4. Autophagy Dysfunction in Respiratory Diseases
A number of recent studies have identified autophagy dysfunction as the central mechanism of elevated inflammatory-oxidative stress, alveolar apoptosis, cellular senescence, and recurrent infections, all of which contribute to the pathogenesis and progression of acute and chronic respiratory diseases [22,35,61,62,70,111]. The inherent ROS in the CS, and the resulting increase in cellular endogenous ROS post-CS exposure, symbiotically contribute to extremely high intracellular ROS levels, which creates an imbalance in the oxidant–antioxidant ratio [22,34,61,112]. This serves as the basic mechanism for lung cellular injury, tissue damage, and the pathogenesis of chronic obstructive or restrictive lung diseases, such as COPD-emphysema (obstructive) and idiopathic pulmonary fibrosis (IPF, restrictive) [37,38,61,113,114,115,116]. Several groups including ours have demonstrated that elevated ROS levels are the key upstream driver of autophagy dysfunction, as treatments with antioxidant drugs rescues the age-related, or smoke, or eCV exposure-induced autophagy defect [15,22,42,61,62].

4.1. Autophagy Defects in Acute Lung Injury (ALI)
Extensive studies utilizing both in vitro and in vivo models of acute lung injury have demonstrated the protective role of autophagy via regulating inflammatory-oxidative stress, apoptosis, and pathogen clearance mechanisms, even though there were some initial opposing studies [117,118,119], the subsequent detailed evaluation validates the protective role as discussed. In general, autophagy is induced upon exposure to common triggers of ALI, such as LPS, bacterial infections, hyperoxia, sepsis, etc. [120,121,122]. There seems to be a consensus that autophagy indeed plays a protective role in LPS-induced acute lung injury and inflammation [82,83,123]. In support of this, LPS-mediated severe lung injury in mice, as quantified by lung edema, elevated leukocyte infiltrations, hemorrhages, and increased inflammatory cytokines (IL-1β and TNFα) in the bronchoalveolar lavage fluid (BALF), was further exacerbated by autophagy inhibition, thereby suggesting its protective role [82]. Mechanistically, the activation of the mammalian target of rapamycin (mTOR) and the resulting autophagy dysfunction has been implicated in promoting LPS-induced lung injury, possibly through the activation of NFκB signaling [124,125]. Further evidence comes from studies demonstrating the utility of autophagy inducers and/or antioxidant drugs in ameliorating LPS-induced acute lung injuries, while treatment with autophagy inhibitors reversed the beneficial effects [125,126]. Autophagy is also protective in sepsis-induced lung injury, as a deficiency of proteins interacting with C-kinase 1 (PICK1) in mice leads to defective autophagy, and more severe acute lung injury in the cecal ligation and puncture (CLP) model of sepsis, as compared to WT animals [120,127]. Additionally, in murine models of hyperoxia-induced ALI, which resembles features of bronchopulmonary dysplasia (BPD), autophagy is proposed as a protective mechanism, and markers of defective autophagy are found in the lungs of human neonates with established BPD [128]. Another recent study describes the suppression of autophagy as a critical mechanism of chronic parenteral nutrition-mediated lung injury, and treatment with the autophagy inducer, rapamycin (an mTOR inhibitor that initiates nucleation, autophagosome elongation, autophagosome maturation, and autophagosome termination), reversed the lung injury features in the animal model of parenteral nutrition [129,130]. Finally, mice deficient in crucial autophagy proteins such as Atg7, Atg5 and Atg4a demonstrate more severe ALI features [83,121,130,131], thus confirming the protective role of autophagy in ALI.

4.2. Autophagy Defects in Acute Respiratory Distress Syndrome (ARDS)
More severe forms of ALI may progress to acute respiratory distress syndrome, or ARDS. Autophagy has been shown to play a critical role in regulating the outcome of ARDS [120]. The clinical manifestations of ARDS are very severe and may lead to rapid lung function decline and death. Using one of most widely used murine models of ALI, the CLP, a recent study demonstrated that autophagy induction by rapamycin was able to improve the survival rate, histological scores, lung wet/dry ratios, PaO2/FiO2, and inflammatory cytokine and myeloperoxidase (MPO) levels in BALF, suggesting a protective role of autophagy in sepsis-induced ALI/ARDS [120]. In another study, the potential of BML-111, a lipoxin A4 receptor antagonist, was evaluated in controlling LPS-induced septic ALI/ARDS in rats. The authors showed that BML-111 inhibited apoptosis and induced autophagy in alveolar macrophages, in response to the LPS challenge, via the suppression of MAPK1 and MAPK8 signaling and was independent of mTOR [132]. Moreover, BML-111 controlled the LPS-induced production of pro-inflammatory cytokines, and reduced apoptosis in the rat lungs, and thus warrants further investigation in ALI/ARDS [132]. Mechanical ventilation (MV)-induced lung injury is another severe form of ARDS, wherein the activation of inflammasome has been shown to mediate ALI symptoms. In a recent article, starvation-induced autophagy augmentation was shown to protect against LPS- and MV-induced ARDS features by reducing IL-1β levels, decreasing lung permeability, and improving arterial oxygenation [133]. Thus, autophagy had a protective role in controlling the inflammasome activation and resolution of the MV-induced production of IL-1β, which plays a pathogenic role through inducing hypoxemia and increasing lung permeability in LPS/MV-induced ALI/ARDS [133]. Although some contrasting reports regarding the role of autophagy in ALI/ARDS were initially reported [134], there has been a consensus on the therapeutic advantage of autophagy augmentation in ALI/ARDS based on significant validation studies [64,120,122]. Thus, proper autophagy function is essential in attenuating the severity of ARDS in patients.

