Barrier disrupting effects of alternaria alternata extract on bronchial epithelium from asthmatic donors

Abstract

Sensitization and exposure to the allergenic fungus Alternaria alternata has been associated with increased risk of asthma and asthma exacerbations. The first cells to encounter inhaled allergens are epithelial cells at the airway mucosal surface. Epithelial barrier function has previously been reported to be defective in asthma. This study investigated the contribution of proteases from Alternaria alternata on epithelial barrier function and inflammatory responses and compared responses of in vitro cultures of differentiated bronchial epithelial cells derived from severely asthmatic donors with those from non-asthmatic controls. Polarised 16HBE cells or air-liquid interface (ALI) bronchial epithelial cultures from non-asthmatic or severe asthmatic donors were challenged apically with extracts of Alternaria and changes in inflammatory cytokine release and transepithelial electrical resistance (TER) were measured. Protease activity in Alternaria extracts was characterised and the effect of selectively inhibiting protease activity on epithelial responses was examined using protease inhibitors and heat-treatment. In 16HBE cells, Alternaria extracts stimulated release of IL-8 and TNFα, with concomitant reduction in TER; these effects were prevented by heat-treatment of the extracts. Examination of the effects of protease inhibitors suggested that serine proteases were the predominant class of proteases mediating these effects. ALI cultures from asthmatic donors exhibited a reduced IL-8 response to Alternaria relative to those from healthy controls, while neither responded with increased thymic stromal lymphopoietin (TSLP) release. Only cultures from asthmatic donors were susceptible to the barrier-weakening effects of Alternaria. Therefore, the bronchial epithelium of severely asthmatic individuals may be more susceptible to the deleterious effects of Alternaria.

Figure 1. Alternaria extract induces a heat-labile increase in IL-8 release and rapid reduction in TER in polarised 16HBE cells.

Polarised 16HBE cells on Transwell inserts were challenged apically with varying doses of Alternaria (Alt) or Cladosporium (Clad) fungal extracts, or heat treated fungal extract. (A) Apical and (B) basolateral supernatants were harvested 24 h post-challenge. IL-8 concentration was determined by ELISA (n = 4–9). TER was measured before challenge and at (C) 1h and (D) 24 h thereafter, and calculated as percentage change from pre-challenge readings (n = 4–15). Bars represent mean ± SEM. Analysis by one way repeated measures ANOVA with Bonferroni correction for pairwise analyses *** p<0.001.

Figure 2. Protease activity of Alternaria extract is reduced by the serine protease inhibitor AEBSF.

FITC-labelled casein was incubated with Alternaria extract (500 μg/ml) alone or in the presence of either AEBSF (2.5 mM), E-64 (500 μM), Pepstatin A (5 μg/ml), or heat-treated (n = 4 separate experiments measured in duplicate). Soluble fluorescence was measured after 24 h, relative to a trypsin standard. Bars represent mean fluorescence relative to inhibitor-free control±SEM. Protease activity of 100 µg/ml Alternaria extract was equivalent to 6.7±2.5 µg/ml trypsin; for comparison, Cladosporium protease activity was equivalent to 3.3±0.0 μg/ml trypsin. Analysis as for Figure 1. Bars represent mean ± SEM; *** p<0.001.

Figure 3. Inhibitors of proteases and p38 MAPK differentially inhibit apical and basolateral IL-8 release after Alternaria challenge.

The effect of Alternaria extract (100 μg/ml) on 16HBE cells was tested alone or in the presence of AEBSF (250 μM), E-64 (50 μM), Pepstatin A (0.5 μg/ml) or SB203580 (25 μM) (n = 3–8). IL-8 release 24 h post-challenge was calculated as “Release (% control)  =  ((Alt_INHIB – No Alt_INHIB)/(Alt_{NO INHIB} – No Alt_{NO INHIB})) ×100”, to correct for any effect of the inhibitors on baseline IL-8 release without Alternaria. Data show mean ± SEM. Analysis as for Figure 1; * p<0.05, ** p<0.01.

Figure 4. ALI cultures of healthy, but not severely asthmatic, donors increase IL-8 release in response to Alternaria.

ALI cultures from healthy (n = 8–12) or severely asthmatic (n = 6–7) donors were differentiated at air-liquid interface, prior to challenge with Alternaria (Alt) or Cladosporium (Clad) fungal extracts. IL-8 release 24 h post-challenge was determined by ELISA. Box shows median and 25^th/75^th percentiles, and whiskers show 10^th/90^th percentiles. Analysis by Friedman's test with Tukey's correction for pairwise analyses; * p<0.05.

Figure 5. ALI cultures from severely asthmatic donors are sensitive to the barrier weakening effect of Alternaria.

ALI cultures from healthy (grey bars; n = 7–9) and severely asthmatic (black bars; n = 6–7) donors were challenged with Alternaria (Alt) or Cladosporium (Clad) fungal extracts. TER was measured pre-challenge and 3 h and 24 h post-challenge. Results are expressed as percentage change in TER relative to pre-challenge and are shown as mean ± SEM. Analysis as for Figure 1; * p<0.05, ** p<0.01.