SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells.

Inflammation and ageing are intertwined in chronic obstructive pulmonary disease (COPD). The histone deacetylase SIRT1 and the related activation of FoxO3 protect from ageing and regulate inflammation. The role of SIRT1/FoxO3 in COPD is largely unknown. This study evaluated whether cigarette smoke, by modulating the SIRT1/FoxO3 axis, affects airway epithelial pro-inflammatory responses. Human bronchial epithelial cells (16HBE) and primary bronchial epithelial cells (PBECs) from COPD patients and controls were treated with/without cigarette smoke extract (CSE), Sirtinol or FoxO3 siRNA. SIRT1, FoxO3 and NF-κB nuclear accumulation, SIRT1 deacetylase activity, IL-8 and CCL20 expression/release and the release of 12 cytokines, neutrophil and lymphocyte chemotaxis were assessed. In PBECs, the constitutive FoxO3 expression was lower in patients with COPD than in controls. Furthermore, CSE reduced FoxO3 expression only in PBECs from controls. In 16HBE, CSE decreased SIRT1 activity and nuclear expression, enhanced NF-κB binding to the IL-8 gene promoter thus increasing IL-8 expression, decreased CCL20 expression, increased the neutrophil chemotaxis and decreased lymphocyte chemotaxis. Similarly, SIRT1 inhibition reduced FoxO3 expression and increased nuclear NF-κB. FoxO3 siRNA treatment increased IL-8 and decreased CCL20 expression in 16HBE. In conclusion, CSE impairs the function of SIRT1/FoxO3 axis in bronchial epithelium, dysregulating NF-κB activity and inducing pro-inflammatory responses.

Di Vincenzo S ，Heijink IH ，Noordhoek JA ，Cipollina C ，Siena L ，Bruno A ，Ferraro M ，Postma DS ，Gjomarkaj M ，Pace E ... -  《-》

Exogenous neutrophil elastase enters bronchial epithelial cells and suppresses cigarette smoke extract-induced heme oxygenase-1 by cleaving sirtuin 1.

An imbalance between oxidative stress and antioxidant activity plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke, a major risk factor of COPD, induces cellular oxidative stress, but levels of antioxidants such as heme oxygenase-1 (HO-1) are reduced in individuals with severe COPD. In this study, we evaluated the molecular mechanism of reduced HO-1 expression in human bronchial epithelial cells. We found that cigarette smoke extract (CSE) increases HO-1 levels via activation of NFE2-related factor 2 (Nrf2). However, pretreating cells with the protease neutrophil elastase (NE) suppressed the CSE-induced expression of HO-1 mRNA and protein. NE also decreased the sirtuin 1 (SIRT1) level, but did not inhibit CSE-induced nuclear translocation and DNA-binding activity of Nrf2. Transfection of cells with a Myc/His-tagged SIRT1 expression vector completely blocked the NE-mediated suppression of HO-1 expression. We further noted that the NE-induced down-regulation of SIRT1 was not due to decreased transcription or proteasomal/lysosomal degradation or loss of solubility. Immunofluorescence staining revealed that NE enters the cell cytoplasm, and we observed that NE directly cleaved SIRT1 in vitro, indicating that SIRT1 levels are decreased via direct degradation by internalized NE. Of note, we observed decreased SIRT1 levels in NE-treated primary human bronchial epithelial cells and in lung homogenates from both smokers and patients with COPD. In conclusion, NE suppresses CSE-induced HO-1 expression by cleaving SIRT1. This finding indicates the importance of cross-talk between oxidative stress and protease responses in the pathogenesis of COPD.

Lee KH ，Jeong J ，Koo YJ ，Jang AH ，Lee CH ，Yoo CG ... -  《-》

Carbocysteine counteracts the effects of cigarette smoke on cell growth and on the SIRT1/FoxO3 axis in bronchial epithelial cells.

