Despite the high morbidity and mortality among patients with extensive cutaneous burns in the intensive care unit due to the development of acute respiratory distress syndrome, effective therapeutics remain to be determined. This is primarily because the mechanisms leading to acute lung injury (ALI) in these patients remain unknown. We test the hypothesis that cutaneous chemical burns promote lung injury due to systemic activation of neutrophils, in particular, toxicity mediated by the deployment of neutrophil extracellular traps (NETs). We also demonstrate the potential benefit of a peptidyl arginine deiminase 4 (PAD4) inhibitor to prevent NETosis and to preserve microvascular endothelial barrier function, thus reducing the severity of ALI in mice. Our data demonstrated that phenylarsine oxide (PAO) treatment of neutrophils caused increased intracellular Ca ²⁺ -associated PAD4 activity. A dermal chemical burn by lewisite or PAO resulted in PAD4 activation, NETosis, and ALI. NETs disrupted the barrier function of endothelial cells in human lung microvascular endothelial cell spheroids. Citrullinated histone 3 alone caused ALI in mice. Pharmacologic or genetic abrogation of PAD4 inhibited lung injury following cutaneous chemical burns. Cutaneous burns by lewisite and PAO caused ALI by PAD4-mediated NETosis. PAD4 inhibitors may have potential as countermeasures to suppress detrimental lung injury after chemical burns.

Vesicants such as arsenicals and mustards produce highly painful cutaneous inflammatory and blistering responses, hence, developed as chemical weapons during World War I/II. Here, using lewisite and sulfur mustard surrogates namely phenylarsine oxide (PAO) and 2-chloroethyl ethyl sulfide (CEES) respectively, we defined a common underlying mechanism of toxic action by these two distinct classes of vesicants. Murine skin exposure to these chemicals causes tissue destruction characterized by increase in skin bifold thickness, Draize score, infiltration of inflammatory cells and appearance of apoptotic epidermal and dermal cells. RNAseq analysis identified ~346 inflammatory genes that were commonly altered by both PAO and CEES along with identification of cytokine signaling activation as the top canonical pathway. Activation of several of these inflammatory genes and pathways are associated with phosphorylation-dependent activation of heat shock protein 90α (p-HSP90α). Topical treatment with known HSP90 inhibitors, SNX-5422 and IPI-504 post PAO or CEES skin challenge significantly attenuated these chemical-induced skin damage (reduction in overall skin injury and clinical scores). In addition, highly upregulated inflammatory genes Saa3, Cxcl1, Ccl7, IL-6, Nlrp3, Csf3, Chil3 etc. by both PAO and CEES, were significantly diminished by the treatment with these HSP90 inhibitors. These drugs not only reduced PAO or CEES-induced p-HSP90α expression but also its client proteins NLRP3, pP38 and expression of their target inflammatory genes. Our data confirm a critical role of HSP90 as a shared underlying molecular signaling of toxicity by these two distinct vesicants and provide an effective and novel medical countermeasure to suppress vesicants -induced skin injury. Significance Statement Development of effective and novel mechanism-based antidotes which can simultaneously block cutaneous toxic manifestations of distinct vesicants is important and urgently needed. Due to difficulties in determining the exact nature of onsite chemical exposure, a potent drug that can suppress widespread cutaneous damage may find a great utility. Thus, we identified HSP90 as a common molecular regulator of cutaneous inflammation and injury by two distinct warfare vesicants: arsenicals and mustards; and HSP90 inhibitors afford significant protection against skin damage.

Lewisite, a chemical warfare agent, induces severe skin injury by oxidative stress and endoplasmic reticulum (ER) dysfunction, necessitating innovative antidote strategies. This study developed chitosan nanoparticle-loaded foam formulations for rapid skin decontamination and sustained topical delivery of 4-phenylbutyric acid (4-PBA), an ER stress-reducing chaperone. Nanoparticles were synthesized via ionic gelation using low (LMW) and medium molecular weight (MMW) chitosan. The optimized formulations, N31 (LMW) and N35 (MMW), achieved drug loadings of 5.04 % and 10.09 % w/w, particle sizes of 141.88 ± 26.31 nm and 176.10 ± 36.97 nm, monodisperse distributions (PDI < 0.3), high entrapment efficiency (>93 %) and good stability with zeta potential of -16.67 mV and -19.37 mV, respectively. Incorporation into foam enabled both effective decontamination (>70 % efficiency) and sustained 4-PBA delivery. In vitro release studies demonstrated sustained drug release over 24 h. Permeation studies using dermatomed human skin revealed that nanoparticle formulations significantly reduced 4-PBA delivery: N35 decreased permeation by 38.4 % (214.35 ± 16.6 µg/cm² vs. 348.10 ± 5.37 µg/cm² for free 4-PBA), while N31 reduced it by 81.35 % (64.90 ± 6.89 µg/cm²). Both formulations retained efficacy in PAO challenged skin, with N35 delivering 158.54 ± 53.93 µg/cm² and N31 138.25 ± 14.72 µg/cm² over 24 h. Furthermore, in vivo studies showed that the optimized formulation with N35 chitosan (4-PBA N35 + N-acetyl cysteine (NAC)) significantly protects against PAO-induced skin injury and inflammatory cytokine production in Ptch1+/-/SKH-1 hairless mice. Thus, the translational feasibility and effective treatment by the foam formulated 4-PBA N35 + NAC against arsenical-induced skin injury is demonstrated.