p19Arf Exacerbates Cigarette Smoke-Induced Pulmonary Dysfunction

Senescent cells accumulate in tissues during aging or pathological settings. The semi-genetic or pharmacological targeting of senescent cells revealed that cellular senescence underlies many aspects of the aging-associated phenotype and diseases. We previously reported that cellular senescence contributes to aging- and disease-associated pulmonary dysfunction. We herein report that the elimination of Arf-expressing cells ameliorates cigarette smoke-induced lung pathologies in mice. Cigarette smoke induced the expression of Ink4a and Arf in lung tissue with concomitant increases in lung tissue compliance and alveolar airspace. The elimination of Arf-expressing cells prior to cigarette smoke exposure protected against these changes. Furthermore, the administration of cigarette smoke extract lead to pulmonary dysfunction, which was ameliorated by subsequent senescent cell elimination. Collectively, these results suggest that senescent cells are a potential therapeutic target for cigarette smoking-associated lung disease.


Introduction
Chronic obstructive pulmonary disease (COPD) is a severe and incurable disease with a relatively high prevalence and is currently one of the leading causes of death worldwide [1]. A major component of COPD is pulmonary emphysema, which is characterized by enlarged alveolar airspaces and alveolar wall collapse. Cigarette smoke (CS) is the dominant cause of the disease, and aging also increases the risk of COPD [2]. COPD is associated with the infiltration of inflammatory cells, which is considered to cause the accumulation of proteinases and lead to alveolar destruction [3,4]. The inhibition of macrophage or neutrophil elastase was previously shown to be sufficient to suppress the pathologies of CS-induced emphysema in mice [5,6]. Cellular senescence is also accelerated in emphysema patients [7]. However, the roles of cellular senescence in this disease have yet to be characterized. Cellular senescence acts as a potent tumor suppression mechanism in mammals by halting the proliferation of cells at risk of malignant transformation [8]. Cellular senescence is mediated by the Cdkn2a locus, which encodes two important tumor suppressors, namely, p16 Ink4a and p19 Arf (p14 ARF in humans) [9,10]. These proteins indirectly activate the Rb and p53 tumor suppressors, respectively, thereby inducing and maintaining cell cycle arrest during cellular senescence. In addition to permanent cell cycle arrest, it has become apparent that senescent cells produce a series of cytokines, chemokines and extracellular proteinases, thereby non-cell autonomously affecting the functions of

Morphometric Analysis
Morphometric analysis was performed as previously described [20]. Lung tissues were fixed with Mildform ® 20N (Wako Pure Chemicals Industries, Ltd., Osaka, Japan) at constant pressure (25 cm H 2 O). Paraffin-embedded section (5 µm thickness) was stained with hematoxylin and eosin. All histopathological analyses were performed in a blinded manner.

Pulmonary Function Test
Pulmonary function tests were performed on a FlexiVent system (SCIREQ, Montreal, Quebec, Canada) as previously described [19,27]. Mice were euthanized by an intraperitoneal injection of excess amount of pentobarbital sodium (100 mg/kg of body weight), and connected to the FlexiVent system after tracheotomy. Diaphragm was removed prior to the spirometry analyses.

Bronchoalveolar Lavage Fluid (BALF) Analysis
BALF cells were analyzed as previously reported [28]. Briefly, cells collected from BALF were attached to slides using StatSpin Cytofuge (Beckman Coulter, Brea, CA, USA), and subjected to modified Giemsa staining using the Diff-Quick stain kit (Sysmex, Kobe, Japan).

