TLR and NKG2D Signaling Pathways Mediate CS-Induced Pulmonary Pathologies

Long-term exposure to cigarette smoke (CS) can have deleterious effects on lung epithelial cells including cell death and the initiation of inflammatory responses. CS-induced cell injury can elaborate cell surface signals and cellular byproducts that stimulate immune system surveillance. Our previous work has shown that the expression of ligands for the cytotoxic lymphocyte activating receptor NKG2D is enhanced in patients with COPD and that the induction of these ligands in a mouse model can replicate COPD pathologies. Here, we extend these findings to demonstrate a role for the NKG2D receptor in CS-induced pathophysiology and provide evidence linking nucleic acid-sensing endosomal toll-like receptor (TLR) signaling to COPD pathology through NKG2D activation. Specifically, we show that mice deficient in NKG2D exhibit attenuated pulmonary inflammation and airspace enlargement in a model of CS-induced emphysema. Additionally, we show that CS exposure induces the release of free nucleic acids in the bronchoalveolar lavage and that direct exposure of mouse lung epithelial cells to cigarette smoke extract similarly induces functional nucleic acids as assessed by TLR3, 7, and 9 reporter cell lines. We demonstrate that exposure of mouse lung epithelial cells to TLR ligands stimulates the surface expression of RAET1, a ligand for NKG2D, and that mice deficient in TLR3/7/9 receptor signaling do not exhibit CS-induced NK cell hyperresponsiveness and airspace enlargement. The findings indicate that CS-induced airway injury stimulates TLR signaling by endogenous nucleic acids leading to elevated NKG2D ligand expression. Activation of these pathways plays a major role in the altered NK cell function, pulmonary inflammation and remodeling related to long-term CS exposure.     

P2X7 receptor and caspase 1 activation are central to airway inflammation observed after exposure to tobacco smoke

Chronic Obstructive Pulmonary Disease (COPD) is a cigarette smoke (CS)-driven inflammatory airway disease with an increasing global prevalence. Currently there is no effective medication to stop the relentless progression of this disease. It has recently been shown that an activator of the P2X7/inflammasome pathway, ATP, and the resultant products (IL-1β/IL-18) are increased in COPD patients. The aim of this study was to determine whether activation of the P2X7/caspase 1 pathway has a functional role in CS-induced airway inflammation. Mice were exposed to CS twice a day to induce COPD-like inflammation and the role of the P2X7 receptor was investigated. We have demonstrated that CS-induced neutrophilia in a pre-clinical model is temporally associated with markers of inflammasome activation, (increased caspase 1 activity and release of IL-1β/IL-18) in the lungs. A selective P2X7 receptor antagonist and mice genetically modified so that the P2X7 receptors were non-functional attenuated caspase 1 activation, IL-1β release and airway neutrophilia. Furthermore, we demonstrated that the role of this pathway was not restricted to early stages of disease development by showing increased caspase 1 activation in lungs from a more chronic exposure to CS and from patients with COPD. This translational data suggests the P2X7/Inflammasome pathway plays an ongoing role in disease pathogenesis. These results advocate the critical role of the P2X7/caspase 1 axis in CS-induced inflammation, highlighting this as a possible therapeutic target in combating COPD.     

Airway Surface Dehydration Aggravates Cigarette Smoke-Induced Hallmarks of COPD in Mice

Introduction

Airway surface dehydration, caused by an imbalance between secretion and absorption of ions and fluid across the epithelium and/or increased epithelial mucin secretion, impairs mucociliary clearance. Recent evidence suggests that this mechanism may be implicated in chronic obstructive pulmonary disease (COPD). However, the role of airway surface dehydration in the pathogenesis of cigarette smoke (CS)-induced COPD remains unknown.

Objective

We aimed to investigate in vivo the effect of airway surface dehydration on several CS-induced hallmarks of COPD in mice with airway-specific overexpression of the β-subunit of the epithelial Na⁺ channel (βENaC).

Methods

βENaC-Tg mice and wild-type (WT) littermates were exposed to air or CS for 4 or 8 weeks. Pathological hallmarks of COPD, including goblet cell metaplasia, mucin expression, pulmonary inflammation, lymphoid follicles, emphysema and airway wall remodelling were determined and lung function was measured.

