Dataset Information

Cigarette Smoke Exposure Inhibits Bacterial Killing via TFEB-Mediated Autophagy Impairment and Resulting Phagocytosis Defect.

ABSTRACT: Cigarette smoke (CS) exposure is the leading risk factor for COPD-emphysema pathogenesis. A common characteristic of COPD is impaired phagocytosis that causes frequent exacerbations in patients leading to increased morbidity. However, the underlying mechanism is unclear. Hence, we investigated if CS exposure causes autophagy impairment as a mechanism for diminished bacterial clearance via phagocytosis by utilizing murine macrophages (RAW264.7 cells) and Pseudomonas aeruginosa (PA01-GFP) as an experimental model.Briefly, RAW cells were treated with cigarette smoke extract (CSE), chloroquine (autophagy inhibitor), TFEB-shRNA, CFTR(inh)-172, and/or fisetin prior to bacterial infection for functional analysis.Bacterial clearance of PA01-GFP was significantly impaired while its survival was promoted by CSE (p < 0.01), autophagy inhibition (p < 0.05; p < 0.01), TFEB knockdown (p < 0.01; p < 0.001), and inhibition of CFTR function (p < 0.001; p < 0.01) in comparison to the control group(s) that was significantly recovered by autophagy-inducing antioxidant drug, fisetin, treatment (p < 0.05; p < 0.01; and p < 0.001). Moreover, investigations into other pharmacological properties of fisetin show that it has significant mucolytic and bactericidal activities (p < 0.01; p < 0.001), which warrants further investigation.Our data suggests that CS-mediated autophagy impairment as a critical mechanism involved in the resulting phagocytic defect, as well as the therapeutic potential of autophagy-inducing drugs in restoring is CS-impaired phagocytosis.

SUBMITTER: Pehote G

PROVIDER: S-EPMC5763241 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Cigarette Smoke Exposure Inhibits Bacterial Killing via TFEB-Mediated Autophagy Impairment and Resulting Phagocytosis Defect.

Pehote Garrett G Bodas Manish M Brucia Kathryn K Vij Neeraj N

Mediators of inflammation 20171228

<h4>Introduction</h4>Cigarette smoke (CS) exposure is the leading risk factor for COPD-emphysema pathogenesis. A common characteristic of COPD is impaired phagocytosis that causes frequent exacerbations in patients leading to increased morbidity. However, the underlying mechanism is unclear. Hence, we investigated if CS exposure causes autophagy impairment as a mechanism for diminished bacterial clearance via phagocytosis by utilizing murine macrophages (RAW264.7 cells) and <i>Pseudomonas aerugi ...[more]

PMID: 29445254

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Project description:Age related macular degeneration (AMD) is one of the leading causes of blindness. Genetics, environmental insult, and age-related factors all play a key role in altering proteostasis, the homeostatic process regulating protein synthesis, degradation and processing. These factors also play a role in the pathogenesis of AMD and it has been well established that cigarette smoking (CS) initiates AMD pathogenic mechanisms. The primary goal of this study is to elucidate whether CS can induce proteostasis/autophagy-impairment in retinal pigment epithelial (RPE) cells. In our preliminary analysis, it was found that cigarette smoke extract (CSE) induces accumulation of ubiquitinated proteins in the insoluble protein fraction (p < 0.01), which was subsequently mitigated through cysteamine (p < 0.01) or fisetin (p < 0.05) treatment. Further, it was verified that these CSE induced ubiquitinated proteins accumulated in the peri-nuclear spaces (p<0.05) that were cleared- off with cysteamine (p < 0.05) or fisetin (p < 0.05). Moreover, CSE-induced aggresome-formation (LC3B-GFP and Ub-RFP co-localization) and autophagy-flux impairment was significantly (p<0.01) mitigated by cysteamine (p<0.05) or fisetin (p<0.05) treatment, indicating the restoration of CSE-mediated autophagy-impairment. CSE treatment was also found to induce intracellular reactive oxygen species (ROS, p < 0.001) while impacting cell viability (p < 0.001), which was quantified using CMH2DCFDA-dye (ROS) and MTS (proliferation) or propodium iodide staining (cell viability) assays, respectively. Moreover, cysteamine and fisetin treatment ameliorated CS-mediated ROS production (p < 0.05) and diminished cell viability (p < 0.05). Lastly, CSE was found to induce cellular senescence (p < 0.001), which was significantly ameliorated by cysteamine (p < 0.001) or fisetin (p < 0.001). In conclusion, our study indicates that CS induced proteostasis/autophagy-impairment regulates mechanisms associated with AMD pathogenesis. Moreover, autophagy-inducing drugs such as cysteamine or fisetin can ameliorate AMD pathogenesis mechanisms that warrant further investigation in pre-clinical murine models.