Cigarette Smoking Induces Arf and Ink4a Expression in Mice
To investigate the roles of p19 Arf -expressing cells in the CS-induced lung pathology, 4-month-old ARF-DTR or wild-type mice were exposed to CS as previously described [29] for 4 weeks (30 min per day, 5 days per week), as shown in Figure 1a. DT was administered twice with a 2-week interval, and initial administration was performed prior to the CS exposure. We initially performed an in vivo imaging analysis to monitor lung luciferase expression, which was controlled by Arf promoter/enhancer activity in ARF-DTR mice [19]. Luciferase activity in the chest region, which represents the expression of luciferase in lung tissue [19], was slightly increased in CS-exposed ARF-DTR mice (Figure 1b). RNA was then extracted from lung tissue and the expression of Arf and Ink4a mRNA was analyzed. CS exposure resulted in an increase in both Arf and Ink4a mRNA levels ( Figure 1c,d), which is in contrast with previous findings obtained from an elastase-induced emphysema model showing no significant change in the mRNA levels of these genes [20]. Consistent with their roles in cellular senescence, the number of cells stained with Sudan Black B (SBB) was increased in CS-exposed lungs, suggesting that cellular senescence is enhanced in these mice (Supplementary Figure S1) [30]. Additionally, p19 Arf , which is generally undetectable in normal tissues but is expressed in a low percentage of mesenchymal cells in the lung of adult or elastase-treated animals [19,20], became apparent in a low number of epithelial cells after CS exposure (Figure 1e,f), suggesting that chronic insult by CS could damage the epithelial cells. Arf mRNA was barely detectable in BALF cell of which major component is macrophage (Supplementary Figure S2).
The administration of DT resulted in significant reductions in Arf and Ink4a mRNA levels (Figure 1c,d). The number of p19 Arf -expressing cells was also decreased by DT treatment (Figure 1e,f). The down-regulation of Ink4a in DT-treated lungs likely reflects the co-expression of p19 Arf and p16 Ink4a in lung (Figure 1g). Consistently, luciferase activities were under the detection limit after the DT treatment ( Figure 1b). SBB staining suggested that senescent cells were eliminated by DT-treatment in CS-exposed animals (Supplementary Figure S1). On the other hand, DT had no effect on Arf or Ink4a mRNA levels in wild-type mice, which confirmed that the expression of these genes was ablated through the transgene (Figure 1c,d).
Biomolecules 2020, 10, 462 5 of 14 treatment (Figure 1b). SBB staining suggested that senescent cells were eliminated by DT-treatment in CS-exposed animals (Supplementary Figure S1). On the other hand, DT had no effect on Arf or Ink4a mRNA levels in wild-type mice, which confirmed that the expression of these genes was ablated through the transgene (Figure 1c,d). In (c), (d) and (f), bars represent means ± SEM. Data were analyzed by a one-way ANOVA and Tukey post-hoc analysis. Student's t-test was performed for the analysis of wild-type mice data and no significance was observed. ** p < 0.01 and *** p < 0.001. In (c), (d) and (f), bars represent means ± SEM. Data were analyzed by a one-way ANOVA and Tukey post-hoc analysis. Student's t-test was performed for the analysis of wild-type mice data and no significance was observed. ** p < 0.01 and *** p < 0.001.

Ablation of Arf Expression Attenuates CS-Induced Lung Pathologies
We analyzed the effects of CS and DT in ARF-DTR mouse lung tissue. After a 4-week exposure to CS, lung tissues were inflated with fixative solution at a constant pressure prior to sectioning. As shown in Figure 2a, partial alveolar wall destruction was observed in the CS-exposed (PBS/CS) ARF-DTR mouse lung, which appeared to be suppressed in DT-treated ARF-DTR mice. To obtain more quantitative data, we evaluated the alveolar size by measuring the mean linear intercept (chord) length. DT did not affect the alveolar size at this age, while it was reduced by DT in the older animals [19] ( Figure 2b). CS exposure resulted in a significant increase in the alveolar size, but no significance was observed in the presence of DT treatment, suggesting that the elimination of Arf -expressing cells protected the tissue from CS-induced alveolar collapse. We previously suggested that the elimination of p19 Arf -expressing cells suppressed elastase-induced alveolar inflammation, thereby protecting tissue from emphysema-associated pathologies [20]. This may also be the case in the CS model because the number of macrophages, which was increased in CS-exposed mice, was maintained in DT-treated animals ( Figure 2c). Matrix metalloproteinase (Mmp)-12 was consistently down-regulated in lung tissue after the DT treatment ( Figure 2d). DT did not markedly affect alveolar sizes, macrophage numbers or Mmp-12 expression in CS-exposed wild-type mice (Supplementary Figure S3). Collectively, these results suggest that similar to the elastase-induced lung injury model, the CS-induced lung pathology was attenuated by the elimination of p19 Arf -expressing cells.

