Algorithmic assessment of cellular senescence in experimental and clinical specimens.

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies-pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells-that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies.

Mitochondrial dysfunction increases pro-inflammatory cytokine production and impairs repair and corticosteroid responsiveness in lung epithelium.

COPD is characterized by chronic lung inflammation and irreversible lung tissue damage. Inhaled noxious gases, including cigarette smoke, are the major risk factor for COPD. Inhaled smoke first encounters the epithelial lining of the lungs, causing oxidative stress and mitochondrial dysfunction. We investigated whether a mitochondrial defect may contribute to increased lung epithelial pro-inflammatory responses, impaired epithelial repair and reduced corticosteroid sensitivity as observed in COPD. We used wild-type alveolar epithelial cells A549 and mitochondrial DNA-depleted A549 cells (A549 Rho-0) and studied pro-inflammatory responses using (multiplex) ELISA as well as epithelial barrier function and repair (real-time impedance measurements), in the presence and absence of the inhaled corticosteroid budesonide. We observed that A549 Rho-0 cells secrete higher levels of pro-inflammatory cytokines than wild-type A549 cells and display impaired repair upon wounding. Budesonide strongly suppressed the production of neutrophil attractant CXCL8, and promoted epithelial integrity in A549 wild-type cells, while A549 Rho-0 cells displayed reduced corticosteroid sensitivity compared to wild-type cells. The reduced corticosteroid responsiveness may be mediated by glycolytic reprogramming, specifically glycolysis-associated PI3K signaling, as PI3K inhibitor LY294002 restored the sensitivity of CXCL8 secretion to corticosteroids in A549 Rho-0 cells. In conclusion, mitochondrial defects may lead to increased lung epithelial pro-inflammatory responses, reduced epithelial repair and reduced corticosteroid responsiveness in lung epithelium, thus potentially contributing to the pathogenesis of COPD.

Cigarette smoke impairs airway epithelial barrier function and cell-cell contact recovery.

Cigarette smoking, the major cause of chronic obstructive pulmonary disease (COPD), induces aberrant airway epithelial structure and function. The underlying mechanisms are unresolved so far. We studied effects of cigarette smoke extract (CSE) on epithelial barrier function and wound regeneration in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) from COPD patients, nonsmokers and healthy smokers. We demonstrate that CSE rapidly and transiently impairs 16HBE barrier function, largely due to disruption of cell-cell contacts. CSE induced a similar, but stronger and more sustained, defect in PBECs. Application of the specific epidermal growth factor receptor (EGFR) inhibitor AG1478 showed that EGFR activation contributes to the CSE-induced defects in both 16HBE cells and PBECs. Furthermore, our data indicate that the endogenous protease calpain mediates these defects through tight junction protein degradation. CSE also delayed the reconstitution of 16HBE intercellular contacts during wound healing and attenuated PBEC barrier function upon wound regeneration. These findings were comparable between PBECs from smokers, healthy smokers and COPD patients. In conclusion, we demonstrate for the first time that CSE reduces epithelial integrity, probably by EGFR and calpain-dependent disruption of intercellular contacts. This may increase susceptibility to environmental insults, e.g. inhaled pathogens. Thus, EGFR may be a promising target for therapeutic strategies to improve mucosal barrier function in cigarette smoking-related disease.

Characterisation of cell adhesion in airway epithelial cell types using electric cell-substrate impedance sensing.

Research on epithelial cell lines and primary epithelium is required to dissect the mechanisms underlying the structural abnormalities in airway epithelium observed for respiratory diseases, including asthma and chronic obstructive pulmonary disease. The novel electric cell-substrate impedance sensing technique was used to monitor cell adhesion/spreading, barrier function and wound healing. Primary bronchial epithelium was compared with airway epithelial cell lines 16HBE14o-, BEAS-2B, NCI-H292 and A549. BEAS-2B, A549 and primary cells form a confluent monolayer more rapidly than do 16HBE14o- cells. In contrast, 16HBE14o- cells form stronger intercellular contacts, with a 10-fold higher resistance than BEAS-2B, A549 and NCI-H292 cells and a five-fold increase over primary cells. Accordingly, expression of the adhesion molecules zona occludens-1 and E-cadherin was highest in 16HBE14o- cells. These molecules were localised in intercellular junctions in both 16HBE14o- and primary cells. Finally, restoration of barrier function upon injury was impaired in BEAS-2B compared to 16HBE14o- cells. In conclusion, epithelial cell types display remarkable phenotypic differences and should, accordingly, be used to address specific research questions. 16HBE14o- cells appear most suitable for studies on barrier formation, whereas resemblance in attachment of primary and BEAS-2B and A549 cells makes the latter more important for translational research on cell-matrix contact.