4.
Discussion
Our study shows that dermal contact with CoCl2 followed
by repeated airway exposure to CoCl2 leads to several
hallmarks of asthma, such as airway hyper-reactivity (AHR) and lung
inflammation, with characteristics of the type 2 adaptive immune
response, in combination with increased cytokines linked to the innate
immune system. Moreover, for the first time we show that in an asthma
model, early changes in the lungs can be identified by µCT imaging in
free-breathing mice.
While the link between skin sensitization and lung susceptibility has
been demonstrated for several chemicals, including diisocyanates, acid
anhydrides and persulfate salts [8–10], this has never been
investigated for metals with sensitizing properties. Cobalt-induced
asthma has been frequently reported in occupations involving inhalation
of cobalt-containing materials, such as hard metal (a composite
consisting mainly of tungsten carbide with cobalt) [14–16].
Previous studies of the respiratory toxicity of cobalt have focused on
histological or immunological changes [17–19]. However, to our
knowledge, no study has investigated the possible role of skin
sensitization in the subsequent development of cobalt asthma. .
In this study, we included an imaging modality for the first time to
investigate whether alterations can be identified in a mouse model of
asthma. The advantage of µCT is that it can provide high-resolution
information in living animals, repeatedly, without causing harm to the
animal, and at relatively low cost. We have previously shown, in mouse
models of lung emphysema (elastase) and fibrosis (bleomycin) [20,
21], that early alterations can be detected by µCT. Moreover, a
low-dose scanning protocol was recently developed, allowing radio-safe
repeated follow-up of disease progression [22]. Our data showed
larger total and aerated lung volumes when mice received
CoCl2 respiratory challenge after skin exposure with
cobalt. We interpret these changes as reflecting early onset lung
alterations associated with inflammation.
Increased airway volume and decreased lung density were observed in both
groups receiving CoCl2 via the airways. This image
pattern, also named paradoxical airway dilation, has been described in
several animal models undergoing bronchoconstriction interventions, such
as mice challenged with methacholine [23] or treated with
intraperitoneal naphthalene [24]. A recent study of hamsters
infected by SARS-CoV-2 also indicated a positive correlation between
bronchial dilation and apoptotic bodies in the bronchial walls [25].
Since we found a correlation between µCT-derived airway enlargement and
BAL airway inflammation (Figure 4A and Table S2), airway dilation could
possible serve as a marker of lung inflammation.
One of the hallmarks of (occupational) asthma is non-specific AHR.
Epidemiological studies show that cobalt may induce or exacerbate AHR in
humans [26, 27]. In our animal model, mice having received both
prior skin exposure and respiratory challenge (the Co/Co-group)
responded more strongly to a methacholine provocation than the other
mice. Thus, the Co/Co-group had a more pronounced decrease in
FEV0.1 under methacholine challenge compared with all
other groups, which is in agreement with the obstructive pattern of
asthmatic murine models [9, 28, 29]. To our knowledge, this is the
first time that FEV0.1 is assessed with methacholine
challenge in an occupational asthma model. In the future, this may lead
to more concordance in assessing lung function of mice in comparison to
human.
Consistent with the increases in airway volume and AHR, mice of the
Co/Co-group exhibited an influx of neutrophils and eosinophils in BAL.
In contrast, mice receiving CoCl2 respiratory challenge
without prior skin exposure (Veh/Co) showed only a minor neutrophilic
influx, indicating that the mixed neutrophilic/eosinophilic inflammatory
response in the Co/Co-group was due to the initial dermal sensitization.
Admittedly, a few mice in the Veh/Co-group responded partly like the
Co/Co-group, and this may have been due to the repeated instillations of
Co.
In the present study, the total dosage of the oropharyngeal challenge (6
μg Co2+ given in five doses) was much lower than that
in previous animal models (13 μg Co2+ for 10 days)
[30], but features of a type 2 allergic inflammation still occurred,
presumably due to the initial dermal exposure. The eosinophilic
inflammatory response in BAL of dermally CoCl2 treated
and CoCl2 challenged group has never been reported in
animal models. The eosinophilic inflammation produced only in animals
with prior dermal contact, even for a short time, strengthens the
conclusion that dermal sensitization increases the airway responsiveness
to the corresponding chemical allergen. We have argued previously that
this general concept is not only of academic importance in terms of
immunological mechanisms, but it has also relevance in the field of
occupational hygiene and the prevention of occupational asthma [7].
Although we observed a mixed neutrophilic/eosinophilic lung
inflammation, we found that only cytokines linked to neutrophilic
inflammation (KC and MCP-1) were increased in the Co/Co-group at the
time of autopsy. Several cytokines have previously been assessed in
studies of cobalt-induced respiratory toxicity, but the results varied
depending on the challenging dosage and frequency. KC, also known as
CXCL1 or GROα, is a chemokine critical to neutrophil recruitment. This
is the first observation in an animal asthma model that BAL KC is
associated with prior skin exposure. While we did not find changes in
IL-4, IL-5, or IL-13 in BAL for all groups, which are linked to type 2
eosinophilic inflammation and airway hyper-reactivity, we found higher
MCP-1 in both groups receiving respiratory cobalt challenge. MCP-1, also
known as CCL2, is a cytokine tightly associated with Th2 reaction. MCP-1
can induce mast cell degranulation, thereby enhancing AHR [31]. The
upregulation of MCP-1 has also been frequently observed in animal models
with skin exposure followed by challenge [7]. The observation in
this study may imply a more important role of MCP-1 in the Th2 reaction
compared with IL4, IL-5 and IL-13. Unexpectedly, the level of MIP-2 was
lower in the Veh/Co-group. MIP-2 (also known as CXCL2) is critical to
neutrophil attraction and secreted mainly by macrophages [32].
