-12 and a new analogue, AR-13, as the small molecule autophagy inducer, can suppress the bacterial multi-drug resistance and the defective host killing via increasing CFTR protein expression and improving autophagy in cystic fibrosis [85]. Moreover, restoring the normal levels of microRNAs Mir17 and Mir20a by specific antagomirs can alleviate clinical symptoms of cystic fibrosis by improving recovering autophagy and CFTR function in macrophages [86, 87]. As described above, the targeting of autophagy may be a new prospect for the treatment of cystic fibrosis lung disease. 4.3 Autophagy regulates the development and progression of silicosis It has been well known that silica dust exposure increased a large amount of collagen fibres in the lung tissue and mediated fibrogenesis in the occurrence and the development of silicosis [88]. Interestingly, silica dust exposure can induce the occurrence of autophagy in the lung tissue of rats [89]. Studies have revealed that the level of autophagy-related proteins LC3 and Beclin1 were closely correlated in lung tissue with different stages of silicosis [90]. More importantly, the ratio of LC3II/LC3I and the expression of Beclin1 were increased in the early stage of silicosis but decreased with the progression of silicosis. Furthermore, autophagy becomes weak with the progression of silicosis. Evidences has shown that autophagy flux was blocked, resulting from injured lysosomal degradation in silicosis [91]. Furthermore, blocking autophagy exacerbates the silica-induced pulmonary fibrosis due to increasing apoptosis in alveolar epithelial cells. These studies have suggested that autophagy may have a protective effect on silicosis. Increasing evidence has supported that autophagy improves the pathological process of silicosis. The therapeutic strategy based on autophagy may be an effective treatment for silicosis. Studies have indicated that the downregulated expression of miR-326 was found in mouse lung tissues of silicosis [92]. The upregulated expression of miR-326 alleviates silicosis by increasing autophagy activity in fibroblasts [93]. The miR-326 promotes autophagy activity of fibroblasts through targeting poly-pyrimidine tract-binding protein 1 (PTBP1). PTBP1, a kind of RNA-binding protein, can bind to the mRNA of ATG10 and negatively regulated the expression of ATG10, which is a key molecule in the formation of autophagosomes [94]. Thus, miR-326 can increase the protein level of ATG10 and autophagy activity via reducing the expression of PTBP1 in fibroblasts. It has been suggested that miR-326 may alleviate pulmonary fibrosis by targeting autophagy. 11 / 24

Dioscin has also been found to alleviate silicosis by inducing the alveolar macrophage-type specific autophagy. Dioscin significantly increases LC3 and BECN1 in alveolar macrophages [95]. Autophagosomes are accumulated in alveolar macrophages [96]. In addition, dioscin limits the level of mitochondrial ROS stimulated and reduces the apoptosis of alveolar macrophage by inducing alveolar macrophage type-specific autophagy in silicosis [95]. Autophagy deficiency in macrophages aggravates apoptosis caused by silica exposure [97]. Dioscin suppresses the generation of inflammatory factors in alveolar macrophages through inducing alveolar macrophage type-specific autophagy. More importantly, dioscin's protective effects and macrophage type-specific autophagy are diminished in alveolar macrophages line MH-S with Atg5 silence or Atg5flox/floxDppa3Cre/+ mice. Thus, this evidence suggests that dioscin protected against pulmonary inflammation and silicosis by inducing alveolar macrophage type-specific autophagy. The decreased inflammatory response also alleviates abnormal collagen repair in silicosis. Futhermore, rapamycin protects alveolar epithelial cells from apoptosis and attenuates silica-induced pulmonary fibrosis through the enhancement of autophagy in the mouse model [91]. Besides, MiR-449a, as a kind of endogenous suppressor of pulmonary fibrosis, has an antifibrotic effect via upregulating autophagic activity and decreasing Bcl2 level in silica-induced pulmonary fibrosis [98]. However, some studies have shown that excessive activation of autophagy promoted the development of silicosis fibrosis. Various studies have shown that autophagy was activated by the monocyte chemotactic protein-1-induced protein 1(MCPIP1) via p53 signalling in macrophages [99], and macrophage autophagy induces pulmonary fibrotic response due to the increase of proliferation and migration of lung fibroblasts in silicosis [100]. Similarly, macrophage autophagy activation by the BCL2-binding component 3 (BBC3) also promotes the proliferation and migration of fibroblasts [101]. Why is the role of autophagy in silicosis contradictory? Mechanically, autophagy-mediated pulmonary fibrosis may be associated with excessive and persistent ER stress [36]. It has been found that macrophage autophagy and ER stress were induced by PPP1R13B, which is an apoptosis-stimulating protein of the p53 family in lung fibroblasts. Activated macrophage autophagy exposed to SiO2 promotes the proliferation and migration of lung fibroblasts, resulting in the development of pulmonary fibrosis in silicosis. Moreover, another study has suggested that autophagy activation through a TLR4-dependent pathway aggravated LPS-induced apoptosis in alveolar macrophages and the development of human silicosis [96]. Inhibition of autophagy reverses the LPS-induced apoptosis by decrease in BAX and CASP3 levels and the increase in Bcl2 in alveolar macrophages during silicosis progression. Overall, it has been suggested that the physiological level of macrophage autophagy may alleviate silicosis progression by reducing pulmonary inflammation. However, 12 / 24