Results

Airway surface dehydration in βENaC-Tg mice aggravated CS-induced airway inflammation, mucin expression and destruction of alveolar walls and accelerated the formation of pulmonary lymphoid follicles. Moreover, lung function measurements demonstrated an increased compliance and total lung capacity and a lower resistance and hysteresis in βENaC-Tg mice, compared to WT mice. CS exposure further altered lung function measurements.

Conclusions

We conclude that airway surface dehydration is a risk factor that aggravates CS-induced hallmarks of COPD.     

Cigarette smoke induces PTX3 expression in pulmonary veins of mice in an IL-1 dependent manner

Background

Chronic obstructive pulmonary disease (COPD) is associated with abnormal inflammatory responses and structural alterations of the airways, lung parenchyma and pulmonary vasculature. Since Pentraxin-3 (PTX3) is a tuner of inflammatory responses and is produced by endothelial and inflammatory cells upon stimuli such as interleukin-1β (IL-1β), we hypothesized that PTX3 is involved in COPD pathogenesis.

Methods and Results

We evaluated whether cigarette smoke (CS) triggers pulmonary and systemic PTX3 expression in vivo in a murine model of COPD. Using immunohistochemical (IHC) staining, we observed PTX3 expression in endothelial cells of lung venules and veins but not in lung arteries, airways and parenchyma. Moreover, ELISA on lung homogenates and semi-quantitative scoring of IHC-stained sections revealed a significant upregulation of PTX3 upon subacute and chronic CS exposure. Interestingly, PTX3 expression was not enhanced upon subacute CS exposure in IL-1RI KO mice, suggesting that the IL-1 pathway is implicated in CS-induced expression of vascular PTX3. Serum PTX3 levels increased rapidly but transiently after acute CS exposure.To elucidate the functional role of PTX3 in CS-induced responses, we examined pulmonary inflammation, protease/antiprotease balance, emphysema and body weight changes in WT and Ptx3 KO mice. CS-induced pulmonary inflammation, peribronchial lymphoid aggregates, increase in MMP-12/TIMP-1 mRNA ratio, emphysema and failure to gain weight were not significantly different in Ptx3 KO mice compared to WT mice. In addition, Ptx3 deficiency did not affect the CS-induced alterations in the pulmonary (mRNA and protein) expression of VEGF-A and FGF-2, which are crucial regulators of angiogenesis.

Conclusions

CS increases pulmonary PTX3 expression in an IL-1 dependent manner. However, our results suggest that either PTX3 is not critical in CS-induced pulmonary inflammation, emphysema and body weight changes, or that its role can be fulfilled by other mediators with overlapping activities.     

Cigarette smoke induces the release of CXCL-8 from human bronchial epithelial cells via TLRs and induction of the inflammasome

COPD is a chronic airway disease associated with inflammation and cigarette smoking. Airway epithelial cells are the first cells exposed to cigarette smoke (CS) and can release CXCL-8 and IL-1β. These cytokines are involved in acute and chronic inflammatory processes in COPD. The aim of this study was to investigate whether toll-like receptors (TLRs) located in/on epithelial cells were involved in cigarette smoke-induced cytokine production. Here we demonstrate that CS induces the release of CXCL-8 and IL-1β from human bronchial epithelial cells (HBE-14o). CS-induced CXCL-8 production was inhibited by an antibody against TLR4 and by inhibitory ODN suggesting the involvement of TLR4 and TLR9. In addition, exposure of HBE-14o cells to TLR4 or TLR9 ligands resulted in the release of CXCL-8 and IL1β. TLR4 and also TLR9 were present on the cell surface and the expression of both receptors decreased after CS exposure. The molecular mechanism of the CS-induced CXCL-8 production by the epithelial cells was further investigated. It was found that P2X7 receptors and reactive oxygen species were involved. Interestingly, the inflammasome activator monosodium urate crystals (MSU) induced the release of CXCL-8 and IL-1β and the caspase-1 inhibitor Z-VADDCB suppressed the CS-induced release of CXCL-8. In addition, CS, CpGODN, lipopolysaccharide and MSU all increased the expression of caspase-1 and IL-1β. In conclusion, our results demonstrate that CS releases CXCL-8 from HBE-14o cells via TLR4 and TLR9 and inflammasome activation. Therefore, inflammasome signaling in airway epithelial cells may play an important role in pathogenesis of diseases like COPD.     

Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells

Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression.     

Exacerbation of cigarette smoke-induced pulmonary inflammation by Staphylococcus aureus Enterotoxin B in mice

Background

Cigarette smoke (CS) is a major risk factor for the development of COPD. CS exposure is associated with an increased risk of bacterial colonization and respiratory tract infection, because of suppressed antibacterial activities of the immune system and delayed clearance of microbial agents from the lungs. Colonization with Staphylococcus aureus results in release of virulent enterotoxins, with superantigen activity which causes T cell activation.

Objective

To study the effect of Staphylococcus aureus enterotoxin B (SEB) on CS-induced inflammation, in a mouse model of COPD.

Methods

C57/Bl6 mice were exposed to CS or air for 4 weeks (5 cigarettes/exposure, 4x/day, 5 days/week). Endonasal SEB (10 μg/ml) or saline was concomitantly applied starting from week 3, on alternate days. 24 h after the last CS and SEB exposure, mice were sacrificed and bronchoalveolar lavage (BAL) fluid and lung tissue were collected.

Results

Combined exposure to CS and SEB resulted in a raised number of lymphocytes and neutrophils in BAL, as well as increased numbers of CD8⁺ T lymphocytes and granulocytes in lung tissue, compared to sole CS or SEB exposure. Moreover, concomitant CS/SEB exposure induced both IL-13 mRNA expression in lungs and goblet cell hyperplasia in the airway wall. In addition, combined CS/SEB exposure stimulated the formation of dense, organized aggregates of B- and T- lymphocytes in lungs, as well as significant higher CXCL-13 (protein, mRNA) and CCL19 (mRNA) levels in lungs.

Conclusions

Combined CS and SEB exposure aggravates CS-induced inflammation in mice, suggesting that Staphylococcus aureus could influence the pathogenesis of COPD.     

Attenuation of Cigarette Smoke-Induced Airway Mucus Production by Hydrogen-Rich Saline in Rats

Background

Over-production of mucus is an important pathophysiological feature in chronic airway disease such as chronic obstructive pulmonary disease (COPD) and asthma. Cigarette smoking (CS) is the leading cause of COPD. Oxidative stress plays a key role in CS-induced airway abnormal mucus production. Hydrogen protected cells and tissues against oxidative damage by scavenging hydroxyl radicals. In the present study we investigated the effect of hydrogen on CS-induced mucus production in rats.

Methods

Male Sprague-Dawley rats were divided into four groups: sham control, CS group, hydrogen-rich saline pretreatment group and hydrogen-rich saline control group. Lung morphology and tissue biochemical changes were determined by immunohistochemistry, Alcian Blue/periodic acid-Schiff staining, TUNEL, western blot and realtime RT-PCR.

Results

Hydrogen-rich saline pretreatment attenuated CS-induced mucus accumulation in the bronchiolar lumen, goblet cell hyperplasia, muc5ac over-expression and abnormal cell apoptosis in the airway epithelium as well as malondialdehyde increase in the BALF. The phosphorylation of EGFR at Tyr1068 and Nrf2 up-regulation expression in the rat lungs challenged by CS exposure were also abrogated by hydrogen-rich saline.

Conclusion

Hydrogen-rich saline pretreatment ameliorated CS-induced airway mucus production and airway epithelium damage in rats. The protective role of hydrogen on CS-exposed rat lungs was achieved at least partly by its free radical scavenging ability. This is the first report to demonstrate that intraperitoneal administration of hydrogen-rich saline protected rat airways against CS damage and it could be promising in treating abnormal airway mucus production in COPD.     

