Senescence of alveolar epithelial progenitor cells: a critical driver of lung fibrosis

Abstract

Pulmonary fibrosis comprises a range of chronic interstitial lung diseases (ILDs) that impose a significant burden on patients and public health. Among these, idiopathic pulmonary fibrosis (IPF), a disease of aging, is the most common and most severe form of ILD and is treated largely by lung transplantation. The lack of effective treatments to stop or reverse lung fibrosis—in fact, fibrosis in most organs—has sparked the need to understand causative mechanisms with the goal of identifying critical points for potential therapeutic intervention. Findings from many groups have indicated that repeated injury to the alveolar epithelium—where gas exchange occurs—leads to stem cell exhaustion and impaired alveolar repair that, in turn, triggers the onset and progression of fibrosis. Cellular senescence of alveolar epithelial progenitors is a critical cause of stemness failure. Hence, senescence impairs repair and thus contributes significantly to fibrosis. In this review, we discuss recent evidence indicating that senescence of epithelial progenitor cells impairs alveolar homeostasis and repair creating a profibrotic environment. Moreover, we discuss the impact of senescent alveolar epithelial progenitors, alveolar type 2 (AT2) cells, and AT2-derived transitional epithelial cells in fibrosis. Emerging evidence indicates that transitional epithelial cells are prone to senescence and, hence, are a new player involved in senescence-associated lung fibrosis. Understanding the complex interplay of cell types and cellular regulatory factors contributing to alveolar epithelial progenitor senescence will be crucial to developing targeted therapies to mitigate their downstream profibrotic sequelae and to promote normal alveolar repair.

NEW & NOTEWORTHY With an aging population, lung fibrotic diseases are becoming a global health burden. Dysfunctional repair of the alveolar epithelium is a key causative process that initiates lung fibrosis. Normal alveolar regeneration relies on functional progenitor cells; however, the senescence of these cells, which increases with age, hinders their ability to contribute to repair. Here, we discuss studies on the control and consequence of progenitor cell senescence in fibrosis and opportunities for research.

INTRODUCTION

Interstitial lung diseases (ILDs) are a group of respiratory conditions characterized by an excessive deposition of extracellular matrix (ECM), mostly fibrillar collagens (type I, III, others), within interstitial spaces. As for most organs, fibrosis within the lung impairs tissue function, which involves gas exchange within the alveolar compartment (respiratory unit). Among ILDs, idiopathic pulmonary fibrosis (IPF) is the most common. IPF is a devastating, progressive chronic respiratory disease of aging with poor clinical outcomes (1, 2) and is increasing in incidence throughout the world (1, 3). Recently, lung fibrosis associated with postacute SARS-CoV-2 infection (PASC) has gained substantial attention (4). Although the long-term clinical outcomes of PASC lung fibrosis are still being uncovered, this condition shares some key pathological features with IPF (5–8). Hence, understanding the fundamental mechanisms of fibrosis will aid in the identification of processes that can be targeted therapeutically to improve outcomes in this spectrum of lung diseases.

In lung and other tissues, such as liver and kidney, repeated epithelial injury, along with aging, genetic, and environmental factors, contributes to defective repair creating a profibrotic environment. Through mechanisms that are still being uncovered, persistent and impaired epithelial repair stimulates the differentiation of interstitial fibroblasts (and possibly other resident cells) into myofibroblasts that produce and deposit an excess of ECM, which is the hallmark of any form of fibrosis. Thus, a defect in the regenerative activity of epithelial progenitor cells likely contributes to the onset of fibrosis.

Epithelial progenitor cells maintain alveolar homeostasis through their capacity for self-renewal and repopulation of alveolar type I (AT1) cells (9, 10). Due to their thin squamous morphology and intimate association with the alveolar capillaries, AT1 cells are specialized for efficient gas exchange. AT1 cells are derived from alveolar type 2 (AT2) cells via an intermediate population (see Transitional Cells and Fig. 1). AT2 cells are abundant, large cuboidal cells that produce pulmonary surfactants and are the critical progenitor population within the alveolus. Failure or exhaustion of their ability to self-renew and to generate AT1 cells leads to impaired alveolar regeneration and repair, defects that are critical to the onset of lung fibrosis (11, 12).

Figure 1.

Alveolar progenitor cells. Various epithelial cells in the distal airways of lungs can generate alveolar type 2 (AT2) cells. These bipotent progenitor cells include club cells, ciliated cells, tuft cells, pulmonary neuroendocrine cells (PNECs), basal cells, and respiratory airway secretory cell (RAS)/alveolar type 0 (AT0) cells. Except for basal cells, which are found in human and not mouse airways, these distal airway progenitor epithelial cells are found in human and mouse lungs, and some can self-renew. AT2 cells in the alveolar compartment can self-renew and differentiate into AT1 cells via a transitional cell type. (Created with BioRender.com with permission).

