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. 2018 Sep 1:293:140-148.
doi: 10.1016/j.toxlet.2018.03.007. Epub 2018 Mar 10.

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

Satyanarayana Achanta  1 , Narendranath Reddy Chintagari  2 , Marian Brackmann  2 , Shrilatha Balakrishna  2 , Sven-Eric Jordt  3
Affiliations

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

Satyanarayana Achanta et al. Toxicol Lett. .

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.

Keywords: CEES; CGRP; Medical countermeasures; Mouse ear vesicant model (MEVM); Skin vesicant injury; Sulfur mustard; TRPA1.

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Conflict of interest statement

Conflict of interest

Sven-Eric Jordt is serving on the Scientific Advisory Board of Hydra Biosciences Inc., a biopharmaceutical company developing TRP ion channel inhibitors for the treatment of pain and inflammation.

Figures

Figure 1
Figure 1. Gross morphological parameters and histopathology in CEES-exposed mice, treated with TRPA1 inhibitors or vehicle
(A) CEES exposure and treatment regimen. TRPA1 inhibitors (HC-030031, i.p or A-967079, p.o) or vehicle (i.p or p.o) was administered at 1, 8, and 16 h after CEES exposure. (B and C) Ear thickness and ear punch biopsy weights. Ear thickness was measured by spring loaded calipers. Ear punch biopsy weights were measured from three 4 mm ear punch biopsy samples. Percent increase or decrease in ear thickness and ear punch biopsy weights over control is presented. (D) Representative H&E stained ear punch biopsy histopathology profiles are presented. e=epidermis; d=dermis; hf=hair follicle; ec=elastic cartilage; black arrows=desquamation and necrosis of epidermis; black arrow heads = infiltration of leucocytes; red arrows = microvesication; Data are presented as mean ± SEM. n=18/vehicle group; 10/treatment group. Statistical significance of the difference between the groups was determined by one-way ANOVA followed by Tukey’s multiple comparison post-hoc test. * p < 0.05; **p < 0.01; ****p<0.0001; ns=not significant.
Figure 2
Figure 2. Effects of TRPA1 inhibitor treatment on pro-inflammatory cytokines in ear punch biopsy samples of CEES-exposed mice
TRPA1 inhibitors (HC-030031, i.p or A-967079, p.o) or vehicle (i.p or p.o) were administered at 1, 8, and 16 h after CEES exposure. (A and B) Quantification of pro-inflammatory cytokines IL-1β and CXCL1/KC in tissue homogenates using ELISA. (C) Levels of MMP-9 in tissue homogenates determined by ELISA. Data are presented as mean ± SEM. n=41/control (dichloromethane only) measurments from all groups; 18/CEES+vehicle (0.5% methyl cellulose), and 9–10/treatment (CEES+HC or CEES+A96). Statistical significance of the difference between the groups was determined by one-way ANOVA followed by Tukey’s multiple comparison post-hoc test. *p < 0.05; **p < 0.01; ****p<0.0001.
Figure 3
Figure 3. Gross morphological parameters in CEES-exposed mice, treated with CGRP antagonist or vehicle
(A) CEES exposure and treatment regimen. CGRP antagonist (MK-8825, p.o) or vehicle (p.o) was administered at 1, 8, and 16 h after CEES exposure. (B) CGRP levels in ear punch biopsy homogenate samples, determined by ELISA (C) Ear thickness, and (D) ear punch biopsy weights. Ear thickness was measured by spring loaded calipers. Ear punch biopsy weights were measured from three 4 mm ear punch biopsy samples. Percent increase or decrease in ear thickness and ear punch biopsy weights over control is presented. Data are presented as mean ± SEM. n= 4–6/group (B), 8–9/group (C and D). Statistical significance of the difference between the groups was determined by two-tailed unpaired t-test. *p < 0.05; ns= non-significant; nd = non-detectable.
Figure 4
Figure 4. Effects of CGRP inhibition on levels of pro-inflammatory cytokines and injury mediators in ear skin of CEES-exposed mice, treated with CGRP antagonist or vehicle
CGRP antagonist (MK-8825, p.o) or vehicle (p.o) was administered at 1, 8, and 16 h after CEES exposure. (A and B) Pro-inflammatory cytokines IL-1β and CXCL1/KC were quantified in tissue homogenates using ELISA (C) Levels of MMP-9 in tissue homogenates, determined by ELISA. Data are presented as mean ± SEM. For MMP-9 and IL-1β: n=20-22/control (dichloromethane only) ears from all groups, 9/CEES+vehicle (methyl cellulose) ears, and 6–8/treatment (CEES+MK-8825) ears; For KC: n=12/control (dichloromethane only) ears, 8/CEES+vehicle (0.5% methyl cellulose) ears, and 7/treatment (CEES+MK-8825) ears. Statistical significance of the difference between the groups was determined by one-way ANOVA followed by Tukey’s multiple comparison post-hoc test. *p < 0.05; **p < 0.01; ****p<0.0001; ns = non-significant.
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