Current biomarkers for sepsis are limited by their non-specificity, short half-life, and insensitive response to therapy. Recently, we have demonstrated that citrullinated histone H3(CitH3) is released into the blood from neutrophil extracellular traps(NETs) in response to severe infection, and CitH3 may be a potential biomarker for sepsis. In the present study, we found that NET components were released in mouse models of both lipopolysaccharide(LPS)-induced shock (LPSS) and hemorrhagic shock (HS). To further quantify CitH3 in the NETs, we established a CitH3 specific enzyme-linked immunosorbent assay. Circulating CitH3 was found to be elevated only in LPSS but not in HS. Importantly, blood CitH3 was detected 30 minutes after LPS insult, and remained elevated for 24 hours (period of the highest mortality). Treatment of endotoxic mice with YW3-56, a peptidylarginine deiminase-2/4 inhibitor, significantly diminished levels of CitH3 in the blood. Interleukin-1β did not respond to LPS early, and interleukin-1β and interleukin-6 fluctuated although they responded to treatment. Procalcitonin reacted to LPS insult late. Compared to CitH3, these biomarkers were non-specifically induced in LPSS and HS. Collectively, our results demonstrate that YW3-56 protects animals from LPSS, and CitH3 is a reliable biomarker due to its early appearance, specificity, duration, and response to therapeutic intervention.

Sepsis results in millions of deaths every year, with acute lung injury (ALI) being one of the leading causes of mortality in septic patients. As neutrophil extracellular traps (NETs) are abundant in sepsis, neutralizing components of NETs may be a useful strategy to improve outcomes of sepsis. Citrullinated histone H3 (CitH3) has been recently shown to be involved in the NET formation. In this study, we demonstrate that CitH3 damages human umbilical vein endothelial cells (HUVECs) and potentiates NET formation through a positive feedback mechanism. We developed a novel CitH3 monoclonal antibody to target peptidylarginine deiminase (PAD) 2 and PAD 4 generated CitH3. In a mouse model of lethal lipopolysaccharide (LPS) induced shock, neutralizing CitH3 with the newly developed anti-CitH3 monoclonal antibody attenuates inflammatory responses, ameliorates ALI, and improves survival. Our study suggests that effectively blocking circulating CitH3 might be a potential therapeutic method for the treatment of endotoxemia.

Immune cell death caused by neutrophil extracellular traps (NETs), referred to as NETosis, can contribute to the pathogenesis of endotoxemia and organ damage. Although the mechanisms by which infection induces NETosis and how that leads to organ dysfunction remain largely unknown, NET formation is often found following citrullination of histone H3 (CitH3) by peptidylarginine deiminase (PAD). We hypothesized that lipopolysaccharide (LPS)-induced activation of PAD and subsequent CitH3-mediated NET formation increases endothelial permeability and pulmonary dysfunction and, therefore, that inhibition of PAD can mitigate damage and improve survival in lethal endotoxemia. Here, we showed that treatment with YW3-56, a PAD2/PAD4 inhibitor, significantly diminished PAD activation, blocked LPS-induced pulmonary vascular leakage, alleviated acute lung injury, and improved survival in a mouse model of lethal LPS-induced endotoxemia. We found CitH3 in the bloodstream 30 min after intraperitoneal injection of LPS (35 mg/kg) into mice. Additionally, CitH3 production was induced in cultured neutrophils exposed to LPS, and NETs derived from these LPS-treated neutrophils increased the permeability of endothelial cells. However, YW3-56 reduced CitH3 production and NET formation by neutrophils following LPS exposure. Moreover, treatment with YW3-56 decreased the levels of circulating CitH3 and abolished neutrophil activation and NET formation in the lungs of mice with endotoxemia. These data suggest a novel mechanism by which PAD-NET-CitH3 can play a pivotal role in pulmonary vascular dysfunction and the pathogenesis of lethal endotoxemia.