4.3. Critical Role of Autophagy in COVID-19 Exacerbations
The ongoing SARS-CoV-2 pandemic has severely impacted quality of life with a significant health care and socio-economic burden globally. In general, the cellular endocytic and autophagy pathways contribute to viral entry and replication, and thus are obvious attractive targets against SARS-CoV-2 and other viral infections [90,91,92]. The SARS-CoV-2 virus, which causes COVID-19, is highly infectious and can cause cytokine-storm leading to pneumonia and severe lung damage in susceptible subjects by triggering an ARDS-like lung disease, with high-risk of mortality [135]. Recent evidence suggests that SARS-CoV-2 may inhibit autophagy, which we anticipate as a potential mechanism for severe COVID-19 lung disease due to impaired viral clearance and immune dysfunction. In a recent study of the SARS-CoV-2 genome, researchers found that the NSP6 protein of the virus binds with greater affinity to the endoplasmic reticulum (ER) [136]. This genetic change may allow the virus to inhibit autophagy via impaired autophagosome processing, which prevents the degradation of viral particles by the lysosome [136]. There is also evidence that PLP2 is over-expressed in SARS-CoV and MERS-CoV cell lines that also allows the virus to inhibit autophagolysosomal formation and autophagy flux and is likely a method of autophagy inhibition in SARS-CoV-2 [96]. These studies suggest potential mechanism by which SARS-CoV-2 inhibits autophagy to infect or circumvent host cells pathogenic clearance pathways and limit an adequate immune response similar to other coronaviruses that warrants further investigation.
The elimination of viruses by autophagy (sometimes termed as virophagy) has been well described for a variety of viral infections [8,84,85,137]. Although, the virus has multiple ways of entry into the cell, autophagy augmentation provides strategic advantage in reducing viral load by promoting its clearance [90,137]. As a proof of concept, recent study demonstrated the utility of three different autophagy-inducing drugs, spermidine, MK02206, and niclosamide, in restricting SARS-CoV-2 propagation [138]. Autophagy induction and related upregulation of overall immunity helps combat exacerbations and is suggested as an immunity boosting strategy as a preventive measure against COVID-19 [139]. In addition to boosting immunity, dampening viral load, and allowing SARS-COV2 clearance, autophagy induction may provide strategic advantage in the treatment of COVID-19 and prevention of negative outcomes, which makes it a subject of ongoing studies. In support of this, autophagy inhibiting drugs, such as hydroxychloroquine (HCQ) that help dampen the immune response in rheumatoid arthritis, malaria, and other illnesses but weakens cellular ability for viral clearance by the critical homeostatic process autophagy. Despite early claims that HCQ may provide benefit in treating COVID-19 [90,91,92], it has since been deemed unsafe for use in COVID-19 treatment by the FDA based on randomized double blind placebo control trials [140,141]. In addition, earlier studies on Middle East respiratory syndrome (MERS) coronavirus [50] provide proof-of-concept data on therapeutic potential of autophagy modulating drugs to combat SARS-COV2 infection, cytokine storm, and the pathogenesis of severe ARDS-like COVID-19 fatal lung disease. Prior studies showing a protective role of autophagy induction in other models of ARDS demonstrate the scope of autophagy augmenting strategies in combating SARS-CoV-2 infections (Figure 1) and thus is part of ongoing validation and rapid clinical development studies that may help limit the burden and spread of this novel virus.