Ablation of Arf Expression Attenuates CS-Induced Lung Pathologies
We analyzed the effects of CS and DT in ARF-DTR mouse lung tissue. After a 4-week exposure to CS, lung tissues were inflated with fixative solution at a constant pressure prior to sectioning. As shown in Figure 2a, partial alveolar wall destruction was observed in the CS-exposed (PBS/CS) ARF-DTR mouse lung, which appeared to be suppressed in DT-treated ARF-DTR mice. To obtain more quantitative data, we evaluated the alveolar size by measuring the mean linear intercept (chord) length. DT did not affect the alveolar size at this age, while it was reduced by DT in the older animals [19] (Figure 2b). CS exposure resulted in a significant increase in the alveolar size, but no significance was observed in the presence of DT treatment, suggesting that the elimination of Arf-expressing cells protected the tissue from CS-induced alveolar collapse. We previously suggested that the elimination of p19 Arf -expressing cells suppressed elastase-induced alveolar inflammation, thereby protecting tissue from emphysema-associated pathologies [20]. This may also be the case in the CS model because the number of macrophages, which was increased in CS-exposed mice, was maintained in DT-treated animals (Figure 2c). Matrix metalloproteinase (Mmp)-12 was consistently down-regulated in lung tissue after the DT treatment (Figure 2d). DT did not markedly affect alveolar sizes, macrophage numbers or Mmp-12 expression in CS-exposed wild-type mice (Supplementary Figure  S3). Collectively, these results suggest that similar to the elastase-induced lung injury model, the CSinduced lung pathology was attenuated by the elimination of p19 Arf -expressing cells.
While macrophages express Ink4a (Supplementary Figure S2) and senescence-associated βgalactosidase [31], Arf and DTR mRNA levels were barely detectable in BALF cells and unchanged by DT, excluding the possibility that DT directly exerts cytotoxicity in macrophages.   Figure S2) and senescence-associated β-galactosidase [31], Arf and DTR mRNA levels were barely detectable in BALF cells and unchanged by DT, excluding the possibility that DT directly exerts cytotoxicity in macrophages.

Arf-Expressing Cells Facilitates CS-Induced Pulmonary Dysfunction
The destruction of alveolar walls by CS or elastase leads to a decline in pulmonary function, which is exemplified by increased lung tissue compliance. Hence, we tested whether the elimination of p19 Arf -expressing cells restores pulmonary function in CS-exposed ARF-DTR mice. Inspiratory capacity (IC) was significantly increased in CS-exposed mice (Figure 3a). Consistently, lung tissue compliance (Cst; static lung compliance, Crs; respiratory system compliance) was significantly increased in CS-exposed mice (Figure 3b-d), while respiratory system resistance (Rrs) was unchanged (Figure 3e). However, the effects of CS were diminished or prevented when DT was administered prior to the CS exposure in ARF-DTR (Figure 3 and Supplementary Figure S4). Tissue damping (G) that reflects the viscoelastic properties of lung tissue was decreased in CS-exposed mice (Figure 3f). These changes were consistent with the phenotypes observed in Timp-3 knockout, SP-D knockout or Notch mutant mice that spontaneously develop pulmonary emphysema [32][33][34]. DT partly suppressed the CS-induced change in tissue damping coefficient in ARF-DTR (Figure 3f and Supplementary Figure S4). Collectively, these results suggest that the presence of Arf -expressing cells exacerbates CS-induced lung dysfunction through enhancements in the recruitment of macrophages, and that elimination of Arf -expressing cells leads to the re-establishment of respiratory function or protection it from CS-induced damage.
Biomolecules 2020, 10, 462 7 of 14 by real-time PCR. Data were normalized to Gapdh in each group. Bars represent means ± SEM. Data were analyzed by a one-way ANOVA and Tukey post-hoc analysis. ** p < 0.01 and ns; not significant.

