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.