Lung effects of inhaled corticosteroids in a rhesus monkey model of childhood asthma

Abstract

Background: The risks for infants and young children receiving inhaled corticosteroid (ICS) therapy are largely unknown. Recent clinical studies indicate that ICS therapy in pre-school children with symptoms of asthma result in decreased symptoms without influencing the clinical disease course, but potentially affect postnatal growth and development. The current study employs a primate experimental model to identify the risks posed by ICS therapy.

Objective: To (1) establish whether ICS therapy in developing primate lungs reverses pulmonary pathobiology associated with allergic airway disease (AAD) and (2) define the impact of ICS on postnatal lung growth and development in primates.

Methods: Infant rhesus monkeys were exposed, from 1 through 6 months, to filtered air (FA) with house dust mite allergen and ozone using a protocol that produces AAD (AAD monkeys), or to FA alone (Control monkeys). From three through 6 months, the monkeys were treated daily with ICS (budesonide) or saline.

Results: Several AAD manifestations (airflow restrictions, lavage eosinophilia, basement membrane zone thickening, epithelial mucin composition) were reduced with ICS treatment, without adverse effects on body growth or adrenal function; however, airway branching abnormalities and intraepithelial innervation were not reduced. In addition, several indicators of postnatal lung growth and differentiation: vital capacity, inspiratory capacity, compliance, non-parenchymal lung volume and alveolarization, were increased in both AAD and Control monkeys that received ICS treatment.

Conclusions and clinical relevance: Incomplete prevention of pathobiological changes in the airways and disruption of postnatal growth and differentiation of airways and lung parenchyma in response to ICS pose risks for developing primate lungs. These responses also represent two mechanisms that could compromise ICS therapy's ability to alter clinical disease course in young children.

Fig. 1

Diagram of experimental protocol for production of allergic airway disease (AAD) in infant rhesus monkeys and their treatment with budesonide (ICS). Infant monkeys were divided into two groups and housed in filtered air (FA) from birth. One group (AAD monkeys) was sensitized to house dust mite allergen (HDMA) at 14 and 28 days of age and exposed to HDMA (days 3–5) with ozone (days 1–5) for eleven 14-day cycles beginning at 28 days of age. The other group (Control monkeys) received FA only. Half of the Control monkeys and half of the AAD monkeys received budesonide daily from the beginning of the sixth exposure cycle.

Fig. 2

Comparison of the histological appearance of alveoli in the lung parenchyma (right middle lobe) of infant rhesus monkeys exposed to filtered air (Ctrl) or house dust mite allergen with ozone to produce allergic airways disease (AAD) and treated with ICS or saline. (a) Control saline. (b) Control ICS. (c) AAD saline. (d) AAD ICS. (e) Alveolar number (Nalv) was estimated by morphometry. *P < 0.05 significantly different from saline groups; one-way ANOVA. There was a significant (P < 0.05) overall effect of ICS treatment on alveolar number by two-way ANOVA. Scale = 50 μm. n = 6 per group.

Fig. 3

Comparison of the bronchial epithelium of midlevel airways in infant rhesus monkeys exposed to filtered air (Ctrl) or house dust mite allergen with ozone to produce allergic airways disease (AAD) and treated with ICS or saline. (a) Morphometric comparison of the mass (volume per surface) of the epithelium (gray bars) and of the histochemically detectable mucin stored in the epithelium (dark bars). (b) Comparison of the relative abundance of stored mucins by composition, determined histochemically based on their reaction to Alcian blue (AB) and Periodic acid Schiff’s (PAS) reagent. (d, e, f, g) Histochemical comparison of the epithelium in midlevel airways of Control saline monkeys (d) or Control ICS monkeys (e) with AAD saline monkeys (f) or AAD ICS monkeys (g). (c) Rate of proliferation of epithelial cells as a percentage of PCNA positive cells in the epithelium. *P < 0.05 significantly different from Control saline group; one-way ANOVA. Scale = 50 μm. Mean ± one standard deviation. n = 6 per group.

Fig. 4

Comparison of the tracheal basement membrane zone (BMZ) of infant rhesus monkeys exposed to filtered air (Ctrl monkeys) or house dust mite allergen with ozone to produce allergic airways disease (AAD monkeys) and treated with saline or ICS. Characterization of the basement membrane zone was based on the relative intensity and distribution of immunoreactive collagen I and perlecan (a, b, c, d). Distribution of immunoreactive collagen I (red signal) in Control saline (a), Control ICS (b), AAD saline (c) and AAD ICS (d) monkeys. The percentage of the basement membrane zone less than 2 μm in thickness (e) was significantly greater in the AAD saline monkeys (P < 0.05) than the AAD ICS monkeys. Expression of immunoreactive perlecan in the basement membrane zone (f) was significantly less in the AAD saline monkeys (P < 0.05) than the AAD ICS monkeys. These data demonstrate that ICS treatment did inhibit the deleterious effects of HDMA + O₃ on the developing BMZ but did not affect the normal development of the BMZ. Scale = 50μm. Mean ± 1 SD. n = 6 per group.

Fig. 5

Comparison of the abundance of intraepithelial nerve fibres associated with postnatal innervation of the mucosal epithelium in midlevel bronchi of infant rhesus monkeys exposed to filtered air (Ctrl) or house dust mite allergen with ozone to produce allergic airways disease (AAD) and treated with ICS or saline. (a, b, c, d) Distribution of immunoreactive Protein Gene Product 9.5 (red signal) in intraepithelial nerve fibres and epithelial cells of Control saline (a), Control ICS (B), AAD saline (c) and AAD ICS (d) monkeys. 50 μm. n = 6 per group.