Arf-Expressing Cells Facilitates CS-Induced Pulmonary Dysfunction
The destruction of alveolar walls by CS or elastase leads to a decline in pulmonary function, which is exemplified by increased lung tissue compliance. Hence, we tested whether the elimination of p19 Arf -expressing cells restores pulmonary function in CS-exposed ARF-DTR mice. Inspiratory capacity (IC) was significantly increased in CS-exposed mice (Figure 3a). Consistently, lung tissue compliance (Cst; static lung compliance, Crs; respiratory system compliance) was significantly increased in CS-exposed mice (Figure 3b-d), while respiratory system resistance (Rrs) was unchanged (Figure 3e). However, the effects of CS were diminished or prevented when DT was administered prior to the CS exposure in ARF-DTR (Figure 3 and Supplementary Figure S4). Tissue damping (G) that reflects the viscoelastic properties of lung tissue was decreased in CS-exposed mice (Figure 3f). These changes were consistent with the phenotypes observed in Timp-3 knockout, SP-D knockout or Notch mutant mice that spontaneously develop pulmonary emphysema [32][33][34]. DT partly suppressed the CS-induced change in tissue damping coefficient in ARF-DTR (Figure 3f and Supplementary Figure S4). Collectively, these results suggest that the presence of Arf-expressing cells exacerbates CS-induced lung dysfunction through enhancements in the recruitment of macrophages, and that elimination of Arf-expressing cells leads to the re-establishment of respiratory function or protection it from CS-induced damage.

Effects of p19 Arf -Expressing Cell Ablation in Cigarette Smoke Extract-Treated Mice
The present results and previous findings suggest that the elimination of p19 Arf -expressing cells prior to the CS exposure or elastase inhalation exerts protective effects against emphysema-associated lung pathologies [20]. However, it currently remains unclear whether the elimination of p19 Arfexpressing cells exerts therapeutic effects after lung injury. To test this possibility, we treated mice with DT after a damage-causing stimulation in ARF-DTR mice. In this experiment, we employed CSE inhalation as a lung injury model. CSE contains fewer chemical components than CS [35], but is

Effects of p19 Arf -Expressing Cell Ablation in Cigarette Smoke Extract-Treated Mice
The present results and previous findings suggest that the elimination of p19 Arf -expressing cells prior to the CS exposure or elastase inhalation exerts protective effects against emphysema-associated lung pathologies [20]. However, it currently remains unclear whether the elimination of p19 Arf -expressing cells exerts therapeutic effects after lung injury. To test this possibility, we treated mice with DT after a damage-causing stimulation in ARF-DTR mice. In this experiment, we employed CSE inhalation as a lung injury model. CSE contains fewer chemical components than CS [35], but is sufficient to induce pulmonary inflammation [36]. Additionally, CSE has been shown to induce cellular senescence in cultured cells [37]. CSE was intranasally administered to ARF-DTR or wild-type mice for 8 weeks (3 times per week), as shown in Figure 4a. In this experiment, DT was administered 4 weeks after the CSE stimulation.
In contrast to the CS model (Figure 1), no marked differences were observed in lung luciferase activity between PBS-and CSE-treated animals (Figure 4b). Real-time PCR analyses consistently revealed that Arf and Ink4a expression remained unchanged in CSE-treated lung tissue (Figure 4c,d). Nevertheless, the DT treatment significantly reduced luciferase activity as well as Arf and Ink4a expression in both control and CSE-treated animals, but had no effect on the expression of these genes in wild-type mice (Figure 4b-d).
Biomolecules 2020, 10, 462 8 of 14 sufficient to induce pulmonary inflammation [36]. Additionally, CSE has been shown to induce cellular senescence in cultured cells [37]. CSE was intranasally administered to ARF-DTR or wildtype mice for 8 weeks (3 times per week), as shown in Figure 4a. In this experiment, DT was administered 4 weeks after the CSE stimulation.
In contrast to the CS model (Figure 1), no marked differences were observed in lung luciferase activity between PBS-and CSE-treated animals (Figure 4b). Real-time PCR analyses consistently revealed that Arf and Ink4a expression remained unchanged in CSE-treated lung tissue (Figure 4c,d). Nevertheless, the DT treatment significantly reduced luciferase activity as well as Arf and Ink4a expression in both control and CSE-treated animals, but had no effect on the expression of these genes in wild-type mice (Figure 4b-d).  and d) The expression of (c) Arf and (d) Ink4a mRNA was analyzed by real-time PCR in the lung tissue of ARF-DTR or wild-type mice. Data were normalized to Gapdh in each group. Bars represent means ± SEM. Data were analyzed by a one-way ANOVA and Tukey post-hoc analysis. Student's t-test was performed for the analysis of wild-type mice data and no significance was observed. * p < 0.05 and ** p < 0.01.

