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Peptidylarginine deiminases (PADs) are a family of calcium-dependent enzymes that are involved in a variety of human disorders, including cancer and autoimmune diseases. Although targeting PAD4 has shown no benefit in sepsis, the role of PAD2 remains unknown. Here, we report that PAD2 is engaged in sepsis and sepsis-induced acute lung injury in both human patients and mice. Pad2-/- or selective inhibition of PAD2 by a small molecule inhibitor increased survival and improved overall outcomes in mouse models of sepsis. Pad2 deficiency decreased neutrophil extracellular trap (NET) formation. Importantly, Pad2 deficiency inhibited Caspase-11-dependent pyroptosis in vivo and in vitro. Suppression of PAD2 expression reduced inflammation and increased macrophage bactericidal activity. In contrast to Pad2-/-, Pad4 deficiency enhanced activation of Caspase-11-dependent pyroptosis in BM-derived macrophages and displayed no survival improvement in a mouse sepsis model. Collectively, our findings highlight the potential of PAD2 as an indicative marker and therapeutic target for sepsis.

Background: The peptidylarginine deiminase (PAD) family converts arginine into citrulline through protein citrullination. PAD2 and PAD4 inhibitors can improve survival in hemorrhagic shock (HS). However, the impact of isoform-specific PAD inhibition in improving survival has not been studied. In this study, we utilize selective Pad2 knockout mice to elucidate loss of function of PAD2 leads to pro-survival effect in HS., Methods: HS: Pad2 and wild-type (WT) mice (n = 5/group) were subjected to lethal HS (55% volume hemorrhage). Survival was monitored over 7 days. Myocardial infarction (MI): Pad2 and WT mice (n = 9/group) were subjected to MI by permanent LAD ligation to examine the effect of ischemia on the heart. After 24 h cardiac function and infarct size were measured., Results: HS: Pad2 mice demonstrated 100% survival compared with 0% for WT mice (P = 0.002). In a sub-lethal HS model, cardiac β-catenin levels were higher in Pad2 compared with WT after 24 h. MI: WT mice demonstrated larger MI (75%) compared with Pad2 (60%) (P < 0.05). Pad2 had significantly higher ejection fraction and fractional shortening compared with WT (P < 0.05)., Conclusions: Pad2 improves survival in lethal HS. Possible mechanisms by which loss of PAD2 function improves survival include the activation of cell survival pathways, improved tolerance of cardiac ischemia, and improved cardiac function during ischemia. PAD2 is promising as a future therapeutic target for the treatment of HS and cardiac ischemia.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Hemorrhagic shock (HS) followed by an infection ("second hit") can lead to severe systemic inflammatory response and multiple-organ failure. Studies have shown that resuscitation with hypertonic saline (HTS) can blunt the inflammatory response. We demonstrated that large doses of valproic acid (VPA, 300 mg/kg), a histone deacetylase inhibitor, improves survival in a rodent two-hit model (HS followed by cecal ligation and puncture [CLP]). In the present study, we examined whether combination of HTS with VPA would allow us to achieve survival advantage at a lower dose of VPA (200 mg/kg)., Methods: Male Sprague-Dawley rats were subjected to HS (50% blood loss) and randomized into five groups (n = 7-8 per group) as follows: (1) isotonic sodium chloride solution (ISCS), (2) 7.5% saline, (3) VPA, (4) ISCS + VPA, and (5) HTS + VPA. After 24 hours, they underwent CLP, followed by the same doses of ISCS, HTS, and/or VPA and were monitored for 10 days. In a parallel experiment, blood, peritoneal irrigation fluid and lung homogenate were subjected to enzyme-linked immunosorbent assay 3 hours and 24 hours after CLP to measure myeloperoxidase activity and proinflammatory cytokines tumor necrosis factor α and interleukin 1β levels. Western blotting was performed to investigate the expression of pentraxin 3 protein in the lung homogenate at 24 hours after CLP. Hematoxylin and eosin staining of lungs at the 24 hours were performed to quantify the degree of acute lung injury., Results: HTS + VPA treatment significantly improved survival (87.5%), compared with the other groups (14.3%; p < 0.05), while attenuating peritoneal myeloperoxidase levels and proinflammatory cytokine tumor necrosis factor α and interleukin 1β levels in the serum, peritoneal cavity, and lung. The degree of acute lung injury and expression of pentraxin 3 in the lung were significantly reduced in the HTS + VPA group., Conclusion: This is the first study to show that VPA and HTS can work synergistically to attenuate inflammation and improve survival in a lethal two-hit model.