Vitamin D attenuates acute skin inflammation following nitrogen mustard exposure by targeting M1 macrophages

Cutaneous exposure to alkylating agents such as nitrogen mustard (NM) causes severe blistering with systemic complications long after the exposure. We have previously shown that a single dose of vitamin D (VD) is sufficient to rescue NM-exposed mice from pancytopenia and death and that early intervention with VD is of critical importance. However, the effect of VD on cellular infiltration and local milieu in the skin is unknown. Here we propose that VD promotes skin recovery by preventing dermal infiltration including pro-inflammatory macrophages to maintain a cellular distribution that accelerates skin repair to restore immune equilibrium. Dermal derived cells isolated from skin of NM-exposed mice were analyzed by flow cytometry for total cell counts and macrophage characterization. Mice treated with VD strongly inhibited cellular infiltration to the inflamed skin on day 2 post exposure (p=0.01) especially F4/80+ macrophages (p=0.02). Decreased infiltration was accompanied by a concomitant decrease in tissue pro-inflammatory cytokine, inducible nitric oxide synthase. On day 5 post NM, VD expanded the number of cells populating the skin suggesting that these cells were conducive to skin repair (p=0.01). To assess the distribution of pro-inflammatory M1 and anti-inflammatory M2 macrophages modulated by VD, F4/80 macrophages were stained for Ly6C and CD206 respectively. The findings demonstrate that compared to NM-exposed mice, VD intervention restrained the expansion of F4/80+Ly6C+ M1 macrophages in the inflamed skin on day 6 (p=0.04). Furthermore, a clear opposing trend is observed between M1 and M2 macrophage populations in the skin of VD-treated mice on day 6 (p=0.005), that is absent in NM-only mice, that sets the foundation for accelerated skin recovery. Taken together our results define a critical role for vitamin D in regulating macrophage infiltration and its phenotype to diminish local inflammation and accelerate skin repair.