CSE Induced Pulmonary Dysfunction in Mice
Similar to the CS exposure, the CSE treatment also leads to impaired pulmonary function [38,39]. We performed pulmonary function tests in CSE-treated mice. The CSE treatment resulted in increases in IC and Cst (Figure 5a-c), although its effect may be much weaker than that of CS. Morphometric analysis also suggest that CSE had weaker effect, as no significant change was observed in alveolar size in CSE-treated animals (Supplementary Figure S5). Other parameters including Crs, Rrs and G were unaffected by the CSE treatment (Figure 5d-f), suggesting that CSE exerts differential effects on pulmonary function or is less effective than in the CS-exposure model. IC and Cst were both restored in the DT-treated group (Figure 5a-c), while DT had no effect on these parameters in CSE-treated wild-type animals (Supplementary Figure S6).
We then analyzed cells in the BALF of these mice. While we and others have previously shown that the total cell number in BALF was significantly increased in the elastase-induced lung injury model [20,28,40], no consistent change was observed in the number of BALF cells (Figure 6a). This was somewhat expected because the continuous inhalation of PBS was sufficient to increase BALF The expression of (c) Arf and (d) Ink4a mRNA was analyzed by real-time PCR in the lung tissue of ARF-DTR or wild-type mice. Data were normalized to Gapdh in each group. Bars represent means ± SEM. Data were analyzed by a one-way ANOVA and Tukey post-hoc analysis. Student's t-test was performed for the analysis of wild-type mice data and no significance was observed. * p < 0.05 and ** p < 0.01.

