Introduction
Chemical nitrobenzene compounds such as 2, 4-dinitrofluorobenzene (DNFB)
and 2, 4-dinitrochlorobenzene (DNCB) have long been known to cause
contact skin irritation and sensitization and they are also used for
establishment of skin inflammatory models in rodents, (Landsteiner and
Chase, 1941; Ren et al., 2019; Tingle et al., 1990). Topical application
of DNFB, commonly called
Sanger’s
reagent used for protein sequencing, mediates contact hypersensitivity
and induces allergic contact dermatitis (ACD) (Garcia-Perez, 1978; Hsieh
et al., 1996). DNFB is also commonly used to sensitize skin and cause
colitis in mice (Rijnierse et al., 2006a; Rijnierse et al., 2006b).
Chemical DNFB as an allergen also elicits immune reactions by inducing
mast cell degranulation and releases of histamine (Ren et al., 2019),
interleukin-1 (IL-1) and prostaglandin E2 (PGE2) (Natsuaki et al.,
1989). However, how DNFB causes skin sensitization and allergic contact
dermatitis at molecular level remains unknown.
A growing number of evidences have indicated that activation of
transient receptor potential ankryn 1 (TRPA1) ion channels is involved
in skin inflammation (Tóth. et al., 2014). TRPA1 is robustly expressed
in primary
sensory
nerve terminals (Mandadi and Roufogalis, 2008) and in numerous
non-neuronal cell types of the skin (Atoyan et al., 2009) and CD4+ T
lymphocytes that play a central role in the adaptive immune response
(Bertin et al., 2017; Li et al., 2020). Activation of TRPA1 by
icilin
in keratinocytes leads to an eluviation of proinflammatory cytokine
interleukin-1 (IL-1), which suggests a role of TRPA1 in promoting
cutaneous inflammation
(Atoyan
et al., 2009). Pharmacological inhibition or deficiency of TRPA1
alleviates inflammation of atopic dermatitis (AD)
(Oh
et al., 2013;
Zeng
et al., 2021). Conversely, specific activation of TRPA1 by agonist
mustard oil (MO) leads to a severe colitis, which is inhibited by
HC-030031 or reduced in TRPA1-/-mice
(Engel
et al., 2011).
TRPA1 is a temperature-sensitive and calcium-permeable cation channel
with a four-fold symmetry around the central ion conductance pathway and
proximal cytoplasmic regions involved in electrophile detection (Liao et
al., 2013; Paulsen et al., 2015; Story et al., 2003). TRPA1 is a sensor
for chemical irritants, such as MO (Jordt et al.,
2004),
acrolein, cinnamaldehyde (CA) (Bandell et al., 2004) and allyl
isothiocyanate (AITC) (Sandor et al., 2016). TRPA1 is also activated by
harmful electrophiles that are recognized by the channel via covalent
modifications of specific cysteine residues located in the cytoplasmic
domain (Suo et al., 2020). All these investigations suggest us that skin
sensitizer DNFB might act on TRPA1 channel that is considered a key
player in skin inflammation.
In this study, we discovered that chemical DNFB that specifically
activates TRPA1 channels through its binding to three key residues
critical in the channel coupling domain for electrophile irritant
sensing using assays of patch clamp recordings in combination with
site-directed mutagenesis and molecular docking. Our findings not only
help explain the mechanistic insights into nitrobenzene DNFB-induced
contact dermatitis, but also provide a molecular tool for further
understanding TRPA1 channel pharmacology in skin-related diseases.