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.

Grant Number: 1R56AR085100-01A1
NIH Institute/Center: NIH
Principal Investigator: Bogi Andersen