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.