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Sepsis is a life-threatening organ dysfunction caused by dysregulated host response to infection that often results in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). An emerging mechanism of sepsis-induced ARDS involves neutrophils/macrophages undergoing cell death, releasing nuclear histones to cause tissue damage that exacerbates pulmonary injury. While published studies focus on unmodified histones, little is known about the role of citrullinated histone H3 (CitH3) in the pathogenesis of sepsis and ALI. In this study, we found that levels of CitH3 were elevated in the patients with sepsis-induced ARDS and correlated to PaO2/FiO2 in septic patients. Systematic administration of CitH3 peptide in mice provoked Caspase-1 activation in the lung tissue and caused ALI. Neutralization of CitH3 with monoclonal antibody improved survival and attenuated ALI in a mouse sepsis model. Furthermore, we demonstrated that CitH3 induces ALI through activating Caspase-1 dependent inflammasome in bone marrow derived macrophages and bone marrow derived dendritic cells. Our study suggests that CitH3 is an important mediator of inflammation and mortality during sepsis-induced ALI. [ABSTRACT FROM AUTHOR]

Production of innate and adaptive immune cells from hematopoietic stem cells, and maturation of T lymphocytes are effective immune responses to fight severe microbial infection. In sepsis, this emergency myelopoiesis is damaged, leading to failure of bacterial clearance, and excessive stress-induced steroids cause immature T-lymphocyte apoptosis in thymus. We recently found that Cl-amidine, a peptidylarginine deiminase (PAD) inhibitor, improves survival in a mouse model of cecal ligation and puncture (CLP)-induced septic shock. In the present study we investigated how Cl-amidine promotes survival, focusing on protective effects of Cl-amidine on immune response. We confirmed survival-improving effect of Cl-amidine and are the first to explore the role of Cl-amidine in immune response. CLP caused bone marrow (BM) and thymus atrophy, decreased innate immune cells in BM. CLP increased levels of cytokines (IL-1β, IL-6, and TNF-α) and bacteria load in blood/liver. In primary splenocyte culture, lipopolysaccharide increased TNF-α production. In contrast, Cl-amidine attenuated these CLP and lipopolysaccharide-induced alterations. Moreover, Cl-amidine increased circulating monocytes. Collectively, our results demonstrate Cl-amidine plays protective roles by significantly decreasing BM and thymus atrophy, restoring innate immune cells in BM, increasing blood monocytes and blood/liver bacteria clearance, and attenuating pro-inflammatory cytokine production in a murine model of lethal sepsis.

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.

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., (Copyright © 2020 Deng, Pan, Alam, Liang, Wu, Liu, Mor-Vaknin, Duan, Williams, Tian, Zhang and Li.)

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.