Clearance of senescent cells reverts the cigarette smoke-induced lung senescence and airspace enlargement in p16-3MR mice

Cigarette smoke (CS) leads to increased oxidative stress, inflammation, and exaggerated senescence, which are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). While the role of cellular senescence in COPD is known, it is not clear if the removal of senescent cells could alleviate the disease symptoms. To test this, we used the novel mouse model-p16-3MR, and studied the effect of ganciclovir (GCV)-mediated removal of senescent cells after chronic CS (3 months) and environmental tobacco smoke (ETS) (6 months) exposure to CS. Our results showed the reversal of CS-induced cellular senescence on the clearance of p16⁺ senesced cells by GCV treatment. Interestingly, the clearance of p16⁺ senescent cells via GCV led to a decrease in the neutrophil counts in the BALF of GCV-treated CS-exposed p16-3MR mice, as well as reversal of CS-mediated airspace enlargement in p16-3MR mice. Mice exposed to low dose ETS caused insignificant changes in the SA-β-Gal⁺ senescent cells and airspace enlargement. Overall, our data provide evidence for the role of lung cellular senescence on smoke exposure and clearance of senescent cells in p16-3MR mice in the reversal of COPD/emphysema pathology with a possibility of senolytics as therapeutic interventions in COPD.     

Long non‐coding RNA TUG1 promotes airway remodelling by suppressing the miR‐145‐5p/DUSP6 axis in cigarette smoke‐induced COPD

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease that is primarily caused by cigarette smoke (CS)‐induced chronic inflammation. In this study, we investigated the function and mechanism of action of the long non‐coding RNA (lncRNA) taurine‐up‐regulated gene 1 (TUG1) in CS‐induced COPD. We found that the expression of TUG1 was significantly higher in the sputum cells and lung tissues of patients with COPD as compared to that in non‐smokers, and negatively correlated with the percentage of predicted forced expiratory volume in 1 second. In addition, up‐regulation of TUG1 was observed in CS‐exposed mice, and knockdown of TUG1 attenuated inflammation and airway remodelling in a mouse model. Moreover, TUG1 expression was higher in CS extract (CSE)‐treated human bronchial epithelial cells and lung fibroblasts, whereas inhibition of TUG1 reversed CSE‐induced inflammation and collagen deposition in vitro. Mechanistically, TUG1 promoted the expression of dual‐specificity phosphatase 6 (DUSP6) by sponging miR‐145‐5p. DUSP6 overexpression reversed TUG1 knockdown‐mediated inhibition of inflammation and airway remodelling. These findings suggested an important role of TUG1 in the pathological alterations associated with CS‐mediated airway remodelling in COPD. Thus, TUG1 may be a promising therapeutic target in CS‐induced airway inflammation and fibroblast activation.     

Unfolded protein response in chronic obstructive pulmonary disease: smoking, aging and disease: a SAD trifecta

Cigarette smoke (CS) is a risk factor for the development of chronic obstructive pulmonary disease (COPD). Oxidative stress is an immediate result of CS exposure and has the ability to modify cellular proteins. The endoplasmic reticulum (ER) is a compartment where early steps of synthesis and folding of membrane and secretory proteins takes place. Oxidative stress has been shown to interfere with protein folding in the ER and elicits the unfolded protein response (UPR). The UPR is a massive endoplasmic reticulum to the nucleus and the cellular kinase cascades signaling pathway. The UPR triggers a series of intracellular events that aim to help cells overcome the consequences of the stress or eliminate rogue cells by altering expression of genes involved in anti-oxidant defense, cell cycle progression, inflammation, and apoptosis. Recent data demonstrate that CS induces the UPR in vitro and in vivo. The timing of UPR induction in smokers and the mechanism of CS-induced UPR are areas of active investigation. The role of UPR in the protection of smoker's lungs from CS-induced oxidative stress, and its contribution to CS-induced apoptosis and inflammation, is beginning to emerge. This review discusses recent data about UPR in COPD and summarizes findings on UPR that have potential relevance to COPD.     