Project description:AimsRecent studies have shown that cigarette smoke (CS)-induced oxidative stress impairs autophagy, resulting in aggresome-formation that correlates with severity of chronic obstructive pulmonary disease (COPD)-emphysema, although the specific step in autophagy pathway that is impaired is unknown. Hence, in this study, we aimed to evaluate the role of master autophagy transcription factor EB (TFEB) in CS-induced COPD-emphysema pathogenesis.ResultsWe first observed that TFEB accumulates in perinuclear spaces as aggresome-bodies in COPD lung tissues of tobacco smokers and severe emphysema subjects, compared with non-emphysema or nonsmoker controls. Next, Beas2b cells and C57BL/6 mice were exposed to either cigarette smoke extract (CSE) or subchronic-CS (sc-CS), followed by treatment with potent TFEB-inducing drug, gemfibrozil (GEM, or fisetin as an alternate), to experimentally verify the role of TFEB in COPD. Our in vitro results indicate that GEM/fisetin-mediated TFEB induction significantly (p?<?0.05) decreases CSE-induced autophagy-impairment (Ub/LC3B reporter and autophagy flux assay) and resulting aggresome-formation (Ub/p62 coexpression/accumulation; immunoblotting and staining) by controlling reactive oxygen species (ROS) activity. Intriguingly, we observed that CS induces TFEB accumulation in the insoluble protein fractions of Beas2b cells, which shows a partial rescue with GEM treatment. Moreover, TFEB knockdown induces oxidative stress, autophagy-impairment, and senescence, which can all be mitigated by GEM-mediated TFEB induction. Finally, in vivo studies were used to verify that CS-induced autophagy-impairment (increased Ub, p62, and valosin-containing protein in the insoluble protein fractions of lung/cell lysates), inflammation (interleukin-6 [IL-6] levels in bronchoalveolar lavage fluid and iNOS expression in lung sections), apoptosis (caspase-3/7), and resulting emphysema (hematoxylin and eosin [H&E]) can be controlled by GEM-mediated TFEB induction (p?<?0.05).InnovationCS exposure impairs autophagy in COPD-emphysema by inducing perinuclear localization of master autophagy regulator, TFEB, to aggresome-bodies.ConclusionTFEB-inducing drug(s) can control CS-induced TFEB/autophagy-impairment and COPD-emphysema pathogenesis. Antioxid. Redox Signal. 27, 150-167.