Senescence is a common feature of progenitor stem cell exhaustion and is characterized by the permanent loss of a cell’s proliferative capability. Cellular senescence is linked to aging, and age is the main risk factor for developing IPF (13). Supporting a functional link between senescence and fibrosis, senescent alveolar progenitor epithelial cells are abundant in both PASC and IPF lungs (14–16). In this review, we summarize the evidence supporting the idea that the senescence of alveolar epithelial progenitor cells contributes to lung fibrosis.

The alveolar environment comprises epithelial cells, endothelial cells, fibroblasts, and immune cells, as well as the fused basement membranes of the epithelium and endothelium and an elastin-rich interstitium (17, 18). Maintaining alveolar homeostasis requires communication among these cell types through cell-cell and paracrine signaling pathways, as well as through biophysical forces via ligation with the ECM (19–21).

As we discuss here, emerging data indicate that the senescence of lung progenitor epithelial cells can initiate lung fibrosis and promote its progression (10, 22, 23). Senescent cells mediate disease pathogenesis by two broad mechanisms: 1) a failure to regenerate alveolar epithelial cells and restore alveolar structure and function creates a profibrotic environment and 2) the paracrine effects of senescence-associated secretory phenotype (SASP) cause an accumulation of senescent cells in the lung microenvironment that furthers impaired repair processes and promotes fibrosis. Interestingly, and as we discuss Senescence of AT2, whereas senescence of alveolar epithelial cells is detrimental, some studies have reported that senescence of nonepithelial cells, such as fibroblasts, appears to be beneficial for effective tissue regeneration (24, 25). The cell type-specific outcomes of senescence on fibrosis create a clear challenge to targeting this process as a therapeutic means to mitigate disease. However, this challenge also provides the opportunity to uncover pro- and antifibrotic mechanisms of distinct senescent cell types, knowledge that could be used to design effective approaches to lessen or reverse fibrosis.

ALVEOLAR EPITHELIAL PROGENITOR CELLS

In recent years, much progress has been made in the identification of lung epithelial stem and progenitor cells (26, 27). Alveolar progenitor cells can be divided into two groups: 1) AT2 cells in the alveolar niche that are capable of self-renewing and differentiating into AT1 cells and 2) bipotent progenitor cells in distal airways that can generate AT2 cells (Fig. 1). In addition, as they differentiate into AT1 cells, AT2 cells assume a transient “prealveolar type-1 transitional” cell state or PATS. As we discuss, compelling evidence from a variety of mouse models and human specimens has linked senescence of AT2 cells to lung fibrosis and IPF (28, 29). In contrast, evidence has not yet emerged to indicate that senescence of the distal airway progenitor cells contributes to fibrosis. Hence, in this article, we focus on the senescence of AT2 cells.

AT2 Cells

In healthy, mature lungs, AT2 serves critical roles necessary for alveolar function and stability. They produce and recycle surfactants and can self-renew and differentiate into AT1 cells (21, 30, 31). A subset of Wnt-responsive AT2 cells marked by Axin2 has been claimed to serve as major facultative progenitor cells in the mouse distal lung (32, 33). In normal human and mouse lungs, AT2 cells are the primary source of AT1 cells (9, 34). Hence, AT2 cells are critical both to maintaining alveolar structure and function during homeostasis and to the regeneration of damaged alveoli following injury and infection. Most studies linking alveolar progenitor cell senescence to fibrosis have focused on AT2 cells.

Transitional Cells

Recent transcriptomic studies have defined epithelial cells with a molecular phenotype suggestive of a transition state between AT2 and AT1 cells, referred to as “transitional cells,” “damage-associated transitional progenitor,” (DATP) or “prealveolar type-1 transitional cells” (PATS) (29, 35–41). In these studies, transitional cells have been defined as either Krt8⁺ (29), Trp53⁺ Krt17⁺ (35), damage-associated transient progenitors (Krt8⁺, Cldn4⁺, and Cdkn1a⁺) (41), double Krt7⁺ and Krt8⁺ cells (42), or transitional Krt5⁻ and Krt17⁺ cells (37). An accumulation of these transitional cells following infection and injury suggests a connection between senescence and abnormal AT2 differentiation (43), leading to impaired lung repair and fibrosis (35). However, more evidence is required to support this notion.

