Conflict of Interest Statement: JEH has related IP and equity interest in and receives consulting fees in the form of equity options from Patrys Ltd, a biotechnology company that has licensed IP that is the basis of this work. ZR, VD, JAC, JZ, and JEH are inventors on IP related to DX1. JZ receives consulting fees in the form of equity options from Patrys Ltd. VD and JAC are employees of and have equity interest in Patrys Ltd. The remainder of the authors declare no competing interests.

Supplemental Figure 1: Flow chart depicting the experimental design used in this study. Mice were separated into 10 groups comprised of a minimum of 6 mice each. At time zero, the right ears were treated with a 5 µl volume of HN2 (0.500 µmol/ear), while the left ears were not treated with HN2 and served as test controls. Next, both the left and right ears received three topical treatments (3X) of DMSO or EB-2 (0.250), (0.500), (0.750), (1.000), (1.250) and (2.000) mg/ear at 15 min, 4 h and 8 h after HN2 exposure. A single treatment (1X) of DMSO or EB-2 (1.000 mg/ear) was also applied 15 min following HN2 exposure alongside the aforementioned 3X treatments. An additional group of 13 mice served as untreated (naïve) controls and did not receive any treatment whatsoever. All ear punch biopsies were collected 24 h after the initial exposure to HN2.

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard gas mimetic which is also used in anticancer therapy. HN2 is associated with skin inflammation and blistering which can lead to secondary infections. The purpose of the present study was to investigate the time-dependent dermatotoxicity of HN2 using the mouse ear vesicant model (MEVM). To this end, our operational definition of dermatotoxicity included tissue responses to HN2 consistent with an increase in the wet weights of mouse ear punch biopsies, an increase in the morphometric thickness of H&E stained ear sections and histopathologic observations including tissue edema, inflammatory cell infiltration and vesication. The ears of male Swiss Webster mice were topically exposed to a single dose of HN2 (0.5 μmol/ear) or DMSO vehicle (5 μl/ear) or left untreated (naive). Mice were then euthanized at 15 min, 1, 2, 4, 8 or 24 hr following HN2 exposure. Compared to control ears, mouse ears exposed to HN2 at all time points showed an increase in wet weights, morphometric thickness, edema, inflammatory cell infiltration and signs of vesication. The incidence in tissue vesication sharply increased between 4 and 8 hr after exposure, revealing that tissue vesication is well established by 8 hr and remains elevated at 24 hr after exposure. It is noteworthy that, compared to control ears, mouse ears treated with DMSO vehicle alone also exhibited an increase in wet weights and morphometric thickness at 15 min, 1, 2 and 4 hr following treatment; however, these vehicle effects begin to subside after 4 hr. The results obtained here using the MEVM provide a more holistic understanding of the kinetics of vesication, and indicate that time points earlier than 24 hr may prove useful not only for investigating the complex mechanisms involved in vesication but also for assessing the effects of vesicant countermeasures.

Nuclear-penetrating anti-DNA autoantibodies have therapeutic potential as delivery agents and in targeting DNA and the DNA damage response (DDR). Derivatives of such Abs have advanced to human testing in genetic disease and are in preparation for oncology clinical trials. DNA release associated with neutrophil extracellular traps (NETs) contributes to immunity, inflammation, and the pathophysiology of multiple diseases. The DDR contributes to mechanisms of NETosis, and we hypothesize that anti-DNA autoantibodies that localize into live cell nuclei and inhibit DNA repair will suppress release of NETs by activated neutrophils. In the current study we evaluated the impact of a nuclear-penetrating anti-DNA autoantibody that interferes with the DDR on decondensation and release of DNA and NETs by activated human granulocyte-like differentiated PLB-985 cells and neutrophils isolated from C57BL/6 mice. The response of cells pretreated with control or autoantibody to subsequent stimulators of NETosis, including PMA and the calcium ionophore ionomycin, was evaluated by DAPI and SYTOX Green stains, measurement of DNA release, analysis of histone citrullination by Western blot, or visualization of NETs by immunostaining and confocal fluorescence microscopy. Autoantibody treatment of the cells yielded significant inhibition of NADPH oxidase–dependent and independent NETosis. These findings establish the concept of nuclear-penetrating anti-DNA autoantibodies as modulators of neutrophil biology with potential for use in strategies to suppress NETosis.

Graphical abstract