Cigarette smoke-induced autophagy impairment accelerates lung aging, COPD-emphysema exacerbations and pathogenesis

Abstract

Cigarette-smoke (CS) exposure and aging are the leading causes of chronic obstructive pulmonary disease (COPD)-emphysema development, although the molecular mechanism that mediates disease pathogenesis remains poorly understood. Our objective was to investigate the impact of CS exposure and aging on autophagy and the pathophysiological changes associated with lung aging (senescence) and emphysema progression. Beas2b cells, C57BL/6 mice, and human (GOLD 0-IV) lung tissues were used to determine the central mechanism involved in CS/age-related COPD-emphysema pathogenesis. Beas2b cells and murine lungs exposed to cigarette smoke extract (CSE)/CS showed a significant ( P < 0.05) accumulation of poly-ubiquitinated proteins and impaired autophagy marker, p62, in aggresome bodies. Moreover, treatment with the autophagy-inducing antioxidant drug cysteamine significantly ( P < 0.001) decreased CSE/CS-induced aggresome bodies. We also found a significant ( P < 0.001) increase in levels of aggresome bodies in the lungs of smokers and COPD subjects in comparison to nonsmoker controls. Furthermore, the presence and levels of aggresome bodies statistically correlated with severity of emphysema and alveolar senescence. In addition to CS exposure, lungs from old mice also showed accumulation of aggresome bodies, suggesting this as a common mechanism to initiate cellular senescence and emphysema. Additionally, Beas2b cells and murine lungs exposed to CSE/CS showed cellular apoptosis and senescence, which were both controlled by cysteamine treatment. In parallel, we evaluated the impact of CS on pulmonary exacerbation, using mice exposed to CS and/or infected with Pseudomonas aeruginosa ( Pa), and confirmed cysteamine's potential as an autophagy-inducing antibacterial drug, based on its ability to control CS-induced pulmonary exacerbation ( Pa-bacterial counts) and resulting inflammation. CS induced autophagy impairment accelerates lung aging and COPD-emphysema exacerbations and pathogenesis.

Keywords: COPD; aging; autophagy; cigarette smoke; cysteamine; emphysema; proteostasis.

Fig. 1.

Tobacco/cigarette smoking induces aggresome-formation and emphysema in chronic obstructive pulmonary disease (COPD) subjects. A and C: ProteoStat aggresome-body dye-based staining of human lung-tissue sections collected from non-emphysema (GOLD 0) and COPD subjects (GOLD I–IV) with emphysema. Nuclei are stained with Hoechst dye. The data show an increase in number of aggresome-bodies that correlates with the severity of emphysema (GOLD I–III) in COPD subjects in comparison to non-emphysema control (GOLD 0) lung sections. However, lung sections collected from severe emphysema-COPD (GOLD IV) subjects exhibit fewer aggresome/autophagy bodies in comparison to less severe emphysema (GOLD III) but it was still higher than control smoker or nonsmoker subjects and was similar to GOLD II. B and D: Sudan B Black staining of human lung-tissue sections collected from smoker and nonsmoker emphysema (GOLD I–IV, B) and non-emphysema (GOLD 0, D) shows that smoking induces senescence in both control and emphysema subjects. Moreover, significant increase is seen in levels of senescent alveolar cells depending on the severity of emphysema (GOLD I–IV). Significant tissue destruction is apparent in GOLD IV subjects that is anticipated to be a consequence of irreversible senescence induced by prolonged smoke exposure. E and F: moreover, the aggresome formation process is clearly smoking exposure dependent, as nonsmoker samples from GOLD-0 or GOLD I–IV did not show significantly elevated levels of aggresome-bodies in comparison to COPD lungs (GOLD I–IV) of smokers. Scale bar 100 μm. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2.