Distal Airway Progenitor Cells

Epithelial progenitors in terminal respiratory bronchioles play essential roles in alveolar repair and regeneration. In lung repair, the expansion of keratin 5-positive (KRT5⁺) cells in severe H1N1 influenza virus and likely COVID-19 infection is a crucial example such that the origin of these KRT5⁺ cells has been traced to distal airway progenitor cells by multiple groups: rare Sox2+ airway progenitor cells (44), Trp63⁺ and Krt5⁺ stem cells (44, 45), embryonic p63⁺ progenitor (46) and Trp63⁺ cells (47, 48), club cell variant (Scgb1a1⁺, Upk3a⁺) (49, 50), lineage-negative epithelial progenitors (LNEP^TRP63−, Itgb4⁺) (51), and H2-K1^high (Itgb4⁺, H2-K1⁺) (52). Specific distal progenitors may be involved in alveolar regeneration: SCGB3A2⁺ cells in the human distal airway [also called respiratory airway secretory (RAS) (50), AT0 cells (53)] can give rise to AT2 cells in vitro three-dimensional (3-D) organoid culture and the Sftpc and Scgb1a1 double-positive cells in mouse bronchioalveolar region [bronchioalveolar stem cells, BASC cells (54)] that can differentiate into AT2 and subsequently into AT1 cells. The dual lineage tracing approach of Sftpc-DreER and Scgb1a1-CreER demonstrated that BASCs become activated and exhibit distinct responses to the specific types of lung injury. Notably, BASCs can differentiate into club cells and ciliated cells in naphthalene exposure and AT2 and AT1 cells in bleomycin injury (55). With additional human studies to validate their roles, the distal airway progenitors play critical functions in maintaining terminal bronchiole and alveolar homeostasis following various insults. The decline of progenitor proliferation during aging indirectly implies the involvement of senescence (56). We also demonstrate the role of p53 in regulating distal airway progenitor cell fates (57).

SENESCENCE AND SASP

Senescence

Cellular senescence increases with age and represents a state of irreversible growth arrest (58, 59). Senescence is characterized by a combination of metabolic changes, acquisition of a distinctive secretome that is commonly referred to as the senescence-associated secretory phenotype (SASP), elevated β-galactosidase activity, oxidative stress, resistance to apoptosis, and morphological changes, such as an enlarged and flattened cell shape, multinucleation, and vacuolization (58, 60). Canonical features of senescent cells include the upregulation of key cell cycle inhibitory proteins, such as p16 and/or p21, cell cycle arrest, and evidence of DNA damage by phosphorylation of H2A histone family member X (γH2AX). A challenge in studying senescence is that the compilation of protein and mRNA markers used to identify senescent cells differs among cell types and is not individually specific to senescent cells (60, 61). Thus, to assure rigor in defining senescent cell types, a range of endpoints must be used.

SASP

The secretome of senescent cells (62) includes proinflammatory cytokines, chemokines, growth factors, proteinases and inhibitors, reactive oxygen species, and other macromolecules (Table 1). Although no individual SASP component can be considered a selective marker of senescence, the combined release of multiple SASP components coupled with the presence of other cell-intrinsic senescence markers/characteristics, especially expression of p16 and/or p21, β-galactosidase activity, and permanent growth arrest, would be confirmatory of a senescent state.

Table 1.

Senescence-associated secretory phenotypes components involved in AT2 cell senescence and lung fibrosis

Classification	Factors	References
Inflammatory factors	IL-1α, IL-1β	(63)
	IL-4	(64)
	IL-6, IL-11	(65, 66, 67–69)
	IL-8	(63)
	IL-13	(70, 71, 72)
	TNFα	(66, 68, 69)
Chemokines	CCL2, CCL5, CCL17	(73, 74)
Cytokines	TGFβ1	(65, 70, 75, 68)
	PDGF	(66, 70)
	IGFBP3, IGFBP4	(76)
Proteinases and inhibitors	MMP-2, MMP-9	(77, 69)
	MMP-9	(63, 69)
	MMP-12	(76)
	PAI-1	(78, 79, 70)
Nonprotein	mitoROS	(80)

AT2, alveolar type 2; CCL, chemokine (CC motif) ligand; IGFBP, insulin-like growth factor binding protein 2; mitoROS, mitochondrial reactive oxygen species; MMP, matrix metalloproteinase; PAI-1, plasma activator inhibitor-1; PDGF, platelet-derived growth factor; TGFβ1, transforming growth factor β1; TNF_a, tumor necrotic factor.

