Effect of environmental irritants on the airway epithelial barrier and airway hypersensitivity

Via inhalation we are continuously exposed to environmental agents, such as gasses, chemicals and particles in the air. The first layer of cells that come into contact with these inhaled agents is the airway epithelium. Airway epithelial cells are closely linked to each other, creating a barrier that prevents entry of inhaled (deleterious) agents to the host. Proper functioning of the airway epithelial barrier is crucial in host defense and the importance of an intact barrier has already implicated in a number of respiratory diseases, such as asthma, rhinitis and chronic obstructive pulmonary disease, where dysfunction of the epithelium occurs.  

Inhalation of irritating agents induces a reversible inflammatory response compromising coughing, wheezing and increased mucus production, persistence of these symptoms is indicative of irritant-induced asthma (IIA). IIA is a subtype of asthma that develops after exposure to irritants, such as chlorine, formaldehyde, strong acids and smoke, without an immunological sensitization towards the causal agent. IIA can arise after acute exposure to high concentrations of respiratory irritants, such as during an explosion, fire or accidental leak. However, continuous exposure to lower levels of irritating agents, such as for example exposure of chlorine to professional swimmers, lifeguards and cleaning personnel, can result in IIA. Currently the mechanisms leading to IIA are poorly known.

The general hypothesis in the dissertation is that environmental irritants can disturb the airway epithelial barrier and that this contributes to the development of airway hypersensitivity, as in IIA. Therefore we first evaluated the effect of different types of irritants on the airway epithelial barrier on an in vitro level. In a second part, we tested in mice the hypothesis that due to airway barrier impairment, inhalation of low levels of irritants can result in airway hypersensitivity.

In chapter 3, a co-culture mimicking the airway epithelial barrier was exposed to different types of environmental irritants. This co-culture existed of bronchial epithelial cells (16HBE) and endothelial cells (HLMVEC) seeded on the opposite sides of a permeable membrane, and monocytes (THP-1) added apically. We found that the nanoparticles graphene and graphene oxide induced an immediate negative effect on the airway epithelial barrier. Hypochlorite only induced a negative effect after 24 hours of exposure and ammonium and sodium persulfate did not alter the barrier function. Using immunofluorescent staining a less connected tight junction network was observed due to graphene and graphene oxide exposure. By means of phosphoproteomics we found that graphene and graphene oxide altered the level of phosphorylation of proteins from the tight junction and adherens junction pathway, hereby providing an explanation of the reduced barrier function following exposure to graphene and graphene oxide.

We evaluated whether mast cells can influence the response of the airway epithelium upon exposure to environmental irritants in chapter 4. Bronchial epithelial cells (16HBE) were seeded on a permeable membrane in absence or in presence of mast cells (HMC-1) on the basolateral or apical side, and were exposed to different types of irritants. Irrespective of the seeding condition, graphene and graphene oxide had an immediate and persistent effect on the airway epithelial barrier, whereas hypochlorite and diesel exhaust particles only induced a temporary decrease in barrier function. After irritant-exposure the bronchial epithelial cells did not release the ‘danger’ signals TSLP, IL-33 or IL-1α and the mast cells were not activated. Due to the inactive (or immature) state of the HMC-1 cells, no additional effects of the airway epithelium following irritant-exposure was determined in the bi-cultures.

In the first in vivo study of this thesis, we combined the mouse model of naphthalene-induced airway damage (after a recovery period) with a single intranasal exposure to the common disinfectant hypochlorite (chapter 5). We demonstrated that naphthalene-induced barrier impairment resulted in temporary enlargement of the lungs and airways, and in a permanent increase in the functional lung volumes. The challenge with hypochlorite induced a sensory irritant response, as well as, development of airway hyperresponsiveness and pulmonary inflammation. The increased airway permeability and epithelial damage after exposure to hypochlorite indicated that due to barrier impairment the epithelium is more vulnerable for the irritant effect of hypochlorite. Moreover, the naphthalene-induced epithelial dysfunction resulted in the recruitment of monocyte-derived epithelial cells towards the lung and increased the type 2 innate lymphoid cell population because of the additional insult with hypochlorite. We confirmed our hypothesis that due to impairment of the airway epithelial barrier, with partial recovery, the mice became more sensitive to exposure of hypochlorite.

In chapter 6, we implemented a more vigorous treatment protocol, in which mice were treated twice with naphthalene in order to impair the airway barrier, followed by repeated endotracheal instillations with hypochlorite. In this model the functional lung volumes of mice treated with naphthalene also increased. In combination with the endotracheal instillations, the repeated barrier impairment resulted in non-specific airway hyperresponsiveness. The increase in neutrophils in the broncho-alveolar lavage fluid and epithelial damage on histologic sections at the end of the experiment indicated that the mice were not fully recuperated from the repeated barrier impairment. Airway exposure to hypochlorite did not augment the airway responses and epithelial damage, but did increase the number of eosinophils in the lung. Using this more vigorous treatment protocol, specific sensitivity towards hypochlorite did not develop, but the mice did become more sensitive to airway exposure in general.

To conclude, in this thesis we demonstrated that different types of environmental irritants have different effects on the airway epithelial barrier in vitro, and that impairment of the airway epithelial barrier can result in the development of airway hypersensitivity in mice. By studying these mechanisms, we demonstrated that the airway epithelial barrier and its interaction with respiratory irritants are crucial aspects in the development of IIA.