The pathogenic mechanisms of IPF are uncertain; however, a currently favored concept is that in a genetically susceptible individual, recurrent environmental factors cause injuries, resulting in increased cell death, aberrant epithelial repair, and dysregulated epithelial and mesenchymal cellular networking, and promoting the activation of residential mesenchymal cells and extracellular matrix (ECM) accumulation [5]. In the lung, the alveolar epithelium lining is maintained by alveolar type I (AT1) and AT2 cells, which are essential for alveolar gas exchange and form a tight barrier to the passive leakage of fluid from the interstitium and vascular compartments into the alveolar space [6]. AT2 cells serve as the progenitor cells for alveolar regeneration during tissue repair and homeostasis, and they differentiate into AT1 cells in pathologic states including DNA repair and senescence [7,8]. Recently, Kobayashi et al. [9] have reported that AT2 cells undergoing differentiation into AT1 cells acquire pre-AT1 transitional cell states via TP53, transforming growth factor (TGF)-β, and DNA damage response signaling after alveolar injury. In human lungs with IPF, a single-cell RNA sequencing analysis revealed high enrichment of SFN, CLDN4, and KRT17 in a distinct cell cluster in pre-AT1 transitional cell states. This finding suggests that pre-AT1 transitional cells in the AT2 and AT1 transition process experience and respond to DNA damage, which is associated with degenerative lung diseases. AT2 cells show high expression of SFTPC and SFTP, which are capable of modulating the biophysical activity of pulmonary surfactant. Recently, mutation or deficiency of SFTPC, SFTPB, and ABCA3 in AT2 cells was shown to cause neonatal respiratory stress or early-onset interstitial lung disease [10]. In familial interstitial pneumonia, the expression of SFTPC with the L188Q mutation was found to alter the phenotype of AT2 cells through upregulation of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), suggesting that UPR activation contributes to fibrotic remodeling in IPF [11]. In I^ER-SP-C^I73T mice, mutation of SPC triggered a rapid increase in mortality, and induced polycellular alveolitis leading to spontaneous fibrotic lung remodeling [12]. Moreover, SP-C^I73T expression induced focal AT2 cell hyperplasia, exhibiting a persistent 40% increase in HA⁺/Ki-67⁺ cells, which may be an upstream driver of early lung injury. These findings support that certain components of the inflammatory response in the epithelium necessarily appear prior to fibrogenesis, and that SFTPC mutants recapitulate pivotal features of the human IPF phenotype. Indeed, patients with IPF frequently exhibit the atypical metaplastic epithelia in the fibrotic lung tissue, and showed increased p53 protein levels and DNA ploidy in the honeycomb region of IPF [13]. Recently, cigarette smoke-mediated AT2 cellular senescence was shown to reduce autophagy and to cause DNA damage-induced PARP1 activation, leading to pulmonary fibrosis [14]. In a comparison between control donor proximal and distal lung tissues and IPF fibrotic lung tissues, CDKN1A/p21, CDKN2A/p16, TP53, MDM2, and CCDN1 were significantly increased in AT2 cell transcriptomes, and the conditional loss of Sin3a was related to silencing of p53 expression, promoting AT2 cell dysfunction and driving progressive fibrosis [15]. Therefore, AT2 cell senescence plays a key role in declining pulmonary function and the pathological features of IPF.

The lung is a representative organ responsible for pathogen clearance and the reinstatement of tissue homeostasis [16,17]. Macrophages are major cellular components that sense pathogens and mediate phagocytosis during all phases of pulmonary fibrosis. Macrophage dysfunction leads to a failure to clear apoptotic cells, resulting in an accumulation of autoantigen and increased sustained inflammation [18]. When pulmonary macrophages were depleted by liposomal clodronate in bleomycin-induced mice, macrophages had no effect during the inflammatory phase [19]. However, depletion of macrophages reduced the surrogate markers of fibrosis α-smooth muscle actin (α-SMA) and Col1A1, indicating that macrophages play a resolution-promoting role during the reversible phase of fibrosis. Gao et al. [20] have shown that netrin-1 derived from macrophages, but not fibroblasts, induces collagen accumulation in ColIα2-CreER mice, and therapeutic α1-adrenoreceptor antagonism suppressed fibrosis. In addition, IPF macrophages showed enriched netrin-1 expression and 22 patients using α1-blockers exhibited significantly better lung function in terms of forced vital capacity and diffusing capacity for carbon monoxide. In Fra-2^Tg mice, alternatively activated macrophages were dominant in cells of the macrophage linage expressing colony-stimulating factor 1 receptor and SPC-positive AT2 cells. They exhibited prefibrotic genes including periostin, α-SMA, and fibroblast-specific protein 1 (S100a4); however, Fra-2^△Ad mice showed limited Fra-2 expression in AT2 cells and milder lung fibrosis progression [21].