excessive and persistent autophagy and subsequent ER stress may promote the development of silicosis fibrosis by promoting the proliferation and migration of lung fibroblasts as well the apoptosis of macrophages. 4.4 The role of autophagy in other types of pulmonary fibrosis Autophagy alleviates smoking-induced pulmonary fibrosis. Tobacco smoke is known to induce the progression of pulmonary fibrosis [102]. Studies have revealed that cigarette smoke exposure caused autophagy impairment via TFEB [103], and autophagy impairment diminishes phagocytosis-mediated bacterial clearance, and then results in the phagocytic defect. Conversely, improving the impaired autophagy attenuates collagen synthesis and cigarette smoking-mediated pulmonary fibrosis [104]. S-nitrosoglutathione augmentation improves cigarette smoking-mediated pulmonary fibrosis by decreasing autophagy impairment [105]. In addition, the singe nucleotide polymorphisms of ATG10 are involved in the development of coal workers' pneumoconiosis [106]. Lysosome-targeted iron chelators ameliorate ionising radiation-mediated pulmonary fibrosis [107]. Autophagy is also implicated in heavy metal nanoparticle-mediated pulmonary fibrosis [108]. Conclusions and Perspectives In this review, the role and signalling mechanisms of autophagy in pulmonary fibrosis disease have been systematically discussed. Furthermore, the therapeutic and diagnostic implications of autophagy in pulmonary fibrosis disease have been elaborated in this paper. Autophagy mitigates numerous pulmonary fibrosis diseases, such as the idiopathic pulmonary fibrosis, cystic fibrosis lung disease, smoking-induced pulmonary fibrosis and ionising radiation-mediated pulmonary fibrosis. Interestingly, autophagy has a different or even opposite effect on silicosis progression, which is related to the level of autophagy flux and different downstream signalling pathways. The physiological level of autophagy alleviates silicosis progression by reducing the secretion of inflammatory factors and pulmonary inflammation chemokines. The physiological level of autophagy also suppresses the apoptosis of alveolar macrophages by decreasing the accumulation of mitochondrial ROS. However, excessive and persistent autophagy and subsequent ER stress promotes the development of silicosis fibrosis by promoting apoptosis of alveolar macrophages as well as the proliferation and migration of lung fibroblasts. Thus, macrophage type-specific autophagy shows an important role in silicosis. Furthermore, regenerating alveolar capillary endothelium and angiogenesis are involved in interstitial pulmonary fibrosis diseases [109]. Inhibited autophagy in pulmonary artery endothelium supresses endothelial cell migration and angiogenesis [110]. Based on these studies, it has been speculated that the dysfunction of autophagy may promote pulmonary fibrosis progression via 13 / 24