As discussed above for other lung diseases, targeting autophagy to prevent the replication of SARS-CoV-2 is not the only potential benefit of autophagy augmentation for the treatment of COVID-19, but it may also allow the fine-tuning of optimal inflammatory responses. As now known, the pathogenesis of SARS-CoV-2-mediated severe COVID-19 involves the activation of numerous pro-inflammatory cytokines as part of the aforementioned cytokine storm causing a hyper-inflammatory state [142]. In addition to the destruction of the lungs and ARDS associated with COVID-19, this inflammatory response can cause damage to the cardiovascular, nervous, renal, hepatic, and gastrointestinal systems with wide ranging immediate and long-term consequences [142]. In support of this, studies have demonstrated the role of autophagy in the inflammatory response within the lungs and other organ systems [61]. As mentioned above, autophagy induction has been demonstrated to attenuate lung inflammation when exposed to a pathogen. Thus, autophagy induction to limit the inflammatory response, in addition to infection, has an immense therapeutic potential as an effective treatment for COVID-19 and decreasing the associated mortality and morbidity.

4.4. Autophagy Defects in COPD
We and others have described autophagy dysfunction as a prime causative factor utilizing in vitro and animal models of smoke- (cigarette and waterpipe) or eCV (nicotine)-induced lung injury and COPD-emphysema [33,34,39,94,109]. Moreover, these were validated in human subjects where defective autophagy was verified using human lung tissues from COPD-emphysema subjects, where classical autophagy impairment features, such as aggresome bodies, were associated with the severity and progression of the disease [22,36]. These aggresome bodies are perinuclear accumulations of misfolded or aggregated proteins, which are poly-ubiquitinated and co-localize with p62 and the autophagy protein microtubule-associated protein 1 light-chain-3B(+) (LC3B+) bodies, and are the key indicators of defective autophagy flux [33,36,143]. Additionally, we and others have also noted the increase in aggresome body formation in aged mice lungs that correlated with alveolar airspace enlargement (emphysema phenotype), indicating that age-related decline in autophagy contributes to COPD-emphysema development, similar to CS exposure [33]. Moreover, an increase in emphysema severity (GOLD 0-IV) in smokers with a minimal age difference [33] also correlated with an increase in alveolar senescence, indicating the presence of accelerated lung aging in severe COPD-emphysema subject lungs. We further validate smoke-induced accelerated lung aging using aging markers and in vitro and murine models of COPD-emphysema. In further studies, a clear mechanistic and protective role of TFEB, the master autophagy regulator, was observed in CS-induced lung disease models where other pathogenic features of COPD-emphysema, such as inflammatory-oxidative stress, senescence, apoptosis, and aggresome formation, were used for the validation of pathogenic roles [34,35]. In fact, the CS-induced sequestration of TFEB protein into aggresome bodies leads to its decreased availability, which prevents its function as a transcription factor to positively regulate the autophagy process [34,35]. Moreover, TFEB-mediated autophagy was shown to be protective against oxidative stress and hepatotoxicity induced by ethyl carbamate (a toxicant in CS) [144], suggesting that TFEB-autophagy is a protective mechanism against CS exposure-induced toxicity, not only in the lungs, but in other vital organs as well. Additionally, TFEB-mediated autophagy has also been shown to control CS-induced cellular senescence, and bacterial phagocytic clearance, thus highlighting its protective role in CS-induced COPD-emphysema [22,34,35]. In addition to TFEB, other mechanistic mediators of autophagy have been shown to participate in the sequential dysfunction or impairment of autophagy processes, contributing as a key mediator of COPD-emphysema pathogenesis. For example, increased levels of bicaudal D1 (BICD1), an adaptor for the dynein–dynactin motor complex, were found in the peripheral lung tissues of COPD patients, which was associated with increased p62 oligomers [145]. Additionally, the exposure of bronchial epithelial cells or mice to CS led to increased BICD1 levels, along with defective autophagosome maturation, and an accumulation of BICD1 with p62 and ubiquitin-associated p62-oligomers, thus confirming the mechanistic role of BICD1 in CS-induced autophagy defects [145].
Dysfunctional autophagy has also been associated with defects in specific cell types of the airway. The secretory cells of the airway, such as the club and goblet cells, play an important role in host defense during infection. Autophagy has been recently shown to be required to maintain the function of club cells, independent of CS exposure [146]. Mice deficient in autophagy protein Atg5, demonstrate a diminished expression of the host defense protein secretoglobulin family 1A member 1 (SCGB1A1) and surfactant proteins A1 and D (Sftpa1 and Sftpd), as well as abnormal club cell morphology [146]. Moreover, a diminished SCGB1A1 expression in club cells correlates with evidence of reduced autophagy in lung tissue from COPD former smokers [146]. CS exposure has also been demonstrated to cause the accumulation of damaged mitochondrial via impaired mitophagy, which has been demonstrated to play a role in COPD pathogenesis disease progression [147,148]. Thus, it can be postulated that CS-induced autophagy dysfunction would further deteriorate the structure and function of club cells, resulting in altered or diminished host defense mechanisms in COPD subjects.