Cigarette smoke (CS) exposure-induced autophagy impairment and aggresome formation activates cellular apoptosis and senescence. A: transmission electron microscopy (TEM) images of Beas2b cells exposed to various concentrations of cigarette smoke extract (CSE) for 6 h. I, air-exposed control cells displaying the nucleus (N) and nuclear membrane (NM) (×5,598 magnification). II, Beas2b cells exposed to 10% CSE show aggresome/autophagy bodies (AB) proximal to the nuclear membrane (NM) (×10,171 magnification). CSE treatment also shows some membrane disintegration along with the thinning of the nuclear membrane (NM) and perinuclear accumulation of aggresome/autophagy bodies (AB). III and IV (scale: 2 µm), higher magnification (×46,422, ×84,190) of I and II (scale: 100 nm). The thin white arrow indicates the nuclear membrane (NM) and thick white arrows indicate the localization of aggresome bodies (AB). B: Western blot analysis illustrating the levels of ubiquitinated proteins, sirtuin1 (Sirt1, senescence marker), and p53 (senescence/apoptosis mediator) in soluble protein fractions of control Beas2b cells and those treated with 250 µM cysteamine and/or 10% cigarette smoke extracts (CSE) for 6 h. Substantial accumulation of ubiquitinated proteins was observed in the insoluble protein fractions of Beas2b cells treated with 10% CSE, suggesting that ubiquitinated proteins are translocated from soluble to insoluble protein fractions upon 6 h of 10% CSE exposure. Cysteamine attenuates the accumulation of ubiquitinated proteins in insoluble fraction and partially restores normal Sirt1 and p53 levels in the soluble protein fraction. β-Actin levels of the soluble protein fraction indicate equal loading of the proteins. C: senescence-associated β-galactosidase (SA-β-gal) activity in Beas2b cells treated with cysteamine (6 h) and/or exposed to 10% CSE overnight was measured using a senescence cells histochemical staining kit. Senescent cells were visualized and identified via blue stain (black arrows), indicating positive SA-β-gal activity. Data are shown as means ± SE (n = 3) of percentage change in SA-β-gal-positive blue senescent cells in comparison to air-exposed controls (bottom) Scale: 56 µm. ***P < 0.001 and ****P < 0.0001.

Fig. 3.

Cysteamine rescues CSE-induced autophagy impairment and aggresome formation. A: Beas2b cells were transduced with Premo autophagy tandem sensor LC3B-RFP-GFP construct. After 24 h posttransfection, Beas2b cells were preincubated with 250 µM cysteamine (Cys) and/or treated with 10% CSE for 6 h. Flow cytometry results show significant increase in GFP fluorescence (autophagosomes), RFP fluorescence (autolysosomes), and their colocalization (autophagosomes) in comparison to air-exposed cells. Preincubation with cysteamine reduced GFP fluorescence, RFP fluorescence, and their colocalization. The x-axis shows the log scale of LC3-GFP and y-axis shows the log scale of LC3-RFP fluorescence signals. The data represent the average (mean ± SE) of three replicates (right). B: Beas2b cells were pretreated with chloroquine (CQ, 90 µM, 12 h) or cysteamine (Cys, 250 µM, 6 h), and/or treated with 10% CSE for 6 h. Fluorescence microscopy images show autolysosomes (marked by the presence of puncta, second column), autophagosomes (marked by the presence of puncta, third column), and autophagosomes (marked by the colocalization of the puncta, fourth column). Scale bars, 56 μm. Fluorescence images were used to count the number of colocalized RFP-LC3B and GFP-LC3B puncta per image (fourth column). The data represent means ± SE of four replicates, and analysis is shown in D and E. Data suggest that CSE and CQ impair autophagy while cysteamine treatment restores CSE-impaired autophagy. C: Beas2b cells were treated with cysteamine (250 µM) and/or 10% CSE for 6 h. Cells were stained with ProteoStat aggresome dye and Hoechst nuclei dye. MG132 (5 μM; 12 h) treatment was used as a positive control for this experiment. Images were captured by ZOE Fluorescent Cell Imager (Bio-Rad) and quantitative analysis of aggresome bodies in each group is shown in D and E. Data suggest that CSE and MG132 induced aggresome/autophagy bodies while cysteamine treatment restored CSE-impaired autophagy, as seen by decrease in the number of aggresome/autophagy bodies. Scale bars, 56 μm. D and E: statistical analysis of microscopy data in B and C. Data are means ± SE (n = 3). **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.