SENESCENCE OF AT2 CELLS IN FIBROSIS

Senescence is observed in various cell types throughout fibrotic lungs, and these different cells can independently or collaboratively promote lung fibrosis. Stemness failure associated with alveolar epithelial progenitor senescence not only inhibits proper alveolar repair and regeneration but can also induce the senescence of healthy progenitors and nonprogenitor in the alveolar niches through the paracrine effects of SASP further deteriorating the fibroproliferative processes (15, 21, 26, 75, 81–84). The loss of alveolar epithelial progenitor cells stemness (i.e., exhaustion) through senescence contributes significantly to the progression of lung fibrosis pathogenesis (75, 85–89). Although the mechanisms are not yet fully understood, impaired alveolar epithelial repair and/or sustained alveolar injury activates pathways, such as TGFβ (75) and Wnt signaling, (90) that promote the differentiation and proliferation of fibroblasts into invasive myofibroblasts, which deposit excessive ECM, and facilitate immune cell dysregulation that can further impair alveolar repair (15, 22, 75, 81, 87, 91).

Numerous external stimuli and internal cellular stress contribute to both age-related and premature senescence of AT2 cells. The accumulation of AT2 senescence in IPF and PASC lung fibrosis, as demonstrated by transcriptomic and immune-staining analyses (16, 65, 75, 80, 92)—as discussed in senescence regulatory pathways—provides compelling evidence that senescence of AT2 cells is a critical effector in these progressive conditions. Indeed, transcriptomic quantitation of lung epithelial senescence by a computational scoring approach for top common senescence genes (http://csgene.bioinfo-minzhao.org) highlights the potential significance of this process in AT2 cells in disease pathogenesis (75, 93). In our work, the spontaneous development of lung fibrosis in mice due to AT2 senescence following the deletion of Sin3a in AT2 cells implicates further the pathogenic role of senescence of AT2 cells in lung fibrosis (75).

The plasticity of AT2 cells in maintaining alveolar homeostasis is partially explained by the presence and accumulation of transitional cells following lung injury in mice and in the progression of IPF in humans (28, 29, 41, 93–95) and COVID-19 lung fibrosis (96). The upregulation of senescent signature genes in PATS and their accumulation in fibrotic regions suggests they have roles in disease pathogenesis. For example, Krt5⁺ transitional cells in IPF express p16 suggesting they are a senescent population in the diseased lung (95). Although the role of senescence in hindering the differentiation of transitional cells into AT1 cells is limited (35–37), transitional cells do show upregulation of canonical senescence markers, indicating some contribution to cellular senescence (27). Both our and public scRNAseq profiling data (28, 29, 35) demonstrate that transitional cells in both control and IPF lungs display a higher senescence score than that of AT2 cells in the same specimens (Fig. 2). Although the impact of senescence on the cellular function of transitional cells remains to be determined, it is reasonable to propose that senescence of these cells hinders their ability to differentiate into AT1 cells, thereby impairing alveolar repair and promoting fibrosis. Indeed, upregulation of senescence markers (p16, CCND1, and GDF15) on transitional cells in IPF and PASC lung fibrosis implicate that senescence of alveolar progenitor differentiation processes is involved in that pathogenesis of these conditions (43, 94, 97).

Figure 2.

Transcriptomic senescence score of alveolar epithelial progenitor cells in idiopathic pulmonary fibrosis (IPF). A: uniform manifold approximation and projection (UMAP) visualization of alveolar progenitor epithelial cells (AT2 and transitional cells). B: average expression of alveolar type 2 (AT2) and transitional cell common markers and transcriptomic senescence score of each cell type in control and IPF. Data are described in two metrics: gene expression and the percentage of expressing cells.

The contribution of SASP from senescent cells to lung fibrosis is established (23, 88). The proinflammatory factors in SASP (Table 1), as discussed in this and impact of at2 cell senescence of other cell types in fibrosis, can activate profibrotic signaling, either directly or indirectly. In addition, SASP can promote the senescence of nearby cells by the paracrine effects of several of the factors released. Thus, in a disease setting like IPF, SASP could function in a deleterious feed-forward manner that promotes senescence, thereby leading to impaired progenitor cell function and interstitial fibrosis.

The regulation of SASP is intertwined with other senescence regulatory pathways (98). Alveolar epithelial progenitors can produce SASP in a senescent microenvironment, thereby augmenting the impact of senescence in hindering proper alveolar repair. Indeed, in the bleomycin model, senescent p21-positive AT2 cells contribute to fibrosis through their SASP-secreted effector proteins (66). Furthermore, the “cytokine storm” concept as a causative event in COVID-19 lung fibrosis makes SASPs relevant as stimulators and effectors of alveolar progenitor senescence (7, 99). Indeed, widespread AT2 senescence and upregulation of SASP genes are observed in lung tissues of patients with fatal COVID-19 and COVID-19 lung fibrosis (100–102). In addition, transcriptomic data, validated with 3-D organoid cultures, provides compelling findings indicating that SASP secreted from senescent AT2 cells and other inflammatory cells are critical culprits in cellular failure in acute and chronic states associated with fibrosis (100–102).

