LAUSR

Learning Objectives: Neutrophil extracellular traps (NETs) are important for local defense against invading pathogens. Excessive systemic NET formation, however, could induced coagulopathy, microcirculatory dysfunction, and organ damage. Consistent with this, NETs have been reported to promote intravascular coagulation during sepsis. Thrombomodulin (TM) plays a pivotal role as an anti-coagulant in vascular endothelial cells. Therefore, recombinant human thrombomodulin (rTM) has been used in Japan to treat coagulopathy induced by sepsis. We have reported that rTM reduces lipopolysaccharide (LPS)-induced NET formation in human peripheral blood in vitro. Consequently, we hypothesized that rTM reduces NET formation in the organs of LPS-induced septic mice. Methods: First, we established an LPS-induced model of sepsis in mice which were later treated with rTM. To induce sepsis, the mice were injected intraperitoneally (i.p.) with 10 mg/kg LPS, which was sufficient to kill 50% of the mice after 72 h. Either rTM (rTM group: 6 mg/kg/day, i.p.) or saline (non rTM group) was administered 1 h after LPS injection. In another experiment, mice were sacrificed at 8 h after injection, and the lungs, liver, and kidneys were collected. NET components were identified through immunohistochemistry (IHC) using an anti-myeloperoxidase antibody and an anti-histone H2A antibody. NET formation was visualized through scanning electron microscopy (SEM). Results: Through IHC, we found that extracellular histones aggregated in the lungs, liver, and kidneys of mice in non rTM group. Compared with non rTM group mice, rTM group mice released fewer extracellular histones within the lungs and the liver in response to LPS. Tissue sample analyzed through IHC were also examined using SEM. NET formation was demonstrated to colocalize with extracellular histone aggregation as observed through IHC. Conclusions: rTM reduced the amount of extracellular histone and inhibited NET formation in the lungs and the liver of LPS-induced septic mice. Thus, rTM could protect organs from damage caused by the reduction of extracellular histones that accompanies NET formation.