“Ciliophagy”

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) associated with respiratory epithelial cell cilia shortening and impaired mucociliary clearance (MCC). The underlying cellular and molecular mechanisms for CS-associated cilia shortening have remained incompletely understood. We have previously demonstrated increased autophagy in the lungs of COPD patients; however, whether or not this process is selective for specific autophagic targets in the lung was not elucidated. Based on observations that increased morphological and biochemical indicators of autophagy correlate with cilia shortening in our models, we posited that autophagy might regulate cilia length in response to CS in the lung. We demonstrate that CS-induced cilia shortening occurs through an autophagy-dependent mechanism mediated by the deacetylase HDAC6 (histone deacetylase 6). Autophagy-impaired (Becn1^+/?, map1lc3b^?/?, or Hdac6^-/Y) mice resist CS-induced cilia shortening. Furthermore, cilia components are identified as autophagic substrates during CS exposure. Assessment of airway cilia function using a 3D MCC assay demonstrates that Becn1^+/?, map1lc3b^?/?, and Hdac6^-/Y mice or mice injected with the HDAC6 inhibitor tubastatin A are protected from CS-associated mucociliary dysfunction. We concluded that an autophagy-dependent pathway regulates cilia length during CS exposure, which identifies new pathways and targets in COPD.     

“Ciliophagy”: The consumption of cilia components by autophagy

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) associated with respiratory epithelial cell cilia shortening and impaired mucociliary clearance (MCC). The underlying cellular and molecular mechanisms for CS-associated cilia shortening have remained incompletely understood. We have previously demonstrated increased autophagy in the lungs of COPD patients; however, whether or not this process is selective for specific autophagic targets in the lung was not elucidated. Based on observations that increased morphological and biochemical indicators of autophagy correlate with cilia shortening in our models, we posited that autophagy might regulate cilia length in response to CS in the lung. We demonstrate that CS-induced cilia shortening occurs through an autophagy-dependent mechanism mediated by the deacetylase HDAC6 (histone deacetylase 6). Autophagy-impaired (Becn1^+/−, map1lc3b^−/−, or Hdac6^-/Y) mice resist CS-induced cilia shortening. Furthermore, cilia components are identified as autophagic substrates during CS exposure. Assessment of airway cilia function using a 3D MCC assay demonstrates that Becn1^+/−, map1lc3b^−/−, and Hdac6^-/Y mice or mice injected with the HDAC6 inhibitor tubastatin A are protected from CS-associated mucociliary dysfunction. We concluded that an autophagy-dependent pathway regulates cilia length during CS exposure, which identifies new pathways and targets in COPD.     

Cigarette smoke-induced pulmonary inflammation and emphysema are attenuated in CCR6-deficient mice

Chronic obstructive pulmonary disease (COPD) is mainly caused by cigarette smoking, and is characterized by an increase in inflammatory cells in the airways and pulmonary tissue. The chemokine receptor CCR6 and its ligand MIP-3alpha/CCL20 may be involved in the recruitment of these inflammatory cells. To investigate the role of CCR6 in the pathogenesis of COPD, we analyzed the inflammatory responses of CCR6 knockout (KO) and wild-type mice upon cigarette smoke (CS) exposure. Both subacute and chronic exposure to CS induced an increase in cells of the innate and adaptive immune system in the bronchoalveolar lavage, both in CCR6 KO and wild-type mice. However, the accumulation of dendritic cells, neutrophils, and T lymphocytes, which express CCR6, was significantly attenuated in the CCR6 KO mice, compared with their wild-type littermates. In the lung tissue of CCR6 KO mice, there was an impaired increase in dendritic cells, activated CD8(+) T lymphocytes, and granulocytes. Moreover, this attenuated inflammatory response in CCR6 KO mice offered a partial protection against pulmonary emphysema, which correlated with an impaired production of MMP-12. Importantly, protein levels of MIP-3alpha/CCL20, the only chemokine ligand of the CCR6 receptor, and MCP-1/CCL2 were significantly increased upon CS exposure in wild-type, but not in CCR6 KO mice. In contrast, CCR6 deficiency had no effect on the development of airway wall remodeling upon chronic CS exposure. These results indicate that the interaction of CCR6 with its ligand MIP-3alpha contributes to the pathogenesis of CS-induced pulmonary inflammation and emphysema in this murine model of COPD.     

