a. Total cell count from lung digests in WT mice. There was an increase in total cell count after HDM challenge, compared to control (** P < 0.01 at ZT23, and P=0.06 at ZT11). There was no time of challenge difference after PBS or HDM challenge. Data is presented as median ± IQR. 1 way ANOVA followed by Tukey’s multiple comparison test, (n=8-11 per treatment group).
b. Eosinophils in lung digests from WT mice significantly increased after HDM challenge (*** P < 0.001 at ZT23 and P < 0.05 at ZT11); there was no time of challenge difference after PBS or HDM challenge. Data is presented as median ± IQR. 1 way ANOVA, followed by Tukey’s multiple comparison test, (n=8-11 per treatment group).
c. HDM challenge at ZT11 and ZT23 caused predominantly eosinophilic inflammation around the bronchioles and blood vessels (haemoatoxylin and eosin (H&E) staining, i-iv) compared to PBS challenge (**** P < 0.0001 at ZT23 and ZT11). There was no time of challenge difference after PBS or HDM challenge. Data is presented as median ± IQR. 1 way ANOVA, followed by Tukey’s multiple comparison test, (n=10-12 per treatment group).
d. Periodic Shift Staining (PAS) shows increased mucus present on the bronchial epithelium in the lungs of WT mice treated with HDM at both ZT11 and ZT23. There was no PAS staining seen in PBS treated mice (i-iv). There was significantly increased PAS scores in HDM challenged mice (**** P < 0.001 at ZT23 and ZT11), compared to controls, but no time of challenge differences. Data is presented as median ± IQR. 1 way ANOVA, followed by Tukey’s multiple comparison test, (n=10-12 per treatment group).