4. Discussion
A better understanding of the molecular and cellular mechanisms
underlying AD is critical for developing new effective, and safe
therapeutic strategies to treat the debilitating skin conditions. In
this study, we investigated the anti-AD potential of TRPA1 by using
TRPA1 knockout mice. Our data suggested that the genetic ablation of the
TRPA1 could reduce the clinical features induced by DNCB in mice.
AD is a chronic inflammatory skin disease mainly characterized by
itching symptoms (observed in 87%-100% patients) [18, 19]. The
vicious cycle of pruritus-scratch induces/aggravates inflammation of AD,
and causes insomnia and fatigue, which seriously affects the patients’
quality of life [20]. TRPA1 is the only member of the TRP channel
anchor protein (TRPA) subfamily, which is widely expressed in sensory
neurons and non-neuronal cells. TRPA1 is involved in the transmission of
several different sensory modes (cold, pain, and pruritus) and in the
mediation of neurogenic inflammation [21]. Current studies have
shown that TRPA1 expression is up-regulated in AD patients, where it has
an important role in histamine-independent pruritus. TSLP, an
inflammatory cytokine, activates TRPA1 by binding it to the receptor
TSLPR on sensory nerve fibers, causing pruritus in mice. Besides, TRPA1
has an essential role in Th2 cell-dependent pruritus mediated by IL-31
receptor expressed on sensory nerves. In the transgenic AD mice model
with overexpression of IL-13, TRPA1 antagonists can significantly
alleviate itching [22-25].
In the present study, we found that TRPA1 knockout reduces the frequency
of itching symptoms and the severity of skin lesions in AD mice.
Besides, the TRPA1 knockout alleviated the pathological change
associated with AD by reducing the number of mast cells, serum IgE,
inflammatory cytokines, and macrophage infiltration, which further
suggests that TRPA1 has an inflammatory positive role in DNCB-induced
AD. Hapten oxazolidone can activate the TRPA1 pathway, up-regulate
inflammatory cytokines, neuropeptide (CXCL-2), and nerve growth factor
(NGF) in chronic dermatitis induced by hapten oxazolidone and can
promote the occurrence of inflammation and pruritus [11]. However,
in allergic contact dermatitis induced by squaric acid dibutylester
(SADBE), TRPA1 participates in chronic pruritus induced by SADBE, while
epithelial skin inflammation persists in TRPA1 knockout mice
[12]. We speculated that
although TRPA1 participates in chronic pruritus induced by different
hapten, its role in skin inflammation may be different. Our findings
suggest that TRPA1 promotes DNCB-induced AD inflammation, which is
consistent with the observation that TRPA1 knockout could reduce
inflammatory cell infiltration and epidermal thickening.
Mast cells have an essential role in the pathogenesis of AD. Those cells
can produce and secrete inflammatory mediators such as histamine, growth
factors, cytokines, and chemokines [26]. A previous study [25]
reported a high expression of TRPA1 in dermal afferent nerves and mast
cells from patients with AD, but not in the skin from healthy subjects.
Besides, the same study found that allyl isothiocyanate, TRPA1 agonist,
increases the calcium signal of those cells [25]. Furthermore, Hoxet al found that TRPA1-dependent activation of sensory neurons
and mast cells regulate the non-allergic airway hyperresponsiveness in
mice [27]. In this study, we found a lower number of mast cells in
DNCB treated- TRPA1-/- mice compared to DNCB treated- WT group. TRPA1
deficiency inhibited the infiltration of mast cells in the dermis of the
DNCB-induced AD model, thus suggesting that TRPA1 may promote AD
symptoms by promoting mast cell infiltration.
Immunological abnormalities of AD are characterized by the imbalance of
Th1/Th2 and abnormal secretion of inflammatory factors. Previous studies
have shown that toluene diisocyanate can activate the TRPA1 channel,
while the antagonize TRPA1 can inhibit Th2 inflammation in allergic
airway inflammation induced by TRPA1 [28]. Oxazolone can activate
the TRPA1 channel, and TRPA1 can up-regulate pre-inflammation and Th2
related cytokines [11]. Our data indicated that TRPA1-/- reduces the
expression of pro-inflammatory factors and Th2 inflammatory cytokines in
DNCB-induced AD mice, thus suggesting that TRPA1 may promote the
symptoms of AD by regulating pro-inflammatory factors (IL-1beta; IL-6),
and Th-2 inflammatory cytokines (IL-4; IL-13).
Activation and sensitization of TRPA1 can regulate skin inflammation by
increasing the release of inflammatory mediators; yet, the role of TRPA1
in immune cells needs to be further explored. In chronic AD, the
increase of macrophage infiltration is closely related to the degree of
skin damage and immune dysfunction [29]. Macrophages, which have a
key role in the immune response, can be divided into two functional
subgroups: macrophages polarized to M1 under the stimulation of
interferon-gamma and lipopolysaccharide; and macrophages polarized to M2
under the stimulation of IL-4 and IL-13 [30]. F4/80 antigen is a
mature mouse cell surface glycoprotein that is highly expressed in both
M1 and M2 macrophages [31]. Our study showed that TRPA1 deficiency
reduced the expression of F4/80 protein and the number of positive
cells, and the expression of iNOS (expressed only M1 macrophages) and
CD206 protein (expressed only M2 macrophages), thus suggesting that
TRPA1 deficiency may inhibit the infiltration of dermal macrophages in
DNCB-induced AD model. In addition, our data suggest that TRPA1 may
promote AD inflammation by promoting macrophage infiltration. Although
TRPA1 activator cinnamaldehyde inhibits the release of IL-1β, IL-6 and
TNF-α
mediated by lipopolysaccharide and lipophosphatidic heterocyclic acid
from mouse macrophages and human monocytes, it suggests that TRPA1 may
have anti-inflammatory effects in monocytes [9, 32]. However, in the
DNCB-induced AD mice model, TRPA1 deficiency inhibits the infiltration
of macrophages, which is consistent with the fact that TRPA1 mediates
the infiltration of macrophages during corneal wound healing and
antagonizing TRPA1 reduces the proportion of M2 macrophages to improve
myocardial fibrosis [33, 34]. It is also consistent with the
decreased expression of inflammatory factors (IL-1β; TNF-α; IL-6)
observed in our study. Yet, the role of TRPA1 in macrophages needs to be
further elucidated.
Nowadays, the small molecule targeted therapy of AD has been attracting
increasing attention [35]. We further explored the therapeutic
effect of TRPA1 antagonist HC-030031 on the DNCB-induced AD model. Our
results showed that HC-030031 can effectively alleviate skin lesions and
pruritus in the AD mouse model. Pathological findings showed that it can
alleviate inflammatory cell infiltration and epidermal thickening,
similar to those observed in TRPA1 deficient mice. At the same time, the
results showed that the TRPA1 antagonist had an anti-AD effect in the
DNCB-induced AD model and could be used as one of the therapeutic
targets of AD.
In conclusion, our study demonstrated that TRPA1 has a crucial role
during AD pathogenesis in mice. TRPA1 deficiency alleviated pruritus and
showed an immunoprotective effect on DNCB-induced AD mice. In addition,
TRPA1 antagonist HC-030031 had a certain therapeutic effect on
DNCB-induced AD mice. These data suggest that TRPA1 can be used as a
potential new target for treating patients with AD.