SENESCENCE REGULATORY PATHWAYS

Pathways that regulate cellular senescence are complex and interconnected. Senescence is regulated by various transcriptional factors and cell cycle-associated genes (103). For example, animals with either AT2-specific loss of function (LOF) of Sin3-a (75), a transcriptional repressor, or global deficiency of B cell–specific Moloney murine leukemia virus insertion region 1 (Bmi-1) (65) developed spontaneous AT2 senescence and lung fibrosis. Furthermore, in the Sin-3a LOF model, administration of a “senolytic cocktail,” which stimulated apoptosis in senescent cells, thereby reducing their numbers, alleviated lung fibrosis, substantiating a causative role of AT2 senescence in promoting fibrosis (76). The “senolytic cocktail” used in this study was a combination of an antioxidant (quercetin) and a multikinase inhibitor (dasatinib). The most studied signaling mechanisms controlling senescence of alveolar progenitor cells are the TP53 and retinoblastoma protein (RB) pathways, which, broadly speaking, function to promote or limit senescence, respectively.

TP53 and p21

The transcription factor p53 (TP53) inhibits cell cycle progression in response to a range of stimuli, such as DNA damage, telomere dysfunction, cell-cell fusion, and SASP (104). As such, TP53 has effects on progenitor cell activity, such as promoting differentiation and hindering self-renewal (105), and via these functions, TP53 promotes senescence. An essential effector molecule of TP53-mediated growth arrest is p21/p21^WAF1/CIP1/CDKN1A (78, 79), which mediates AT2 senescence-induced lung fibrosis (103, 106). In our work, we found that conditional silencing of Sin3a in AT2 cells leads to spontaneous lung fibrosis by activating p53 and p21 that, in turn, promoted AT2 cell senescence (75). Recently, Kobayashi et al. (35) reported that TP53 signaling hinders the differentiation of transitional cells into AT1 cells.

RB and p16

In contrast to TP53, Rb protein (pRb) is a tumor suppressor whose normal activity regulates cell cycling (107). Unphosphorylated pRb binds E2F1, a transcriptional factor that promotes cell division. When bound to pRb, E2F1 is held in the cytosol leading to G1 arrest (108, 109). Phosphorylation of pRb by cyclin-dependent kinases, CDK1 and CDK4, allows dissociation of E2F1 that then enters the nucleus and drives the transition of cells from G1 to S phase (107). An important player in pRb signaling is p16/CDKN2A, which inhibits CDK1 and CDK4. Thus, by preventing phosphorylation of pRb, p16 promotes senescence by causing cell cycle arrest. Consistent with this idea, compared with wild-type mice, p16-null mice have greater AT2 cell expansion and renewal during adulthood and better alveolar regeneration in response to injury (25), phenotypes that imply reduced senescence. Similar findings were demonstrated in elastase-induced alveolar damage in emphysema (110). These observations suggest that the pRb/p16 pathway functions to moderate senescence and to promote AT2 cell self-renewal and differentiation into transitional cells and transitional cells into AT1 cells.

Sirtuin Pathways

Impaired autophagy and mitochondria dysfunction can lead to cellular senescence, and these processes are affected by the Sirtuin pathway. For example, SIRT1 activity is downregulated by cigarette smoke and is associated with impaired autophagy, greater mitochondria dysfunction, and senescence of AT2 cells than in room-air controls (80). Pharmacological stimulation of SIRT1 activity with SRT1720 or minimization of mitochondria dysfunction with mitoROS both reversed these disease responses (80). Similarly, stimulating Sirtuin 1 by H₂S inhalation reduces AT2 senescence (111). In addition, H₂S attenuates bleomycin-induced lung fibrosis in rats (112), although the mechanism of its protective effect is not known. Furthermore, extracellular vesicles released by lung fibroblast contain miR-23b-3p and miR-494-3p that can downregulate SIRT3 (113), leading to mitochondria dysfunction-induced AT2 senescence and enhanced lung fibrosis.

Recent work from our center uncovered a novel mechanism linking the Sirtuin pathway to AT2 cell senescence and fibrosis (114). They found that downregulation of the zinc transporter ZIP8/SLC39A8 (ZIP8) in both IPF lungs and lungs of old mice is associated with AT2 cell senescence and enhanced lung fibrosis and that these phenotypes are dependent on SIRT1 (114). Replenishment with exogenous zinc and SIRT1 activation promoted self-renewal and differentiation of AT2 cells from lung tissues of patients with IPF and old mice. Therapeutic strategies to restore zinc metabolism and appropriate SIRT1 signaling could improve the progenitor function of AT2 cells, thereby moderating ongoing fibrogenesis. Overall, several studies suggest that the Sirtuin pathway functions to mitigate cell senescence and, in turn, fibrosis.

