Suppression of Hyperactive Immune Responses Protects against Nitrogen Mustard Injury

Abstract

DNA alkylating agents like nitrogen mustard (NM) are easily absorbed through the skin and exposure to such agents manifest not only in direct cellular death but also in triggering inflammation. We show that toxicity resulting from topical mustard exposure is mediated in part by initiating exaggerated host innate immune responses. Using an experimental model of skin exposure to NM we observe activation of inflammatory dermal macrophages that exacerbate local tissue damage in an inducible nitric oxide synthase (iNOS)-dependent manner. Subsequently these activated dermal macrophages reappear in the bone marrow to aid in disruption of hematopoiesis and contribute ultimately to mortality in an experimental mouse model of topical NM exposure. Intervention with a single dose of 25-hydroxyvitamin D3 (25(OH)D) is capable of suppressing macrophage-mediated iNOS production resulting in mitigation of local skin destruction, enhanced tissue repair, protection from marrow depletion, and rescue from severe precipitous wasting. These protective effects are recapitulated experimentally using pharmacological inhibitors of iNOS or by compounds that locally deplete skin macrophages. Taken together, these data highlight a critical unappreciated role of the host innate immune system in exacerbating injury following exposure to NM and support the translation of 25(OH)D in the therapeutic use against these chemical agents.

Oral Vitamin D Rapidly Attenuates Inflammation from Sunburn: An Interventional Study

“…However, excessive production of iNOS perpetuates tissue damage, retards the resolution of inflammation, and prevents tissue repair (Bronte and Zanovello, 2005, Mills, 2012). We and others have shown using murine models that vitamin D 3 inhibits the production of TNF-α, and is capable of attenuating skin inflammation by reducing macrophage-specific iNOS production (Au et al, 2015, Zhang et al, 2012). …”

“…RNA was extracted from fresh frozen tissue using the RNeasy Lipid Mini Kit (Qiagen, Redwood City, CA), and tissue mRNA expression of TNF-α and iNOS were quantified as previously described (Au et al, 2015). A fold change representing the difference in RNA expression between the investigative and control phases of the study was calculated for TNF-α and iNOS [(RNA 48hr ) invest /(RNA 48hr ) control ].…”

“…To analyze the expression of the arginase-1 protein in skin, freshly frozen biopsy samples embedded in optimal cutting temperature compound were cut into eight micrometer sections and stained immunofluorescently as previously described (Au et al, 2015). CD163 was used as a marker for skin macrophages, and 4′-6-diamidino-2-phenylindole (DAPI) was used to stain nuclear DNA.…”

“…Vitamin D has pleiotropic effects on the immune system, including the enhancement of anti-microbial responses, induction of autophagy, and suppression of pro-inflammatory mediators, including tumor necrosis factor-α (TNF-α) (Di Rosa et al, 2012, Liu et al, 2006, Zhang et al, 2012). We recently demonstrated that intervention with a single dose of vitamin D is capable of rapidly attenuating an inflammatory response in a mouse model of chemical induced skin injury through inhibition of inducible nitric oxide synthase (iNOS) and TNF-α by activated macrophages (Au et al, 2015). Therefore, we designed a pilot, proof-of-principle interventional study in humans, modeled after a randomized, double-blinded, placebo-controlled clinical trial, to test the hypothesis that a single high dose of oral vitamin D 3 (cholecalciferol) would be capable of rapidly attenuating experimental sunburn induced by simulated solar radiation (SSR).…”

Defining the timing of 25(OH)D rescue following nitrogen mustard exposure

“…Vitamin D (VD), an endocrine hormone responsible for maintenance of musculoskeletal health, is emerging as a powerful immunomodulatory drug responsible for suppressing inflammation 8–10 . We have recently demonstrated the efficacy of 25(OH)D to suppress early inflammation, mitigate tissue destruction and prevent systemic toxicity including death in animals exposed to NM 9,11 . Intervention with a single dose of 25(OH)D administered in a mouse model of NM-mediated cutaneous injury 1 h following exposure suppressed inflammatory mediators tumor necrosis factor-a and inducible nitric oxide synthase in the skin, promoted wound healing and ensured animal survival.…”