Chronic and subchronic CS exposure induces aggresome formation and age-related changes in murine lungs. A: Western blot analysis illustrating the levels of ubiquitinated protein, the impaired autophagy marker p62, and valosin-containing protein (VCP) in soluble and insoluble protein fractions of mouse lung lysates collected from air- or chronic-CS-exposed C57BL/6 mice that were intraperitoneally injected with a daily dose of 0.01 M cysteamine for 10 days and/or infected intranasally with PA01-GFP (2 × 10⁶) for 5 days. Expression of the above proteins was normalized to β-actin loading control. B: Western blot analysis illustrating the levels of ubiquitinated protein, the impaired autophagy marker p62, and VCP and Sirt1 in soluble and insoluble protein fractions of mouse-lung lysates collected from C57BL/6 mice exposed to air or subchronic-CS and/or intraperitoneally injected with a daily dose of 0.01 M cysteamine for 10 days. Expression of the above proteins was normalized to β-actin loading control. The old mice (~12 mo) were used as a positive control. Densitometry analysis (of B) is shown in right panel. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.

Cysteamine rescues CS-induced aggresome formation, senescence, and alveolar space enlargement. A–C: ProteoStat-RED aggresome-dye was used for staining of lung sections from C57BL/6 mice exposed to air or chronic-CS, infected with PA01-GFP, and/or treated with cysteamine. The lung sections from mice exposed to chronic-CS [or c-CS and Pseudomonas aeruginosa (Pa)] display increased number of aggresome bodies in comparison to air-exposed lungs. The chronic-CS-exposed mice (or CS and Pa) treated with 0.01 M cysteamine for 10 days exhibit reduction in CS-induced aggresome-bodies in comparison to chronic-CS-exposed mice (B). Nuclei were stained using Hoechst dye. Scale bars, 100 µm. Sudan B Black (SBB) and hematoxylin and eosin (H&E) staining of longitudinal lung sections shows senescence and alveolar space enlargement in lung sections collected from chronic-CS-exposed mice in comparison with lung sections of room-air-exposed controls. The lung sections of mice treated with cysteamine display mitigation of chronic-CS-induced senescence and alveolar space enlargement. The lung sections from PA01-GFP (2 × 10⁶)-infected mice were used as a positive control for pulmonary exacerbation. The lung sections from chronic-CS-exposed mice infected with Pa display increased senescence and alveolar space diameter in comparison to samples collected from mice exposed to chronic-CS or Pa alone. Moreover, lung sections from chronic-CS-exposed mice infected with Pa and treated with 0.01 M cysteamine exhibit reduced senescence and alveolar space enlargement in comparison to samples collected from chronic-CS-exposed mice infected with Pa. D, E, and F: ProteoStat-RED dye was used for aggresome staining of lung sections from C57BL/6 mice exposed to air or subchronic CS (sc-CS) and/or treated with cysteamine. The old mice (~12 mo) were used as a control group. The lung sections from mice exposed to subchronic CS or from old mice display an increase in number of aggresome bodies in comparison to air-exposed mice. The lung sections of subchronic-CS-exposed mice treated with 0.01 M cysteamine for 10 days exhibit reduction in CS-induced aggresome-bodies. Sudan B Black (SBB) and hematoxylin and eosin (H&E) staining of longitudinal lung sections shows senescence and alveolar space enlargement in lung sections collected from subchronic-CS-exposed and old mice in comparison with lung sections of room-air-exposed controls. The lung sections of mice treated with cysteamine display mitigation of subchronic-CS induced senescence and alveolar space enlargement. The lung sections from old mice were used as a positive control for senescence and alveolar space enlargement. The data analysis of microscopy figures in left panel (A and D) is shown in the right panel (B, C, E, and F). Nuclei were stained using Hoechst dye. Scale bars, 100 µm. ****P < 0.0001.