Other Pathways

Caveolin-1, a membrane-associated scaffolding protein of caveolae, mediates cell cycle arrest in stromal cells through TP53/p21 (115). In AT2 cells, caveolin-1 mediates senescence by impairing autophagy via a p53/p21-dependent mechanism (116). Furthermore, Cav1^−/− mice have less senescent AT2 cells and reduced fibrosis in response to bleomycin injury compared with wild-type mice, which was linked to enhanced pRB/p16 levels (77). These findings indicate that caveolin-1 promotes fibrosis by being permissive for the onset of senescence.

Other signaling pathways that may regulate senescence include WNT/β-catenin, NF-κB, insulin-like growth factor (IGF), and other tyrosine kinases. Although Wnt signaling is important for maintaining AT2 stemness, chronic stimulation of WNT/β-catenin signaling through WNT3a promotes AT2 senescence in lung fibrosis (117). The loss of PTEN can activate IκB, IKK, and NF-κB (63) and Akt leading to AT2 senescence (81). Moreover, inhibiting NF-κB by gene silencing or drugs hinders the onset of senescence of AT2.

Studies on the IGF pathway have shown that different signaling components can have distinct effects on cellular senescence. For example, whereas IGF1/PI3K/AKT signaling promotes senescence of AT2 cells in IPF (118), IGF binding protein 2 (IGFBP2) hinders AT2 cell senescence in bleomycin-injured mice (119). The TGF-β1/IL-11/MEK/ERK (TIME) pathway has been reported to promote AT2 senescence and lung fibrosis (65). In this model, the TIME pathway was stimulated by deletion of the gene for BMI-1, a polycomb repressor complex 1 protein essential for stem cell self-renewal (65). Hence, this study supports the general concept that impaired progenitor cell function leads to senescence and creates a profibrotic environment.

PROCESSES THAT INITIATE SENESCENCE IN AT2 CELLS

Senescence is initiated by various processes, such as genetic and epigenetic dysregulation, stress- or oncogenic-induced, infection-associated diseases, etc. These factors contribute to alveolar progenitor senescence via both paracrine-affected and autocrine-affected means.

Telomere Dysfunction

In familial pulmonary fibrosis and IPF, shortened telomere length in AT2 cells arises due to genetic abnormalities and/or to age-related vulnerability of the DNA damage response (120, 121). In these and other lung fibrotic diseases, senescent AT2 cells are proximal to fibrotic lesions and, overall, are more abundant than other senescent cell types (120, 121). Mouse models of AT2 cell-conditional telomere dysfunction demonstrated that shortened telomere length leads to senescence of AT2 cells and spontaneous fibrosis (121–124). In contrast, telomere dysfunction in fibroblasts did not lead to lung fibrosis (121–124)—possibly by impairing the differentiation into myofibroblasts—and a recent preclinical study confirmed this conclusion (125). Furthermore, telomere uncapping associated with F-box and WD40 repeat domain-containing 7 binding to telomere protection protein 1 accelerates AT2 senescence and promotes lung fibrosis (126). Moreover, downregulating of telomerase reverse transcriptase (TERT) by overexpression of Krüpple-like-factors 4 reduces AT2 senescence and minimizes bleomycin-induced lung fibrosis (127). Together, these studies indicate that telomere pathology is a significant risk factor for AT2 senescence and fibrosis. To date, it remains unclear if telomere attrition contributes to the accumulation and/or senescence of PATS cells.

ER Stress

Some studies have indicated a causal link between ER stress and AT2 senescence leading to spontaneous lung fibrosis (128, 129). For example, activation of the unfolded protein response (UPR) in AT2 cells due to overexpression of mutant surfactant protein C increases ER stress and induces senescence markers (43, 87, 130). Stimulating ER stress in AT2 cells by reducing or loss of glucose-regulated protein 78 (GRP78), an ER stress chaperone protein, causes senescence and spontaneous lung fibrosis (131). A recent study demonstrated that activation of the UPR via IRE1-α signaling following bleomycin injury promotes AT2 cell senescence, an accumulation of PATS cells, and fibrosis (42). Blocking IRE1-α signaling reduced senescence and promoted the differentiation of PATS cells to AT1 cells resulting in less bleomycin-induced lung fibrosis (42). These data provide compelling evidence for a mechanistic association among ER stress, senescence of AT2 cells, and the onset of lung fibrosis. In addition, these findings suggest that senescence of AT2 cells does not hinder their ability to transition into PATS cells but does impair the ability to fully differentiate into AT1 cells. Additional research is needed to determine how AT2 cell senescence affects PATS cell function.

