*p <0.05 (two-tailed), ** p <0.01, ***p <0.001, **** p <0.0001 analyzed by Spearman correlations of selected individual responses including all animals (n= 6-9)
Figure 1 Experimental protocol. Balb/c mice received skin exposure to dimethyl sulfoxide (DMSO) or 5% CoCl2*6H2O (25μL per ear) on days 1 and 8. On day 15, 17, 19, 22, 24, mice received oropharyngeal challenge with saline or 0.05% CoCl2*6H2O. μCT was performed on day 1 (before the skin exposure), day 9, day 16 and day 25 (before the pulmonary function measurement).
Figure 2. Longitudinal micro-computed tomography (µCT) quantification of lung response. Mice were scanned with µCT on day 1, 9, 16 and 25. Value changes of each time point compared with day 1 were calculated. A) Change of total lung volume. B) Change of aerated lung volume (Hounsfield unit, HU < -188).C) Change of non-aerated volume (HU > -188).D) Change of alveolar volume (-565 < HU < -188). E) Change of airway volume (HU < -565).F) Change of cross-sectional area at the level of first airway bifurcation. G) Change of total lung density. H)Change of aerated lung density. Data are shown as mean. *p <0.05 compared with the Veh/Veh control group, when the interaction between type of treatment and time of µCT assessment was examined with two-way ANOVA with repeated measures. n =6-9 per group.
Figure 3. Lung function measurement and inflammatory response in bronchoalveolar lavage (BAL). A) Forced expired volume at 0.1sec (FEV0.1) in response to increasing concentrations of methacholine (0-20 mg/mL) were measured 24 h after the last time of oropharyngeal instillation. B) Concentration of methacholine causing a 20% drop (PC20) in FEV0.1.C) Airway resistance in response to increasing concentrations of methacholine. D) Percentage of neutrophils, eosinophils and macrophages were calculated from the BAL obtained 24 h after the last time of oropharyngeal instillation. E) Levels of keratinocyte chemoattractant (KC), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein 2 (MIP-2) in BAL were determined by MSD U-Plex Assay.
Data are shown as mean. *p <0.05, **p <0.01, *** p <0.001, ****p <0.0001 compared with the Veh/Veh control group;#p<0.05,##p<0.01 compared with the Veh/Co-group, when the interaction between type of treatment and response to methacholine was examined via two-way ANOVA with repeated measures (3A & 3C) or when data were analyzed by Kruskal–Wallis test (3B, 3D & 3E). n =6-9 per group for 3A-C; n =12-17 per group for 3D-E.
Figure 4. Representative scatter plots between skin and lung endpoints. A) Correlation between airway volume change (measured by µCT) on day 25 and percentage neutrophils in bronchoalveolar lavage (BAL). B) Correlation between NCR-ILC3 cells in lung and percentage neutrophils in BAL. C) Correlation between ILC2 in lung and CD3+CD4+ cells in auricular lymph node (ALN). D) Correlation between ILC2 in lung and serum IgE. Each point represents data from a single animal (total n=6-9). Coefficients are calculated by Spearman correlation. Note that data with a skewed distribution are expressed on a logarithmic scale (Ln).
Figure 5. Dendritic cells (DCs) and innate lymphoid cells (ILCs) in the lung . A) Percentage of DCs (CD45+, low-autofluorescent, CD11c+, MHCII+) in leukocytes. B) Percentage of DC subpopulations in live leukocytes are shown as type 1 conventional DC (cDC1, CD11b-, CD103+), type 2 conventional DC (cDC2, CD11b+ ,CD103-), monocyte-derived DC (moDC, CD11b+, CD103-, CD64+), and plasmacytoid DC (pDC, CD11b- Siglec-H+). C)Percentage of ILCs (CD45+, lineage negative, CD90.2+, CD127+) in leukocytes.D) Per mille of ILC subpopulations in live leukocytes are shown as ILC1 (KLRG1-, NKp46+, ROR γT-), ILC2 (KLRG1+), NCR+ILC3 (KLRG1-, NKp46+, ROR γT+) and NCR-ILC3 (KLRG1-, NKp46-, ROR γT+).
Data are shown as mean. *p <0.05, **p <0.01, *** p <0.001, ****p <0.0001 compared with the Veh/Veh control group;#p <0.05 compared with the Veh/Co-group as analyzed by Kruskal–Wallis test. n =6-8 per group.