Fig. 6.

Chronic or subchronic CS exposure impairs autophagy/proteostasis and induces inflammation in bronchoalveolar lavage fluid (BALF) cells. A: flow cytometry analysis of CD4 (cluster of differentiation 4, T cell marker) and Mac-1 (macrophage 1 antigen, macrophage marker) positive cells in BALF harvested from C57BL/6 mice exposed to chronic-CS and/or infected with PA01-GFP. Significant increase in CD4⁺ T cells and Mac-1⁺ macrophages is displayed in BALF samples collected from mice exposed to chronic-CS and/or PA01-GFP infection. BALF cells from mice treated with 0.01 M cysteamine for 10 days exhibit reduction in the number of CD4⁺ T cells and Mac-1⁺ macrophages in comparison to BALF collected from mice exposed to chronic-CS. B: flow cytometry analysis of BALF harvested from C57BL/6 mice exposed to room-air or chronic-CS and/or infected with Pa. BALF collected from mice exposed to chronic-CS or Pa displayed increased coexpression of ubiquitinated-protein (Ub) and the impaired-autophagy marker, p62. In addition, BALF collected from chronic-CS-exposed or Pa infected mice treated with 0.01 M cysteamine for 10 days attenuated induced coexpression of Ub and p62. C: flow cytometry analysis of BALF cells harvested from old (~12 mo) and room-air or subchronic-CS-exposed adult (5 mo) C57BL/6 mice. BALF collected from subchronic-CS exposed mice displays an increase in number of CD4⁺-T cells and Mac-1⁺ macrophages in comparison to room-air exposed control mice. BALF collected from subchronic-CS exposed C57BL/6 mice treated with cysteamine exhibits decreased number of subchronic-CS induced CD4⁺ T cells and Mac-1⁺ macrophages in comparison to subchronic-CS exposed murine BALF cells. Moreover, BALF cells harvested from old mice show an increase in the number of Mac-1⁺ macrophages while no change was observed in CD4⁺ T cells in BALF cells collected from room-air-exposed control.

Fig. 7.