Autophagy and Mitochondria Dysfunction

Autophagy can both activate and inhibit cellular senescence—and, in turn, fibrosis—depending on the setting and on the presence and selectivity of autophagy regulatory molecules (132). For example, both inhalation of cigarette smoke and single-walled carbon nanotubes impairs autophagy and induces AT2 senescence and lung fibrosis (80, 133). Although these and other studies suggest that impaired autophagy promotes cellular senescence and fibrosis, the senescence-suppressor protein SIRT1 can be degraded through the autophagy-lysosome pathway indicating functional autophagy can limit senescence (134).

Mitochondria dysfunction is tightly correlated with cellular senescence (135). For example, dysfunctional and dysmorphic mitochondria, as evident by the accumulation of mitochondrial DNA (mtDNA), result from the downregulation of macroautophagy and are associated with AT2 senescence in IPF (136, 137). Using aged mice, Bueno et al. (137) demonstrated that PINK1 (PTEN-induced putative kinase 1) deficiency in AT2 cells induces mitochondria dysfunction and AT2 senescence and enhances susceptibility to virus-mediated fibrosis. As reported by Summer et al. (138), another characteristic of mitochondrial dysfunction in senescent cells is activation of the mTOR/PGC-1α/β pathway (mammalian target of rapamycin/peroxisome proliferator-activated receptor gamma, coactivator 1α/β), which promotes mitochondria synthesis, increased oxidative phosphorylation, and mtROS production, which can all further facilitate AT2 senescence. Of interest, Summer et al. (138) found that PINK1 activity was elevated in senescent cells—which contradicts the findings of Bueno et al. (137)—and independent mTOR activity. They found that although rapamycin-mediated inhibition of mTOR activity blocked senescence, it did not reduce elevated PINK1 levels (137). Thus, additional research is needed to determine if and how PINK1 functions in senescence. Furthermore, as seen in both IPF and bleomycin-injured mouse lungs, upregulation of CD38, a nicotinamide adenine dinucleotide (NAD) hydrolase, in AT2 cells leads to NAD deficiency and mitochondria dysfunction promoting AT2 senescence and fibrosis (139).

Metabolic reprogramming of alveolar epithelial progenitor cells through the autophagy-mitochondria axis contributes greatly to progenitor senescence-induced lung fibrosis. This notion is supported by studies showing that AT2 senescence is mitigated in vivo and in vitro by human mesenchymal stromal cells (MSCs) through nicotinamide phosphoribosyl transferase (NAMPT)-mediated NAD metabolism (140); specifically, MSCs upregulated NAMPT expression, increasing NAD⁺ level in AT2 cells. Thus, targeting metabolic pathways could prevent mitochondria dysfunction in and senescence of AT2 cells and, in turn, fibrosis.

IMPACT OF AT2 CELL SENESCENCE OF OTHER CELL TYPES IN FIBROSIS

Although eliminating senescent epithelial cells with senolytic agents or blocking senescence regulatory pathways alleviates lung fibrosis and improves survival in mouse models (15, 75, 76, 141–145), not all senescent cell types are profibrotic. In addition, not all senescent cells release SASP factors, and the onset of cellular senescence does not require SASP from other senescent cells (146). Another important confounding issue is that some senescent cell types and some SASP products—none of which are specific to senescent cells—can promote repair. For example, p16⁺ fibroblasts promote airway epithelial repair after naphthalene injuries via macrophage-derived IL-1β (24), and PDGF-AA secreted from senescent fibroblasts and endothelial cells promotes cutaneous wound healing (147), and other studies have concluded that senescent mesenchymal cells can contribute to tissue repair in injured lung, heart, and muscle (24, 148, 149). Along with the many studies demonstrating the profibrotic effects of senescent AT2 cells, as discussed in the preceding section, these studies indicate opposing consequences mediated by the senescence of different cell types in different tissue compartments. For example, senescent interstitial cells, like fibroblasts, could be beneficial in tissue regeneration, possibly by limiting their ability to be fibrotic, senescence of epithelial cells, particularly progenitor cells, would be detrimental by hindering reepithelialization and restoration of tissue function. Thus, understanding the unique mechanisms controlling senescent in specific cell types would be needed to design effective therapeutic approaches.

Senescent AT2 cells can recruit and activate immune cells, which can promote fibrosis and disease progression. Senescent AT2 cells in mice expressing a mutant surfactant protein C due to activation of UPR and ER stress and stimulate the recruitment of granulocytes (43). Similarly, deficiency of leucine-rich repeat kinase 2 in AT2 cells promotes bleomycin-induced senescence of these cells, which secrete CCL2 to recruit profibrotic macrophages (73). SASP components, such as IL-4 and IL-13, released by injury-induced senescent AT2 cells, can promote the transition of alveolar macrophages to a profibrotic phenotype (70, 150, 151). Findings from a preclinical model of SARS-CoV-2 pneumonia suggest that the virus caused senescence of AT2 cells that stimulated the recruitment of granulocytes and macrophages, which then caused lung injury (14). These findings indicate that the interplay between senescent AT2 cells and immune cells, mainly through SASP components, plays a crucial role in developing and progressing pulmonary fibrosis.

