Figure 1. Pathogenesis and perpetuation of fibrosing interstitial lung diseases. Environmental risk factors, age-related cell perturbations, genetic predisposition, persistent exposure to antigens and autoimmune diseases are identified as important factors that increase susceptibility to epithelial lung injury acting as initial triggers of the disease. In an early phase, shown in the left part of the figure (1), epithelial type 2 cells (AEC2) show several markers of stress, activation, and senescence, as they fail to respond to stress resulting in an impaired capacity of the lung to regenerate. In a later phase, and especially after repeated alveolar damage, sustained aberrant activation and senescence of the epithelium (2) leads to the hyperactive secretion of high levels of pro-fibrotic growth factors, cytokines, chemokines and matrix metalloproteinases, collectively known as senescence-associated secretory phenotype (SASP) factors (3), that promote cellular senescence of the adjacent AEC2 (4). This initial event is encompassed in a process called primary senescence, which is thought to be characterized by the appearance of a senescent lung epithelium which persists by adapting its metabolic pathways and becoming resistant to apoptosis. In this later phase, a dysregulated crosstalk between the senescent epithelium and the mesenchymal cells through SASP factors will give rise to the accumulation of fibroblasts and myofibroblasts, resulting in an increased production and deposition of extracellular matrix components (5). A considerable part of fibroblasts and myofibroblasts will present a stressed and senescent phenotype, including resistance to apoptosis, which entails a process called secondary or paracrine senescence. As a consequence, in the context of senescence in lung injury and repair, the accumulation of extracellular matrix components in the interstitium of the lung will lead to lung fibrosis.

3. Lung Susceptibility to Cellular Senescence

Under normal conditions, AEC2s can self-renew and differentiate into type I alveolar epithelial cells and therefore are considered as alveolar progenitor cells [47,48]. However, in age-related diseases such as lung fibrosis, a decrease of the renewal capacity and disfunction of distinct lung’s cell types, as well as AEC2s or lung fibroblasts, is a widely known phenomenon. Cellular senescence of AEC2s and lung fibroblasts of patients with IPF determine a cellular phenotype with the hyperactive secretion of a set of pro-fibrotic SASP’s cytokines, growth factors, angiostatic and procoagulant mediators, and proteases that promote an abnormal wound healing response [20,49,50]. Activated AEC2s express several mediators (platelet-derived growth factor, transforming growth factor β1 [TGFβ1], tumour necrosis factor, osteopontin and CXC chemokine ligand 12, and endothelin-1), promoting profibrotic responses and inducing the migration of mesenchymal cells of different origins such as fibrocytes and resident fibroblasts [51]. Stress-induced senescent fibroblasts and myofibroblasts in the IPF lung secrete excessive amounts of ECM components, mainly fibrillar collagens and fibronectin, and are resistant to apoptosis, thus promoting the development of irreversible damage and lung fibrosis [49].

4. Pathological Impact of SASP in Fibrotic Lungs

Alterations in the secretome of senescent cells with a chronic increment of production of several inflammatory secretory proteins, including plasminogen activator inhibitor-1 (PAI-1), matrix remodelling proteins (MMPs), growth factors such as TGFβ1, as well as other initial inflammatory stimuli, including IL-6, IL-8 and IL-1α, are present in IPF lungs. The SASP of these cells also has the potential to induce ER stress and enhances senescence through autocrine activity, but also can promote adjacent healthy cells to undergo senescence through paracrine senescence.  There is increasing evidence that paracrine effects of the molecular pathways that regulate SASP can be manipulated and several experimental drugs are being tested, however, most studies are now at early preclinical phases.

5. Aged Fibroblasts and Extracellular Matrix Changes

It has been shown that the senescent phenotype of IPF lung fibroblasts is associated with increased levels of senescent markers (p16Ink4a and p21) and positive β-gal staining. While p21 expression is driven by telomere shortening-induced p53 activity, p16Ink4a is upregulated by telomere-independent unknown mechanisms [70]. Activating caspase-8 moiety in the Ink-Attac (INK-linked apoptosis through targeted activation of caspase) suicide gene product, which is expressed only in p16-positive senescent cells, significantly improved health and lifespan after initiation of the combined therapy with dasatinib plus quercetin by promoting the clearance of senescent cells [71]. Navitoclax (ABT-263, a specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL), effectively cleared senescent cells in young p16-3MR exposed to sublethal doses of radiation that induced a significant increase in senescent cells [72]. Therefore, improvements in lifespan after clearance of senescent cells suggests that persistent cellular senescence may contribute to several age-related diseases, including pulmonary fibrosis. It also has been shown that in normally proliferating cells, such as human fibroblasts, there is a dynamic feedback loop in which the long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and reactive oxygen species (ROS) production, which is considered necessary for the establishment of the senescent phenotype [73]. In fibroblasts from IPF patients, this process particularly affects redox balance, controlled by NADPH oxidase 4 (NOX4) which has a crucial role in DNA damage, and is the key mediator of cellular antioxidant response NEF2-related factor 2. High expression of the NOX4 enzyme induces senescence in myofibroblasts by histone modifications and interferes with redox balance. It reduces reactive oxygen species, produces apoptosis of senescent myofibroblasts, and reverses established lung fibrosis in mice [8].

6. Loss of Epithelial Integrity

It has long been recognized that patients with sporadic or familial IPF present a dysfunctional alveolar epithelium associated with mutations and stress-induced senescence, which has a pivotal role in the aberrant lung injury-remodelling process. AEC2s in the IPF lungs express markers of senescence and apoptosis, which may indicate that the replacement of these cells might be a useful approach to attempting tissue repair and regeneration. It has been also proposed that stressed AEC2s in patients with lung fibrosis undergo an ineffective regeneration of the lung, so another pool of progenitor cells (KRT5+ Δp63+ cells) with persistent activation of the Notch signalling pathway mediates the repairment promoting micro-honeycombing rather than the regeneration of the normal alveoli epithelium [74].

A recent study demonstrated that although alveolar macrophages are long-lived lung-resident cells, changes in their number and transcriptional identity with age-related processes are not cell autonomous, although they instead might be shaped by interaction with the alveolar microenvironment independent of signalling in circulation [79]. Collectively, these findings suggest that strategies targeting the aging lung epithelial microenvironment may be crucial for restoring the alveolar macrophage-AECs axis function in aging.

7. Stem Cell Dysfunction