Alternaria alternata-induced inflammation and mucus overproduction in C57Bl/6 and BALB/c mice is limited by C391
We have reported that intranasal administration A. alternataincreased PAR2-dependent inflammation, mucus production and mucus cell hyperplasia in wild type but not β-arrestin-2-/-C57Bl/6 mice (Yee et al., 2018). To determine if A. alternata-induced inflammation could be controlled pharmacologically with C391, we examined hallmarks of cellular inflammation after the same exposure to 8 µg A. alternata filtrate (3 times over 8 days), with or without C391. As previously reported, histological grading of Hematoxylin and Eosin (H & E) -stained lungs (Figure 4A-F; K-L, n = 3) , revealed that A. alternata sensitization and challenge increased inflammation (histological score 3.3 ± 0.2 compared to 1.3 ± 0.3 in HBSS-treated controls) and epithelial thickening (2.4 ± 0.28-fold over saline controls). Concurrent administration of 2.5 nmoles C391 withA. alternata significantly reduced inflammation and epithelial thickening (histological scores of 1.2 ± 0.1, p<001 and 0.98 ± 0.0.9, p=.0012, respectively). A lower dose (0.25 nmoles) of C391 reduced inflammation to a lesser extent (histological score of 2.3 ± 0.25) but did not significantly reduce epithelial thickening. A second hallmark of asthma is the increased production of mucus and A. alternata filtrate increased the number of epithelial cells staining positive for mucus (80.2 ± 3.1% compared to 13.7 ± 4.2% in saline controls), which was reduced by administration of C391 (42.8 ± 6.4% with 0.25 nM and 33.9 ± 2.3% with 2.5 nM C391, p<.001 for both concentrations; Figure 4G - J, M, n = 3).
While the C57Bl/6 mice from above provided comparison to studies that used genetically manipulated mice (Nichols et al., 2012; Yee et al., 2018), BALB/c mice represent a strain that is more prone to allergen response. H & E stains demonstrated extensive lung inflammation in response to A. alternata in BALB/c mice (Figure 5B ) that was reduced in the presence of C391 (Figure 5C ). A. alternata histology scores showed increased inflammation over control that was significantly reduced when the PAR2 antagonist C391 was included in the A. alternata filtrate (Figure 5D ; n = 4, p < 0.05) . Mucus staining and mucus cell hyperplasia in the BALB/c mice were evaluated via Periodic Acid Schiff (PAS) staining (Figure 5E - G ). A. alternata treatments significantly increased PAS staining from a limited baseline scoring (0 on a scale of 5; see Methods) to a robust mucus score (4.2 ± 0.2;Figure 5F ). Inclusion of C391 in the A. alternatatreatment significantly reduced the PAS score (2.6 ± 0.4, p < 0.05; Figure 5G ), although this remained significantly above control scores (Figure 5E, H , p < 0.05) .