Estado del Arte

The pseudostratified epithelial layer in the healthy, normal large airways provides both a barrier to foreign particles and bacteria, but also plays a key role in clearing particulates and debris from the lungs [19, 238]. The epithelial cells in the large airways possess cilia on their apical surface, which beat in time in order to move mucus and particles up the airways and out into the trachea, where they can be swallowed or otherwise expelled from the body [19]. In order to facilitate this, and to trap pathogens and unwanted substances, the epithelial layer has numerous goblet cells and sub-mucosal glands, and the mucus these cells and glands produce encapsulates pathogens and particulates, allowing them to move easily along the airway. Typically there is a minimal immune presence in both the airway walls and the lumen, as the airway epithelium is considered to be a site of ‘immune privilege’, similar to the testes [245]. However, in COPD, the epithelial layer is dysregulated and is the site of a number of pathological changes.

Squamous metaplasia, the thickening of the airway epithelium, is among the easiest COPD- realted change to spot, as is the proliferation and hyperactivity of mucus producing goblet cells and sub-mucosal glands [20-23]. There is also a decrease in cilia length and a

corresponding reduction in airway clearance [24, 25]. Many of the more characteristic changes of the airway, however, such as immune cell activation and airway wall thickening,

Useful abbreviations

NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist

NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells

COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1

COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract

133 occur in either the lumen or beneath the epithelial layer. It was not until 2010 that Sohal and colleagues proposed that the airway epithelium may be playing a different, more active role in COPD pathology, via the process of EMT [31]. Since 2010, it hass been demonstrated that the airway epithelium, particularly the basal cell layer, is highly dysregulated [26-30],

expressing a number of mesenchymal markers such as S100A4, vimentin and α-smooth muscle actin [31-34], in addition to being highly activated via pathways such as the TGF-β related Smad pathway [207, 240, 241] and the Wnt signalling pathway [242]. Furthermore, the epithelial layer overexpresses viral and bacterial adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and platelet activating factor receptors (PAFr) [243-252]. Not only is the epithelium dysregulated in vivo, but it has also been demonstrated that epithelial cells taken from people with airflow limitation exhibit sustained pathological differences from cells taken from non-smokers.

Useful abbreviations

NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist

NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells

COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1

COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract

134

4.1.2.Differences between epithelial cells isolated from non-smokers and people with airflow limitation in vitro

In vitro, epithelial cells taken from the airways of people with COPD do not proliferate as rapidly as those taken from non-smokers, nor do they survive and differentiate as well as cells from non-smokers when grown at an air-liquid interface (ALI) [26]. In 2010 Milara and colleagues were able to show that primary epithelial cells taken from the bronchi of people with COPD exhibited a sustained EMT phenotype in culture [33]. Fibroblasts taken from the airways of people with COPD have also shown that they secrete currently unknown factors into the surrounding environment which can cause epithelial cells to undergo EMT [34]. This suggests that bronchial epithelial cells taken from COPD airways exhibit a changed

phenotype which endures in culture, potentially making them a valuable resource for identifying pathways and mechanisms.

In vitro cell models need to mimic the in vivo disease as closely as possible, and ALI cultures are considered good for examining the airway epithelium since they allow differentiation of the cells into a full epithelial layer with secretory and ciliated cells [26]. However, there is evidence that epithelial cell dysregulation in COPD begins and stems primarily from issues with the basal progenitor cells in the epithelium, which are not terminally differentiated and comprise about 50% of the epithelial cell population in a pseduostratified layer [253]. TraditionalT submerged culture does not allow a differentiated epithelium to form, instead causing proliferation of undifferentiated basal cells, making it potentially more useful for studying airway epithelial dysregulation in the sub-population of cells it affectsa most than ALI culture. In light of this, it is important to not dismiss submerged culture, even though it

Useful abbreviations

NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist

NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells

COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1

COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract

135 does not produce a differentiated airway epithelium. However, it is crucial to check, if

possible, that a culture model accurately reflects both the disease and the non-pathogenic state which is seen in vivo, since an inaccurate model is of no use to anyone.

Useful abbreviations

NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist

NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells

COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1

COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract

136