In recent studies, nintedanib and pirfenidone have been pursued to treat mild to moderate IPF. However, the major treatments are limited due to side effects; moreover, the mechanisms of these drugs mainly focus on anti-fibrotic effects. IPF is a progressive and sustained fibrotic lung disease that primarily affects older adults [22]. Aged lungs undergo a degenerative process through the accumulation of extrinsic and intrinsic damage, which results in altered cellular homeostasis and apoptosis. Moreover, aging causes the decline of mitochondrial function and the mitochondrial respiratory chain, while increasing reactive oxidative stress (ROS) production, which is associated with mitochondrial DNA damage [23]. Damaged mitochondria with PINK1 deficiency and insufficient mitophagy accelerated the fibrotic progression of AT2 cells [24]. In alveolar macrophages, mitochondrial calcium uniporter, which triggers a Ca2⁺ influx, caused profibrotic polarization and augmented mitochondrial ROS production [25]. In patients with IPF, oxidative phosphorylation is decreased in primary alveolar macrophages, while the production of mitochondrial ROS is increased [26]. Although LC3B (an autophagy marker) and the selective autophagy receptor p62 were not changed in IPF alveolar macrophages, an imbalance in mitochondria biogenesis and degradation is involved in the pathogenesis of IPF.

Recent studies have found that age-related changes are associated with the differential severity of pneumonia in older patients. In the alveolar macrophage transcriptome, advanced age was associated with a reduced expression of cell-cycle related genes and the aged microenvironment of macrophages conferred resistance to the granulocyte macrophage colony-stimulating factor axis [27]. When tissue-resident alveolar macrophages from old and young adult mice were analyzed, aging-associated changes in the transcriptome of macrophages were entirely attributable to the alveolar environment, indicating that the cellular interactions of macrophages and AT2 cells are essential to restore youthful responses in the aged lung.

Cigarette smoking, which is known as a major cause of COPD, results in a high level of ROS, which activate airway/alveolar inflammation and contribute to disease development [32]. Prolonged high levels of ROS can exacerbate COPD pathogenesis. Indeed, ROS signaling is intimately associated with cell damage, including cell death, apoptosis, aging, and mutations [33]. Cigarette smoke-induced oxidative stress regulates redox-sensitive transcription factors, such as mitogen-activated protein kinase (MAPK) family, nuclear factor kappa-B (NF-κB), and signal transducer and activator of transcription 1 (STAT1) [34]. These factors stimulate them, leading to the further recruitment of inflammatory cells in the alveoli. In COPD patients, excessive numbers of inflammatory cells, especially macrophages, are observed in the bronchoalveolar lavage fluid (BALF) and airways. They produce pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, tumor necrosis factor-alpha (TNF-α), and monocyte chemoattractant protein-1.

In addition, IL-1β increases the expression of neutrophilic cytokines and matrix metalloproteinases (MMPs), which are proteolytic enzymes that degrade the matrix components [35,36]. The pathogenesis of COPD is related to imbalanced protease/antiprotease levels, which are essential for maintaining the elastin framework [34]. It has been suggested that the destruction of connective tissue by various proteases contributes to emphysema. In normal tissue, MMPs are tightly regulated by tissue inhibitor of metallopeptidase, which maintains a well-balanced state [37]. It has been suggested that disruption of the balance between apoptosis and replenishment of structural cells in alveoli contributes to the destruction of lung tissue in response to injury [38].

As discussed above, pulmonary emphysema is characterized by epithelial cell death. In emphysema, destruction of epithelial cells caused by apoptosis leads to airspace enlargement, in which gas exchange fails, resulting in dyspnea [39]. Alveolar epithelial apoptosis is mediated by several mechanisms, including disruption of the balance between proteolytic and anti-proteolytic molecules, inflammation, oxidative stress, inhibition of vascular endothelial growth factor (VEGF) receptor, regulation of autophagy, and cell cycle arrest [38,40-42]. A VEGF-receptor 2 inhibitor, SU5416, led to a progressive increase in caspase-3 activity in rat lungs [40]. Caspase-3, a cascade protein, is considered to be a hallmark of apoptosis [43]. Furthermore, autophagy, a homeostatic process of intracellular degradation system, has been reported to be deeply involved in apoptosis signaling [44]. According to other research, autophagic cell death is caused by ROS resulting from autophagic degradation by catalase [45]. In general, autophagy is considered to play a protective role in normal tissue; however, prolonged and excessive autophagy has also been associated with autophagic cell death [46]. It has been reported that autophagy plays a functional role in stress responses to cigarette smoke exposure in vitro [42]. Both in vitro and in vivo, cigarette smoke-induced autophagy in alveolar epithelial cells. Qin et al. [47] showed that nuclear receptor 77 mediated CS-induced autophagy, which increased apoptosis in A549 cells and mouse.

