Postexposure Application of Fas Receptor Small-Interfering RNA to Suppress Sulfur Mustard–Induced Apoptosis in Human Airway Epithelial Cells: Implication for a Therapeutic Approach

Abstract

Sulfur mustard (SM) is a vesicant chemical warfare and terrorism agent. Besides skin and eye injury, respiratory damage has been mainly responsible for morbidity and mortality after SM exposure. Previously, it was shown that suppressing the death receptor (DR) response by the dominant-negative Fas-associated death domain protein prior to SM exposure blocked apoptosis and microvesication in skin. Here, we studied whether antagonizing the Fas receptor (FasR) pathway by small-interfering RNA (siRNA) applied after SM exposure would prevent apoptosis and, thus, airway injury. Normal human bronchial/tracheal epithelial (NHBE) cells were used as an in vitro model with FasR siRNA, FasR agonistic antibody CH11, and FasR antagonistic antibody ZB4 as investigative tools. In NHBE cells, both SM (300 mM) and CH11 (100 ng/ml) induced caspase-3 activation, which was inhibited by FasR siRNA and ZB4, indicating that SM-induced apoptosis was via the Fas response. FasR siRNA inhibited SMinduced caspase-3 activation when added to NHBE cultures up to 8 hours after SM. Results using annexin V/propidium iodidestained cells showed that both apoptosis and necrosis were involved in cell death due to SM; FasR siRNA decreased both apoptotic and necrotic cell populations. Bronchoalveolar lavage fluid (BALF) of rats exposed to SM (1 mg/kg, 50 minutes) revealed a significant (P , 0.05) increase in soluble Fas ligand and active caspase-3 in BALF cells. These findings suggest an intervention of Fas-mediated apoptosis as a postexposure therapeutic strategy with a therapeutic window for SM inhalation injury and possibly other respiratory diseases involving the Fas response.

Mustard vesicant-induced lung injury: Advances in therapy

“…Non-coding RNA-based therapeutics are currently under investigation for treatment of lung diseases and pathologies including injury caused by mustards. Keyser et al (2013) analyzed the effects of siRNA targeting the Fas receptor (FasR) on the toxicity of SM in human bronchial/tracheal epithelial cells. FasR siRNA was found to inhibit SM-induced caspase 3 activation via the extrinsic pathway; this was associated with significant decreases in both apoptosis and necrosis.…”

Mustard Gas Inhalation Injury

“…A recent report has also demonstrated that the use of RNA interference against the Fas receptor, applied up to 8 hours postexposure in vitro, was able to significantly reduce apoptosis 24 hours after SM exposure. 24 This use of a highly selective antagonist of the Fas-mediated pathway of apoptosis added further evidence for an important role of this pathway in SM-induced apoptosis. Other proteins in the extrinsic apoptosis pathways (eg, cFLIP, DR-4, and DR-5) could play a role in SM-induced apoptosis.…”

“…[5][6][7][8][9][10][11][12][13] The multiplicity of the different cellular components (DNA, proteins, thiol compounds, etc) and the pathways affected by SM have complicated the efforts to establish a clearly defined mechanism-based therapeutic approach for SM exposure. As schematically indicated in Figure 1, some of the toxic effects of SM and the half mustard (2-chloroethyl sulfide, CEES) include oxidative stress, [14][15][16][17][18][19][20] proinflammatory pathways, 21,22 apoptosis, 19,[23][24][25] and DNA damage. 26,27 In addition to these toxic effects, low-level exposure to SM causes weight loss and sensory irritation.…”