Cigarette smoke may accelerate cellular senescence by increasing oxidative stress. Altered proliferation and altered expression of anti-aging factors, including SIRT1 and FoxO3, characterise cellular senescence. The effects of carbocysteine on the SIRT1/FoxO3 axis and on downstream molecular mechanisms in human bronchial epithelial cells exposed to cigarette smoke are largely unknown. Aim of this study was to explore whether carbocysteine modulated SIRT1/FoxO3 axis, and downstream molecular mechanisms associated to cellular senescence, in a bronchial epithelial cell line (16-HBE) exposed to cigarette smoke. 16HBE cells were stimulated with/without cigarette smoke extracts (CSE) and carbocysteine. Flow cytometry and clonogenic assay were used to assess cell proliferation; western blot analysis was used for assessing nuclear expression of SIRT1 and FoxO3. The nuclear co-localization of SIRT1 and FoxO3 was assessed by fluorescence microscopy. Beta galactosidase (a senescence marker) and SIRT1 activity were assessed by specific staining and colorimetric assays, respectively. ChiP Assay and flow cytometry were used for assessing survivin gene regulation and protein expression, respectively. CSE decreased cell proliferation, the nuclear expression of SIRT1 and FoxO3 and increased beta galactosidase staining. CSE, reduced SIRT1 activity and FoxO3 localization on survivin promoter thus increasing survivin expression. In CSE stimulated bronchial epithelial cells carbocysteine reverted these phenomena by increasing cell proliferation, and SIRT1 and FoxO3 nuclear expression, and by reducing beta galactosidase staining and survivin expression. The study shows for the first time that carbocysteine may revert some senescence processes induced by oxidative stress due to cigarette smoke exposure.

Pace E ，Di Vincenzo S ，Ferraro M ，Bruno A ，Dino P ，Bonsignore MR ，Battaglia S ，Saibene F ，Lanata L ，Gjomarkaj M ... -  《-》

Down-regulated peroxisome proliferator-activated receptor γ (PPARγ) in lung epithelial cells promotes a PPARγ agonist-reversible proinflammatory phenotype in chronic obstructive pulmonary disease (COPD).

Lakshmi SP ，Reddy AT ，Zhang Y ，Sciurba FC ，Mallampalli RK ，Duncan SR ，Reddy RC ... -  《-》

Resveratrol attenuates cigarette smoke extract induced cellular senescence in human airway epithelial cells by regulating the miR-34a/SIRT1/NF-κB pathway.

Chronic obstructive pulmonary disease (COPD) is characterized by accelerated lung aging. Smoking is the critical risk factor for COPD. Cellular senescence of airway epithelial cells is the cytological basis of accelerated lung aging in COPD, and the regulation of microRNAs (miRNAs) is the central epigenetic mechanism of cellular senescence. Resveratrol (Res) is a polyphenol with anti-aging properties. This study investigated whether Res attenuates cigarette smoke extract (CSE)-induced cellular senescence in human airway epithelial cells (BEAS-2B) through the miR-34a/SIRT1/nuclear factor-kappaB (NF-κB) pathway. BEAS-2B cells were treated with Res, CSE and transfected with miR-34a-5p mimics. Cellular senescence was evaluated by senescence -related β-galactosidase (SA-β-gal) staining and expression of senescence-related genes (p16, p21, and p53). The expressions of miR-34a-5p, SIRT1, and NF-κB p65 were examined using quantitative real time polymerase chain reaction and western blotting. The senescence-associated secretory phenotype (SASP) cytokines (IL-1β, IL-6, IL-8, TNF-α) were assessed by enzyme-linked immunosorbent assay. The binding between miR-34a-5p and SIRT1 was confirmed by dual-luciferase reporter assay. The results showed that CSE dose-dependently decreased cell viability and elevated cellular senescence, characterized by increased SA-β-gal staining and senescence-related gene expressions (p16, p21, and p53). Further, CSE dose-dependently increased the expression of miR-34a-5p and SASP cytokines (IL-1β, IL-6, IL-8, TNF-α) in BEAS-2B cells. Pretreatment with Res inhibited CSE-induced cellular senescence and secretion of SASP cytokines (IL-1β, IL-6, IL-8, TNF-α) in a dose-dependent manner. Moreover, Res reversed the CSE-induced down-regulation of SIRT1 and up-regulation of miR-34a-5p and NF-κB p65. SIRT1 is a target of miR-34a-5p. Overexpression of miR-34a-5p via transfection with miR-34a-5p mimic in BEAS-2B cells attenuated the inhibitory effect of Res on cellular senescence, accompanied by reversing the expression of SIRT1 and NF-κB p65. In conclusion, Res attenuated CSE-induced cellular senescence in BEAS-2B cells by regulating the miR-34a/SIRT1/NF-κB pathway, which may provide a new approach for COPD treatment.

Zeng XL ，Yang XN ，Liu XJ 《-》