Though the reason for decreased MIP-2 in the Veh/Co-group remains to be
verified, a possible explanation is that the immune reaction shifted
away from Th1-macrophage axis, thereby leading to downregulated
macrophage and MIP-2 downstream.
Dendritic cells and innate lymphoid cells have been investigated
extensively in ovalbumin and house dust mite asthma models [33, 34],
yet this has never been done in mouse models of asthma caused by
chemical or metal exposure. The analysis of DCs in lung revealed
elevated cDC1 in both groups having received cobalt skin exposure. On
the other hand, pulmonary cDC2, moDC and total dendritic cells increased
only in the Co/Co-group. cDC1 has been demonstrated to modulate Th17
immune responses [35], though its full role to enhance Th2 reaction
remains controversial. cDC2s and moDCs are known to facilitate Th2 and
Th17 cell differentiation [36, 37], which is also in accordance with
the cytokine pattern from our study. Though some studies have evaluated
the shifting of pulmonary DC subtypes after respiratory exposure to
sensitizing agents, the shifting after solely skin exposure has never
been reported. Accumulation of cDC2 and moDC in mediastinal lymph node
has been revealed in mice receiving solely respiratory exposure to HDM
[37]. Our team has previously examined pulmonary DC subtypes in
mice, and observed elevated moDC after sequential skin and airway
exposure to TDI [38], but the effect of exclusive skin exposure was
not assessed.
As is the case for dendritic cells, the innate lymphoid cells have not
been investigated in a skin/lung model. In this study, pulmonary ILC2
numbers increased in both groups receiving skin exposure, but not in the
group receiving solely respiratory challenge. This finding might explain
the presence of the eosinophilic inflammation in BAL from the
Co/Co-group without an elevation of Th2-related cytokines, such as IL-5,
and imply an association between skin exposure and ILC2/Th2/eosinophil
axis. The role of ILC3 in asthma has rarely been studied in animal
models compared with ILC2. One ILC3 subtype, NCR-ILC3, is known to produce IL-17 [39]. In our study, respiratory
CoCl2 challenges irrespective of prior dermal contact
showed a trend to increase the level of NCR- ILC3 in
CD45+ pulmonary cells, admittedly not at a significant
level. Only in the group receiving both skin and airway exposure
(Co/Co), did NCR- ILC3 increase significantly. As
shown in Figure 4B, the neutrophil proportion in BAL was positively
associated with NCR-ILC3. Together with the observed
inflammatory patterns in BAL and lung, the results imply that the
respiratory exposure to cobalt is associated with ILC3/Th17/neutrophil
axis. In spite of the mixed type 2/type 3 inflammatory reaction in
airway, other related cytokines in BAL did not increase altogether. We
did not observe increasing IL4/IL5/IL13, which are related to
ILC2/Th2/eosinophil reaction. Likewise, IL-17A and IL-17F did not
increase despite the elevation of KC and prominent neutrophilic reaction
in BAL. Other cytokines associated with asthma, such as IL-22, IL-25,
IL-33 might also serve as potential influential factors [40, 41].
Though the mice in the Co/Co-group presented a more marked inflammation
than those in the other groups, the intensity of the inflammatory
responses varied considerably between animals and some mice did not show
inflammation at all. We hypothesize that these “non-responding” mice
had not been successfully sensitized via the skin, possibly because the
cobalt solution (in DMSO) did not always persist long enough on the ears
(in our previous studies we always used a mixture of acetone and olive
oil to dissolve the test chemicals, but CoCl2 did not
dissolve in this vehicle). This may also explain why we failed to
observe significant cell proliferation of the auricular lymph nodes with
concordant cytokine release, and consistent increases in total serum IgE
in the mice dermally sensitized and pulmonary challenged with
CoCl2. The absence of these responses contrasts with our
previous extensive experience with chemical sensitizers in this
skin/lung model, since we always observed pronounced signs of skin and
systemic sensitization after applications of TDI, trimellitic anhydride
or ammonium persulfate [8–10]. The possibility that some animals
were in fact not dermally sensitized to Co is borne out by our
correlation analysis showing that in spite of the relatively low average
changes at group level, T helper cells
(CD3+CD4+) in the auricular lymph
nodes and serum IgE were positively associated with numbers of cDC1 and
ILC2 cells in the lung and other immunological and physiological
responses in individual animals (Table 1, Figure 4C-D). The
intra-individual consistency between the various responses strengthens
our conclusion that dermal sensitization to cobalt enhanced the
susceptibility of the airways to subsequent challenges with cobalt.
In conclusion, prior skin exposure to cobalt modulates immune cells in
lung, induces airway hyper-reactivity and a mixed
neutrophilic/eosinophilic inflammation upon respiratory challenge with
Co, which is accompanied by dendritic cells and innate lymphoid cells of
the type 2/type 3. This observation with cobalt strengthens the evidence
that asthma caused by low-molecular weight agents is not only related to
sensitizers reaching the lungs via airway exposure, but also to
chemicals sensitizing the body via skin exposure, which increases the
susceptibility of the lungs to inhaling sensitizers.