TRPA1 and CGRP antagonists counteract vesicant-induced skin injury and inflammation

Abstract

The skin is highly sensitive to the chemical warfare agent in mustard gas, sulfur mustard (SM) that initiates a delayed injury response characterized by erythema, inflammation and severe vesication (blistering). Although SM poses a continuing threat, used as recently as in the Syrian conflict, no mechanism-based antidotes against SM are available. Recent studies demonstrated that Transient Receptor Potential Ankyrin 1 (TRPA1), a chemosensory cation channel in sensory nerves innervating the skin, is activated by SM and 2-chloroethyl ethyl sulfide (CEES), an SM analog, in vitro, suggesting it may promote vesicant injury. Here, we investigated the effects of TRPA1 inhibitors, and an inhibitor of Calcitonin Gene Related Peptide (CGRP), a neurogenic inflammatory peptide released upon TRPA1 activation, in a CEES-induced mouse ear vesicant model (CEES-MEVM). TRPA1 inhibitors (HC-030031 and A-967079) and a CGRP inhibitor (MK-8825) reduced skin edema, pro-inflammatory cytokines (IL-1β, CXCL1/KC), MMP-9, a protease implicated in skin damage, and improved histopathological outcomes. These findings suggest that TRPA1 and neurogenic inflammation contribute to the deleterious effects of vesicants in vivo, activated either directly by alkylation, or indirectly, by reactive intermediates or pro-inflammatory mediators. TRPA1 and CGRP inhibitors represent new leads that could be considered for validation and further development in other vesicant injury models.

Accelerating Inflammation Resolution to Counteract Chemical Cutaneous Injury

“…Sulfur mustard analog-induced mouse ear vesicant model was developed as described in Achanta et al, 2018 (6). Briefly, the right ears of male 8 week old C57BL/6 mice were exposed to a total single dose of 0.2 mg (in 20 µL, ten microliters on each side of right ear) of 2-chloroethyl-ethyl-sulfide (CEES), a sulfur mustard analog following sevoflurane anesthesia.…”

“…Ear thickness was measured using spring-loaded electronic calipers (Mitutoyo QUICKmini, Japan). Three 4 mm ear punch biopsies were excised (4 mm Biopsy Punch, Miltex Inc., York, PA, USA) and weighed to determine edema as described previously (6). One punch biopsy was used to determine concentrations of pro-inflammatory mediators known to contribute to the vesicant injury.…”

“…Exposures to chemical threat agents oftentimes induce strong inflammatory tissue responses that contribute to morbidity and delayed tissue repair and recovery. Skin blistering agents (vesicants) such as sulfur mustard (HD or SM) in mustard gas, or its analog, 2-chloroethyl-ethyl-sulfide (CEES), while not immediately irritating, induce delayed inflammatory responses that contribute to the separation of skin layers and blister formation hours after exposure (1-6). This response is initiated by the alkylating activity of HD and CEES, followed by induction of oxidative stress and the formation of lipid peroxidation products.…”

“…Within a short time, pro-inflammatory cytokines and chemokines can be detected, including IL-1α/β, IL-6, IL-8, TNF-α, GM-CSF and KC, and inflammatory cells infiltrate the exposed skin, including neutrophils, mast cells, and macrophages. Proteases such as MMP-9 are induced that contribute to edema formation, tissue destruction and blistering (6). Sulfur mustard wound often shows incomplete healing, with patients burdened by chronic skin inflammation and pruritus even decades after exposure (2, 7).…”

“…The actual concentrations of incapacitating agents exposed to civilians and law enforcement authorities are highly variable, which further complicates the triaging of clinical symptoms and medical management. While some of the potential therapeutic drug candidates are being tested in pre-clinical studies and are promising, none of the compounds were approved by US FDA (6, 8, 18, 19, 24-28). The lack of effective antidotes for SM and CS tear gas-induced skin injuries constitute the need for continued screening and development efforts.…”