P32-14 Immune and inflammatory mechanism in mustard vesicating agents induced long-term skin toxicity

Vesicating agents sulfur mustard (SM) and nitrogen mustard (NM), lewisite (L) and phosgene oxime (CX) are potential chemical warfare and terrorist weapons with both military and terrorist potential. SM has been the most extensively used warfare agent in numerous conflicts and causes severe acute injuries and long-term effects, such as blistering, systemic toxicity, and potentially life-threatening secondary infections. Even though SM causes debilitating skin injuries, its mechanism of action leading to the long-term effects is not fully understood and targeted therapies are lacking. In our study, we seek to establish a mouse model of long-term injury arising from acute nitrogen mustard (NM, an analog of SM) exposure and to identify key biomarkers. Male C57BL/6 mice were topically exposed to 0.5 mg NM in 100 µL of acetone. Clinical assessments were carried out, and the tissues were collected at various timepoints for over a 3-month period post-exposure. Histological injury assessments and PCR analysis for inflammatory, fibrotic, and antioxidant markers was carried out in the skin tissue samples. Significant weight loss was observed in mice at 7 days post-NM exposure, with chronically suppressed weight gain even after recovery to pre-exposure weights. Erythema was observed during the first 14 days post exposure, while edema was observed throughout the study period. Necrotic patches on the skin were first seen at 3 days post exposure that remained throughout the study period. Histopathological lesions, such as scabs, hyperkeratosis, eschars, acanthosis, necrosis and micro vesications, were observed in NM-exposed mice. Interestingly, the skin pathology of NM-exposed mice at later study time points resembled that of psoriatic mice. A bimodal mast cell activation was observed during the first week, a drop at 14 days, that again elevated up to 84 days post NM exposure. Heme oxygenase-1, an antioxidant marker, and Inflammatory markers [Interleukins (IL-6, IL-1β), matrix metalloproteinase-9, cyclooxygenase-2, and Tumor Necrosis Factor alpha, C-C motif ligand 2] as well as fibrotic and psoriatic markers (transforming growth factor-beta and interleukins-17 and -23) were elevated in NMexposed mice 1-3 months post NM exposure. These results indicate that a single acute topical exposure to NM can cause long-term inflammatory lesions like psoriasis and fibrosis mimicking human toxic effects. Further investigations into mast-cell related mechanism in long-term consequences of mustard agent exposure are being explored using mast cell deficient (MCD) mice. The preliminary results from our studies show significantly greater survival and less body weight loss in MCD mice compared to wild type mice post NM-exposure. Further molecular and spatial transcriptomic studies are being carried out to elucidate if mast cells could be important therapeutic targets in vesicating agents induced skin inflammation and long-term consequences.