Disulfiram inhibits neutrophil extracellular trap formation and protects rodents from acute lung injury and SARS-CoV-2 infection
Jose M. Adrover, Lucia Carrau, Juliane Daßler-Plenker, Yaron Bram, Vasuretha Chandar, Sean Houghton, David Redmond, Joseph R. Merrill, Margaret Shevik, Benjamin R. tenOever, Scott K. Lyons, Robert E. Schwartz, Mikala Egeblad
Jose M. Adrover, Lucia Carrau, Juliane Daßler-Plenker, Yaron Bram, Vasuretha Chandar, Sean Houghton, David Redmond, Joseph R. Merrill, Margaret Shevik, Benjamin R. tenOever, Scott K. Lyons, Robert E. Schwartz, Mikala Egeblad
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Research Article COVID-19 Immunology

Disulfiram inhibits neutrophil extracellular trap formation and protects rodents from acute lung injury and SARS-CoV-2 infection

Abstract

Severe acute lung injury has few treatment options and a high mortality rate. Upon injury, neutrophils infiltrate the lungs and form neutrophil extracellular traps (NETs), damaging the lungs and driving an exacerbated immune response. Unfortunately, no drug preventing NET formation has completed clinical development. Here, we report that disulfiram — an FDA-approved drug for alcohol use disorder — dramatically reduced NETs, increased survival, improved blood oxygenation, and reduced lung edema in a transfusion-related acute lung injury (TRALI) mouse model. We then tested whether disulfiram could confer protection in the context of SARS-CoV-2 infection, as NETs are elevated in patients with severe COVID-19. In SARS-CoV-2–infected golden hamsters, disulfiram reduced NETs and perivascular fibrosis in the lungs, and it downregulated innate immune and complement/coagulation pathways, suggesting that it could be beneficial for patients with COVID-19. In conclusion, an existing FDA-approved drug can block NET formation and improve disease course in 2 rodent models of lung injury for which treatment options are limited.

Authors

Jose M. Adrover, Lucia Carrau, Juliane Daßler-Plenker, Yaron Bram, Vasuretha Chandar, Sean Houghton, David Redmond, Joseph R. Merrill, Margaret Shevik, Benjamin R. tenOever, Scott K. Lyons, Robert E. Schwartz, Mikala Egeblad

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Figure 1

Disulfiram blocks neutrophil extracellular trap (NET) formation, and TRALI is a model of NET-driven lung injury.

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Disulfiram blocks neutrophil extracellular trap (NET) formation, and TRA...
(A) Ex vivo NET formation assay of mouse neutrophils sorted by FACS, unstimulated/untreated (NT) or stimulated with 100 nM of PMA or PMA + 10 μM disulfiram (PMA+DS). NET frequency (NET counts normalized to neutrophil counts, with NETs defined by the triple colocalization events of DNA, myeloperoxidase [MPO], and citrullinated histone H3 [citH3]). n = 18 random fields from 4 mice per condition. (B) Ex vivo NET formation assay of human neutrophils from RBC-lysed blood, unstimulated or stimulated with PMA or PMA + 10 μM disulfiram (PMA+DS). n = 18 random fields from 3 healthy donors per condition. Scale bar: 50 μm. (C) Experimental design used to induce TRALI. (D) Absolute number of neutrophils (PMNs) infiltrated to the lung upon TRALI, determined by flow cytometry. n = 4 mice per group. (E) Protein content in the bronchoalveolar lavage fluid (BALF) as a measure of endothelial integrity. n = 6 control and 8 TRALI mice. (F) Representative longitudinal CT scan of a mouse subjected to TRALI showing edema formation over time (representative of CT scans from 11 independent mice). (G) Whole mount tissue clearing images (left, showing CD31 and NETs, defined as the triple colocalization channel of DNA, MPO, and citH3). Quantification (right) of NETs in the lungs of mice 40 minutes after TRALI induction or in mice treated only with LPS. n = 6 lungs per group. Scale bar: 100 μm. (H) Survival of mice after TRALI induction and treatment with Cl-amidine, a PAD4 inhibitor able to block NET formation, or vehicle. n = 27 (vehicle) and 24 (Cl-amidine) mice. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; as determined by unpaired 2-tailed t test analysis (D, E, and G), 1-way ANOVA with Tukey’s multiple comparison test (A and B), or log-rank (Mantel-Cox) test (H). Arrows indicate NETs in A, B, and G.