3. Results

3.1 Longitudinal µCT

The changes in longitudinal µCT results are provided in Figure 2 (Absolute values are provided in the supplementary Figure S2). Compared with the Veh/Veh-group, Co/Co treatment significantly modified the total lung volume growth rate (Figure 2A), which was mainly attributed to increases in aerated lung volume. The Veh/Co-group also exhibited a larger aerated lung volume growth than the Veh/Veh-group (Figure 2B). Compared with the aerated lung volume, the contribution of the non-aerated lung in the total lung volume change was minor (Figure 2C). None of examined mice showed visual evidence of lung infiltration.
The changes in aerated lung volume were mainly due to increases in alveolar volume for cobalt-instilled groups (Figure 2D). The airways of these groups were enlarged, with the change of airway volume in the Co/Co-group significantly larger than the Veh/Veh-group (Figure 2E).
In accordance to the airway volume change, the trachea surface at the first bifurcation showed a trend of larger increase in both groups receiving Co via the airways (Veh/Co and Co/Co) compared with the Veh/Veh control group (Figure 2F). These two groups also showed a trend of lower lung density in both total lung and aerated lung area (Figure 2G & 2H).

3.2 Pulmonary hyper-reactivity and inflammation

When assessed on the basis of FEV0.1, the Co/Co-group showed higher responses to methacholine compared with either Veh/Veh or Veh/Co-group (Figure 3A). The Co/Co-group also showed a lower PC20 (Figure 3B). When the response to methacholine was assessed by measuring resistance, only the Co/Co-group, not the Veh/Co-group, clearly showed a higher reactivity than the Veh/Veh. (Figure 3C).
The total number of BAL cells did not differ between the four groups, but the completely CoCl2 treated mice (Co/Co-group) showed significantly higher proportions of neutrophils (8±7 %, mean±SD) compared with Veh/Veh control (1±1 %), but not compared with the Veh/Co-group (4±5 %) (Figure 3D). The Co/Co-group also showed higher proportions of eosinophils (4±7 %) than either the Veh/Veh (0±0 %) or the Veh/Co-group (0±0 %).
In terms of the cytokines in BAL, the Co/Co-group had higher levels of KC and MCP-1 compared with the Veh/Veh-group (Figure 3E). When compared with the Veh/Co-group, the Co/Co-group showed higher levels of KC and MIP-2. The Veh/Co-group had increased MCP-1 but decreased MIP-2; KC in this group did not differ from the Veh/Veh control. The levels of IFN-γ, IL-4, IL-5, IL-10, IL-13, IL-17A and IL-17F did not differ among groups.
Although some mice from Veh/Co and Co/Co presented with inflammation in bronchovascular areas, the semi-quantitative histological scores did not differ among groups (Figure S3).
As seen in representative scatterplots Figure 4A and Table S2, when considering all individual data, cellular markers of inflammation in BAL (percentages of neutrophils and eosinophils) correlated with lung imaging features (airway volume change assessed by µCT), KC and MCP-1 in BAL, and also with and physiological parameters (airway resistance and FEV0.1 response to methacholine).

3.3 DC and ILC subtypes in lung

In whole lung tissue homogenates, we assessed subsets of DCs and ILCs. The Co/Co-group showed an increased amount of DC compared with the Veh/Veh-group (Figure 5A). In the analysis of DC subtypes, the Co/Co-group showed significantly increased cDC1, cDC2 and moDC compared with Veh/Veh control (Figure 5B), whereas the Co/Veh-group showed increased cDC1. Plasmacytoid DC (pDC) showed a tendency to decrease in the Co/Co and the Co/Veh-groups, but without reaching statistical significance.
The ratio of ILCs of the CD45+ live cells was significantly higher in both the Co/Co and the Co/Veh-groups, compared with the Veh/Veh control (Figure 5C). This was mainly due to an increase of the ILC2 subset in these two groups (Figure 5D). Furthermore, the Co/Co-group had higher NCR-ILC3 compared with the Veh/Veh control group, and higher ILC1 compared with the Veh/Co-group.

3.4 Immunological indicators from auricular lymph nodes and serum

The analysis of lymphocyte subtypes from auricular lymph nodes did not show differences in the numbers of B cells, T cells, or subtypes of T cells. The cytokine levels in the supernatants from lymph nodes did not change significantly in the Co/Co-group. Total serum IgE, IgG1 and IgG2 did not differ between groups.
Although no significant differences were observed in immune responses in auricular lymph nodes and serum, the skin-lung association was apparent from the association analysis. As shown in Table 1 and Figure 4 (B, C and D), individual levels of serum IgE and helper T cells (CD3+CD4+) in auricular lymph nodes were positively associated with cDC1 and ILC2, and negatively associated with pDC in the lung. On the other hand, regulatory T cells (CD3+CD4+CD25+), cytotoxic T cells and B cells in auricular lymph nodes were associated with pDC and ILC1 in the lung.