CSE Induced Pulmonary Dysfunction in Mice
Similar to the CS exposure, the CSE treatment also leads to impaired pulmonary function [38,39]. We performed pulmonary function tests in CSE-treated mice. The CSE treatment resulted in increases in IC and Cst (Figure 5a-c), although its effect may be much weaker than that of CS. Morphometric analysis also suggest that CSE had weaker effect, as no significant change was observed in alveolar size in CSE-treated animals (Supplementary Figure S5). Other parameters including Crs, Rrs and G were unaffected by the CSE treatment (Figure 5d-f), suggesting that CSE exerts differential effects on pulmonary function or is less effective than in the CS-exposure model. IC and Cst were both restored in the DT-treated group (Figure 5a-c), while DT had no effect on these parameters in CSE-treated wild-type animals (Supplementary Figure S6). neutrophil number in both PBS and CSE-treated ARF-DTR mice by DT (Figure 6d), but not in CSEtreated wild-type mice (Supplementary Figure S7). Consistently, neutrophil elastase (Elane) levels were also decreased by DT treatment in ARF-DTR mice (Figure 6f). Collectively, these results suggest that CS and CSE differentially induce lung pathologies, while both stimulations lead to pulmonary dysfunction exemplified by increased lung tissue compliance, and the ablation of Arf-expressing cells exerts beneficial effects, even after the damage-inducing stimulation.  neutrophil number in both PBS and CSE-treated ARF-DTR mice by DT (Figure 6d), but not in CSEtreated wild-type mice (Supplementary Figure S7). Consistently, neutrophil elastase (Elane) levels were also decreased by DT treatment in ARF-DTR mice (Figure 6f). Collectively, these results suggest that CS and CSE differentially induce lung pathologies, while both stimulations lead to pulmonary dysfunction exemplified by increased lung tissue compliance, and the ablation of Arf-expressing cells exerts beneficial effects, even after the damage-inducing stimulation.  We then analyzed cells in the BALF of these mice. While we and others have previously shown that the total cell number in BALF was significantly increased in the elastase-induced lung injury model [20,28,40], no consistent change was observed in the number of BALF cells (Figure 6a). This was somewhat expected because the continuous inhalation of PBS was sufficient to increase BALF cells [24]. Consistent with this observation, the number of macrophages, which accounts for more than 80% of the total BALF cell number, was unchanged by the CSE or DT treatment (Figure 6b). Other cells, including neutrophils, lymphocytes and eosinophils, were also unaffected by the CSE treatment (Figure 6c-e). While the administration of DT reduced the macrophage number in CS-treated ARF-DTR mice ( Figure 2) and the elastase-induced emphysema model [20], it had no effect in CSE-treated ARF-DTR mice (Figure 6b). Nevertheless, we observed a significant decrease in the neutrophil number in both PBS and CSE-treated ARF-DTR mice by DT (Figure 6d), but not in CSE-treated wild-type mice (Supplementary Figure S7). Consistently, neutrophil elastase (Elane) levels were also decreased by DT treatment in ARF-DTR mice (Figure 6f). Collectively, these results suggest that CS and CSE differentially induce lung pathologies, while both stimulations lead to pulmonary dysfunction exemplified by increased lung tissue compliance, and the ablation of Arf -expressing cells exerts beneficial effects, even after the damage-inducing stimulation.