Senescent AT2 cells can modulate phenotypic changes in interstitial cells, such as fibroblasts, and vice versa. For example, SASP components released from senescent AT2 stimulate collagen production from fibroblasts in vitro (63). In addition, single-walled carbon nanotubes-induced AT2 senescence facilitated lung fibrosis by promoting the differentiation of lung fibroblasts to become myofibroblasts (133). Similarly, bleomycin-induced AT2 senescence activated lung fibroblasts through Nanog-WNT/β-catenin signaling pathway to promote lung fibrosis (152). Moreover, the loss of PTEN from AT2 cells promoted their senescence via NF-κB activation (63).

CONCLUSIONS

Senescence of alveolar epithelial progenitors, largely AT2 cells, plays an essential role in promoting progressive lung fibrosis. Current data suggest that alveolar epithelial progenitors are vulnerable to becoming senescent and orchestrating pathological processes in the lung microenvironment. Persistent alveolar injury and/or dysfunctional alveolar repair, such as due to progenitor cell exhaustion, are key initiating mechanisms in fibrotic lung diseases. Cellular senescence is likely the key causative process that impairs stemness capacity of alveolar epithelial progenitor cells, thereby negatively impacting effective lung repairing processes.

In contrast, the beneficial function of senescence during development and the role of senescent (p16⁺) interstitial cells in promoting repair and/or limiting fibrosis in lung, skeletal muscle, and liver (149) indicates that senescence is not universally bad. The somewhat opposing outcomes of senescence of different cell types in different tissue compartments (i.e., epithelium vs. interstitium) illustrate the complexity of targeting senescence regulatory networks as therapeutic interventions. In addition, these observations underscore the need for specific markers that can distinguish pathological senescent cells from beneficial or benign ones.

Nonmodifiable processes, such as aging and genetic mutation, and modifiable risk factors, including oxidative stress and mitochondria dysregulation, contribute to alveolar progenitor stemness exhaustion in IPF. Controllable risk factors are potential targets for effective prevention or therapeutic intervention, and metabolic reprogramming is emerging as an intervenable senescence regulatory mechanism (153). For example, the ability of zinc replenishment to reverse impaired self-renewal ability of AT2 cells from IPF lungs, which lack a zinc transporter (114), suggests that even approved supplements could improve AT2 progenitor function and, in turn, mitigate ongoing fibrogenesis. Alternative approaches include eradicating senescent cells or inhibiting the downstream effects of senescence. Since some senescent cells convey beneficial roles in lung regeneration, more targeted senescence elimination, either cell-specific or function-specific, would be more effective.

A challenge in preventing alveolar progenitor exhaustion is a lack of cell type-specific modifiable predisposing factors and targets in the early stages of senescence. Developing more appropriate animal models to recapitulate human pathology and using human biospecimens that represent better early cellular and molecular events are essential steps. In addition, the management of alveolar epithelial progenitor senescence should prioritize prevention or containment rather than the total elimination of senescent progenitors. Moreover, efforts should be made to explore more specific approaches to eliminate senescent cells. A first step toward selective eradication would be the identification of specific markers to differentiate pathological senescent cells from nonpathological senescent cells. Because such markers are currently not known, identification of specific features of disease-causing senescent cells would provide a significant advance in our understanding of and treatment of progressive fibrotic conditions.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants K08 HL141590 (to T.P.), R01 HL159953 and R01 HL155759 (to P.C.), P01 HL108793 (to P.W.N. and B.R.S.), R01 HL159953 (to W.C.P.), R01 HL151160 (to B.R.S.), and R35 HL150829 (to P.W.N.); National Institute on Aging Grant R01 AG078655 (to J.L. and P.W.N.); Foundation for the National Institute on Health Grant UCLA CTSI KL2 TR001882 (to T.P.); Parker B. Francis Foundation (to C.Y.); UCLA | Clinical and Translational Science Institute, University of California, Los Angeles (CTSI) Grant KL2-NCATSKL2TR001882 (to C.Y.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

T.P. and C.Y. prepared figures; T.P. and C.Y. drafted manuscript; T.P., P.C., B.R.S., J.L., D.J., P.W.N., W.C.P., and C.Y. edited and revised manuscript; T.P., P.C., B.R.S., J.L., D.J., P.W.N., W.C.P., and C.Y. approved final version of manuscript.