Inhibition of peptidylarginine deiminase alleviates LPS-induced pulmonary dysfunction and improves survival in a mouse model of lethal endotoxemia

Abstract

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.

Citrullinated Histone H3 as a Therapeutic Target for Endotoxic Shock in Mice

“…After validation of the CitH3 mAb (4 Cit), we evaluated whether the new antibody could improve survival in a mouse model of LPS-induced endotoxic shock. It has been shown previously that CitH3 appears within 30 min of endotoxic shock (18,23). In the present study, mice were intravenously administrated anti-CitH3 mAb immediately after LPS injection.…”

“…Unlike histones, the role of citrullinated histones in diseases remains largely unknown. CitH3 release has been detected in blood and peritoneal fluid in sepsis (17,18,23); however, the effects of CitH3 production and its pathophysiologic roles in sepsis are unclear.…”

“…Administration of a pan-PAD Inhibitor, Cl-amidine, improves survival in both lethal and sub-lethal models of murine sepsis, increases bacterial clearance, and ameliorates thymus and bone marrow atrophy (16,17). YW3-56, another pan-PAD inhibitor, has been shown to increase survival in mice with lipopolysaccharide (LPS)induced endotoxic shock (18). Administration of DNase has been demonstrated to markedly reduce cell-free DNA and improve outcomes in both Escherichia coli-induced and CLP-induced sepsis (19,20).…”

“…Citrullinated histone H3 (CitH3) has been shown to be highly involved in the process of NETosis (4,6,18,22). As such, CitH3 is considered a good biomarker for the diagnosis of endotoxic shock due to its early appearance (as early as 30 min) in the blood, long half-life, high specificity, and response to treatment (23).…”

Site-specific opening of the blood-brain barrier by extracellular histones

“…In addition, arginine residues on histones H3 and H4 may undergo deimination by peptidyl deiminase 4 (PAD4) in neutrophils, converting arginine to citrulline which facilitates chromatin decondensation during NET formation [ 51 ]. Moreover, citrullinated histones are found in the extracellular space as the most abundant protein component of NETs [ 52 , 53 ]. Previous studies from our laboratory demonstrated that recombinant citrullinated histone 3 (CitH3) caused plasma leakage in the splanchnic microcirculation and promoted endothelial AJ opening via contractile cytoskeleton reorganization [ 54 ].…”