Skin cell heterogeneity in sensitivity to mustard-induced cell death - Abstract Sulfur mustard, also known as mustard gas, is a threat in chemical warfare and terrorism. Although major advances have been made in understanding mustard-induced skin injury, effective countermeasures are lacking. Under an R34 grant, we have been investigating shared and unique mechanisms of mustard- and arsenical- induced skin injury with single cell tanscriptomics and fluorescence lifetime imaging microscopy. Using nitrogen mustard as a surrogate for sulfur mustard, we unexpectedly found that dermal fibroblasts are damaged early after mustard exposure and that CDK8, a kinase component of the Mediator transcription complex, is a potential new treatment target in mustard-induced skin injury. We also found that the vitamin B12 analog cobinamide, a new and potent antioxidant that neutralizes both reactive oxygen and nitrogen species, effectively counters the skin and systemic toxicity of nitrogen mustard and phenylarsine oxide, a common research surrogate for lewisite. Furthermore, we found that cobinamide is more effective than N-acetyl-cysteine (NAC) in countering nitrogen mustard-induced skin damage. The goals of this project are to understand the mechanisms of mustard-induced skin injury and how cobinamide affects these mechanisms. Ultimately, insights from this research could be used to develop new countermeasures that will be effective on their own or in combination with cobinamide. The Specific Aims are: 1. To understand the variable sensitivity of different skin cells to mustard-induced cell death. This Aim focuses on the early loss of fibroblasts and tests the hypothesis that different skin cells have differential sensitivity to mustard-induced injury: whereas some cells initiate a cell death program, other cells are more resistant and may ultimately recover. We will combine single cell RNA sequencing with spatial transcriptomics and we will test whether CDK8 contributes to mustard-induced injury. The studies will be done on SKH-1 mice, initially with nitrogen mustard and then with sulfur mustard. 2. To understand mustard-induced effects on skin fibroblasts and their interactions with immune cells. This Aim tests the hypothesis that skin fibroblasts play an important role as early sensors of mustard exposure and that they activate and recruit neutrophils and macrophages. We will use multiphoton intravital microscopy to study the dynamics of myeloid cell accumulation, and as in Aim 1, studies will be done with nitrogen mustard in mice. In both Aims, we will test how cobinamide affects the mechanisms under study. This research is innovative because cellular heterogeneity in response to mustard-induced skin injury and the role of dermal fibroblasts during mustard-induced injury are under-investigated. Moreover, we are using state-of-the-art techniques, we have assembled a talented multi- disciplinary team, and we are testing cobinamide, a new type of antioxidant that is bifunctional for reactive oxygen and nitrogen species and has not been previously evaluated in vesicant-induced skin injury. The studies are significant because no effective countermeasures exist against mustard-induced skin injury.