Discussion
The present study demonstrated that the elimination of Arf -expressing cells ameliorated CS-induced lung pathologies using ARF-DTR mice. In contrast to INK-ATTAC and p16-3MR mice [41,42], ARF-DTR mice express the transgene (DTR and luciferase) under the control of the Arf promoter/enhancer [19]. While the role of ARF in human cellular senescence and aging remains controversial, Arf is essential for senescence in rodent cells [43]. Additionally, the DT treatment decreased Ink4a as well as Arf expression, indicating Arf -and Ink4a-positive senescent cells were eliminated from the lung tissue of ARF-DTR mice. The elimination of Arf -expressing cells prior to the CS inhalation trended toward protection against CS-induced alveolar enlargement, macrophage infiltration and pulmonary dysfunction. Additionally, the present results obtained with the CSE inhalation model suggest that the elimination of Arf -expressing cells after the stimulation still exerted beneficial effects on lung function.
The etiology of COPD is complex because many factors contribute to the development of this disease [1,2] and its biology remains poorly understood. While cigarette smoking is the predominant risk factor for COPD, a relatively small proportion of smokers develop the disease. This underscores other factors in addition to CS influencing susceptibility to the disease. The incidence of COPD is increased by aging irrespective of the smoking history [44], which highlights aging-associated changes in pulmonary and/or other organs facilitating the development of this disease. Cellular senescence may be a key factor that promotes disease-associated pathologies. Cellular senescence is accelerated in emphysema patients [7], and we previously suggested that cellular senescence plays a role in CS-induced lung pathologies [29], while aging-associated changes other than cellular senescence may also contribute to the CS-induced pathology [45].
We previously reported that the elimination of Arf -expressing cells protected against elastase-induced emphysema [20]. Elastase inhalation is a facile and highly reproducible model for the induction of pulmonary emphysema, and a single shot of elastase is sufficient to induce alveolar collapse. However, the elastase treatment results in panlobular (panacinar) emphysema, which is a rare disease in humans. In humans, centrilobular (centriacinar) emphysema is the most common type of pulmonary emphysema, the main cause of which is smoking. Therefore, we investigated the effects of senolysis in the CS mouse model in the present study. In contrast to the elastase model, CS exposure resulted in weak, but chronic damage to the lung tissue, which leads to impaired pulmonary function. We also employed CSE inhalation for the lung injury model. CSE contains fewer chemical components than CS [35]. However, in contrast to the CS model in which the whole body is exposed to CS, the CSE inhalation model may more specifically target the tissue/organ. The present results suggest that CSE inhalation was sufficient to induce pulmonary dysfunction; however, it did not induce notable histological changes as previously documented [36]. In both models, the elimination of Arf -expressing cells was proven to ameliorate lung damage, suggesting that senescent cells also play an important role in CS-induced pathologies.
Inflammation in lung tissue is a critical step for the development of disease. The inactivation of macrophage or neutrophil elastase has been shown to confer resistance to CS-induced emphysema in mice [5,6]. The results obtained herein indicate that CS and CSE differentially affect alveolar inflammatory cells. Nevertheless, the elimination of Arf -expressing cells suppressed the expression of elastolytic enzymes, suggesting that senescent cells exacerbate CS/CSE-induced lung pathologies by enhancing alveolar inflammation. Hall and colleagues recently reported that p16 Ink4a is expressed in non-senescent macrophages [31,46]. These findings suggested that the DT treatment ablated non-senescent macrophages, thereby affecting pulmonary inflammation and the associated phenotypes. However, this is unlikely because endogenous Arf levels are extremely low and the expression of the transgene (DTR and luciferase) was not detected in the macrophages of ARF-DTR mice (Supplementary Figure S2) [19]. Furthermore, our previous findings indicated that p19 Arf was present in the mesenchymal cells of lung tissue; however, the population of Arf -expressing cells was estimated to be as small as 2% of all mesenchymal cells in lung tissue. Senescent cells produce chemokines/cytokines as part of SASP, which have a marked impact on the inflammatory process [47]. Hence, it is reasonable to assume that the effects of DT were attributed to the clearance of senescent cells that may facilitate the inflammatory response through SASP.
We previously demonstrated that the elimination of Arf -expressing cells exerted preventive effects against elastase-induced pathologies [20]. The present results obtained with the CSE model strongly suggest that senolysis also exhibits therapeutic efficacy because DT partly restored pulmonary function in ARF-DTR mice in which lung injury was induced by CSE in advance. Thus, these results suggest the potential of the application of senolytic drugs as emphysema therapy. In this regard, it is important to note that the oral administration of ABT-263/Navitoclax exerted beneficial effects against elastase-induced emphysema [20]. While ABT-263/Navitoclax has side effects [48,49] and may not be directly applied to senolysis in humans, the targeting of senescent cells will be an effective approach. Further studies are required to assess the therapeutic efficacy of senolysis and senolytic drugs for the treatment of emphysema.

Conclusions
Elimination of p19 Arf -expressing cells ameliorated the CS-and CSE-induced pulmonary dysfunction in mice. These findings suggest emphysema-associated pathologies are exacerbated by cellular senescence, and that targeting senescent cells may be an effective approach for COPD therapy.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/3/462/s1, Figure S1: Effects of CS and DT on Sudan Black B staining, Figure S2: Expression of Ink4a, Arf and DTR/Luc mRNA in BALF cells, Figure S3: DT had no effect on alveolar size, macrophage numbers, or Mmp-12 expression in wild-time mice, Figure S4: DT had no effects on the pulmonary function of CS-exposed wild-type mice, Figure  S5: CSE had no effect on lung morphology in ARF-DTR mice, Figure S6: DT had no effect on the pulmonary function of CSE-treated wild-type mice, Figure S7: DT had no effects on the BALF cell population in CSE-treated wild-type animals.