2. Materials and
Methods
2.1 Animals
Male BALB/cAnNCrl mice (6-8 weeks old) were purchased from Charles
Rivers Laboratories (Germany). All mice were housed in a conventional
animal house with 14h light/10h dark cycles, in individually ventilated
cages (n=3-4 per cage). They received water and pelleted food ad
libitum . All animal procedures were approved by the KU Leuven Ethical
Committee for animal experiments (P135/2017).
2.2 Reagents
Cobalt (II) chloride hexahydrate
(CoCl2*6H2O) (CAS 7791-13-1), dimethyl
sulfoxide (DMSO) (CAS 67-68-5), and acetyl-β-methylcholine
(methacholine) were obtained from Sigma-Aldrich (Bornem, Belgium). The
concentration of CoCl2*6H2O is given as
percentage (w/v) in dissolving vehicle (1%
CoCl2*6H2O=42mM).
2.3 Experimental protocols
The choice of concentrations (and vehicle) for skin application and
oropharyngeal instillations were based on preliminary dose-finding
experiments described in a supplement (Figure S1). Based on these
results (mainly increased IgE) and literature data [13], we chose a
concentration of 5% CoCl2*6H2O in DMSO
to achieve dermal sensitization. We chose a concentration of 0.05%
CoCl2*6H2O in saline for oropharyngeal
challenges because this concentration caused only mild airway
inflammation (<10% neutrophils in BAL) 24 h after a single
administration, thus allowing us to distinguish inflammation caused by
an allergic response from that caused by nonspecific irritation.
We did two series of experiments, each following similar treatment
protocols (Figure 1) and having 6 to 9 mice per group. Mice were divided
into four groups based on whether they received 1) initial skin exposure
to CoCl2 (Co) or vehicle (Veh) and/or 2) oropharyngeal
instillation of CoCl2 or vehicle. On days 1 and 8, the
mice received 25 μL of 5% CoCl2*6H2O
(310 μg Co2+) or vehicle (DMSO) on each ear. On days
15, 17, 19, 22 and 24, the mice received an oropharyngeal instillation
of 50 µL of 0.05% CoCl2*6H2O (6 μg
Co2+) or vehicle (saline). This led to the following
four groups: Veh/Veh, Veh/Co, Co/Veh, and Co/Co.
In the first series, we performed sequential µCT, and assessed pulmonary
function, immune cell distribution in the auricular lymph nodes, and
lung histology. Micro-CT was conducted on day 1 (before the dermal
sensitization), day 9 (24h after the second dermal sensitization), day
16 (24h after the first challenge) and day 25 (24h after the last
challenge and before the measurement of pulmonary function). In the
second series, we analyzed the innate lymphoid cells (ILCs) and the
dendritic cells (DCs) in lung tissue. In both series, we evaluated total
IgE, IgG1 and IgG2 in the serum, and cytokines and cell subtypes in BAL.
The complete description of the materials and methods is available in
the online supplement. The gating strategy of DCs and ILCs is provided
in supplementary Table S1.
2.4 Data analysis
Data were analyzed by non-parametric Kruskal–Wallis test with
independent variables being four groups: Veh/Veh, Co/Veh, Veh/Co and
Co/Co. When there was significant effect from the Kruskal–Wallis test,
a multiple comparison post-hoc test was conducted. The associations
between two variables were analyzed by nonparametric Spearman
correlation test.
To analyze results of sequential measurements (µCT and methacholine
test) obtained within the same animals, we used two-way ANOVA with
repeated measures to test the interaction between 1) type of treatment
and time of µCT assessment, or 2) type of treatment and response to
methacholine. A level of p<0.05 (two tailed) was considered
significant.