mediating angiogenesis. However, autophagy still shows a therapeutic potential in pulmonary fibrosis diseases. The targeting of autophagy may be a novel avenue for therapeutic intervention of pulmonary fibrosis diseases. Further studies should focus on the relationship between the autophagy and pulmonary fibrosis diseases. Author contributions Hong Zhao conceived and wrote the manuscript. Yiqun Wang reviewed and edited the manuscript. Pingbo Yao and Wei Liu designed and guided this paper. All authors read and approved the manuscript. Acknowledgements This study was funded by grants from the Natural Science Foundation of Hunan Province (2018JJ2339, 2018JJ6122), the 13th Five-Year Plan of Hunan Province Education Science (XJK17BGD070). I am profoundly grateful to Yao Pingbo, whose illuminating instruction and expert advice have guided me in the completion of this review. Conflict of interest The authors declare that there are no conflicts of interest. This article does not contain any studies with human participants or animals performed by any of the authors. References [1] L. Krustev, T. Taschev, A. Pokrovskij, W. Tuteljan, I. Apostolov, L. Krawtschenko, A. Popov, [Effects of reduced diet and physical stress on liver lysosomes. Morphological and biochemical studies], Die Nahrung 22(3) (1978) 309-13. [2] B.H.M. Hunn, S. Vingill, S. Threlfell, J. Alegre-Abarrategui, M. Magdelyns, T. Deltheil, N. BengoaVergniory, P.L. Oliver, M. Cioroch, N.M. Doig, D.M. Bannerman, S.J. Cragg, R. Wade-Martins, Impairment of Macroautophagy in Dopamine Neurons Has Opposing Effects on Parkinsonian Pathology and Behavior, Cell reports 29(4) (2019) 920-931 e7. [3] C. He, D.J. Klionsky, Regulation mechanisms and signaling pathways of autophagy, Annual review of genetics 43 (2009) 67-93. [4] I. Tanida, Autophagosome formation and molecular mechanism of autophagy, Antioxidants & redox signaling 14(11) (2011) 2201-14. [5] K. Mizumura, S. Cloonan, M.E. Choi, S. Hashimoto, K. Nakahira, S.W. Ryter, A.M. Choi, Autophagy: Friend or Foe in Lung Disease?, Annals of the American Thoracic Society 13 Suppl 1 (2016) S40-7. [6] P. Jara, J. Calyeca, Y. Romero, L. Placido, G. Yu, N. Kaminski, V. Maldonado, J. Cisneros, M. Selman, A. Pardo, Matrix metalloproteinase (MMP)-19-deficient fibroblasts display a profibrotic phenotype, American journal of physiology. Lung cellular and molecular physiology 308(6) (2015) L511-22. [7] S. Bahri, F. Mies, R. Ben Ali, M. Mlika, S. Jameleddine, K. Mc Entee, V. Shlyonsky, Rosmarinic acid potentiates carnosic acid induced apoptosis in lung fibroblasts, PloS one 12(9) (2017) e0184368. [8] M.L. Sosulski, R. Gongora, S. Danchuk, C. Dong, F. Luo, C.G. Sanchez, Deregulation of selective autophagy during aging and pulmonary fibrosis: the role of TGFbeta1, Aging cell 14(5) (2015) 774-83. 14 / 24

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Table1 Some autophagic proteins are involved in the process of pulmonary fibrosis Name Protein level Model Role Reference Beclin1 Decrease Specimens from eight Caused autophagy [14] fatal cases paraquat dysfunction poisoning-induced lung fibrosis Decrease In fibroblasts of Caused autophagy [15] idiopathic pulmonary and apoptosis fibrosis system dysfunction LC3B Decrease In human pulmonary Impeded TFEB[9] fibrosis tissues induced autophagic flux ATG4B Deficient In alveolar epithelial Impaired autophagic [16] cells of in Atg4bactivity deficient mice Deficient In alveolar and Disrupted autophagy [17] bronchiolar epithelial and augmented cells of bleomycinapoptosis induced lung fibrosis ATG7 Deficient In endothelial cellsImpaired autophagic [18] specific ATG7 knockflux loss of out mice endothelial and induced endothelialto-mesenchymal transition

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Highlights: 1. This review focuses on the role and therapeutic potential of autophagy in pulmonary fibrosis diseases including idiopathic pulmonary fibrosis (IPF), cystic fibrosis lung disease, silicosis and smoking-induced pulmonary fibrosis. 2. Targeting autophagy may open a novel avenue of therapeutic intervention to pulmonary fibrosis diseases.

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