Wei et al. [48] presented the gene-expression profile in COPD. They performed a microarray analysis using COPD patient samples to investigate the gene-expression patterns of COPD and to explore possible therapeutic targets. As described in the paper, the upregulated genes were involved in the immune response, antigen processing and presentation, positive regulation of immune response, and negative regulation of caspase activity, while the downregulated genes were significantly related to the functions of response to drugs, regulation of transcription, and especially steroid metabolic processes. In particular, HLA-A expression was significantly increased in COPD; therefore, they suggested that HLA-A may serve as a diagnostic marker for COPD. In our previous study, we found that the receptor for advanced glycan end products (RAGE) pathway was related with COPD pathology [49,50]. Then, we showed that a RAGE antagonist, FPS-ZM1, can be therapeutic agent via blockade of RAGE-damage-associated molecular patterns (DAMP) signaling.

In animal disease models, several studies have used porcine pancreatic elastase (PPE) as a COPD inducer. PPE is a protease that can cause a protease-antiprotease imbalance and has been applied to induce emphysema and COPD in animals [51]. Although PPE successfully induced COPD-like pathology in mice, as shown by the inflammatory response, loss of epithelial cells, ROS levels, and COPD-related gene expression, it has limitations for mimicking human COPD. Recently, β₂-agonists, long-acting muscarinic antagonists, and inhaled corticosteroids have been developed as drugs for COPD [52]. The above-mentioned drugs are mostly bronchodilators; thus, although attenuate symptoms of COPD such as phlegm, cough, and dyspnea, disease progression is not affected. Additionally, a selective phosphodiesterase 4 inhibitor, roflumilast, was also approached as COPD drug with an anti-inflammatory effect [53]. Although many studies of COPD cures have been tried, no therapy has successfully repaired the damaged alveoli or airway. Three-dimensional (3D) organoid systems are expected to be used in the fields of disease modeling, drug screening, and pre-clinical research for human COPD in vitro.

Recent studies have shown that dysregulated AT2 cells are highly involved in fibrotic progression [55]. It has been demonstrated that the stimulated AT2 cells have an impaired function of differentiation into healthy and mature AT1 cells, and that alveolar remodeling is initiated via a sub-fibrotic cell state from AT2 cells as a repair process leading to fibrosis [9,56]. Indeed, direct conversion into myofibroblasts, known as the epithelial-mesenchymal transition (EMT), is also considered a key IPF characteristic. During EMT, AT2 cells lose their typical cell functions, including differentiation, proliferation, migration, and cell-cell junction [57]. Previously, we reported that a mediator of IPF in vivo, bleomycin, induced apoptosis in AT2 cells with increased levels of Schlafen 1 (SLFN1), SLFN2, and SLFN4, which are involved in cell cycle arrest and apoptosis [58]. Wu et al. [55] demonstrated that impaired regeneration of AT2 after damage resulted in sustained mechanical tension. This pathological stimulation activated TGF-β signaling, a well-known factor related to the EMT, leading to an accumulation of myofibroblasts. At an early state of IPF progression, inflammation has been observed in animal models. Recent studies have shown that many inflammatory cells, such as macrophages, neutrophils and lymphocytes, contribute to the pathogenesis of IPF [59]. As inflammatory responses are mediated by various routes, AT2 cells repair damaged tissue via remodeling, which contributes to physiological changes, including accumulation of ECM, contraction of fibroblasts, tissue tension, and stiffness [60,61]. Sun et al. [61] showed that tankyrase inhibition could impair AT2-AT1 differentiation via TAZ inhibition using an AT2 organoid system. They found decreased RAGE expression, furnishing evidence of a chronically inadequate capacity to regenerate AT2 in IPF in a system that could even be representative of human patients. Although AT2 cells play a critical role in lung repair, direct molecular targets remain to be elucidated.