Autophagy induction by cysteamine rescues CS-induced inflammatory responses. A: supernatants separated from BALF harvested from mice exposed to chronic-CS and/or treated with cysteamine. Chronic CS-exposed mice showed significant (****P < 0.0001) increase in the inflammatory cytokine IL-6 levels. Treatment with cysteamine restored (****P < 0.0001) IL-6 levels as observed in air-exposed or cysteamine treated mice. B: analysis of BALF samples from mice infected with PA01-GFP showed extremely significant (****P < 0.0001) increase in IL-6 levels, and cysteamine treatment significantly (****P < 0.0001) alleviated IL-6 levels as compared with PA01-GFP-infected mice. No significant change in IL-6 levels was observed in air-exposed or cysteamine treated mice. C: chronic CS-exposed mice infected with PA-01-GFP induced increase (***P < 0.001) in IL-6 levels, which was significantly reduced (***P < 0.001) upon treatment with cysteamine. D: in addition to IL-6 levels, a significant (****P < 0.0001) increase in inflammatory IL-1β was observed in chronic CS-exposed mice. Treatment with cysteamine reduced the CS-induced IL-1β levels significantly (****P < 0.0001) in comparison to air-exposed control levels. E: infection with PA01-GFP significantly (***P < 0.001) increased IL-1β levels, which were restored (***P < 0.001) back to control levels with cysteamine treatment. F: cysteamine treatment reduced (****P < 0.0001) IL-1β levels in mice exposed to chronic-CS that showed significant (****P < 0.0001) increase in IL-1β. G: significant (**P < 0.01) increase in the inflammatory cytokine IL-1β was observed in mice exposed too subchronic CS. Levels of IL-1β were reduced (**P < 0.01) upon treatment with cysteamine. Old mice (~12 mo) also showed slight elevation (**P < 0.01) in IL-1β levels. H: chronic CS-exposed mice were infected with PA01-GFP (2 × 10⁶) and/or treated with cysteamine followed by bacterial survival assay using the lung homogenate. Significantly (~4-fold) higher bacterial load (colony-forming units, CFU) was observed in mice exposed to chronic-CS (****P < 0.0001) and/or infected with PA01-GFP mice (****P < 0.0001) in comparison to the room-air controls (n = 4). Treatment with cysteamine significantly (~2-fold) reduced the bacterial load in PA01-GFP (***P < 0.001) and chronic CS-exposed mice (****P < 0.0001). I: neutrophil activity was measured by myeloperoxidase (MPO) assay in plasma samples obtained from 6 wk young and 19 mo older C57BL/6 mice. We found significant (*P < 0.028, n = 3) increases in MPO activity in aged (19 mo older) mice in comparison to adult (young) mice, indicating age-related basal changes in MPO activity. J: moreover, we found significant constitutive increases (*P = 0.01, n = 3) in caspase-3/7 activity in aged (older) mice relative to adult (young) mice, thus indicating age-related susceptibility to mortality that can be exacerbated by chronic smoking and/or infection.

Fig. 8.

Cysteamine attenuates cigarette smoke (CS)- and age-related aggresome formation. A: fluorescence microscopic analysis for detection of ubiquitinated protein, p62 and VCP in lung sections from C57BL/6 mice exposed to air or chronic CS and/or treated with a daily dose of 0.01 M cysteamine for 10 days and intranasally infected with PA01-GFP (2 × 10⁶) for 5 days as indicated. Samples collected from mice exposed to chronic CS and/or infected with PA01-GFP exhibit perinuclear localization of ubiquitinated protein, p62, and VCP within aggresome bodies. Nuclei were stained with Hoechst dye. Scale bar, 100 µm. B: fluorescence microscopic analysis for detection of ubiquitinated protein, p62, and Sirt1 in lung sections from C57BL/6 mice exposed to air or subchronic CS (2-mo old + 3 mo exposure) and/or treated with a daily dose of 0.01 M cysteamine for 10 days. Lung sections were also collected from old mice (~12 mo) as a positive control. The mice exposed to subchronic CS exhibit perinuclear colocalization of ubiquitinated protein and p62 within aggresome/autophagy bodies. The lung sections collected from old mice also display autophagy bodies and substantial (P < 0.05) decrease in Sirt1 expression similar to CS exposure. Nuclei were stained with Hoechst dye. Scale bar, 100 µm. C: fluorescence microscopic analysis for detection of ubiquitin and VCP in lung sections collected from adult (6 mo, YNG) and aged/older (19 mo, old) C57BL/6 mice. In comparison to the adult mice, lungs collected from the aged/older mice display an increase in perinuclear ubiquitin accumulation and VCP colocalization; this is indicative of an age-related increase in aggresome-formation. Nuclei were stained with Hoechst. Scale bar, 100 µm.

Fig. 9.

Schema describing tobacco-smoke-mediated proteostasis/autophagy decline as a mechanism for accelerated lung aging. Tobacco/cigarette smoke (CS) exposure and aging can induce proteostasis and autophagy impairment, serving as a central mechanism to induce inflammatory-oxidative stress. Chronic-CS-exposure-mediated steep decline in proteostasis/autophagy accelerates inflammatory-oxidative stress, apoptosis, and senescence, serving as a mechanism for accelerated lung aging and emphysema progression.