4.5. Autophagy Defects in Cystic Fibrosis
Cystic fibrosis is a chronic obstructive lung disease which is marked by recurrent infections, chronic inflammatory-oxidative stress, and mucus overproduction that contributes to severe airway obstruction. Initial seminal studies by Luciani A et al. established the link between defective CFTR and the presence of aggresome bodies, lung inflammation, and ROS-mediated autophagy inhibition [53]. We and others have not only validated that defective CFTR-mediated ROS-TG2 pathway drives the crosslinking of Beclin-1, which results in the accumulation of misfolded ΔF508-CFTR into p62+HDAC6+ aggresome bodies leading to autophagy dysfunction, but we have also demonstrated the key central role of autophagy in regulating CF pathogenesis and exacerbations [42,58,149,150]. These studies describe the specific role of defective autophagy in CF-related chronic infections and resulting inflammatory-oxidative stress. In addition, CF macrophages demonstrate impaired phagocytic activity and thus CF patients are more prone to bacterial infections, such as P. aeruginosa and Burkholderia cenocepacia (B. cenocepacia) [45,51,58,106,151,152,153]. A recent study investigated the precise mechanism of weak autophagic activity in CF macrophages. Using the technique of reduced representation bisulfite sequencing (RRBS) to determine the DNA methylation profile, the authors found that the promoter regions of Atg12 in CF macrophages are significantly more methylated as compared to the control WT cells, thereby elucidating a novel mechanism for reduced autophagy activity in CF immune cells [154].
In a separate study, an increased expression of the microRNA (Mir)c1/Mir17-92 cluster was identified as a potential negative regulator of autophagy in CF macrophages when compared to normal control cells [105]. Furthermore, the in vivo downregulation of Mir17 and Mir20a, partially restored autophagy gene expression and improved the clearance of B. cenocepacia [105]. These studies highlight the importance of autophagy as a key protective mechanism in CF exacerbation, and lung disease pathogenesis and progression.

4.6. Autophagy Defects in IPF
IPF is a chronic, progressive, and frequently fatal disease associated with aging and dysfunctional autophagy. It is accepted that accelerated epithelial cell senescence plays a vital role in IPF pathogenesis by virtue of atypical epithelial–mesenchymal interactions, and insufficient autophagy is attributed as a mechanism of accelerated epithelial cell senescence and myofibroblast differentiation in IPF [155]. Bleomycin is widely used as a model of drug-induced lung fibrosis. The first study to describe the protective role of autophagy in bleomycin-induced lung fibrosis used the Atg4b-deficient mice model [156]. After 7 days of bleomycin treatment, these mice demonstrated a significantly higher neutrophilic infiltration and inflammatory cytokine production as compared to untreated mice [156]. Additionally, after 28 days of bleomycin treatment, mice developed extensive lung fibrosis, which was accompanied by an elevated collagen deposition and deregulated expression of extracellular matrix genes [156]. Similarly, in mice deficient in LC3B (LC3B−/−), bleomycin-mediated lung injury and fibrotic changes were more pronounced, suggesting the protective role of autophagy in bleomycin-induced lung injury and the resulting development of fibrotic lung disease in mice [157]. Another recent study describes the protective role of the anti-inflammatory cytokine IL-37 in the IPF murine model [158]. A further mechanistic delve into the mechanism of IL-37-mediated protection showed that it induces Beclin-1-dependent autophagy while downregulating TGFβ1-mediated lung fibroblast proliferation [158]. Moreover, IL-37 also decreased inflammation and collagen deposition in bleomycin-treated murine lungs while the protective effect was reversed by treatment with 3-methyladinine (3MA), an autophagy inhibitor [158]. Thus, it is plausible that a decrease in IL-37-mediated autophagy might be involved in the progression of IPF. Moreover, the protective effects of autophagy are apparent from this and the other mechanistic studies mentioned above.
Additional mechanistic evidence comes from a study that showed bleomycin directly binds to annexin A2 (ANXA2) in lung epithelial cells, thereby preventing the nuclear translocation of TFEB; thus, there is an inhibition of the autophagy flux resulting in fibrotic lung disease pathogenesis [159]. Moreover, torin1-mediated TFEB activation restores autophagy flux and ameliorates bleomycin-induced pulmonary fibrosis [159]. Autophagy dysfunction is also reported in human lung fibroblasts from IPF patients, and it is believed that defective autophagy is required to maintain a cell death-resistant phenotype in IPF fibroblasts, suggesting that autophagy dysfunction is a profibrotic mechanism and promotes IPF pathogenesis [64]. Hence, in age-related IPF pathogenesis, autophagy declines with age and the resulting imbalance of inflammatory-oxidative responses is anticipated to mediate the initiation of fibrotic pathophysiology. However, further clinical evaluation is needed to evaluate the therapeutic potential of autophagy augmentation in IPF patients.