*3.1. Elevated Levels of G-CSF in the Steroid-Resistant Neutrophilic Airway Inflammation Model*

First, we established a murine model for steroid-resistant neutrophilic airway inflammation as described previously with some modifications [10,32]. Mice were intranasally sensitized with 10 μg of LPS and 75 μg of OVA on days 0, 1, 2, and 7, followed by a challenge with 50 μg of OVA on days 14, 15, 21, 22, 28, and 29. The mice were euthanized 6 h after the last challenge. Inhibitors were i.p. injected 1 h before each challenge (Figure 1A). To confirm whether the mouse model was steroid-resistant, we examined the effect of corticosteroid (dexamethasone) treatment. Dexamethasone administration did not significantly reduce the recruitment of immune cells, airway inflammation (H&E), or mucus secretion (PAS) induced by LPS/OVA treatment (Figure 1B,C). Similarly, myeloperoxidase (MPO) and G-CSF levels in BALF were not reduced by dexamethasone treatment (Figure 1D,E). Neither the number of total cells nor neutrophils in BALF were affected by dexamethasone treatment (Figure 1F). Immunofluorescence staining analysis showed that the levels of MPO and G-CSF expression increased by LPS/OVA treatment were not attenuated by dexamethasone treatment (Figure 1G,H). Taken together, our experimental murine model exhibited a steroid-resistant neutrophilic airway inflammation phenotype with elevated G-CSF levels.
