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.