In distal region of lung tissue, the alveolar epithelium (where gas exchange typically occurs) protects against the external environment, including PM, viral or bacterial infections, and even harmful factors from cigarette smoke. Chronic respiratory injuries result in a loss of epithelial cells, leading to shortness of breath. This pathological condition is known as emphysema, which is a component of COPD [62]. Cigarette smoking, a major risk factor for COPD, causes high levels of free radicals, which increase oxidative stress in AT2 cells [50]. ROS are believed to be a mediator in the development of COPD, through their involvement in apoptosis, aging, mitochondrial damage, and inflammation in AT2 cells [63].

Previously, we established a two-dimensional (2D) in vitro COPD model involving cigarette smoke extract (CSE). CSE exposure induced inflammation and ROS production. Interestingly, we identified high expression of RAGE via DAMP-Nrf signaling in COPD-like damaged AT2 cells. In both in vivo and in vitro studies, a RAGE antagonist (FPS-ZM1) showed an efficient curative effect via blocking RAGE-mediated Nrf2/DAMP signaling in damaged AT2 cells [49,50]. Likewise, it is important to find a novel disease-associated target that might allow a curative approach in vitro. Since 3D organoids can recapitulate the cellular diversity of complex lung tissues, unlike 2D systems, many researchers have expected that 3D lung organoid systems will serve as a useful modeling tool for pathologic or therapeutic studies in the field of pulmonary diseases. Nevertheless, it is necessary to develop models with a greater similarity to the human lung environment to recapitulate disease conditions or homeostasis against damage in vitro.

As discussed above, IPF occurs when repetitive alveolar injuries induce the activation of mesenchymal cells and fibroblasts and the differentiation of alveolar epithelial cells into myofibroblasts. Repeated alveolar epithelial injuries cause an aberrant wound healing process leading to fibrotic plaque formation and excessive accumulation of ECM. In this process, macrophages acquire a phenotype that promotes fibro-proliferation in injured site. In particular, alveolar macrophages actively participate in ECM remodeling via increased MMPs of neutrophils and accumulation of collagen. Moreover, the activated alveolar macrophages release ROS and TGF-β. In the pathogenesis of IPF, sustained inflammation triggers the initiation of fibrotic responses with increased numbers of Ly6C^hi monocytes. In macrophages of interferon regulatory factor 5 (IRF-5) deficient mice, the inhibition of IRF-5 associated with TGF-β led to fibrotic responses in adipose tissue, indicating the dual role of M1 macrophages in wound repair and fibrotic responses [69]. In general, infiltration of M2 macrophages is recognized as a critical regulator of fibrogenesis. In mice treated with bleomycin, loss of Chop significantly attenuated TGF-β production, decreased the number of M2 macrophages, and reduced ER stress [70]. Recently, Cui et al. [71] reported that apolipoprotein E, produced primarily by monocyte-derived alveolar macrophages, promoted type I collagen phagocytosis and interacted with low-density lipoprotein receptor-related protein 1 leading to the resolution of lung fibrosis in IPF mice. They demonstrated that monocyte-derived alveolar macrophages have distinct functions in the early fibrogenic and fibrotic resolution phases. In contrast, interstitial macrophages are rapidly positioned in the interstitium to influence lung fibrotic processes, rather than alveolar macrophages. In bleomycin- and radiation-induced mice, CD206-expressing interstitial macrophages acquired a profibrotic phenotype and mediated the fibrogenic process, accelerating the myofibroblast differentiation. Interstitial macrophages isolated from tissues with radiation-induced lung fibrosis showed highly increased levels of arginase-1, and inhibition of colony-stimulating factor receptor-1 was significantly associated with a decrease of radiation-induced lung fibrosis [72]. Thus, alveolar and interstitial macrophages in the lungs show contrasting and distinctive phenotypes, and they play separate roles depending on the phase of IPF.

It is widely accepted that alveolar macrophages play a critical role in the pathogenesis of COPD. In patients with COPD, markedly increased numbers of alveolar macrophages were found in airways, lung parenchyma, BALF, and septum. The patients with COPD have high number of macrophages. In COPD mice, alveolar macrophages released elastolytic enzymes, proteases, MMP-2, MMP-9, and cathepsin K, leading to the destruction of alveoli [73]. Impairment of alveolar macrophages leads to an increased susceptibility to bacterial colonization and exacerbation by respiratory viruses or bacteria. M1/M2 macrophage polarization has been reported to determine the impact of COPD progression. A high ratio of nitric oxide-expressing M1 macrophages was observed in COPD airways, while arginase-expressing M2 macrophages were present in COPD mice. Hence, a better understanding is needed of the M1/M2 macrophage polarization state and the roles of macrophages in the pathogenesis of COPD. In COPD, the functions of interstitial macrophages are veiled compared with alveolar macrophages.