Project description:Introduction/rationaleIn chronic obstructive pulmonary disease (COPD), defective macrophage phagocytic clearance of cells undergoing apoptosis by efferocytosis may lead to secondary necrosis of the uncleared cells and contribute to airway inflammation. The precise mechanisms for this phenomenon remain unknown. LC3-associated phagocytosis (LAP) is indispensable for effective efferocytosis. We hypothesized that cigarette smoke inhibits the regulators of LAP pathway, potentially contributing to the chronic airways inflammation associated with COPD.MethodsBronchoalveolar (BAL)-derived alveolar macrophages, lung tissue macrophages obtained from lung resection surgery, and monocyte-derived macrophages (MDM) were prepared from COPD patients and control participants. Lung/airway samples from mice chronically exposed to cigarette smoke were also investigated. Differentiated THP-1 cells were exposed to cigarette smoke extract (CSE). The LAP pathway including Rubicon, as an essential regulator of LAP, efferocytosis and inflammation was examined using western blot, ELISA, flow cytometry, and/or immunofluorescence.ResultsRubicon was significantly depleted in COPD alveolar macrophages compared with non-COPD control macrophages. Rubicon protein in alveolar macrophages of cigarette smoke-exposed mice and cigarette smoke-exposed MDM and THP-1 was decreased with a concomitant impairment of efferocytosis. We also noted increased expression of LC3 which is critical for LAP pathway in COPD and THP-1 macrophages. Furthermore, THP-1 macrophages exposed to cigarette smoke extract exhibited higher levels of other key components of LAP pathway including Atg5 and TIM-4. There was a strong positive correlation between Rubicon protein expression and efferocytosis.ConclusionLAP is a requisite for effective efferocytosis and an appropriate inflammatory response, which is impaired by Rubicon deficiency. Our findings suggest dysregulated LAP due to reduced Rubicon as a result of CSE exposure. This phenomenon could lead to a failure of macrophages to effectively process phagosomes containing apoptotic cells during efferocytosis. Restoring Rubicon protein expression has unrecognized therapeutic potential in the context of disease-related modifications caused by exposure to cigarette smoke.

Project description:IntroductionCigarette smoking adversely affects multiple aspects of human health including eye disorders such as age-related macular degeneration, cataracts and dry eye disease. However, there remains a knowledge gap in how constituents of cigarette smoke affect vision and retinal biology. We used zebrafish to assess effects of short-term acute exposure to cigarette smoke extract (CSE) on visual behaviour and retinal biology.MethodsZebrafish larvae with a developed visual system at three days post-fertilization (dpf) were exposed to CSE for 4, 24 or 48 hours. Visual behaviour, hyaloid vasculature morphology, retinal histology, oxidative stress gene expression and outer segment phagocytosis were investigated using visual behavioural optokinetic and visual motor response assays (OKR and VMR), microscopy (light, fluorescence and transmission electron microscopy), and real-time PCR.ResultsIn zebrafish larvae, 48 hours of CSE treatment resulted in significantly reduced visual behaviour. Larvae treated with 10, 15 or 20 μg/mL CSE showed an average of 13.7, 10.7 or 9.4 saccades per minute, respectively, significantly lower compared with 0.05% DMSO controls (p=0.0093, p=0.0004 and p<0.0001, respectively) that exhibited 19.7 saccades per minute. The diameter of intraocular vessels increased from 4.833 μm in 0.05% DMSO controls to 5.885 μm in the 20 μg/mL CSE-treated larvae (p=0.0333). Biometry analysis highlighted a significant axial length elongation in 20 μg/mL CSE-treated larvae (216.9 μm, p<0.0001) compared to 0.05% dimethyl sulfoxide (DMSO) controls (205.1 μm). Larvae exposed to 20 μg/mL CSE had significantly (p=0.0002) higher numbers of RPE phagosomes compared to vehicle controls (0.1425 and 0.093 phagosomes/μm RPE, respectively).ConclusionsZebrafish larvae with a developed visual system display apparent defects in visual behaviour and retinal biology after acute exposure to CSE, establishing a valuable in vivo model to investigate ocular disorders related to cigarette smoke.

Dataset Information

Cigarette Smoke Exposure Inhibits Bacterial Killing via TFEB-Mediated Autophagy Impairment and Resulting Phagocytosis Defect.

Publications

Cigarette Smoke Exposure Inhibits Bacterial Killing via TFEB-Mediated Autophagy Impairment and Resulting Phagocytosis Defect.

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