Cigarette smoke-induced pulmonary inflammation is attenuated in CD69-deficient mice

Cluster of differentiation 69 (CD69) has been identified as a lymphocyte early activation marker, and recent studies have indicated that CD69 mediates intracellular signals and plays an important role in various inflammatory diseases. Cigarette smoke (CS) is a strong proinflammatory stimulus that induces the release of proinflammatory mediators by recruiting macrophages and neutrophils into the lung tissue, and is one of the main risk factors for a number of chronic diseases. However, the potential role of CD69 in CS-induced pulmonary inflammation has not been determined. To address to this question, CD69-deficient (KO) and wild-type (WT) mice were subjected to CS-induced acute pulmonary inflammation. After the exposure with CS, the expression of CD69 in the lung of WT mice was significantly induced, it was predominantly observed in macrophages. In conjunction with this phenomenon, neutrophil and macrophage cell counts, and expression of several cytokines were significantly higher in the bronchoalveolar lavage fluid (BALF) of CS-exposed WT mice compared with air-exposed WT mice. Likewise, the CS-induced accumulation of inflammatory cells and cytokines expression were significantly lower in CD69-KO mice than in WT mice. These results suggest that CD69 on macrophages is involved in CS-induced acute pulmonary inflammation.     

Cigarette smoke-induced pulmonary inflammation is attenuated in CD69-deficient mice

Cluster of differentiation 69 (CD69) has been identified as a lymphocyte early activation marker, and recent studies have indicated that CD69 mediates intracellular signals and plays an important role in various inflammatory diseases. Cigarette smoke (CS) is a strong proinflammatory stimulus that induces the release of proinflammatory mediators by recruiting macrophages and neutrophils into the lung tissue, and is one of the main risk factors for a number of chronic diseases. However, the potential role of CD69 in CS-induced pulmonary inflammation has not been determined. To address to this question, CD69-deficient (KO) and wild-type (WT) mice were subjected to CS-induced acute pulmonary inflammation. After the exposure with CS, the expression of CD69 in the lung of WT mice was significantly induced, it was predominantly observed in macrophages. In conjunction with this phenomenon, neutrophil and macrophage cell counts, and expression of several cytokines were significantly higher in the bronchoalveolar lavage fluid (BALF) of CS-exposed WT mice compared with air-exposed WT mice. Likewise, the CS-induced accumulation of inflammatory cells and cytokines expression were significantly lower in CD69-KO mice than in WT mice. These results suggest that CD69 on macrophages is involved in CS-induced acute pulmonary inflammation.     

Critical role of CFTR-dependent lipid rafts in cigarette smoke-induced lung epithelial injury

Apoptosis of lung epithelial and endothelial cells by exposure to cigarette smoke (CS) severely damages the lung tissue, leading to the pathogenesis of emphysema, but the underlying mechanisms are poorly understood. We have recently established a direct correlation between decreased lipid raft CFTR expression and emphysema progression through increased ceramide accumulation. In the present work, we investigated the role of membrane CFTR in regulating apoptosis and autophagy responses to CS exposure. We report a constitutive and CS-induced increase in the number of TUNEL-positive apoptotic cells in Cftr(-/-) murine lungs compared with Cftr(+/+) murine lungs that also correlated with a concurrent increase in the expression of ceramide, NF-κB, CD95/Fas, lipid raft proteins, and zonula occludens (ZO)-1/2 (P < 0.001). We also verified that stable wild-type CFTR expression in CFBE41o(-) cells controls constitutively elevated caspase-3/7 activity (-1.6-fold, P < 0.001). Our data suggest that membrane CFTR regulates ceramide-enriched lipid raft signaling platforms required for the induction of Fas-mediated apoptotic signaling. In addition, lack of membrane CFTR also modulates autophagy, as demonstrated by the significant increase in constitutive (P < 0.001) and CSE-induced (P < 0.005) perinuclear accumulation of green fluorescent protein-microtubule-associated protein 1 light chain-3 (LC3) in the absence of membrane CFTR (CFBE41o(-) cells). The significant constitutive and CS-induced increase (P < 0.05) in p62 and LC3β expression in CFTR-deficient cells and mice corroborates these findings and suggest a defective autophagy response in the absence of membrane CFTR. Our data demonstrate the critical role of membrane-localized CFTR in regulating apoptotic and autophagic responses in CS-induced lung injury that may be involved in the pathogenesis of severe emphysema.