*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.